Full text of "The Biological bulletin"

See other formats

', ;

C oj&ts? I*/

THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

E. G. CONKLIN, Princeton University
E. N. HARVEY, Princeton University
SELIG HECHT, Columbia University
LEIGH HOADLEY, Harvard University
L. IRVING, Swarthmore College
M. H. JACOBS, University of Pennsylvania

FRANK R. LILLIE, University of Chicago
CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
A. C. REDFIELD, Harvard University

H. S. JENNINGS, Johns Hopkins University F. SCHRADER, Columbia University

H. B. STEINBACH, Washington University
Managing Editor

VOLUME 84

FEBRUARY TO JUNE, 1943

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE &, LEMON STS.

LANCASTER, PA.

THE BIOLOGICAL BULLETIN is issued six times a year at the
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn-
sylvania.

Subscriptions and similar matter should be addressed to The
Biological Bulletin, Marine Biological Laboratory, Woods Hole,
Massachusetts. Agent for Great Britain : Wheldon and Wesley,
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
W. C. 2. Single numbers, $1.75. Subscription per volume (three
issues), $4.50.

Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Massachusetts, between July 1 and October 1, and to the Depart-
ment of Zoology, Washington University, St. Louis, Missouri,
during the remainder of the year.

Entered as second-class matter May 17, 1930, at the post office at Lancaster,
Pa., under the Act of August 24, 1912.

LANCASTER PRESS, INC., LANCASTER, PA.

CONTENTS

No. 1. FEBRUARY, 1943

PAGE

LOEB, LEO, H. D. KING AND H. T. BLUMENTHAL

Transplantation and Individuality Differentials in Inbred Strains of Rats 1

BODENSTEIN, DIETRICH

Factors Influencing Growth and Metamorphosis of the Salivary Gland

in Drosophila 13

BODENSTEIN, DIETRICH

Hormones and Tissue Competence in the Development of Drosophila . . 34

GOODCHILD, CHAUNCEY G.

The Life-History of Phyllodistomum Solidum Rankin, 1937, with Obser-
vations on the Morphology, Development, and Taxonomy of the
Gorgoderinae (Trematoda) 59

MENDOZA, GUILLERMO

The Reproductive Cycle of the Viviparous Teleost, Neotoca Bilineata,

a Member of the Family Goodeidae. IV. The Germinal Tissue 87

SCHALLEK, WILLIAM

The Reaction of Certain Crustacea to Direct and to Diffuse Light .... 98

JOHNSON, MARTIN W.

Studies on the Life History of the Marine Annelid Nereis Vexillosa. . . . 106

ROBINSON, EDWIN J., AND ROBERTS RUGH

The Reproductive Processes of the Fish, Oryzias Latipes 115

No. 2. APRIL, 1943

DAY, M. F.

The Function of the Corpus Allatum in Muscoid Diptera 127

ROMANOFF, ALEXIS L., AND FREDERICK W. HAYWARD

Changes in Volume and Physical Properties of Allantoic and Amniotic
Fluids under Normal and Extreme Temperatures 141

VON BRAND, THEODOR

Physiological Observations upon a Larval Eustrongylides. IV. In-
fluence of Temperature, pH and Inorganic Ions upon the Oxygen Con-
sumption 148

HUNGATE, R. E.

Further Experiments on Cellulose Digestion by the Protozoa in the

Rumen of Cattle 157

BROOKS, MATILDA MOLDENHAUER

Methylene Blue, Potassium Cyanide and Carbon Monoxide as Indi-
cators for Studying the Oxidation-Reduction Potentials of Developing
Marine Eggs 1 64

m 56666

iv CONTENTS

PAGE

COE, WESLEY R.

Development of the Primary Gonads and Differentiation of Sexuality in
Teredo Navalis and other Pelecypod Mollusks 178

SUMNER, F. B., AND PETER DOUDOROFF

An Improved Method of Assaying Melanin in Fishes 187

SUMNER, F. B.

A Further Report upon the Effects of Visual Environment on the
Melanin Content of Fishes 195

No. 3. JUNE, 1943

ROOT, R. W., AND LAURENCE IRVING

The Effect of Carbon Dioxide and Lactic Acid on the Oxygen-Com-
bining Power of Whole and Hemolyzed Blood of the Marine Fish,
Tautoga Onitis (Linn.) 207

BROOKS, S. C.

Intake and Loss of Ions by Living Cells. I. Eggs and Larvae of Arbacia
Punctulata and Asterias Forbesi Exposed to Phosphate and Sodium Ions 213

BROOKS, S. C.

Intake and Loss of Ions by Living Cells. II. Early Changes of Phos-
phate Content of Fundulus Eggs 226

EARNER, DONALD S.

Biliary Amylase in the Domestic Fowl 240

CORNMAN, IVOR

Acceleration of Cleavage of Arbacia Eggs by Hypotonic Sea Water. . . . 244

EVANS, HIRAM J.

The Independent Differentiation of the Sensory Areas of the Avian
Inner Ear 252

WILLIAMS, CARROLL M., LEWIS A. BARNESS AND WILBUR H. SAWYER
The Utilization of Glycogen by Flies During Flight and Some Aspects of
the Physiological Ageing of Drosophila 263

PARKER, G. H.

Methods of Estimating the Effects of Melanophore Changes on Animal
Coloration. 273

THE LIBRARY STAFF

MRS. PRISCILLA B. MONTGOMERY, LIBRARIAN
DEBORAH LAWRENCE MARY A. ROHAN S. MABELL THOMBS

FOREWORD

The Library of The Marine Biological Laboratory is represented in this serial
list, not only as four floors of journals and books reporting most of the scientific
efforts of the world, but as a pleasant place in which to work, with unlimited facili-
ties at one's fingertips. It is open to the use of everyone in attendance at the Lab-
oratory and a limited number of readers may be accommodated with desk space
upon approval of the Director, and payment of a fee.

A staff of librarians is in attendance, but it is felt that maximum value is
obtained from the Library by free access to the stacks. Numerous tables give ample
opportunity for work in close proximity to the collections. A large reading room
with extensive racks and shelves for current journals, encyclopedias, maps, indexes
and new books is provided with unlimited reading space.

The journals are arranged, beginning with the top floor, alphabetically as they
appear in this list. Here they are listed under every possible title, and cross-
referenced, and it is hoped that this revised edition of the library holdings will
prove invaluable to investigators.

On the first level of the stacks a very large collection of separata, reprints and
duplicate volumes is maintained. This is arranged in alphabetical order by authors,
and these reprints may be charged out for use in the individual laboratory rooms
on the cards provided at the main desk.

Reports of scientific expeditions published independently, and volumes of non-
serial literature are shelved on the second stack level near the entrance from the
reading room. These may be charged out in the same manner as reprints.

The Library has previously co-operated in granting Inter-library loans, but it
is proposed now to reduce this as much as possible. There has been installed a
microfilm apparatus suitable for copying from volumes of the usual quarto and
octavo sizes. Copies of publications will be made upon the request of qualified
individuals, at the lowest practicable rates. Information may be secured from the
Librarian, and orders should be directed to her.

CHARLES PACKARD, Director of the Laboratory
PRISCILLA B. MONTGOMERY, Librarian

It is with deep regret that the Biological Bulletin

records the death, on January 4, 1943, of

Dr. Gary Nathan Calkins, a member of

the Editorial Board since 1927.

[!±MU8RARY

Vol. 84, No. 1 NTA/ ^^^TFebruary, 1943

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

TRANSPLANTATION AND INDIVIDUALITY DIFFERENTIALS
IN INBRED STRAINS OF RATS

LEO LOEB, H. D. KING, AND H. T. BLUMENTHAL

(From the Laboratory of Research Pathology,* Oscar Johnson Institute, Washington University
School of Medicine, St. Louis, and from the Wistar Institute, Philadelphia')

In two previous papers (1927, 1931), Loeb and King have reported on the
individuality differentials of rats inbred in the Wistar Institute for a considerable
number of years by Dr. Helen Dean King. In this paper we shall report on a
third series of experiments, carried out during the last two or three years, in
which rats from inbred generations 102 to 106 were used. There was added,
also, a small series of experiments with rats from inbred generations 91 and 92.
However, in order to evaluate the changes which have taken place in the course
of the continued inbreeding it will be necessary to give also the main data obtained
in the first and second series of transplantations made with the earlier inbred
generations. A comparison can be readily made if instead of discussing isolated
data, we group experiments of a similar kind together by adopting a system of
grades and determining the average grades obtained in each case. We have
already adopted, in previous papers, such a system of approximately quantitative
grading of the findings, and we have discussed these grades more fully on former
occasions. Here, the principles underlying the choice of these grades will be
only briefly explained. To simplify this task, we shall consider mainly the
results obtained in transplantation of thyroid, cartilage, fat tissue, and also of
the other organs often associated with these, namely, parathyroid, bone, bone
marrow, striated muscle. It will not be necessary at this time to enter into a
discussion of the corresponding reactions shown in all types of tissues used in
our grafting experiments. The grades used range between 1 and 3+ (3.25);
3+ and 3 are the grades characteristic of autogenous transplantations. In this
latter, the tissue — in particular, thyroid gland— is well preserved, and while at
first there may be some irregularities in the structure of the graft, it gradually
assumes the structure of the normal organ. Marked lymphocytic infiltration is
lacking, but at early periods some very small collections of lymphocytes may be

* The work from the Laboratory of Research Pathology of Washington University School of
Medicine was carried out previous to June 1, 1941, when this laboratory was discontinued. The
experiments mentioned in this paper were done with the aid of a grant from The International
Cancer Research Foundation.

LOEB, KING AND BLUMENTHAL

seen; subsequently, these usually disappear. Likewise, the connective tissue
ingrowth is restricted and an invasion of the fat tissue by small vacuolated cells
and by fibrous tissue is absent. Grades 3- (2.75) and 2+ (2.25) are given if
the tissues are, on the whole, well preserved but if a reaction is definitely notice-
able. Various degrees of lymphocytic infiltration and a somewhat increased
activity of the connective tissue may appear and cause a limited injury to the
transplant. Such reactions may be seen when donor and host are related. If
these reactions are more marked and lead to a partial destruction of the transplant,
the grade 2 is given; this indicates a somewhat greater strangeness of the -indi
viduality differentials. In typical, more severe homoio-reactions the grades
range between 2- (1.75) and 1. Grade 2— is given if the thyroid gland is
severely invaded by fibrous tissue and a considerable part of the acinar tissue
has been destroyed ; but at least one-half of the organ has escaped destruction at
the time of examination, which in most cases is between 20 and 30 days following
transplantation. There is, in these cases, a definite lymphocytic infiltration,
provided injury to the tissue has not led to a marked diminution in the effective-
ness of the individuality differential substances. Grade 1+ (1.25) signifies the
survival of only a small part of the thyroid gland. The reaction in the fat
tissue is very severe. Grade 1 is applied to results in experiments in rats and
guinea pigs in which the thyroid has been entirely destroyed and the fat tissue
is largely replaced by fibrous tissue.

The changes in other organs or tissues, on the whole, correspond to those
taking place in thyroid and fat tissue and this correspondence makes possible the
grading of the results obtained in all tissues from the same donor and transplanted
into the same host. For instance, if ovaries are transplanted together with
thyroid, cartilage and fat tissue, we find corresponding intensities of reaction
and injuries of the various constituent structures of this organ. In the most
favorable cases, large follicles, and even corpora lutea, are found. In somewhat
less favorable cases only medium-sized or small follicles develop; a still more
unfavorable reaction is indicated if merely primordial follicles survive, without
undergoing further growth processes. If the reaction is still more severe, no
follicles are seen, but merely a cyst and ducts of the germinal epithelium, medul-
lary ducts, spindle-cell connective tissue, and interstitial gland tissue, together
with necrotic remnants of the transplanted ovary. At last, only some interstitial
gland may be found, or even this tissue may be lacking and necrotic material
with fibrous tissue may be all that is left. However, under all circumstances it
is necessary to make allowances for the occurrence of accidental injuries to the
transplant. This can be recognized in many instances, but even if it should be
difficult, errors in the appraisal of the reaction can be avoided by making a series
of experiments instead of a single one and then relying on an evaluation of the
total results. While this method of grading can claim only approximate exact-
ness, still it is very helpful in comparing the results in the interaction of different
types of individuality differentials and in making possible a concise and suffi-
ciently accurate expression of a large number of otherwise separate data.

The principal results obtained in the first series of experiments (1927) are
as follows:

INDIVIDUALITY DIFFERENTIALS IN RATS 3

SERIES I
Inbred Families A and B

In subseries 1, generations 37 and 38, 40 and 41, in subseries 2, generations 46 and 47
were used.

Subseries 1, family A (different litters): grade 1.96 (20 rats)
Subseries 2, family A ( " ): " 1.12 (4 rats)

Total family A ( " ): " 1.82 (24 rats)

Subseries 1, family B (different litters): grade 2.1 (22 rats)
Subseries 2, family B ( " " ): " 1.55 (5 rats)

Total family B ( " ): " 1.92 (27 rats)

Families A and B combined: grade 1.87 (51 rats)

Subseries 1, family A to family B: grade 1.74 (11 rats)
Subseries 2, family A to family B: " 1.57 (7 rats)
Total family A to family B: " 1.67 (18 rats)

Subseries 1, family B to family A: 1.61 (16 rats)

Subseries 2, family B to family A: " 1.33 (3 rats)
Total family B to family A: " 1.57 (19 rats)

Total inter-family A and B transplantations: Combined grade 1.62 (37 rats)

Exchange of tissues between members of the same litters (brothers and sisters]

in families A and B

Subseries 1, family B: grade 2.87 (17 rats) (40th and 41st generations)
Subseries 2, family A: grade 2 (6 rats) (42nd generation)
Total families A and B (40-42 generations): grade 2.5 (23 rats)

Subseries 3, family A: grade 1.37 (6 rats) (46th and 47th generations)

Subseries 4, family B: grade 1.78 (7 rats)

Total families A and B (46-47 generations): grade 1.59 (13 rats)

Total families A and B (members of same litters): grade 2.26 (36 rats)
2 subseries of family A: grade 1.68 (12 rats)
2 subseries of family B: " 2.55 (24 rats)

These data are summarized in Table I.

In evaluating these grades, we must attach greater importance to those
experiments in which the figures represent the averages of a large number of
rats than to those in which the figures are based on a relatively small number;
in the latter, variable factors of an accidental nature may preponderate. This
is a point to be considered if there is some discrepancy between the figures in
related series of experiments. However, on the whole the figures here agree
very well with each other.

The transfer of tissues from members of family A to members of family B,
and vice versa, should correspond about to ordinary homoiogenous transplanta-
tions. The average grade is, here, 1.62, which is perhaps very slightly better

LOEB, KING AND BLUMENTHAL
TABLE I (Series I)

Donor and host Grades Combined grades

Family A to family A 1.82 (24 rats)]

(different litters)

1.87 (51 rats)
Family B to family B 1.92 (27 rats) j

(different litters)

Family A to family A 1.68 (12 rats) ]

(litter mates)

2.26 (36 rats)
Family B to family B 2.55 (24 rats) j

(litter mates)

Family A to family B 1.67 (18 rats)]

1.62 (37 rats)
Family B to family A 1.57 (19 rats)

than might be expected. This grade may be compared with that of transplanta-
tions between members of family A: 1.82, and between members of family B:
1.92, or an average grade of both kinds of intra-family transplantations: 1.87.
There is a slight improvement, a slight diminution in the severity of the reactions
in these intra-family transplantations as compared with the inter-family trans-
plantations. Still somewhat higher is the average grade of transplantations
between litter mates (brothers and sisters) in family A as well as in family B.
Here the total average grade is 2.26 (in the larger series of experiments in family
B the average grade is 2.55 and in the smaller series in family A it is 1.68). We
may then conclude that as a result of close inbreeding for 37 to 47 generations
in families A and B, only a very slight progress towards a homozygous condition
has been accomplished.

Series II. In this series of transplantations, rats inbred for 60 to 67 genera-
tions were used. The following is a summary of the principal results in this
series.

If we compare the grades in the first and second series, we find that trans-
plantations between different litters in family A, and again in family B, show no
improvement in series II over the corresponding grades in series I; on the con-
trary, the average grades in series II happen to be somewhat lower. This may
perhaps have been due to a smaller number of brother-sister matings which
different litters in series II had in common. On the other hand, in the latter
series the grades between litter mates in family A, as well as in family B, are
not only better than the grades obtained in transplantations between different
litters, but they are also better than the transplantations between litter mates
in series I. If the results of transplantations between brothers and sisters are
compared in inbred rats, the variable factor of the branching-off of certain lines
at different levels of brother-sister matings is eliminated and the improvement
in series II in this type of grafts may thus indicate a certain progress towards a
homozygous condition, although this cannot be maintained with certainty.
Exchange of tissues between families A and B (inter-family transplantations)

INDIVIDUALITY DIFFERENTIALS IN RATS
TABLE II (Series II)

Donor and host

Grades

Combined grades

Family A to family A
(different litters)

Family B to family B
(different litters)

Family A to family A
(litter mates)

Family B to family B
(litter mates)

Family A to family B, or
family B to family A

Homoiotransplantation in
non-inbred families

Hybrids (AxB)F4 (or F5)

to hybrids (AxB)F4

(different litters)

Hybrids (AxB)F4 (or F5)
to hybrids (AxB)F4
(litter mates)

Family A or family B to
hybrids (AxB)F4

Hybrids (AxB)F4 to
family A or family B

1.16 (16 rats)
1.65 (33 rats)J
2.60 (17 rats)
2.81 (19 rats) :
1.37 (32 rats)
1.36

1.29 (12 rats)

1.80 (26 rats)
1.50 (13 rats)
1.39 (13 rats)

1.49 (49 rats)

2.71 (36 rats)

corresponds to a severe homoio-reaction, as might be expected. Similar are the
grades of transplantations between hybrids (AxB)F4. These grades are im-
proved to a certain degree if hybrids which are litter mates are used. But in
these transplantations the results are not so good as in brother-to-brother trans-
plantations in family A or in family B. Such a difference might be expected,
because in hybrids there is a greater chance for unlike genes to accumulate in
brothers when both A and B may contribute to the genes of the fertilized germ
cells.

Transplantations from parent to hybrid give somewhat better results than
the reciprocal transplantations, but both elicit severe homoiogenous reactions as
an indication that a homozygous genetic constitution has not yet been reached
in either family A or family B. It is of interest to note that in both series I
and II the grades in family B are higher than in family A. On the whole, then,
while it is possible that a further slight progress towards a homozygous condition
in family A and in family B has been made in continued propagation by brother-
sister matings in the interval between the 37th to 47th generations and the 60th
to 67th generations, this progress, if present at all, is not very marked.

O LOEB, KING AND BLUMENTHAL

Series III. This is the series of experiments which has been carried out
during the last few years and it will be discussed, therefore, in somewhat more
detail. A small number of experiments were made with rats of the 91st and
92nd generations. These were young animals and at the end of the experiments,
weighed between 125 and 140 grams. Transplantations into three litter mates,
serving as hosts, gave the grade 2.95 ; very closely approaching the results obtained
in autogenous transplantations. In seven transplantations from family B to
family A the grade was 1.48, if the examination took place after 20 days, and
it was 1.84 in 7 rats after 12 days. The grade after 20 days was very similar to
the grades obtained in the corresponding transplantations in series I and II.
No essential changes have taken place in this type of grafting between two
different families in the course of long-continued brother-sister matings. The
grades for transplantations between litter mates are somewhat better than in the
corresponding ones in series II, and they are distinctly better than in series I,
indicating further progress to a homozygous condition after 91 to 92 continuous
brother-sister matings. However, in evaluating the grades obtained in trans-
plantations between litter mates, the small number of experiments, of which
this figure represents the average, has to be considered.

In generations intermediate between the 92nd and 104th generations, family
B died out and from then on only family A and hybrids (AxB)F4 or (BxA)F4
were available for experiments. A number of transplantations were carried out
with generations 104 to 106 of family A and with the hybrids. Younger rats,
one month and one or two weeks of age, or somewhat older ones, four and a half
to five and a half months of age and weighing between 185 and 200 grams at the
beginning of the experiments were used for this purpose. Examination as a
rule took place after 20 days. Usually three pieces, namely, thyroid, cartilage
with fat tissue, and striated muscle tissue were transplanted, and in some experi-
ments the transplants from one kind of donor were transferred to the right side,
and those from another kind of donor were transplanted to the other side of the
same host.

Group A . Transplantations of organs in young rats

(a) Transplantations between members of different litters in family A:
average grades, 2.81 and 2.83; (6) between litter mates: average grades, 3.15;
3.15; 3.12 and 3.07; (c) from hybrids F4 between families A and B to family A:
average grades, 3.12 and 3.10; (d) between litter mates of hybrids (AxB)F4 or
(BxA)F4: average grade, 3.12 — the same grade was found between hybrids
which belonged to different litters. There is no difference, or only a very slight
one, in the reactions obtained in these various transplantations, the grades
between non-litter mates being somewhat lower than those between litter mates;
but the significance of this difference is not great, because only four transplanta-
tions were carried out between members of different litters. On the other hand,
even the grades for transplantations from hybrids F4 to family A, which might
have been expected to lead to more severe reactions, were as good as those
between litter mates of strain A. We must therefore assume that the young
age of these rats helped to diminish the severity of the reactions, which was also
diminished by the long-continued inbreeding, causing an approximately autog-
enous condition of the individuality differentials in family A and in those

INDIVIDUALITY DIFFERENTIALS IN RATS 7

members of family B which were components of hybrids (AxB)F4. However,
in one experiment the transplantations from hybrids F4 to family A, in accordance
with expectations, gave a somewhat more severe reaction; the average grade
was here slightly below 2. In this case, the host reacted definitely against two
transplants which contained strange B genes; the threshold which determines
an antagonistic reaction had evidently been passed.

Group B. Transplantations in somewhat older rats

These rats varied in age between four and a half and seven and a half months
and belonged to the 102nd, 103rd, or 104th generation. The kind of transplanta-
tions were about the same as in the younger animals. Transplantations were
made (a) betwreen litter mates of family A: grade 3.04 in rats aged 4^ to 5|
months, and grade 2.56 in rats aged 6^ to 1\ months; (b) between different
litters of family A: grade 2.93 (age of rats 4^ to 5| months), and grade 2.4 (age
6^ to 7 months). The grades between litter mates are only very slightly better
than those between members of different litters in family A. These grades are
not as good as those in the younger rats, and also among the older rats the
severity of the reaction increased somewhat with increasing age; transplantations
were also made (c) between hybrids (BxA)F4 and hybrids (A"xB)F4: grade 2.18;
(d) from family A to hybrids (AxB)F4: grade 2.39; (e) from hybrids (AxB)F4
or (BxA)F4 to family A: grade 2.23. The grades in transplantations between
hybrids themselves and between hybrids and members of family A, in accordance
with expectations, are less good than those between members of family A. These
differences come out in the older rats, while they are not present in the corre-
sponding transplantations in younger rats. However, also in the older rats
there are no very sharp differences between these various groups.

We shall now give brief abstracts of our microscopic findings in some of these
transplantations, which may serve as examples. (1) Transplantations in young
rats, family A, inbred for 104 or 105 generations. These rats were 37 to 44 days
old. at the time of transplantation ; examination took place 20 days after trans-
plantation. Pieces from litter mates were transplanted to the right side and
pieces from different litters to the left side of the host. There was no definite
difference between pieces from the two sides. The transplant of striated muscle
tissue was well preserved. There were well-preserved, thin muscle fibers, either
close together or separated by some hyaline tissue. Some thick fibers were
surrounded by hyaline tissue and in certain instances also by strands of fibro-
blasts. Necrotic muscle fibers were, in places, surrounded by foreign-body giant
cells. There were well-developed nuclear chains, but there was less tendency to
the formation of nuclear masses, less crowding, and better cross-striation of
muscle fibers in the rat than in the mouse. On the whole, the multiplication of
nuclei in fibers was moderate. On the average, the least proliferation of muscle
nuclei was found where the thickness of the muscle fibers was greatest and where
the cross-striation was best. Where the muscle fibers were very thin and much
drawn-out, they often resembled fibroblasts. Some muscle fibers might be
changed into a light material, which did not stain with eosin.

The thyroid transplants were well preserved. There was a ring of two to
three layers of acini of medium or small size. The acinar epithelium was flat
cuboidal, or, occasionally, cuboidal in shape; the center consisted of fibrillar-

LOEB, KING AND BLUMENTHAL

cellular connective tissue, in which there were some lymph and blood vessels.
There were also some ducts lined with squamous epithelium, or there were some
epithelial pearls in the center of the transplant, and here, too, fat cells were
found, indicating a favorable result of the transplantation.

Cartilage and fat tissue were well preserved. The strands of fibrous tissue
observed in the fat tissue presumably were caused by the injury sustained during
operation. Otherwise there was no connective tissue ingrowth, no infiltration
with lymphocytes, and no accumulation of small vacuolated cells between the
fat cells. Necrosis occurred in a small piece of cartilage — probably as the result
also of injury at the time of transplantation — and surrounding the latter was a
plate of regenerated perichondral cartilage; but in the piece from the left side of
the host, some necrotic cartilage, without regenerated cartilage plate, was seen.

As to the behavior of the lymphocytes in the muscle tissue from the right side
(litter mates), there were merely a few lymph vessels filled with lymphocytes.
In the piece from the left side (non-litter mates) there were some similar lymph
vessels, and, in addition, some diffuse lymphocytic infiltration. In the thyroid
transplant from the right side the lymphocytic infiltration was very moderate;
there were a few lymph vessels filled with lymphocytes, and at the two poles,
small collections of lymphocytes were seen. In the thyroid transplant from the
left side a dense lymphocytic infiltration was noted in the center of the gland
around squamous cell nests. In places, lymphocytes penetrated between acini
and separated them. Lymph vessels were stuffed with lymphocytes. Also, the
parathyroid showed lymphocytic infiltration. In the cartilage-fat tissue trans-
plants of the right side (litter mates) there were no lymphocytes. On the left
side (non-litter mates) there was some lymphocytic infiltration in the fat tissue
along the living cartilage. This experiment exemplifies well the correspondence
between multiple transplants from the same donor into the same host.

(2) Transplantation in young rats from (BxA)F4 to A (right side), and from
(AxB)F4 to A (left side). In the muscle transplants on the right and left sides
there was some lymphocytic infiltration in various places between groups of
muscle fibers. On the right side the muscle was less well preserved, more necrosis
and more lymphocytic infiltration had taken place, and many nuclear masses
were seen. Around some cloudy muscle fibers, foreign-body giant cells had
formed. In both thyroid transplants there was intense lymphocytic infiltration;
the ring of acini was incomplete; some groups of acini were separated by lympho-
cytic masses. Lymphocytes penetrated also into the colloid, where they disinte-
grated. The masses of lymphocytes had destroyed a number of acini. In the
cartilage-fat tissue, cartilage was well preserved. On the left side there was
more fat tissue replaced by fibrous tissue than on the right side. In both trans-
plants there was some lymphocytic infiltration along the perichondrium and, in
some places, also between the fat cells. In these experiments the reaction is
more severe than in transplants from A to A.

(3) In the somewhat older rats, the grades in some of the transplantations
within the inbred family A approached those of autogenous reactions. There
were occasionally slight lymphocytic infiltrations in the thyroid, in which there
were several layers of acini, but there were no lymphocytes between the acini.
The muscle transplant was well preserved, with much cross-striation and with
nuclear chains and masses. The fibers with medium thickness were best pre-

INDIVIDUALITY DIFFERENTIALS IN RATS 9

served as a rule. Some muscle fibers became hyaline and the preserved fibers
were usually embedded in hyaline tissue. There were occasionally some small
collections of lymphocytes in the transplant. In the cartilage-fat-tissue trans-
plant some thickened septa with a small number of lymphocytes were observed.

(4) (BxA)F4 to (AxB)F4 (older rats — experiment 8). The muscle transplant
showed very intense lymphocytic infiltration throughout, and this destroyed
the fibers. Certain muscle fibers disintegrated, others became hyaline. There
were some nuclear chains. The thyroid ring was incomplete. Single acini might
be separated by connective tissue and lymphocytes. Much lymphocytic infiltra-
tion had taken place and lymph vessels might be stuffed with lymphocytes.
The lymphocytes penetrated between the acinar cells into the colloid. The
greater part of the thyroid had been destroyed. In the cartilage-fat-tissue
transplant some cartilage had become necrotic and dense fibrous tissue surrounded
it. Much fat tissue was destroyed, but there were still some groups of fat cells.
In some places more fat tissue was preserved, with fibrous strands and definite
lymphocytic infiltration in the hyaline connective tissue. Lymph vessels were
stuffed with lymphocytes in the connective tissue and fat tissue.

(5) Experiment 61b. 91st and 92nd generations, family B to family A;
(20 days). In the thyroid the results varied between those in which no thyroid
was left, to those in which the remnant of the transplant consisted mainly of
compressed acini, with much lymphocytic infiltration and some connective tissue
ingrowth. In the cartilage-fat-tissue transplant, much fat tissue, and in some
places almost all of it, was replaced by fibrous tissue. If much lymphocytic
infiltration occurred in the thyroid, usually a certain amount was found also in
the fat tissue. Bone marrow was not preserved, but as a rule some transplanted
muscle fibers were preserved. Some lymphocytic infiltration had taken place
in this transplant.

The principal results obtained in series III are summarized in Table III.

Table III shows that the grades in the younger group of rats are consistently
higher than in the somewhat older rats; this consistency in the difference between
the grades in these two groups makes it very probable that this difference is
significant, even in cases in which the averages are based on a small number of
individuals. The differences are, however, not very great in transplantations
within the inbred family A; they are greater if we compare the transplantations
between hybrids and from hybrids to parent-family A in the old and young groups.
The grades in transplantations from family B to family A, both belonging to
the 91st and 92nd generations, are very low; as was to be expected, they corre-
spond to homoiogenous reactions.

If we compare the grades obtained in series III with those of series II, we
notice that they are much better in the former. In transplantations within
family A, the grades have advanced from 1.16 in series II to about 2.75 in series
III. Among litter mates within family A they have advanced from 2.60 in
series II to about 2.90 in series III. This improvement is not so great as in the
transplantations between non-litter mates, which is due to the fact that there
was already a considerable improvement among the grafts between litter mates
in series II, as compared to those in series I with a corresponding grade of 1.68.
There has been also a great improvement in the grade of transplantations between
hybrids F4 (non-litter mates). The corresponding grades are 1.29 in the II

10 LOEB, KING AND BLUMENTHAL

TABLE III (Series III)

Donor and host Grades Grades

A to A (different litters) 2.82 (7 rats) 2.72 (10 rats)

A to A (litter mates) 3.10 (22 rats, including 2.77 (7 rats)

3 from the 91st and
92nd generations)

(AxB)F4and (BxA)F4
(different litters as hosts
and donors in various
combinations) 3.12 (6 rats) 2.18 (2 rats)

(AxB)F4to (AxB)F4)

(litter mates) 3.12 (6 rats)

(BxA)F4to (BxA)F4J

Ato(AxB)F4 2.39 (4 rats)

(AxB)F4 or (B XA)F4 to A 2.87 (10 rats) 2.23 (4 rats)

B to A (91-92 generations) 1.48 (7 rats) 20 days

1.84 (7 rats) 12 days

series and about 2.50 in series III. Transplantations from hybrids to family A
(or B) show a considerable improvement from grade 1.39 in series II to about
grade 2.50 in series III; and between litter mates of hybrids there is a change
from 1.80 (series II) to 3.12 (young rats) in series III. In transplantations from
family A to hybrids the advance is marked, in series II the grade being 1.50,
in series III, 2.39 (older rats). Where in series III there were grades available
in both groups, namely, in those of old and of young rats, a somewhat arbitrary
intermediate grade was chosen, but one nearer to the grade of the older rats.
In contrast to the change which has taken place in the interaction between the
individuality differentials of the members of inbred strains, in the course of
long-continued brother-sister matings, no essential change has occurred if the
individuality differentials interacted between members of family A and of
family B. In this case, the grade (1.48) obtained in series III, when the exami-
nation took place after 20 days, indicates a typical homoiogenous reaction. As
was to be expected, the reaction was not yet fully developed in its full strength
and the grade therefore was somewhat higher (1.84) if the examination took
place after 12 days.

DISCUSSION

A comparison of the reactions observed in these three series of transplantations
shows that a continuous progress to a homozygous condition has been made.
In the first series, there was only a slight indication of an improvement in grades
over the grades in ordinary homoiogenous and syngenesious transplantations.
A further slight progress was noted in the II series, but the greatest advance was
made in the interval between the II and III series. The first series comprised

INDIVIDUALITY DIFFERENTIALS IN RATS 11

the 37th to 41st generations; the second series, the 60th to 67th generations; and
the III series, the 91st and 92nd generations as well as the 102nd to 107th genera-
tions. After about forty generations, there was then only a very slight progress
towards an autogenous character of the individuality differentials; some advance
was made after 60 to 67 consecutive brother-sister transplantations, and still
more in the 102nd generation; but even at that time, no completely homozygous
condition had been attained. This finding is indicated especially by the trans-
plantations into which the hybrids entered; but it is noticeable also in the trans-
plantations within the inbred family A. Of interest in the third series is also
the difference in the grades in the group of the young and the somewhat older
rats, which agrees with the general observation that when donors and hosts are
very young, the reactions are milder than in older animals. This difference
cannot be due to a lack of individuality differentials in the former, because they
are present; but it is due, rather, to a lesser sensitiveness to strange individuality
differentials or to a not yet fully developed mode of reaction in the younger
animals. In addition, the fact must be taken into account that younger tissues
grow more vigorously than older ones, and this condition is associated with a
greater ability to overcome the effect of the antagonistic reactions of the host;
it may also be that tissues growing more rapidly do not give off individuality
differential substances in as large amounts as the more differentiated tissues,
which metabolize in carrying on their function. In accordance with these
considerations, we noticed that in the group of younger rats the grades are higher
even in transplantations from hybrids to an inbred parent strain, where the
derivatives of strange genes are introduced into the host. Another important
conclusion to be drawn from these experiments is that although so large a number
of consecutive brother-sister transplantations were carried out, a completely
homozygous condition has not yet been reached in these families, as is shown by
the results of transplantations within the inbred family. This conclusion agrees
with the findings obtained also in transplantations in inbred families of guinea
pigs and mice. In none of these, even after years of consecutive brother-sister
matings, has a completely autogenous state of the individuality differentials
been attained. This effect, which is contrary to expectation if merely the
distribution of chromosomes during fertilization is considered, is presumably
due to the occurrence of random mutations in the germ cells, which prevent a
complete homogeneity between the various individuals of an inbred strain.
However, these mutations are able merely to delay but not entirely to prevent
an approach to a completely homozygous condition, although this desired
endstage, on account of such mutations, may never be fully reached by close
inbreeding. There was, however, a difference in the rapidity with which progress
in the direction towards this endstage was accomplished; it was more rapid in
inbred guinea pigs than in inbred rats. In rats, a selection was made of the
strongest members of each litter for the purpose of propagation. Such a selection
was not made in the case of the guinea pigs. But, it is not probable that this
factor would have been so influential as to cause the difference between the results
of inbreeding in these two species. Such a difference may possibly be due to
differences in the rate of mutations in guinea pig and rat. There is no indication
that the sensitiveness of the host for and his reactivity against homoiogenous
individuality differentials is greater in the rat than in the guinea pig.

12 LOEB, KING AND BLUMENTHAL

CONCLUSION

In the course of more than one hundred consecutive brother-sister matings
in family A, and of somewhat less than one hundred brother-sister matings in
family B of King's inbred albino rats, a gradual increase in the homozygous
condition of the inbred families has taken place, as indicated by the lessened
severity of the reactions of the hosts against the individuality differentials of
various members of these families. However, the advance was slight in the
course of the first 67 generations of brother-sister matings; it was greater between
the 67th and the 102nd generations; but even at the latter point a completely
homozygous condition in the inbred family A had not yet been attained. These
results were obtained in testing the interaction of the individuality differentials
in litter mates, in members of different litters, as well as in hybrids of these
inbred strains.

LITERATURE CITED

LOEB, LEO, AND H. D. KING, 1927. Transplantation -and individuality differentials in strains of

inbred rats. Amer. Jour. Path., 3: 143.
LOEB, LEO, AND H. D. KING, 1931. Individuality differentials in strains of inbred rats. Arch.

of Path., 12: 203.

FACTORS INFLUENCING GROWTH AND METAMORPHOSIS OF
THE SALIVARY GLAND IN DROSOPHILA

DIETRICH BODENSTEIN *

(Department of Zoology, Columbia University, New York City)

INTRODUCTION

The present investigations are concerned with the development of the salivary-
glands in Drosophila. The larval salivary glands are strictly larval organs and
grow by increase in cell size. They are completely histolysed during the early
part of pupal life. The salivary glands of the adult fly, on the other hand, are
imaginal organs which develop from imaginal discs situated at the extreme
proximal end of the larval salivary gland, and grow by cell multiplication.
Thus both larval and imaginal salivary glands undergo a period of growth during
larval life, while metamorphosis leads to the differentiation of the imaginal and
the destruction of the larval salivary glands. From the work of Hadorn (1937)
we know that the principle causing pupation in Drosophila is a hormone released
by the ring gland, a small glandular organ situated dorsally between the two
hemispheres of the larval brain. The role of the ring gland in the further differ-
entiation of the various organs involved in the process of metamorphosis is,
however, still obscure. Furthermore, virtually nothing is known about the causal
factors concerned in the growth of organs during larval life. The present study-
attempts to analyze some of the causes which underlie the visible expression of
growth and differentiation in the development of the salivary glands.

MATERIAL AND METHODS

Drosophila virilis was used for this investigation. Experimental animals
were kept at a constant temperature of 25° ± 0.5° C. Instead of the usual
method of transplanting larval tissues into larvae, the body of the adult fly was
used as a carrier of the larval transplant. The larval tissues transplanted into
the body cavity of the adult fly live in their new environment for a long time,
perhaps indefinitely. They do not lose their developmental potencies; they grow
and differentiate normally when provided with the appropriate stimulus. The
adult hosts withstand the operation, which is simple in method, very well. The
mortality rate is negligible even when the same host is used for continued trans-
plantations. Indeed this new method approximates tissue culture more closely
than any other so far known for insects. Its great advantage lies in the fact
that one is able to study the developmental behavior of larval tissues outside
their own larval environment. All histological observations on the salivary
gland are based on orcein-stained material which was examined either in total
mounts or in smear preparations. For certain developmental characteristics the
glands were also examined in the living condition.

* Fellow of the John Simon Guggenheim Memorial Foundation.

DIETRICH BODENSTEIN

It gives me great pleasure to extend my sincerest thanks to Drs. L. C. Dunn,
Th. Dobzhansky, F. Schrader and S. Hughes-Schrader for many stimulating
discussions concerning this work and for their continued interest in the progress
of these investigations. I also wish to express my appreciation of the assistance
given by my wife.

NORMAL DEVELOPMENT OF THE SALIVARY GLAND

In the normal development of the salivary gland one is confronted with a
sequence of developmental steps, each of which is characteristic for a definite
stage of development. Since the normal development of the salivary gland is
necessary for the understanding of the experimental part of this paper, it will be
discussed briefly. However, only such features of development will be set forth
as are of importance for our special problem.

The salivary glands are paired organs. They develop as invaginations on
either side of the anterior ectoderm. As development proceeds these invagina-
tions elongate, grow inward, and unite medially into a common duct leading to
the pharyngeal cavity. (Sonnenblick, 1940.) When the embryo hatches, the
number of cells in each gland is, according to Makino (1938), about 115 and it
does not change during larval life. The growth of the larval gland is, as already
mentioned, the result of increase in cell size rather than cell multiplication.
The development of the salivary gland has been staged from the time the larva
leaves the egg to the time the gland is histolysed. Eleven successive stages of
development are shown in Plate I, which represent photomicrographs of salivary
gland total mounts, stained with orcein. In Plate II the distal portion of the
same glands is shown at a higher magnification. Camera lucida drawings at a
magnification of 85 X were made for each gland of this normal series, as well as
for the glands used in the experiments, and their circumference measured with a
planimeter. The size values thus obtained together with special histological
landmarks characteristic for the different developmental stages, assured great
accuracy in the determination of the various stages. In the following the
measurements of the circumferences are given for the normal series of development.

stage of development

1st
molt

2nd

molt

10-11

circumference

(X 85) in cms.

2.5

4.0

6.0

9.0

11.0

12.5

14.0

17.0

23.0

28.0

41.0

meta .

The histological changes that occur during the development of the salivary
gland are now set forth. The observations are based on orcein-stained total
mounts.

Stage 1 (Plate I, Figure 1 and Plate II, Figure 1').

The cell size is uniform throughout the gland. The cells are very small and
the cell borders not clear. No details are visible in the nucleus.

SALIVARY GLAND IN DROSOPHILA

-t

— >
00

a
•-

^ bo

— ' _C

o 5

•" o

- s

tn •-**

<L> O

b/J ^

a o
o j-

S t" O

O 4-> 4_)

" nS X
<U 4-> QJ

£ M *

rt c ; rt
j_, a>

ill

a-o M
^ > >,

5 « §

^H O

1-1 a

16 DIETRICH BODENSTEIN

Stage 2 (Plate I, Figure 2 and Plate II, Figure 2').

The cell size is uniform throughout the gland. The chromatin forms a fine
net-work around the edge of a clear area in the nucleus. The chromocenter is
visible in all cells and stains deeply.

First molt. The cells are larger than in stage 2 ; otherwise there is no change.

Stage 3 (Plate I, Figure 3 and Plate II, Figure 3').
The cells are larger; otherwise unchanged.

Stage 4 (Plate I, Figure 4 and Plate II, Figure 4').

The cells are larger; they are still uniform in size throughout the gland.
The chromatin forms a net-work consisting of fine strands around the edge of a
clear area in the nucleus. The chromocenter is visible in all cells and stains
deeply. At the proximal tip of the gland, where it borders the duct, a small
number of tiny cells becomes visible; these represent the imaginal anlage of the
adult salivary gland. (See Plate I, Figure 4, arrow.)

Second molt. The cells in the distal portion of the gland have become larger
in size than those of the proximal portion. The chromosome strands, especially
those in the nuclei of the distal gland portion, have become wider; alternating
deeply stained areas and lightly stained areas are clearly distinguishable within
the individual strands. The deeply staining chromocenter is still visible in all
cells. The imaginal anlage of the adult gland has become very clear and its
cells form a ring-like structure around the salivary duct.

Stage 5 (Plate I, Figure 5 and Plate II, Figure 5')-

The cells in the proximal gland portion are much smaller than in the distal;
this difference is very pronounced. The chromosome strands have become wider
and the chromatin bands more distinct. The chromosome strands on the
periphery of the nucleus embrace the clear area in the center of the nucleus like
octopus arms. The chromocenter is still visible in all cells. The imaginal ring
cells have increased in number.

Stage 6 (Plate I, Figure 6).

The cells are larger; otherwise unchanged.

Stage 7 (Plate I, Figure 7 and Plate II, Figure 7')-

The difference in cell size between the proximal and distal gland portions is
very clear. The chromosome strands are broad and the chromatin bands very
distinct. The chromocenter has become invisible in the cells of about one-half
of the distal portion of the gland. To avoid misunderstanding this point has
to be made clear. The staining capacity of the chromocenter at this stage has
by no means changed considerably, as smear preparations show. It has become
invisible in total mounts because the chromosome strands in the distal cells have
grown so much in width as to eliminate the former discrepancy in width between
chromocenter and strands. Therefore if we speak in the following of the disap-
pearance of the chromosome center, we mean just this fact. The disappearance

SALIVARY GLAND IN DROSOPHILA

PLATE II. Figures 1' to 9': the distal portion of the same glands as shown in Plate I, Figure, 1
at a higher magnification. (For explanation see text.)

18 DIETRICH BODENSTEIN

of the chromocenter is hence a good indication of the nuclear size. The imaginal
ring cells have increased in number.

Stage S (Plate I, Figure 8).

The difference between the cell size in the proximal and distal part of the
gland is very pronounced. The proximal cells have reached a size which corre-
sponds approximately to the cell size in the distal part of the preceding stage.
At no time, however, do we find any sharp separation into a proximal and distal
portion of the gland as far as cell size is concerned. On the contrary, the cells
increase gradually in size in a proximal-distal direction. Because of this differ-
ential growth, the salivary gland acquires its characteristic shape. The form
and size of the salivary gland seem thus to be determined by two different growth
rates in two different directions; one correlated with age affects the gland as a
whole, and the other constitutes a proximal-distal gradient which determines
the size of the cells throughout the length of the gland. While up to this time
cell growth took place by an increase in size of the nucleus and cytoplasm simul-
taneously, at this stage a remarkable change occurs in the cells of the distal half
of the gland. The nuclei in these large distal cells cease to grow, or grow only
very little indeed, yet the cytoplasm increases immensely. A comparison of
(Plate II) Figure 7' with 9' illustrates this point quite clearly. The chromocenter
has become invisible in the distal two-thirds of the gland and the chromatin
bands in the chromosome strands especially of the distal cells stain very distinctly.
The imaginal ring has increased in size.

Stage 9 (Plate I, Figure 9 and Plate II, Figure 9').

The larvae are full grown, they have left the food and are ready to pupate.
In the living condition the cytoplasm of the glands has become slightly opaque
instead of being transparent as in previous stages; this condition is the first
indication of metamorphosis. The opaqueness is usually somewhat more
pronounced distally. The proximal cells are still smaller than the distal ones.
The cells in the distal two-thirds of the gland are extremely large, due to an
increase in their cytoplasm, while their nuclei are of about the same size as in
stage 8. Although the nuclei are unaltered in size, their condition has changed.
The clear area in the center of the nucleus has disappeared. The chromosome
strands have become more compact, winding their way through the whole
nucleus. The individual strands show their characteristic banding most per-
fectly. The cytoplasm stains less heavily than in previous stages, while the
nuclei take the stain exceedingly well. One of the characteristic features of this
stage is that in total mounts the nuclei stand out clearly against a lightly stained
background (Figures 9 and 9'). The chromocenters are visible only in the
proximal one-fifth portion of the gland. The imaginal ring has become larger;
the chromosome strands stretch very well in smear preparations, in contrast to
earlier or later stages. This stage is hence best suited for studies on the arrange-
ment of bands in the chromosome strands.

Stage 10 (Plate I, Figure 10).

Histolysis of the glands begins about 10 hours after puparium formation.
In various regions, usually first in the distal part of the gland, the cells begin to

SALIVARY GLAND IN DROSOPHILA 19

vacuolate and the cell walls rupture, while the nuclei are still intact. The
cytoplasm in these regions of degeneration stain poorly. The chromosome strands
on the other hand stain very deeply. They are clumped in the center of the
nucleus and are surrounded by a clear spherical area (Figure 10). In living
conditions the glands appear much more opaque, while milky-white zones
indicate the regions of advanced histolysis. The chromocenter is by now visible
only in a few cells at the extreme proximal end of the gland. The imaginal
ring has increased in size.

Stage 10-11 (Plate I, Figure 11).

The next stages of histolysis proceed very rapidly. The degenerating regions
within the gland which stain poorly, and which in life appear milky, extend and
become more frequent. The nuclei become picnotic. The basement membrane
which surrounds the gland breaks down. Finally, about 25 hours after puparium
formation, with the probable help of phagocytosis, the larval gland is dissolved.
The proximal part is the last to disappear. The differentiation of the imaginal
ring begins and is completed during the rest of the pupal period, leading to the
formation of the adult salivary gland.

EXPERIMENTS

The induction of premature metamorphosis.

The description of normal development has revealed that the metamorphosis
of the salivary gland consists of the histolysis of the larval and the differentiation
of the imaginal salivary gland. The question now arises whether the character-
istic developmental behavior of the gland during metamorphosis is dependent on
or independent of the conditions prevailing in the animal during metamorphosis.
This can be tested by transplanting young salivary glands into older hosts, thus
exposing the gland prematurely to the metamorphosis factors.

Salivary glands of stage 5 were transplanted into the abdomen of older
larvae shortly before pupation. The transplanted glands were dissected and
their condition studied after they had remained in the hosts for various lengths
of time. The results of the experiments are summarized in Table I A. The
first animals in this series were dissected two days after the operation, when the
hosts were about one-day old pupae. The transplanted glands had reached
stage 10, i.e. they showed clear signs of histolysis. Their state of development
corresponded closely to that of the normal host gland. Yet if the transplanted
glands had been left in their own environment, they would have developed
certainly not further than stage 8 by this time. Thus, under the influence of
the metamorphosis factors of the host, the grafted glands have metamorphosed
prematurely. The transplanted glands reached an advanced stage of histolysis
when they were left three days in the host, and they disappeared completely
when left in the host for six days or longer.

In these transplantations the anlage of the imaginal ring was included in the
larval graft. Now it was found (Table I A), that the imaginal gland, like the
larval gland, is able to metamorphose precociously when exposed to the meta-
morphosis factors prematurely. This becomes evident from the observation
that the transplanted imaginal anlage in the newly emerged host, i.e. six to seven

DIETRICH BODENSTEIN

days after the operation, is already completely differentiated into the imaginal
salivary gland (Plate III, Figure 4), while donor controls by this time would
have been two days old pupae with quite undifferentiated imaginal glands.

In summarizing the experiments at this point, we find that the metamorphosis
of purely larval structures, as well as imaginal structures, is not autonomous,

TABLE I A

Transplantation of young salivary glands into larvae shortly before pupation.
A. Transplantations of larval glands of stage 5.

Circumference of
transplanted
gland

Days transplant
remains in
host

Host
stage
dissected:

Stage of
transplanted
larval gland

Stage of
transplanted
adult gland

14.0

pupa

stage 10 +

larval

15.0

pupa

stage 10 —

larval

15.0

pupa

stage 10 +

larval

15.5

pupa

stage 10 —

larval

15.5

pupa

stage 10 —

larval

13.5

pupa

stage 11 —

larval

13.5

pupa

stage 11 —

larval

15.5

pupa

stage 11 +

larval

15.5

pupa

stage 1 1 —

larval

15.5

pupa

stage 11 —

larval

13.5

adult

completely histolysed

adult gland formed

13.5

adult

completely histolysed

adult gland formed

14.0

adult

completely histolysed

adult gland formed

13.5

adult

completely histolysed

adult gland formed

13.5

adult

completely histolysed

adult gland formed

15.5

adult

completely histolysed

adult gland formed

TABLE I B

B. Transplantations of larval glands of stage 3.

7.5

pupa

stage 6

larval

7.5

pupa

stage 10-

larval

7.5

pupa

stage 10 —

larval

9.0

pupa

stage 1

larval

7.5

adult

stage 1 —

larval

7.5

adult

stage 1 -

larval

7.5

adult

stage 1

larval

7.5

adult

stage 1 -

larval

9.0

adult

stage 1

larval

9.0

adult

stage 1

larval

9.0

adult

stage 1

larval

9.0

adult

stage 1

larval

7.5

adult

stage 1

larval

7.5

adult

stage 1 1

larval

but is caused by some factors in the organic environment of the host during the
period of metamorphosis.

The next question to be answered is whether salivary glands younger than
stage 5 are competent to respond to the metamorphosis factors. For this,

SALIVARY GLAND IN DROSOPHILA 21

salivary glands of stage 3 were transplanted into older larvae shortly before
pupation. The region of the imaginal discs was again included in the graft, but
in contrast to the previous series of experiments the imaginal ring as such was
not yet morphologically visible. The results of these experiments summarized
in Table I B show that histolysis of the young transplanted glands begins about
two days after the operation, but is never completed, although the glands may
remain as long as 15 days in the host. Young glands which have remained for
three days in the host only begin their histolysis, while older glands are at this
time quite extensively histolysed (Cff. Table I A with I B). Thus the young
glands are presumably unable to respond at a time when the metamorphosis
factors are most effective. \Ye must assume that the metamorphosis factors
have become less efficient when the glands have finally reached their responsive
stage, and are hence unable to induce complete metamorphosis, because it is
difficult to understand why the glands should not be histolysed completely if the
metamorphosis factors were still active in the late pupal or adult stage.

The inability of the young organ to respond to the metamorphosis factor is
demonstrated in the behavior of the adult gland (Table IB). In spite of the
fact that the young imaginal gland discs have remained in some cases for a
considerable length of time in the host, they show no signs of metamorphosis
(Plate III, Figure 3). Although the anlagen have developed well beyond their
stage of transplantation and have acquired their typical ring-shaped form, they
remain larval and never surpass this stage. Their state of development corre-
sponds to stages 10 and 11 of the normal developmental series, yet this stage,
as previous experiments have shown, by far exceeds the stage at which the adult
gland anlage is able to react. These observations lead necessarily to the conclu-
sion that the larval development of the disc is not interrupted in its new environ-
ment, but that by the time the disc has reached its reactive stage the factors
necessary for promoting metamorphosis are absent or not effective enough.
The specific results of this experimental series may then be briefly summarized
as follows: the larval glands, as well as the analgen of the adult glands, must
reach a certain developmental stage before they are able to respond to the
metamorphosis factors. If they reach this stage after the active period of
metamorphosis they are unable to metamorphose and persist as larval structures
in an imaginal environment (Plate III, Figure 3).

In examining prematurely-metamorphosed young salivary glands, one has the
impression that the nuclei in these glands are histolysed before they have attained
their fully normal size. The same observation was made in prematurely-
metamorphosed young salivary glands which were grown in the body of adult
hosts. It is difficult to decide whether this impression is real, since the reduction
of the nuclear size is only slight and probably within the limit of normal variation.
If true, however, it would mean that premature metamorphosis causes an early
cessation of nuclear growth, which is not at all -unlikely. In the light of these
considerations, it seemed interesting to investigate whether older glands trans-
planted into younger hosts would grow larger than normally, because under these
experimental conditions they would be exposed to the metamorphosis factor later
than normally. Salivary glands of stage 7 were therefore transplanted into
younger host larvae, the salivary glands of which were at stage 4 at the time of
the operation. In other words, salivary glands of the last larval instar were

DIETRICH BODENSTEIN

transplanted into hosts of the second larval instar. This experimental series
consisted of six hosts which were dissected two days after the operation when
the transplanted and host salivary glands were compared. It was found that
the host salivary glands had developed to stage 8 — . They were still transparent,
and showed no signs of metamorphosis. The transplanted glands on the other
hand were found to be at stage 9+ and had begun to metamorphose, as indicated
by their intensely opaque appearance. Moreover, the chromosome strands of
the transplanted gland were distinctly wider than those of the host glands.
Yet the strands were definitely not larger than normally, i.e. their size was
characteristic for a normal salivary gland of stage 9 -f- • Thus the original question
whether it is possible to obtain salivary glands larger than normal has been
answered by this experiment in a negative way. Some further conclusions
which might be drawn here will be discussed on page 30 in a somewhat different
connection.

The effect of the ring gland on the development of the salivary gland.

Until now we have dealt almost entirely with specific reactions of the salivary
gland. So far nothing has been said about the factors which govern the processes
of growth of the gland during larval life and those of metamorphosis during
pupation. In the light of our knowledge of hormone-controlled insect meta-
morphosis, we expect these factors to be hormonal in nature. The search for
the activating principle resolves itself into the problem of locating an organ or
organs of internal secretion and demonstrating their action on the developing
organ system. There are obviously two alternatives in attacking this problem
experimentally. One may attempt to remove systematically various organs of
supposedly endocrine nature from the larvae and thus hope to locate the re-
sponsible organ by testing the developmental behavior of the operated animal.
The difficulty involved in a study of this kind is mainly a technical one, for it is
very difficult indeed and at the present time seemingly impossible to perform
such an operation in the larvae of Drosophila. The other possible approach
open to the investigator is to dissect various organs from a donor larva which
can be sacrificed and transplant them to a second larva and then test their effect
on the development of the host. Although technically quite feasible, this
procedure has one great disadvantage. The effect of the transplanted gland on
the host may be counteracted or blurred to a great extent by the hormone supply
of the host itself, and thus not detectable. The introduced hormone, moreover,
may upset the host system to such an extent that the situation, instead of being
clarified, is rather obscured. Apart from these difficulties there is the additional
one that both approaches are rather indirect, since the experiments are designed

PLATE III, FIGURE 3. Adult salivary gland, developed from the imaginal salivary gland
anlage which was transplanted at stage 6 into a host larva shortly before pupation. Transplant
dissected from the emerged host six days after the operation.

FIGURE 4. Larval salivary gland persisting as larval structure in the adult host; obtained
by transplanting salivary glands at stage 3 into the abdomen of host larvae shortly before pupation.
Note: undifferentiated imaginal ring cells (arrows).

FIGURE 5. Paired salivary gland transplantation. Glands transplanted at stage 6 into
adult hosts and dissected two days after the operation, a. Partner transplanted alone: note
bloated appearance of gland and the small nuclear size. b. Other partner transplanted together
with two ring glands. Note the large, very well stained nuclei with chromosome strands.

SALIVARY GLAND IN DROSOPHILA

PLATE III

24 DIETRICH BODENSTEIN

for testing the hormone action on the whole organism rather than on the special
organ. We have hence to seek a method by which the hormone action may be
more directly studied.

An effort in this direction was made by using the abdominal cavity of the
adult male fly as a place for culturing larval tissue in vitro. This environment
is supposedly neutral as far as the progress of development is concerned, for the
larval salivary glands cease to grow after the transplantation, but continue to
live indefinitely. The transplanted tissues do not suffer from a lack of nutrition
nor are they unable to utilize the nutritive components of the new environment,
as is shown by the following experiment. \Yhen young Drosophila larvae are
starved, they use the fat stored in their fat bodies and, if starvation is continued,
they exhaust their food reserves in the fat bodies completely. Such exhausted
fat bodies are strikingly different from the fat bodies of feeding larvae. Now
it was found that starved exhausted larval fat bodies restore their food reserves
again and become indistinguishable from fat bodies of normally fed larvae when
transplanted into the body cavity of adult flies. For these experiments a small
strip of fat body closely attached to the salivary gland was transplanted together
with the gland. The gland, important in this instance only as a marker, enables
one to find and distinguish the transplanted fat body from the fat bodies of the
host. The inability of the larval organs to develop in the adult fly is hence not
caused by a lack of nutrition but obviously by the lack of some other factor.

Before considering the nature of this developmental factor, one further point
has to be cleared up. It has to be showrn whether the inability of the gland to
develop is typical for glands of all ages or only characteristic for glands of a
particular stage of development. In order to elucidate this point, salivary
glands of stages 2 to 6 were transplanted into the abdomen of adult male flies.
The grafted organs were allowed to remain for various lengths of time in the host
before they were dissected out and their developmental condition examined.
The results of these experiments summarized in Table II show clearly the inability
of the gland to develop in the body of the adult fly regardless of their age at the
time of the transplantation and of the length of time they remained in the host.
The only perceptible way in which the transplanted glands seemed to be changed
while in the adult host was that they became greatly inflated, this being caused
by the accumulation of a clear watery fluid in the lumen of the gland. The
longer the glands remained in the host, the more fluid accumulated and the more
bloated the glands became. The accumulated fluid presumably represents saliva,
which is secreted by the gland cells into the lumen of the gland and is unable to
escape, since the outlet is closed off in dissecting the gland from the donor larvae.
Incidentally, should this fluid really prove to be saliva, we have here a method
of accumulating it in order to study its chemical properties.

The foregoing experiments have shown that the larval salivary glands depend
for their development upon some factor missing in the body cavity of the adult fly.
This factor was found to be the larval ring gland, which when transplanted
simultaneously with the salivary glands into the abdominal cavity of the adult
fly, caused the latter to continue their development leading to histolysis. The
progress of development of the salivary glands was indicated by changes in the
nuclei, cell growth and certain characteristic staining reactions. All the typical
developmental stages found and described in the normal developmental series

SALIVARY GLAND IN DROSOPHILA

may be observed, but they proceed at a much lower rate. The salivary glands
are also able to metamorphose, i.e. they become gradually histolysed if left
long enough in the adult host. The ring glands used for these experiments
came from donor larvae which were ready to pupate. The number of ring
glands implanted into one host varied from one to four. Salivary glands of
stage 2 to stage 6 were tested for their reaction to the ring gland factor. The
transplanted salivary glands remained in the host for various lengths of time.
These experiments, summarized in Table II, led to the discovery of a number of
essential facts concerning the development of the salivary glands. Glands of all
ages tested respond to the ring gland factor. The older the gland is at the time
of transplantation, the earlier metamorphosis occurs. This implies that at least
younger glands do not metamorphose right away, but grow to a certain point
before metamorphosis begins. In the study of normal development we have
seen that the first signs of metamorphosis are noticeable in glands of stage 9,
as indicated by a slight but distinct opaqueness of the gland in living condition.
At this time the chromocenter has become invisible in the distal portion of the
gland. We find in glands which develop in adult hosts some variability in the
relationship between the onset of opaqueness, i.e. metamorphosis, and the
disappearance of the chromocenter. The degree of opaqueness observed in the
transplanted glands is indicated in Table II by the Roman numerals I to III
placed next to the number which designates the stage of development. The
number I represents an opaque condition of stage 9 and the number III of stage
11— in normal development. When no Roman numerals are given, the relation
between metamorphosis and developmental stage is normal. The transplanted
glands become opaque at an earlier developmental stage. This is especially true
in the transplantation of younger glands. For instance, glands which have
developed in the adult host to stage 7-- show an opaque condition which corre-
sponds to stage 9 or 10 in normal development. Since we know from normal
development that the chromocenter becomes invisible because of the thickening
of the chromatin threads, it follows that the glands begin their metamorphosis
before the strands have developed to their normal size. It was also noticed in
many cases that the nuclear size in advanced prematurely metamorphosed glands
seemed smaller than normal. This would indicate that they degenerated before
they had reached their full size. These facts are in agreement with the experi-
ments described on page 19 where young glands in old larval hosts were made
to metamorphose precociously. The number of ring glands transplanted seems
to be of no great importance for the development of the salivary gland, since
one or three ring glands produce about the same effect.

In all these experiments where adult flies were used as hosts the imaginal
salivary anlage usually transplanted together with the larval gland remained
larval. Its cell number, however, increased considerably when transplanted
together with ring glands. The amount of increase depended upon the time the
anlage remained in the host. However, no sign of differentiation could be
noticed, in spite of the fact that in some cases more than four ring glands were
present and the disc remained for a considerable length of time in the host.
In the effort to obtain a more precise comparison between the developmental
behavior of larval salivary glands with and without ring glands, the two glands
of one donor were compared with each other. For this experiment the paired

DIETRICH BODENSTEIN

gland of a single donor was dissected, the two glands separated and one partner
transplanted into one adult host without ring glands and the other partner
transplanted into a second adult host together with two to four ring glands.

TABLE II

Transplantation of salivary glands of various ages; alone and together -with different numbers of ring

glands into the abdomen of adult male flies. Roman numerals indicate state of

opaqueness of the gland. (For explanation see text.}

Number of ring glands transplanted

Stage

Days

trans-

None

One

Two

Three

Four

trans-

plant

planted

remains

salivary
gland

in
host

Num-
ber

Stage
of

Num-
ber

Stage

Num-
ber

Stage

Num-
ber

Stage
of

Num-
ber

Stage
of

develop-

cases

ment

cases

ment

cases

ment

cases

ment

cases

ment

I 9

I 7-

4 +

I 7-

O ~~~

II 9 +

I 7 +

II 8

III 10

5 +

3 +

I 7

I 8-

I 7

II 8 +

4 +

II 8

II 9

I 7

II 9-

10 +

11-

9 +

10-

III 9 +

10 +

11-

Number of

cases

The gland in the first host thus served as a control for the partner gland in the
second host. At the desired time, both hosts were killed simultaneously, the
two glands dissected and compared. Twenty-three such pairs are available;

SALIVARY GLAND IN DROSOPHILA

they are recorded in Table III. As in the previous experiments we find the
salivary glands unable to develop when transplanted alone, while the partner
glands with added ring gland implants continue development. Figure 5 of
plate III illustrates such a paired transplantation. This pair was transplanted
at stage 6 and left for two days in the adult host. The partner (a) transplanted
alone, has become greatly inflated, yet has not developed beyond the stage of
transplantation. The other partner (6) which was transplanted together with

TABLE III

Paired transplantation of salivary glands of different age into the abdomen of adult male flies,
one host carries salivary gland alone, while second host carries partner
salivary gland and ring gland grafts.

Note:

Stage of transplanted
salivary gland

Number of ring
glands transplanted

Days graft remains
in host

Condition of transplanted salivary gland

Partner without ring
gland stage:

Partner with ring
gland stage:

I 74-

II 8

II 9

5 +

I 8-

4 +

II 8 +

III 9 +

II 9 +

10-

5 +

10 +

11-

9 +

6 +

9 +

10-

10 +

two ring glands has continued its development and has reached stage
Plate III, Figure 5b illustrates clearly the characteristic large nuclei at this
stage which stand out against a lightly stained background (Cf. Plate III,
Figure 5b with stage 9 of the normal developmental series). The chromocenter
has become invisible in at least two-thirds of the gland. The chromosome
strands are broad and the chromatin bands within them very distinct. The
single cells are extremely large, due to an increase in the volume of the cytoplasm.
In the living condition the cytoplasm is opaque in contrast to the transparent

DIETRICH BODENSTEIN

appearance of the partner gland. Moreover, the salivary gland which has
undergone development is not swollen as is the case with the partner gland.
This fact can be seen in all salivary glands which are transplanted together with
ring glands. The ring gland must hence interfere with the secretory function of
the salivary gland. On turning again to Table III we find that the first response
to the ring gland is obviously a growth response. This becomes evident if one
compares glands of the same age which were left for increasingly longer periods
in the host. (See Tables II and III.) For example, glands transplanted at
stage 4 grow in one day to stage 5 + , in four days to stage 8— and reach stage
9+ when left for seven days in the host. After growth has continued for a
certain length of time, the physiological condition of the glands must have
changed in some way, since they now respond with metamorphosis to the ring
gland factor. The younger the glands are at the time of transplantation, the
later they respond with metamorphosis, for glands transplanted at stage 2
develop in five days only to stage 4 + , while glands transplanted at stage 4 show
indications of metamorphosis after four days. Metamorphosis is far advanced
in four days when glands of stage 6 are used for the transplantation. The
transplanted glands, especially the younger ones, metamorphose precociously,
for the opaque metamorphosis condition is noticeable in transplanted glands as
early as stage 7 + , thus at a considerable earlier stage than in normal development.
(See also Table II.) This abnormal relationship between the stage of develop-
ment and the onset of metamorphosis becomes less pronounced as increasingly
older glands are used.

The different responsiveness of younger and older salivary glands has been
demonstrated very clearly in a further experiment especially designed for testing
this point. Two salivary glands, one of stage 3 and the other of stage 5, were
transplanted together with two ring glands into the abdomen of a single adult
host. This series consisted of four hosts, each of which contained a young and
an old salivary gland, as well as two ring glands. Six days after the operation,
the first host was examined. The young salivary gland had developed to stage
7 + and was slightly opaque (I), while the older gland was at stage 9+ and
distinctly more opaque. In two hosts examined nine days after the operation,
the younger glands \vere found to be at stage 8 to 9 and the older glands at
stage 10 + . While the younger glands appeared intensely opaque, the older
glands were obviously in an advanced state of metamorphosis, as indicated by
large milky regions of degeneration. The last host examined 13 days after" the
operation revealed the young gland at stage 10 and the older one at stage 10+.
Again histolysis had progressed further in the older gland.

In a second series of this kind, consisting of six animals, salivary glands of
stages 2 and 3 were transplanted together with three ring glands into a single host.
The results of these experiments are essentially the same as in the foregoing
series; they show again that older glands are always further metamorphosed
than young ones, although, as pointed out before, the onset of metamorphosis in
the young glands is premature, relative to their state of development.

It has been seen that the development of the salivary gland in the adult
host with the support of the ring gland is considerably slower than in normal
development. However, the question as to how much development is slowed
down has not as yet been examined. In order to elucidate this point, one partner

SALIVARY GLAND IN DROSOPHILA

of a pair of glands of stage 5 was transplanted into the abdomen of an adult
host together with 3 ring glands, while the other partner was transplanted into
the abdomen of an old larva shortly before pupation. In this way it was possible
to make a direct comparison of the same gland in an adult and larval environment.
From six such experimental pairs, five were examined two days after the operation.
At this time the larval hosts pupated. In one pair the gland in the larval host
was found to be at stage 10, showing clear regions of degeneration, while the
partner gland in the adult host had only developed to stage 9 and appeared but
slightly opaque. In two other pairs only the proximal part of the gland in the
pupated larval host could be found, since the distal gland region was completely
histolysed. The partner glands in the adult host in one of these pairs had
developed to stage 9, being slightly opaque, while the gland in the other adult
host had reached only stage 8 and was still transparent. The glands in the
pupated larval hosts of the two remaining pairs could not be found, presumably
because they were already completely histolysed. The partner glands in the
adult hosts were found to be transparent and had reached stage 8. The last
pair of this series was examined seven days after the operation. At this time the
larval host had already emerged. Its graft could not be found and was appar-
ently completely histolysed. The partner graft in the adult host was at stage
10 — , showing advanced signs of histolysis.

The results of these experiments bring into focus one interesting point.
We have seen from the previous experiments that growth and metamorphosis of
the salivary gland is controlled by the ring gland. The salivary glands in the
larval hosts are under the influence of only one ring gland, namely that of the
host, while in the adult hosts the glands are exposed to the effects of two ring
glands. In spite of this, we find the progress of development of the salivary
glands much more rapid in the larval than in the adult host. This shows that
the ring gland factor is more effective in a larval than in an adult environment.

Before closing the experimental part of this paper it should be mentioned
that the ring gland factor is not species-specific, since ring glands of melanogaster
cause the development of virilis salivary glands in both virilis and melanogaster
hosts. Conversely, melanogaster salivary glands develop under the influence of
virilis ring glands in virilis or melanogaster hosts.

DISCUSSION

The present data prove without doubt the necessity of the ring gland for the
metamorphosis of the salivary gland. The responsible ring gland factor is
presumably hormonal in nature, since the transplanted ring glands have no
contact with the test organs which are nevertheless compelled to metamorphose.
The ring gland is thus the source of the metamorphosis hormone in Drosophila
and is not responsible only for puparium formation as previously assumed. In
fact, puparium formation has to be considered as the first step in metamorphosis.
The observations of Hadorn and Neel (1938) that ring glands implanted into
young larvae bring about premature puparium formation but have no effect
on the metamorphosis of the larval organs can be explained by the incompetence
of the young organs to respond to the metamorphosis hormones. This is sup-
ported by the observation that young salivary glands react more slowly to the
ring gland hormone than older ones and that the salivary glands must reach a

30 DIETRICH BODENSTEIN

certain developmental stage before they are able to respond to the metamorphosis
hormone. The inability of organs to respond with metamorphosis before a
certain stage of development is reached is not peculiar to salivary glands but
has been observed for eye discs (Bodenstein, 1939a and b), and for ovaries
(Vogt, 1941). Inasmuch as the time at which larval glands and imaginal gland
anlagen reach their state of responsiveness is concerned, we find that both
structures become responsive at about the same time. This, however, is true
only of grafts into larval hosts; in adult hosts we find the larval glands able to
metamorphose, but not the imaginal glands, in spite of the presence of several
ring glands. This indicates clearly that the larval glands respond much more
readily than the imaginal glands. In comparing the ring gland action in a
larval and an adult environment, we find the hormone more active in the former.
Whether one ring gland in the larval host is able to produce more hormone and
thus assure a higher hormone concentration than four ring glands in an adult
host is questionable, but would explain easily why in the adult host the imaginal
anlage is unable and the larval gland is able to respond; and why in the larval
host both glands are able to metamorphose synchronously. On such an hy-
pothesis the low hormone level in the adult host is able to induce metamorphosis
only in the. readily responsive larval gland but not in the imaginal gland anlage,
which needs a higher hormone concentration for the same task. In the larval
environment on the other hand, where the hormone concentration is presumable
higher, it is sufficient to induce metamorphosis in the imaginal gland also. In
the light of these considerations it becomes evident that any difference in the
competence of the reactive tissue might be detectable only when low hormone
concentrations are used. The quantitative action of the metamorphosis hormone
has also been demonstrated by Hadorn and Neel (1938), who found that pupation
in Drosophila occurs sooner when three instead of one ring gland are implanted
into the larval host. The question of hormone concentrations was not directly
tested in our experiments because there seemed to be no difference in the effect
of two or more ring glands. There is, however, some indication that one ring
gland is not as effective as four; this can be seen if one compares (Table II)
salivary glands which were transplanted at stage 4 together with one and with
four ring glands and which were then left for six days in the adult host. The
salivary glands (two cases) transplanted together with one ring gland have
developed to stage 7 and the salivary glands transplanted together with four
ring glands to stage 10 (three cases). Similar experiments on other organ discs
not reported here gave the same results. Thus it seems quite certain that the
hormone concentration is a decisive factor in the development of the salivary
gland.

One of the most important facts revealed by the present investigations is
that the first salivary gland response to the ring gland hormone is a growth
response. It is only after the salivary glands have grown to a certain size that
the hormone elicits the metamorphosis response in the salivary gland. The
younger the salivary glands are at the time of transplantation the longer the
growth period persists. In other words, younger glands begin their metamor-
phosis later than older glands when under the influence of the same number of
ring glands, yet the onset of metamorphosis of young glands in adult hosts under
ring gland influence is definitely premature when compared with normal develop-

SALIVARY GLAND IN DROSOPHILA 31

ment. However young the salivary glands are when transplanted, the onset of
their metamorphosis is never earlier than stage 7 — , as compared with stage 9 in
normal development. This shows that the young glands are able to respond with
growth only they have reached stage 7 — . This observation leads us directly to
the question why in normal development the onset of metamorphosis takes place
at stage 9 and not at stage 7 — . The answer to this may be found in the following
considerations. The experiments have shown that the growth of the salivary
glands depends on the ring gland hormone. This must obviously also be the
case in normal development. Now in normal development the salivary gland is
under the influence of the ring gland during the entire larval period. However,
the ring gland of younger larval stages is much smaller than the ring gland of
mature larvae and thus probably produces much less hormone. The hormone
concentration of younger larvae is consequently expected to be much lower than
in older larvae. The hormone level in younger larvae, we might argue, is thus
not high enough to assure metamorphosis, but is sufficient to promote growth.
As development proceeds, the ring gland grows and thus produces more hormone;
at the same time however, we find the competence of the salivary gland changing.
At a given time, therefore, when hormone level and responsiveness of glands are
in a definite relationship with each other, metamorphosis occurs. In normal
development this time is reached when the salivary glands have developed to
stage 9. We may now ask why, under experimental condition, we are able to
metamorphose glands which have reached only stage 7 — . The answer to this
lies very probably in the fact that we have changed the hormone concentration.
In the adult host the hormone level produced by several ring glands is presumably
much higher than in younger larvae and is thus able to induce metamorphosis in
glands as young as stage 7 — . Since metamorphosis proceeds at a very slow
rate, we must conclude that the experimentally established hormone level in
adult animals, while higher than in younger larvae, is not as high as in full-
grown larvae, where young glands metamorphose much more rapidly. Whether
the ring glands produce less hormone in the adult fly, or whether some other
factors are responsible for the fact that we are unable to raise the adult hormone
level to the equivalent of the level existing in the old larvae is, however, still
questionable.

There is one further point of considerable interest. We have seen on page 22
that old salivary glands transplanted into younger larvae metamorphose before
the glands of the host larvae show any signs of metamorphosis. Since the cell
size of the grafted glands was found to be not larger than normal, there seems
to be a limit beyond which nuclear growth is impossible. The question now
arises, what exactly do we mean by this limit? Judging from the results cited
earlier, we have to assume that the salivary glands become more and more
responsive to metamorphosis as they increase in age. The older gland in the
young host is therefore at a much more advanced stage of responsiveness than
the host glands, and is hence able to react with metamorphosis to a hormone
level much lower than that needed for the same reaction by the younger gland.
In assuming, as we have, that the hormone level increases gradually during the
larval period, the growth limit is nothing more than the expression of a definite
relationship between cell competence and hormone level. In the light of this
it is theoretically possible to obtain cells larger than normal, when older glands

DIETRICH BODENSTEIN

are affected by a hormone level too low for metamorphosis but high enough for
growth; granted, however, that a hormone level low enough for a highly
responsive gland could be found.

Logically this conception is based on the assumption that the ring gland
produces its hormone during the entire larval life. Only with the demonstration
of this does the present hypothesis become meaningful. Actually there is really
good evidence available which shows that ring glands from larvae as young as
the first instar are able to promote growth in certain organ discs. More precise
information concerning this point will be given in a later communication.

SUMMARY

1. The normal development of the salivary gland of Drosophila virilis is
described. Eleven successive stages of development have been distinguished.

2. Larval salivary glands of different ages were transplanted into the abdo-
mens of older larvae and thus exposed prematurely to the metamorphosis factor.
It was found that the metamorphosis of the transplanted glands is not autonomous
but depends upon some factor in the host.

3. Glands as young as stage 3 are unable to react to the metamorphosis factor
and persist as larval tissue in the adult fly. However, glands transplanted at
stage 5 metamorphose synchronously with the host and hence undergo a pre-
mature metamorphosis. In these cases the transplanted larval glands are
completely histolysed, and the simultaneously transplanted anlagen of the
imaginal salivary gland differentiate into adult salivary glands.

4. Salivary glands of older larval donors transplanted into younger hosts
metamorphose before the host glands show any signs of metamorphosis.

5. Larval salivary glands of various ages were transplanted into the body
cavity of adult male flies. The thus transplanted glands ceased to grow and
remained unchanged even when left for a considerable length of time in their
adult environment. If, however, ring glands of old larvae are transplanted
together with salivary glands into the adult host, the growth of the salivary
glands is restored, leading finally to metamorphosis. These facts have been
demonstrated very clearly by using salivary glands of a single donor and trans-
planting one partner into one host without ring glands and the other partner
into a second host together with ring glands.

6. The number of ring glands implanted is of no great importance for the
development of the salivary glands, since two ring glands have about the same
effect as four ring glands. However, one ring gland is presumably somewhat
less effective than four ring glands.

7. The rate of metamorphosis of the salivary gland in adult hosts is decidedly
slower than in normal development.

8. Younger salivary glands metamorphose later than older salivary glands
under the influence of the same number of ring glands.

9. Although the young glands metamorphose later, their metamorphosis is
premature as far as their state of development is concerned.

10. A comparison of the time of metamorphosis of salivary glands in adult
and larval hosts shows that metamorphosis proceeds much more rapidly in larval
hosts in spite of the fact that in the adult host the salivary glands may be under
the influence of as many as four ring glands.

SALIVARY GLAND IN DROSOPHILA

11. The ring gland factor is presumably hormonal in nature, and is not
species-specific.

12. The role of hormone concentration and tissue competence in the determi-
nation of the various stages of growth and differentiation in the development of
the salivary glands is discussed.

LITERATURE CITED

BODENSTEIN, D., 1939a. Investigations on the problem of metamorphosis. IV. Developmental

relations of interspecific organ transplants in Drosophila. Jour. Exp. Zoo!., 82: 1-30.
BODENSTEIN, D., 1939b. Investigations on the problem of metamorphosis. V. Some factors

determining the facet number in the Drosophila mutant bar. Genetics, 24: 494-508.
BODENSTEIN, D., 1942. Hormone controlled processes in insect development. Cold Spring

Harbor Symp. on Quant. Biol., 10: (in press).

Fujli, S., 1936. Salivary gland chromosomes of Drosophila virilis. Cytologia, 7: 272-275.
HADORN, E., AND NEEL, J., 1938. Der hormonelle Einfluss der Ringdriise (corpus allatum) auf

die Pupariumbildung bei Fliegen. Roux Arch.f. Entw. niech., 138: 281-304.
MAKING, S., 1938. A morphological study of the nuclei in various kinds of somatic cells of

Drosophila virilis. Cytologia, 9. 272-282.
Ross, F. B., 1939. The postembryonic development of the salivary gland of Drosophila melano-

gaster. Jour. Morph., 65: 471-495.
SONNENBLICK, B., 1940. The salivary glands in the embryo of Drosophila melanogaster.

Genetics, 25: 137.
VOGT, M., 1940. Ztir Ursache der unterschiedlichen gonadotropen Wirkung der Ringdriise von

Drosophila funebris und Drosophila melanogaster. Roux Arch. f. Entw. mech., 140:

525-546.

HORMONES AND TISSUE COMPETENCE IN THE DEVELOPMENT

OF DROSOPHILA

DIETRICH BODENSTEIN*

(Department of Zoology, Columbia University, New York)

The larvae of Drosophila molt twice, and change with the third molt into the
pupal stage, during which the larval organism is gradually made over into the
final adult insect. It was found (Bodenstein, 1936) that the initiation of pupation
depends upon some factor in the anterior part of the larva which becomes active
shortly before pupation and which is presumably hormonal in nature. Hadorn
(1937) has located and analyzed this factor more precisely. He brought forward
conclusive experimental evidence that a hormone causing pupation in Drosophila
is produced by the ring gland, a small organ of internal secretion situated dorsally
between the two brain hemispheres of the larvae. Although responsible for
pupation, the ring gland was seemingly unable to initiate further pupal develop-
ment, i.e. the differentiation of the larval organ anlagen to imaginal completion.
For larval abdomens which, as a result of the removal of the anterior part,
remain constantly larval could be caused to pupate when one or more ring glands
were transplanted into them (Hadorn and Neel, 1938). Yet only puparium
formation but no further development could be induced. Likewise, trans-
plantation of several ring glands into younger larvae brought about only pre-
cocious puparium formation but again no subsequent development (Hadorn and
Neel, 1938). In the light of these facts it appeared highly probable that some
other hormone than that for puparium formation governed imaginal differentia-
tion. The following observations seem to verify this assumption. The imaginal
differentiation of pupal abdomens proceeds to imaginal completion when the
anterior pupal part is cut off about 20 hours after pupation but the abdomen
remains pupal when the anterior part is removed earlier (Bodenstein, 1938 and
1939a). The imaginal differentiation of organ anlagen, for example, eye discs,
also depends upon this factor in the anterior part. On the basis of this rather
indirect evidence a special differentiation hormone was postulated (Bodenstein,
1938). However, attempts to localize this factor in the anterior pupal part
failed completely (Bodenstein, 1939a and c). Pupal abdomens, the anterior
part of which was cut off before the imaginary hormone was released and which
consequently were expected to remain pupal, continue their development to
imaginal completion when placed in a pure oxygen atmosphere (Bodenstein,
1939c). This observation made the existence of a special hormone for differentia-
tion very doubtful. Moreover it was shown (Bodenstein, 1939c), that the
inability of the pupal abdomen to develop could be correlated with disturbances
in the development of the tracheal system. These experiments, then, indicated
that abnormalities in the functional development of the tracheal system rather
than the lack of a special hormone was the cause of the inability of the abdomens

* Fellow of the John Simon Guggenheim Memorial Foundation.

HORMONES OF DROSOPHILA 35

to develop. While these considerations do not disprove the existence of a
differentiation hormone completely, they make its assumption quite unnecessary.
The main object of this paper is to bring forward more conclusive evidence that
the ring gland is responsible not only for pupation but also for differentiation.
Actually we have to consider pupation as the first step in the process of imaginal
differentiation.

MATERIAL AND METHODS

The experiments reported here were performed on Drosophila melanogaster
(Ore. R + ) and Drosophila virilis (wild stock). Both of these species were used
as donors and hosts. Various organ discs were transplanted into the body cavity
of adult flies and the development of the transplant in its new environment was
studied. This new method (see Bodenstein, 1943b, in press) of using the body
cavity of adult flies as a culture medium for larval tissues proved to be very
successful and was used throughout this investigation. The mortality rate in
these experiments was negligible. All the experimental animals were kept at a
constant temperature of 25° ± 0.5° C.

I am greatly indebted to Dr. L. C. Dunn and Dr. Th. Dobzhansky for many
stimulating discussions and for their continued interest in this work. I also
wish to thank Mrs. E. Sansome for helpful criticism during the preparation of
this paper.

EXPERIMENTS

When larval eye or leg discs of Drosophila are transplanted into the body
cavity of adult flies, the grafted organ is unable to develop. Although left for
many days in the adult environment the graft remains unchanged as far as its
morphological appearance is concerned. This observation is in agreement with
earlier experiments of this kind (Bodenstein, 1938, p. 497). From this it was
assumed that the adult environment is a medium unsuitable for the development
of larval organs. It was therefore rather unexpected when it was found that
larval eye discs which were transplanted simultaneously with two ring glands of
mature larvae into adult flies had grown well beyond their original size. This
experiment was repeated as follows: Eye discs of melanogaster donor larvae of
equal age were transplanted either alone or together with ring glands into adult
melanogaster hosts. Three days after the operation the grafted eye discs were
dissected and compared. It was found that the eyes in hosts with ring glands
were larger by far than those which were in hosts without ring glands. A great
number of similar experiments was then performed, consisting of 243 cases where
organ discs were transplanted together with ring glands into adult hosts and
156 control cases where the organ discs were transplanted alone. The bulk of
this material comprises many different series; the series varied as to the time the
organs were allowed to remain in the adult host, the number of ring glands
transplanted into one host, the kind of organ disc used (eye and leg) and the
kind of host used (melanogaster and virilis). In comparing the discs in hosts
with and without ring glands it was invariably found that the organ discs in the
hosts with ring glands had become much larger than the control discs in the
hosts without ring glands. Further proof for the initiation of growth by the ring-
gland was obtained by experiments in which the two partners of a single

DIETRICH BODENSTEIN

organ pair were compared one with the other. For this a pair of eye or leg
discs was dissected from a single donor larva and one partner disc transplanted
into one host together with two to four ring glands, and the other partner
transplanted alone into a second host. The results of these experiments con-
sisting of 34 individual pairs are summarized in Table I, where it can be seen

•

TABLE I

Paired transplantation of eye and leg discs into two adult hosts. One host receives disc alone, -while

other receives partner disc and two to four ring glands.

Transplant

Number of pairs

Days pairs remain
in hosts

Number of pairs
where the disc is
larger in hosts
with ring glands

mel. eye

mel. leg

mel. eye

viril. leg

mel. leg

viril. leg

mel. eye

mel. leg

viril. leg

that in each pair the disc which was transplanted together with ring glands had
become larger than its partner. Figure 1 (a, b) illustrates very clearly the
enormous size difference between two partner discs which were dissected and
photographed five days after the operation. This particular pair is a melano-
gaster leg pair. One partner disc (a) was transplanted alone and the other

PLATE I

FIGURE 1. Melanogaster leg disc pair five days after the operation. One partner disc
(a) was transplanted into an adult melanogaster host; the other partner disc (b) into an adult
melanogaster host together with four ring glands. Note: the enormous difference in size between
the two discs.

FIGURE 2. b: virilis leg disc four days after the operation, showing the first signs of meta-
morphosis, i.e. the beginning of evagination. The disc was transplanted together with four
ring glands into an adult virilis male host, a: Virilis leg disc at the time of transplantation.
c: a normal pre-pupal leg disc beginning to evaginate. Note: the similarity in the process of
evagination between the transplanted (b) and the normal (c) leg.

FIGURE 3. A virilis leg disc transplanted together with three ring glands into an adult
virilis female host, 13 days after the operation. The leg disc is completely differentiated. Note:
the well-formed dark brown chitinous tarsus segments, with hairs and claws well developed.

FIGURE 4. Virilis leg disc pair transplanted into two adult virilis male hosts six days after
the operation. Note: same size of both discs.

FIGURE 5. Virilis leg disc pair four days after the operation. One partner disc (a) was
transplanted into an adult virilis male host and the other partner disc (b) into an adult virilis
female host. Note: disc in female host (b) has become much larger than the partner disc (a) in
the male host, which has not grown at all.

HORMONES OF DROSOPHILA

PLATE I

DIETRICH BODENSTEIN

partner (b) together with four ring glands into the abdomen of adult melanogaster
hosts.

In normal development we notice that a short time after puparium formation
many organ discs undergo a characteristic change of form; they evaginate and
obtain thus their typical pupal shape. This evagination process is one of the
first visible signs of metamorphosis of the organ discs. Now we find that the
transplanted leg discs in the body cavity of adult flies also evaginate under the in-
fluence of the ring gland after they have grown to a certain size. This induced
evagination process is not quite complete, presumably because of mechanical
difficulties, but is nevertheless very clear. This is illustrated in Figure 2 (a, b),
Figure 2a shows a leg disc at the time of transplantation; Figure 2b a leg disc of
a normal young pupa which has started to evaginate, and Figure 2c a disc which
was transplanted into an adult host together with ring glands and which was
dissected four days after the operation. In comparing Figure 2b with Figure 2c
one may notice the similarity between normal and induced evagination. This
observation clearly proves that the ring gland is able to induce the first stages
of metamorphosis in the transplanted leg anlage. Moreover the ring gland is
able to induce complete metamorphosis in the leg, if the leg is left in the adult
host long enough. In these cases we find a completely differentiated imaginal
leg with femur, tibia and tarsus segments as well as well-formed and dark brown
chitinized hairs, bristles and claws in the abdominal cavity of the fly (Figure 3).
From these experiments it becomes evident that the ring gland is not only re-
sponsible for an early initiation of growth, but also for the imaginal differ-
entiation of the organ discs.

While it is true that organ discs transplanted together with ring glands were
always larger than the control discs transplanted without ring glands, there was
nevertheless a certain variability in the growth of the discs. In some cases
where the discs v/ere transplanted alone into adult hosts it was found that they
had not grown at all, although they had remained for ten days or longer in these
hosts. In other cases the discs had grown quite wrell and even showed signs of
metamorphosis although no ring glands were present. Similarly, discs of equal
age gro\vn for the same length of time in the presence of the same number of
ring glands could vary quite extensively in size. Now it has to be realized that
in experiments of the kind described one deals with three different developmental
systems which together determine the outcome of the experiment. These systems
are: 1. The adult host environment; 2. The activating system, i.e. the ring
glands; 3. The reacting system, i.e. the test organ discs. Thus in order to
clarify the observed discrepancies in the experiments, a more thorough investi-
gation of these three systems was undertaken. For this purpose experiments
were designed in such a way that two of the systems were held constant and
the third one varied. In doing this for each system in turn a clear understanding
of the part played by each system was obtained.

/. The adult host environment

Under this general heading we will discuss a number of experiments in which
the adult flies used as hosts were varied. As an indicator for possible differences
between the various hosts wre used only the early growth reaction of the test
organs. This method is very sensitive, for even small differences reflect them-

HORMONES OF DROSOPHILA 39

selves very clearly in the growth of the test organ discs, especially when the two
partners of a single organ pair are compared.

a. Growth variability test in male hosts without ring glands.

This series was designed to test whether there is any difference in the growth
of test organs in different host individuals of the same sex. For this purpose,
pairs of leg discs were dissected from virilis larvae and one partner disc trans-
planted into one and the other partner disc into a second adult virilis male host.
The two hosts were then reared and dissected together. From eight such pairs
two pairs were dissected two days, one pair three days and five pairs five days
after the operation. The partner discs in all pairs were found to be the same
size (Figures 4a and 4b). This proves that there is no detectable difference in
the environment of the different individual male hosts as far as the test organs
are concerned. Moreover, the leg discs remained unchanged in size, which
indicates that no growth had occurred from the time of transplantation until
they were dissected five days later. In order to obtain more information on
this point, four of these disc pairs were again transplanted into adult male hosts.
Seven days later they were dissected and found to be unchanged. Thus we
must conclude that the virilis organ discs are unable to grow in an adult virilis
male environment.

b. Growth tests in hosts of different age.

The question whether there is any difference in hosts of different age has been
tested in the following way. The two partners of a pair of virilis leg discs were
transplanted, one into a one-day old virilis male host and the other into a 29-day
old virilis male host. From five such pairs one was dissected four days, and
four pairs six days after the operation. In all cases it was found that the partner
discs of the single pairs were of the same size. There was also no growth in
either partner discs during the time they remained in the hosts.

In a second series consisting of six pairs, the discs were transplanted into
female hosts instead of into male hosts. One female host was two days old and
the other 30 days old. One pair, dissected three days and five pairs dissected
five days after the operation revealed again that the two discs of one pair were
of equal size. Vet in contrast to the previous series each of the discs had grown
during the time it remained in the host.

These experiments prove that there is no difference between young and old
hosts. They confirm the previous observation that the environment of each
individual male host is the same, and extend the information in showing that
this is also true for the environment of each individual female host. The ob-
servation that no growth takes place in discs transplanted into male hosts is also
confirmed. However, when one compares female and male environment one
finds the discs able to grow in the former but not in the latter environment.

c. Growth tests in male and female hosts.

It is evident from the foregoing experiments that male and female environ-
ments are different as to their effect on the growth of the grafted organ discs.
Decisive evidence for this is provided by the following experiments. The two

40 DIETRICH BODENSTEIN

partners of the virilis leg disc pairs were transplanted, one into a male, the other
into a female virilis host. Five such pairs were dissected three days, and eight
pairs four days after the operation. In all pairs it was found that the discs in
the female hosts were much larger than their partner discs in the male hosts,
which had not grown at all. Figure 5 (a, b) illustrates this effect very clearly.

Similar results were obtained in another series of experiments (five pairs dis-
sected three days after the operation), where leg discs pairs of virilis were trans-
planted into female and male melanogaster hosts.

In transplanting the two partners of virilis eve disc pairs into virilis male
and female hosts (three pairs dissected three days after the operation), we again
find the discs in the female hosts larger than their partners in the male hosts
(Figures 6a and 66). The eye discs in the male hosts had ceased to grow, being
of the same size at the time of dissection as at the time of transplantation.

Finally, in a last series of this kind, the two partners of a pair of salivary
glands of virilis larvae were transplanted, one into a male and the other into a
female virilis host. From eight such pairs three were dissected in four days,
two, seven days and three, eight days after the operation. Again, as in the
case of the organ discs, it was found that the glands in the male hosts had not
developed while their partners in the female hosts were all in an advanced stage
of development.

d. Growth test in different host species.

The object of this group of experiments was to test for possible species differ-
ences between virilis and melanogaster hosts. To this end the two partner
discs of a virilis leg pair were transplanted, one into a virilis male host and the
other into a melanogaster male host. From six pairs available, two were dis-
sected three days, two, four days and two, six days after the operation. The
transplanted discs were found to be of the same size in both hosts in all pairs.
There is evidently no difference between the melanogaster and virilis environment,
as far as it affects the graft.

In a second series of experiments, comprised of seven pairs, which were
dissected three days after the operation, the two virilis leg pair partners were
transplanted into a melanogaster and a virilis female host. Being in a female
environment, the discs in both hosts had, of course, grown; in one pair the discs
were of the same size, in three pairs the discs in the melanogaster hosts were

PLATE II

FIGURE 6. Virilis eye disc pair three days after the operation. One partner disc (a) was
transplanted into an adult virilis male host and the other partner disc (b) into an adult virilis
female host. Note: disc in female host has become much larger than partner disc in male host,
wjiich has remained unchanged.

FIGURE 7. Virilis leg disc pair three days after the operation. One partner disc (a) was
transplanted together with two ring glands into an adult virilis male host and the other partner
disc (b) was transplanted together with two ring glands into an adult melanogaster male host.
Note: disc (b) in the melanogaster host has become much larger than its partner (a) in the virilis
host.

FIGURE 8. Virilis leg disc pair four days after the operation. One partner disc (a) was
transplanted into an adult virilis male host together with four ring glands and its partner (b) into
an adult virilis male host together with eight ring glands. Note: both discs have grown the same.

HORMONES OF DROSOPHILA

PLATE II

42 DIETRICH BODENSTEIN

slightly larger and in the last three pairs they were somewhat larger in the virilis
hosts. The fact that the discs may be larger in melanogaster female as well
as in virilis female hosts indicates that there is no significant difference between
the environment of both host species.

e. Growth test in male and female hosts in the presence of ring glands.

It has been shown before that organ discs grow larger in female hosts than
in male hosts. The question now arises, how the growth of equal discs is affected
when they are under the influence of the same number of ring glands in both
environments. For this, two partner discs of a virilis leg pair were transplanted,
one into a virilis male and the other into a virilis female host, while at the same
time each of the two hosts in two of such pairs received five ring glands, and in
two other pairs four ring glands from mature larvae. In dissecting the two pairs
with five ring glands three days, and the two pairs with four ring glands four
days after the operation, it was found that the discs in the female hosts of all
pairs were larger than their partner discs in the male hosts. However, in contrast
to the earlier experiments, where the discs were grafted alone, into male and
female hosts, both discs in this experimental combination had grown. Moreover,
the discs in the female hosts had become much larger in the presence of ring
glands than discs which had grown in female hosts without ring glands.

/. Growth tests in different host species in the presence of ring glands.

If the experiments where virilis partner leg discs are transplanted into two
hosts of different species (melanogaster male and virilis male) are repeated,
but each host receives in addition two ring glands from mature larvae, then the
results obtained are quite different. The transplanted organ discs are much
larger in the melanogaster hosts (Figures la and 7b). This was observed in
nine out of 12 pairs four of which were dissected three days, six four days and
two six days after the operation. Only in three pairs left for four days in the
host were the discs found to be alike in both hosts. Moreover in another pair,
where one partner disc was transplanted into a virilis male host together with
five ring glands, and the other partner disc into a melanogaster male host together
with four ring glands, it was found again that the disc in the melanogaster host
was much larger. Finally, in two additional pairs in which one partner disc
was transplanted together with four ring glands, into a virilis male host, and the
other partner into a melanogaster male host together with only two ring glands,
it was again observed that, three days after the operation, the discs in the melano-
gaster hosts were much larger than their partners in the virilis hosts. Thus two
ring glands in melanogaster hosts are able to induce more growth in the test
organs than four ring glands in virilis hosts. Since in all these cases virilis ring
glands were used as grafts we witness the peculiar fact that virilis ring glands
are more effective in a foreign than in their own species environment.

//. The activating system

Experiments described in this section are designed to further the understanding
of the ring gland action.

HORMONES OF DROSOPHILA

a. The effect of different numbers of ring glands on organ growth.

\Ye have seen that organ discs transplanted into adult male hosts are able to
grow only when under the influence of simultaneously transplanted ring glands.
It remained to be shown, however, as to how main' ring glands are actually
needed to assure maximum growth of the organ disc. For this, eye discs of
virilis larvae of equal size were transplanted from virilis male hosts while in
addition, each of these hosts received a different number of ring glands from
mature larvae. The results of the experiments are summarized in Table II,

TABLE II

Effect of different number of ring glands on eye growth.

Transplant remains in hosts for

Experiment

Number of ring
glands

three days

four days

Number of cases

Size of eyes

Number of cases

Size of eyes

no growth

larger than A

larger than B

larger than C

where it can be seen that two ring glands produce a greater growth effect in the
test organs than one ring gland, but that the effect of four or eight ring glands
is the same as that produced by only two ring glands. This evidence is further
supported by experiments in which the two partners of single virilis leg pairs
subjected to the influence of a different number of ring glands in virilis male
hosts are compared. Table III summarizes the results obtained from six such

TABLE III

The effect of different numbers of ring glands on leg growth.

Number of

ring glands
compared

0 and 1

2 and 4

4 and 8

2 and 8

Days leg pairs
remain in

hosts

Size of
leg pairs

larger in
ring gland
host

same in
both hosts

pairs, showing again that the growth of the test organ is the same whether two,
four or eight ring glands are present (Figures 8a and 8fr). In summarizing the
results, we must conclude that one ring gland is apparently unable to raise the
hormone concentration in the adult male fly to a level high enough to assure
maximal growth in the test organs. Yet the hormone concentration produced
by two ring glands must have reached the level of saturation as far as the growth

44 DIETRICH BODENSTEIN

of the test organ is concerned, since more than two ring glands have no greater
effect than only two ring glands.

Evidence that the number of ring glands is also of importance for the time
of imaginal differentiation of the test organ is provided by the following experi-
ments. Melanogaster eye discs of equal size were transplanted into adult
melanogaster female hosts, (a) alone, (6) together with two ring glands, (c)
together with three ring glands, (d) together with four ring glands. All hosts
were dissected eight days after the operation. It was found that in the hosts
without ring glands the eye discs had grown, but were still white and showed no
sign of imaginal differentiation (seven cases). In the hosts with two ring glands,
reddish pigmented regions could be seen in the transplanted discs, showing that
the ring gland had brought about pigment differentiation in the eye disc (six
cases). The eyes in hosts with three ring glands (four cases) and in hosts with
four ring glands (four cases), had developed to the same stage of pigmentation.
In a further set of experiments melanogaster eye discs were transplanted alone
into melanogaster female hosts but left for 16 days (two cases), 17 days (one case),
and 22 days (one case), in the hosts before they were dissected. By this time
pigmentation had also started in these eyes but was in a much less advanced
stage of development. This is indicated by the slight yellow coloration, as
contrasted with the reddish color, developed in eyes grown in hosts with ring
glands. These experiments show that color development can take place in eye
discs transplanted into female hosts without the support of ring glands, but that
it is much less rapid than in female hosts in the presence of ring glands. The
onset and degree of eye pigmentation in the presence of two, three or four ring-
glands in female hosts is about the same. Thus as in the experiments where the
effect of different numbers of ring glands on the early growth of the eye discs
was tested we find that for the later processes of differentiation also two ring
glands produce the maximal effect.

b. The effect of ring glands of different age on organ growth.

Until now we have studied only the effects of mature ring glands, that is,
of ring glands from larvae shortly before pupation. It remains to be seen,
however, whether there is any difference in the effects produced by younger or
older ring glands. Single pairs of virilis leg discs were thus transplanted, one
partner alone and the other partner together with ring glands, into two virilis
male hosts. The virilis ring glands used for each pair were of different age.
In this way progressively younger ring glands were tested as to their effect on
the growth of the organ discs. In two series of this kind, each host received
three ring glands, in a third series, four ring glands. All pairs in the three
separate series were dissected five days after the operation and the growth of the
disc in each pair compared. The results of the experiments are summarized
in Table IV, where it can be seen that ring glands of all ages, even when coming
from larvae as young as five and a half clays before pupation, i.e. young first
instar larvae, are able to promote growth in the transplanted leg test disc. This
proves that the ring gland can produce its groxvth hormone during the entire
larval period of the animal. Whether there may be any interruption in the
hormone production of the ring gland during this period, as the few negative
cases might indicate (see Table IV), is not known, and needs further investigation.

HORMONES OF DROSOPHILA

c. Differences in hormone production of young and old ring glands.

The question as to the amount of hormone produced by young and older
ring glands was tested in the following way: melanogaster leg discs of an average
diameter of 13 units were divided into three lots. One set of legs was transplanted

TABLE IV

The effect of ring glands of various age on organ growth. In all cases the test organ remained for

five days in the host. Positive indicates that the leg partner in the host with ring glands is

larger than its partner in the host without ring glands; while negative

indicates that both leg partners in the two hosts

compared are of the same size.

Number of ring
glands transplanted

Ring gland donor. Age in days
before pupation

Ring gland donor,
larval stage

Result

before pupation

positive

before pupation

positive

negative

positive

before pupation

positive

before pupation

positive

negative

positive

negative

positive

negative

positive

negative

positive

51A

positive

sy2

positive

into melanogaster females alone, the legs of the second set into melanogaster
females each together with one melanogaster ring gland from an old larva shortly
before pupation, and the legs of the third set into melanogaster females each
together with one two-day younger melanogaster ring gland. Three days after
the operation the hosts of these three groups were dissected and the transplanted

46 DIETRICH BODENSTEIN

leg discs measured and compared. The legs in the hosts without ring glands
(eight cases) had grown to an average diameter of 16 units. The legs in the
second group with one young ring gland (five cases) were found to average 21
units in diameter and in the last group with one old ring gland, the average
diameter of the legs (four cases) was 24 units. It was noticed, moreover, that
the legs in the last group had begun to evaginate, which was not the case in the
other two groups.

Now one may test, although in a somewhat different way, the amount of
hormone produced. If it is true, as the above mentioned experiments indicate,
that one young ring gland produces less hormone than one old ring gland, one
might expect equal discs transplanted at the same time, and left long enough in
the adult host, to be advanced further in their metamorphosis in the presence of
old ring glands than in the presence of the same number of younger ring glands.
In such an experiment we use the state of metamorphosis rather than differences
in growth as an indicator for the hormone concentration. In order to elucidate
this point, melanogaster eye discs of equal age were transplanted into adult
hosts, some with four ring glands from larvae shortly before pupation and others
with four one day younger ring glands. Eight days after the operation the hosts
were dissected and the discs compared. The eyes in hosts with four ring glands
had developed yellow-red pigment (four cases) while the eyes in hosts with four
younger ring glands were much less advanced in their differentiation. Although
they had grown extensively in the presence of the younger ring glands, they
were still white, showing no trace of pigmentation (four cases). From these
two groups of experiments we may thus conclude with reasonable certainty that
young ring glands produce less hormone than old ring glands.

d. Species differences in ring glands.

Qualitatively the ring glands of virilis and melanogaster are the same. This
has been shown many times in experiments where the action of virilis or melano-
gaster ring glands has been tested as to its effect on the growth and differentiation
of melanogaster or virilis organ discs. The question whether there is any quanti-
tative difference in the amount of hormone output during a given time between
the ring glands of these two species is, however, not so clear. Since the ring
gland of virilis is larger than that of melanogaster one might expect it to produce
more hormone. If quantitative differences between melanogaster and virilis
ring glands are present, they are at least not large, as the following experiment
indicates. Equal melanogaster eye -discs were transplanted into adult melano-
gaster female hosts together with two ring glands from a melanogaster larva
shortly before pupation (three cases) and together with two virilis ring glands
from larvae shortly before pupation (three cases). The dissection of these cases
eight days after the operation showed that in the hosts with melanogaster ring
glands, one eye disc had developed slight yellow pigment and two eye discs
yellow-red pigment. In the hosts with two virilis ring glands, two eye discs
had become slightly yellow and one eye disc yellow-red pigmented. We thus
observe about the same amount of development under the influence of the same
number of melanogaster or virilis ring glands, indicating that there is no difference
in the quantity of hormone production between the ring glands of these two
species tested.

HORMONES OF DROSOPHILA

e. The time of action of ring glands in adult hosts.

The question as to how long transplanted ring glands in adult hosts continue
to produce hormone was tested as follows: virilis ring glands from larvae shortly
before pupation were transplanted into adult virilis males. The glands were
left in these hosts for a certain length of time, then dissected out and re-trans-
planted into a second adult host together with one partner disc of a leg pair.
The other leg partner was transplanted alone into another virilis male host.
After several days the pair of hosts was dissected, the growth of the leg discs
compared and the ring gland grafts recovered. The recovered ring glands were
now grafted for the third time into a virilis male host together with new test
leg discs, the partners of which were again transplanted as the growth control
into virilis male hosts alone. Several days later the pairs were dissected, the leg
discs compared and the retransplantation procedure of the recovered ring glands
using new test organs and new hosts repeated once or twice more. Since it was
not easy to recover such a small organ as the ring gland from the body of the
adult fly, two or four ring glands were usually transplanted together into one
host. If one ring gland was lost in the dissection, the remaining ring glands
could be used to continue the test. Table V shows the results of these experi-

TABLE V

The time of action of ring glands in adult hosts. (For explanation see text.) Positive indicates that
the retransplanted ring gland has stimulated the growth of the test organ.

Experiment

Days ring gland remains in first host

Days ring gland retransplanted together with
test organ remains in second host.
Condition of test organ

5
positive

6
positive

Days ring gland retransplanted together with
test organ remains in third host.
Condition of test organ

4
positive

5
positive

6
positive

Days ring gland retransplanted together with
test organ remains in fourth host.
Condition of test organ

5
positive

4
negative

Days ring gland retransplanted remains in
fifth host.
Condition of test organ

3
positive

ments. Each of the five columns (A to E) represents one case of successive
re-transplantation of the same original glands. Now, as it can be clearly seen
from Table V, it was found that ring glands after being in adult hosts for 22
days, during which time they had been three times retransplanted and found
to be active, were still active in a fourth transplantation (Table V B). Simi-
larly, ring glands which were left for 22 days in one host before they were tested
in a second host for their activity were still able to induce growth in the test

48 DIETRICH BODENSTEIN

organ (Table V E). From these experiments we must conclude that ring glands
transplanted into adult hosts secrete their hormone continuously for a long time.

///. The reacting system

It has to be realized that the various kinds of organ discs may differ as to
their responsiveness towards the same hormone level. We may also expect
differences in the responsiveness between old and young organ discs. Experi-
ments which investigate these possibilities are presented in the following.

a. The differentiation capacity of different discs in adult hosts.

In comparing the first growth effect of such organs as eye and leg discs,
one finds both very responsive to the hormone of the ring gland. Even in
female hosts without ring glands, which must be considered the least favorable
environment, the growth effect of both discs is considerable. Thus there seems
to be no appreciable difference in the responsiveness between leg and eye discs.
Yet when one compares the further development of these discs in the adult
environment a marked difference between these organs becomes evident. One
finds the leg discs able to differentiate in the adult host to imaginal completion
under the influence of ring glands, but not the eye discs, which never continue
their differentiation beyond the first stages of pigmentation. The leg discs in
their final state of differentiation show typical imaginal characteristics; i.e. dark
brown chitinized leg segments covered with chitinized hairs and bristles and with
a blackish chitinized end claw on the distal tarsus segment. In the eye discs on
the other hand, we find that the pigment is the only component which differ-
entiates to an appreciable extent. There is however some doubt whether even
pigmentation reaches its final imaginal stage. The development of hairs,
bristles, lenses, or the darkening of the chitinous eye parts has never been observed
in eye grafts. Although a more detailed histological examination of these
partially developed eyes is still missing, there can be no doubt that differentiation
is incomplete, since it would have been easy to detect chitinous structures in
total mounts if they were present. It was found, moreover, that the anlage of
the genital apparatus, when transplanted into adult hosts, is unable to differentiate
at all, even in melanogaster female hosts in the presence of four ring glands,
thus in an environment where the ring glands are most effective. Independent
of the time these genital discs remain in the host, they never develop beyond a
stage corresponding to the stage the discs would have reached in normal develop-
ment at the time of puparium formation. In Table VI we have summarized a
number of experiments in which different organ discs were transplanted together
with ring glands into different adult hosts. Only such cases are recorded where
the grafts were left for more than seven days in the host. We find, for example,
that a melanogaster leg disc in a melanogaster female host in the presence of
only one ring gland has already differentiated imaginal characters eight days
after the operation, while a melanogaster eye disc in the same host in the presence
of as many as four ring glands has developed only to the stage of pigment forma-
tion 19 days after the operation. Since about the same amount of pigment is
present in melanogaster eyes which were left for eight days in female hosts
together with two ring glands, it follows that the differentiation in the 19-day
old eye has not progressed much beyond that observed in the eight-day old eye.

HORMONES OF DROSOPHILA

Thus in a melanogaster female host environment under the influence of two or
more ring glands, the eye discs reach their limit of differentiation about ten days
after the operation. As far as the genital discs are concerned, we find them to
grow somewhat beyond their stage of transplantation. Their growth, however,

TABLE VI

The differentiation capacity of different discs in adult hosts.

Num-

Number

Days

ber

Transplanted

Donor

Host

of trans-

transplant

Result

organ

planted

remains

cases

ring glands

in host

9 genital disc

viril.

viril. 9

no clear change

9 genital disc

mel.

mel. 9

no clear change

9 genital disc

viril.

viril. 9

prepupal

9 genital disc

mel.

mel. 9

prepupal

eye

mel.

mel. 9

light yellow spots; no hairs

eye

mel.

mel. 9

light yellow spots; no hairs

eye

mel.

mel. 9

light yellow spots; no hairs

eye

mel.

mel. 9

red yellow spots; no hairs

eye

mel.

mel. 9

reddish spots; no hairs

leg

mel.

mel. 9

brownish hairs and chitin diff.

9 genital disc

viril.

mel. 9

little growth; prepupal

9 genital disc

viril.

viril. 9

little growth; prepupal

9 genital disc

viril.

.mel. 9

little growth; prepupal

9 genital disc

viril.

mel. cf

little growth; prepupal

9 genital disc

mel.

mel. 9

little growth; prepupal

9 genital disc

viril.

viril. cf

little growth; prepupal

9 genital disc

viril.

viril. cf

little growth; prepupal

9 genital disc

viril.

viril. 9

little growth; prepupal

9 genital disc

viril.

viril. cf

little growth; prepupal

9 genital disc

viril.

mel. cf

little growth; prepupal

9 genital disc

mel.

mel. 9

little growth; prepupal

9 genital disc

viril.

viril. 9

little growth; prepupal

eye

mel.

mel. 9

yellow red spots; no hairs

eye

mel.

mel. 9

yellow red spots; no hairs

eye

mel.

mel. 9

yellow red spots; no hairs

eye

mel.

mel. 9

large reddish spots; no hairs

eye

mel.

mel. 9

large reddish spots; no hairs

eye

mel.

mel. 9

large reddish spots; no hairs

leg

viril.

viril. cf

large; no hairs

leg

mel.

mel. 9

brownish hairs and chitin diff.

leg

viril.

mel. 9

brownish hiiirs and chitin dirt.

leg

viril.

viril. 9

hairs diff.; still white

leg

viril.

viril. 9

brownish hairs and chitin diff.

leg

viril.

viril. cf

hairs diff.; still white

leg

viril.

viril. 9

brownish hairs and chitin diff.

leg

viril.

viril. cf

brownish hairs and chitin diff.

leg

viril.

viril. cf

brownish hairs and chitin diff.

is very much slower than that of leg or eye grafts, even in their early growth
effect. They never surpass, as said before, a prepupal stage, although they
may be as long as 17 days in a female environment under the influence of four
ring glands.

DIETRICH BODENSTEIN

13
53

o
•••^

a
"I
8

>—< K

53
J

a^l

aaaaaaaaaaa

33333333333

aaaaaaaaaaa

<J — i u i- u C! u d i

aaaaaaaaaaa

I I I

bbbbbbbbooo

C UUUUUliUUUI-'V"

t/5

u
</) cfl cn

.= .=. .=

rt rt cj
£ J5 J5 J= O

•3

y
cd

c 'o'o'oOOOOOOOOOy'OO

S . ^ aaaccclcccccc^C!::

o * ^ ^ "

c
_o

•5

c
o

.5 c c

tn tr. x js tn tn js tn tn tn tn u; j

.5 .3 .Jj CJ _|- u o ^- t- i- t- i- cj

— -^ -M -4-J .W

T3 '" '~ — 1 !° fr •/! •- •— f-0

i i i .2 c c '> '> .22 'g "S •> •> •> ^
...c ^^ffc^f'^f'-'c.

03 4J Wl

= c , c £
!° cfl ^'3 S o
H"5. I -^

»~ ,V ">

c o 2 S wo

|b||-s|

^^•""a M

OOCM-OOOOOOOOO
6

j o 4) W W "C "C "C "C "C C u "C "C "C "C "C

Q c

CJCJOCJOUOUCJOCJ

< PQ U Q

O E H-

HORMONES OF DROSOPHILA 51

The capacity of the three imaginal discs tested to differentiate in adult hosts
in the presence of ring gland thus decreases in the order leg, eye and genital disc.
This can be demonstrated conclusively by a somewhat different experiment, as
follows: a leg, eye and genital disc from the same donor larva were transplanted
into a single host, together with ring glands. In this way all the three discs
are under the influence of the same number of ring glands in the same host
environment and can thus be compared more directly than in the previous
experiments. Fifteen such cases are shown in Table VII, where it can be seen
that the differentiation capacity of eye, leg and genital discs of the same animal
differs markedly under the same hormonal conditions.

Before closing this section, one further point of importance should be men-
tioned. It has been found that there are also differences in the differentiation
capacity in the various regions of the same organ disc. The clearest example of
this phenomenon is provided in the differentiation of the leg disc. While it is
true that leg discs are able to differentiate to imaginal completion in adult hosts,
this statement must be modified somewhat, because it applies only to the distal
leg disc portions. It is known that the larval leg disc not only includes the
presumptive tissue of the actual adult leg, but also some material which gives
rise to the ventral body wall in the nearest neighbourhood of the leg. This
proximal portion of the leg disc never differentiates completely, while the distal
leg portions consisting of femur, tibia and tarsus develop to imaginal completion.
The differentiation capacity of the leg parts seems to increase in a proximal
distal direction, since we find the tarsus segments always to be the first structures
which become imaginal, and only if the discs are left longer in the host do we
find tibia and femur completely differentiated. However, we have not been able
to compel the proximal leg disc portions to become completely differentiated,
although the discs have remained for a considerably longer time in the host
than that needed for the complete differentiation of the distal leg parts. A very
similar situation prevails in the eye discs, where we find that material which
gives rise to pigment cells is able to express its differentiation tendencies, resulting
in the formation of well-differentiated red pigment, while the material destined
to form hairs or lenses is unable to differentiate to any great extent. Moreover,
there seem to be regional differences in the eye for pigment formation also, for
we observe most frequently that only certain eye regions are pigmented while
others are still white. It seems possible that this last phenomenon might be
correlated with the position of the graft in the host as well as with the arrangement
of folding of the developing eye, which in turn may affect the oxygen supply in
the different eye regions, and thus promote or inhibit, as the case may be, the
oxidation of pigment.

b. Differences between young and old discs.

Young and old discs in the same hormonal environment differ as to their
time of differentiation. This has been shown by the following experiment.
Young and old leg or eye discs were transplanted simultaneously into single adult
hosts together with one or more ring glands. The grafts were left in the host for
not less than nine days; they were then dissected and compared. In all cases it
was found that the older graft was further differentiated than the younger graft
(see Table VIII).

DIETRICH BODENSTEIN

TABLE VIII

Differences between young and old discs, transplanted together into one host.

Num-

Days

trans-

Condition of organ at dissection

Ex-

ber

plant

peri-

Transplant

Do-

nor

.Host

trans-
planted

re-

ment

mains

ring

glands

in
host

Old disc

Young disc

young and old eye

mel.

mel. 9

large reddish spots; no hairs

large white; no hairs

young and old eye

mel.

mel. 9

large reddish spots; no hairs

large white; no hairs

young and old leg

mel.

mel. 9

large, with white hairs

large; no hairs

young and old leg

mel.

mel. 9

large, with white hairs

large; no hairs

young and old leg

viril.

viril. 9

large, with white hairs

large; no hairs

young and old leg

viril.

viril. 9

large, with white hairs

large; no hairs

young and old leg

viril.

viril. 9

brownish hairs and chitin

large; no hairs

young and old leg

viril.

viril. 9

brownish hairs and chitin

large; no hairs

young and old leg

viril.

viril. 9

brownish hairs and chitin

yellowish hairs and chitin

c. The responsiveness of organs of different species.

If one compares (Tables VI and VII) the final developmental condition of
melanogaster eyes with virilis eyes which were left for the same length of time
in an approximately equal adult environment, one observes that the melanogaster
eyes have developed further than the virilis eye discs. This indicates a difference
in the competence of virilis and melanogaster eyes to respond to the same hor-
monal conditions. The same indication is seen in another set of experiments
where virilis leg discs were transplanted into virilis female hosts together with
three ring glands (five cases) and melanogaster leg discs (four cases) into virilis
females together with three ring glands. Eleven days after the operation two
melanogaster legs had developed hairs which were, however, still white; the two
other melanogaster legs were completely differentiated, showing yellow-brown
chitinous structures and hairs. Three of the virilis legs were without any hairs,
one had hairs but was white, and -one was completely differentiated. Although
these observations speak for the assumption that virilis organs respond with
greater difficulty to the same hormonal environment, there is one further point
to be taken into consideration. Virilis and melanogaster differ in their time of
development. The larval as well as the pupal period of virilis is much longer
than that of melanogaster. Therefore it is possible that in the above-mentioned
experiments, leg discs of unequal age were compared, especially since no accurate
record was made of the exact age of the donor discs. The observed difference in
the time of differentiation between virilis and melanogaster organs may thus not
really reflect species differences but rather age differences. Even if we assume
that the organs in question were of the same age, this would mean only that they
were alike in their chronological age but not in their physiological age. In the
light of these considerations it becomes evident that it is rather difficult in
experiments of this kind to be quite sure whether any discrepancies in the time
of differentiation between discs of two species are caused by species-specific
responses or age effects.

DISCUSSION

The present investigations have brought forward a number of pertinent facts
concerning the relationship between hormone actions and tissue competence in

HORMONES OF DROSOPHILA 53

the development of Drosophila organ discs. It has been shown that the organ
discs depend for their growth as well as for their imaginal differentiation upon
the action of the ring gland which functions as a gland of internal secretion.
As judged from their effect on test organs, young ring glands produce qualitatively
the same hormone as ring glands of a mature larva. The quantity of hormone
produced by young ring glands is presumably less than that produced by old
ring glands. Equal ring glands differ as to their effect in hosts of different species
and in the two sexes of the same species. The amount of organ growth during a
given time and the speed at which differentiation proceeds depends upon the
number of ring glands, i.e. on the amount of hormone available as well as on the
competence of the organs to respond. Different organ discs as well as discs of
different ages and different regions within the same organ disc differ as to their
competence to respond. These facts reflect very clearly the highly relative
nature of conditions which find their expression in the processes of growth and
differentiation. They show that we cannot ascribe absolute values to either
organ competence or hormone concentration but rather that we have to measure
one in terms of the other.

We have now to consider in more detail certain aspects of the problem of
hormone-controlled growth and differentiation which have arisen in the course of
these investigations. For this it seems best to discuss separately the principal
points in question, and after we have estimated their value to try to fit them
into the framework of the general concept.

A. Relationship between hormone concentration and effective level.

Of particular interest is the observation that organ discs transplanted into
adult female hosts are able to grow even in the absence of ring glands. Since
we know that the growth of the transplanted organ is under the control of the
ring gland hormone we might assume that female hosts, in contrast to male
hosts, either produce or have stored some hormone. We know further that two
ring glands have the same effect as four glands. In the presence of two or four
ring glands, therefore, the environment of either female or male hosts must be
considered saturated with hormone as far as the growth of the organ is concerned.
We should thus expect the hormonal environment of female and male hosts to
be the same, i.e. saturated when both hosts are supplied with five ring glands
each. Consequently, we should also expect the growth response of identical
organs grown in such a saturated male and female environment to be the same.
This, however, is not the case, as the experiments show (see p. 42). The
discs in the female environment grow much better than their partners in the
male environment, although both were in a hormone-saturated environment.
This suggests that the ring gland hormone does not act directly but, rather,
indirectly by the intervention of some factors in the host. Limited by the lack
of further knowledge on this point we might assume for the time being that the
ring gland hormone establishes what might be called an "effective level" in the
host, which in turn is responsible for the various reactions of the test organs.
This assumption is supported by the fact that we observe similar differences in
the reaction of the test organs under the influence of the same number of ring
glands in different species. In these cases, too, a different growth effect is
produced when the hormone concentration has saturated 'the environment.

54 DIETRICH BODENSTEIN

For example, we find that two ring glands in melanogaster hosts have a greater
effect on the growth of the test organ than four ring glands have in virilis hosts.
However, there is a definite relationship between the hormone concentration and
the effectiveness of this level. We find that a low hormone concentration
produced by one ring gland is unable to raise the host level to its most effective
state, while the hormone concentration produced by two ring glands already
brings the level to its highest state of effectiveness. Although the effective level
cannot be raised above a certain threshold even when higher hormone concen-
trations, i.e. more ring glands, are used, its peak effectiveness is nevertheless
higher in females than in males and in melanogaster than in virilis. Yet there
is no apparent difference in the effective level of the females in these two species
when tested without ring glands. The difference between the species becomes
evident only when their levels are elevated by the ring gland hormone. Whether
the low effective level of female hosts is caused by the presence of a small amount
of hormone is as yet still obscure, but of course possible. In any event, it seems
unlikely that some hormone is stored, since in this case we would expect that the
stored hormone would gradually decrease as the flies become older. The experi-
ments show, however, that young and old flies are equally affected. Now, when
one follows the thread of implications connecting these various points it becomes
evident that one may obtain different effective host levels either by varying up
to a certain point the hormone concentration, or by varying the host animals.
For example, the lowest effective level prevails in female hosts without ring
glands. The effective level is somewhat higher in male hosts with one ring gland.
In melanogaster and virilis male hosts with two or more ring glands, the effective
level is lower than in the virilis female hosts with two or more glands, while in a
melanogaster female with two or more ring glands the effective level is highest.
If in the following we speak of hormone concentration, it should be understood
that we always refer to a host level of a certain effectiveness, produced by a
definite concentration of ring gland hormone in a definite host.

It is characteristic that organ discs are unable to grow in adult male hosts
without the support of ring glands. The male host environment was thus
considered neutral. Now we know only that the adult male environment is
neutral as far as the larval discs are concerned. Whether pupal organs which
are presumably much more responsive than larval organs are also unable to
develop in male hosts is not known so far. Actually it would be very difficult
to prove that such an environment is neutral in an absolute sense, i.e. for all
larval as well as all pupal tissues. If we should find, for example, that pupal
discs, but not larval discs, would develop in adult male hosts and from this
conclude that the pupal discs have attained the capacity of independent develop-
ment, this conclusion could well be erroneous. We must take into consideration
that the effective level in the male hosts, although too low for the growth of the
larval discs, might well be high enough to assure the development of the highly
responsive pupal discs. This argumentation brings us directly to one further
aspect of the problem. In an earlier paper (Bodenstein, 1939a) it was shown
that eye discs of young pupae continue their development when transplanted into
larvae the anterior parts of which were cut off by means of a ligature. From
these experiments the conclusion was drawn that pupal eye discs, which already
had been stimulated by the differentiation-promoting hormone, are able to

HORMONES OF DROSOPHILA 55

develop independently in an environment lacking the differentiation stimulus.
At the time these experiments were performed we did not know that the ring-
gland is the source of the hormone which promotes differentiation, nor that this
hormone is produced in younger larval stages. The larval host therefore was
expected to contain no differentiation hormone. Although the source of the
hormone supply in these earlier experiments was cut off by the ligature, and
thus no hormone coming from the ring gland could have reached the transplant,
it is highly probable that enough hormone was left in the rear part to account
for the continued development of the transplanted organ. Since we must
assume that even a very low hormone concentration is sufficient to affect the
very responsive older eye discs, this experiment does not prove the independent
development of the pupal eye. Ephrussi (1943) has recently performed a similar
experiment. He transplanted eye discs from mature larvae into the abdomen
of young larvae and observed that these discs developed synchronously with the
host organs. However, in another series of experiments where he transplanted
eye discs of one-day old pupae into young larvae he found the transplanted eyes
to develop heterochronously. In these cases the transplanted pupal eye had
already formed red pigment while the hosts were still in their larval stage.
These experiments also do not prove the independence of eye development,
for the hormone concentration in the young larvae, although not high enough
for the differentiation of the larval host organs, might have been sufficient for the
differentiation of the pupal eye. In the light of these considerations, it is very
difficult indeed to be sure whether one is dealing with dependent or independent
development. Again we are confronted with the fact that development is not
the reflection of absolute conditions, but is highly relative indeed; it is the
expression of a very delicate balance between the activating and reacting systems
involved.

B. The effective level and tissue competence.

It takes about eight days for a leg disc to differentiate to imaginal completion
in a very effective female environment obtained by a hormone concentration
produced by two or more ring glands, while in normal development in the presence
of only one ring gland the leg disc completes its differentiation in four days.
This shows that the effective level in the normal pupal environment must be
much higher than that of the most effective adult environment. This low level
in the adult environment is very fortunate for the understanding of the responsive
capacity of the test organs, since it has brought to light real differences in the
responsiveness of different test organs and of different regions within identical
organs. For example, if we compare different discs as to their capacity to
differentiate, we find in the most effective adult environment only the distal
parts of leg discs are able to complete their imaginal differentiation, while under
the same conditions, eye discs differentiate only partially and genital discs not
at all. These differences in the responsive capacity of the different discs are not
detectable if we grow them in a pupal environment under the influence of a very
effective level. For, if we transplant legs, eyes and genital discs into larvae
shortly before pupation, all these discs become mature in complete synchrony
with the host organs and there seems to be no difference between them as far as
their responsiveness is concerned. We have demonstrated that young and old

56 DIETRICH BODENSTEIN

discs grown in a highly effective adult environment differ in their time of onset
of differentiation. The young leg discs begin and complete their differentiation
considerably later than older leg discs. When finally even the young discs have
attained imaginal character they are of approximately the same size as the older
discs. In other words, the young discs grow to a certain size before their differ-
entiation leading to imaginal completion begins. This seems in disagreement
with the results of earlier experiments (Bodenstein, 1939b) where it was found
that young eye discs transplanted into older larval hosts differentiated pre-
maturely, that is, before they had reached their full larval size, and as a conse-
quence were finally smaller than normal eyes. When we recall that the effective
pupal level is much higher than even the most effective level in an adult environ-
ment, we realize how we can explain the observed discrepancies between the
results of our earlier and present experiments. Obviously, the pupal level is
high enough to induce premature differentiation in the young organ while the
adult level is able only to promote growth in the young organ. Only after the
young disc in the adult environment has grown to a certain stage and has thereby
become more readily responsive is the low effective adult level able to induce
differentiation also into the young disc. Experiments in which the responsiveness
of young and old salivary glands was tested (Bodenstein, 1943a in press) yield
the same results. These experiments show that differentiation takes place only
when both organ-responsiveness and effective level together attain a sufficient
value. The difference between the responsive capacity of young and old organ
discs is also clearly demonstrated by experiments (Bodenstein, 1939a and b) in
which very young eye discs were transplanted into larvae shortly before pupation.
In these cases the very young eye discs wrere only partly differentiated at the time
the host emerged, although they had been under the influence of the very effective
pupal level. This shows that even the very effective pupal environment is
unable to bring about complete differentiation in test organs which are very
young and hence possess a very low responsive value.

If we list the different organ discs as to their capacity to differentiate in the
most effective adult environment, we find them arranged in the following order:
legs, larval salivary glands, eyes, adult salivary glands and genital discs. Under
the influence of the same effective adult level we thus find that the value for the
differentiation response is highest in the leg disc and lowest in the genital and
adult salivary gland discs, while the values for the other discs tested fall between
these extremes. However it seems that the larval skin is more readily responsive
than all the organ discs, as the following experiments by Hadorn and Neel (1938)
indicate. The authors transplanted ring glands into young larvae of the early
third instar and found that under the influence of the ring gland grafts puparium
formation took place prematurely, yet these prepupae failed to develop further.
This indicates that the larval skin is very responsive indeed, since it responded
to the increased hormone level with puparium formation, before the organ discs
\\rre able to respond and hence failed in their differentiation.

Viewing the specific results of the investigations we conceive the following
general picture of the mode of action of the ring gland in the development of
Drosophila. The larval ring gland of Drosophila is an organ of internal secretion
which produces its hormone during the entire larval period. This hormone
controls the growth of organ discs during larval life. In the course of larval

HORMONES OF DROSOPHILA 57

development the ring gland becomes larger and produces more hormone, while
at the same time the responsiveness of the organ discs increases as they grow
older. When the hormone concentration and the responsiveness of the organ
discs have reached a certain value, the ring gland hormone controls imaginal
differentiation also. The evagination of the organ discs is the first indication
that they have reached a differentiation phase. In normal development this
stage is reached at the time of pupation. Pupation is thus nothing more than
the first step in the process of differentiation. The kind of organ response, i.e.
whether the organ discs respond with growth or differentiation to the ring gland
hormone depends upon a definite relationship between hormone concentration
and organ responsiveness. It is very probable that the ring gland hormone has
no direct effect on the reacting organ systems, but that it rather acts indirectly
through the intervention of some as yet unknown mechanism. If these conclu-
sions deduced from experimental results are correct, it should follow that extirpa-
tion of the ring gland in the larval stage should prevent the growth of the organ
anlagen. This experiment, technically not possible in Drosophila, has actually
been performed by Burtt (1938) on Calliphora larvae, with the result that the
growth of the organ disc was arrested in larvae which had their ring glands
removed. These experiments then provide further evidence that the ring gland
hormone controls not only differentiation but also the processes of organ growth
during the larval period. The general interpretation of the problem under
discussion is in contrast to Hadorn's view; he maintained that only ring glands
from mature larvae produce hormone and that this hormone controls solely the
processes of puparium formation, but has no effect on the growth or differentiation
of the organ discs. On the basis of our experimental evidence, Hadorn's con-
ception seems to be no longer tenable.

SUMMARY

A variety of organ discs of Drosophila was transplanted together with or
without ring glands into the body cavity of adult flies and their developmental
behavior in their new surroundings studied. The specific results of these investi-
gations are briefly summarized as follows:

1. Organ discs transplanted into adult male hosts cease to develop but remain
alive presumably indefinitely. The transplanted discs do not lose their develop-
mental potencies, although development may be arrested for a long time.

2. Organ discs transplanted into adult male hosts will grow and finally
differentiate to imaginal completion when under the influence of simultaneously
transplanted ring glands.

3. Organ discs transplanted into adult female hosts continue their develop-
ment at a very slow rate even in the absence of ring glands.

4. There is no difference in the organic environment of different species as
far as the development of test organs is concerned. If however, different host
species are provided with the same number of ring glands it is found that the
ring glands have a greater effect on the development of the test organs in melano-
gaster than in virilis hosts.

5. Ring glands of all larval ages, even from larvae only 12 hours old, are able
to induce growth in the transplanted test organ.

58 DIETRICH BODENSTEIN

6. The amount of hormone produced by young larval ring glands is less than
that produced during the same time interval by ring glands of mature larvae.

7. Different organ discs differ as to their capacity to differentiate in adult
hosts under the influence of ring glands.

8. Different regions within the same organ disc also differ as to their differ-
entiation capacity.

9. Under the same hormonal environment it takes the young organ discs a
considerably longer time to complete differentiation than it takes the old organ
discs.

10. The ring gland hormone, apparently, does not affect the reacting organ
directly, but acts rather through the intervention of some as yet unknown factors
in the host.

11. The kind of organ response, that is, whether the organ disc responds with
growth or differentiation to the ring gland hormone depends upon the relationship
between hormone concentration and organ responsiveness.

12. The problem of growth and differentiation in the development of Droso-
phila is discussed. It is pointed out that development is not the reflection of
absolute conditions but that it is highly relative indeed; it is the expression of a
very delicate balance between the activating and reacting systems involved.

LITERATURE CITED

BODENSTEIN, DM 1936. Das Determinationsgeschehen bei Insekten mit Ausschluss cler friih-

embryonalen Determination. Ergeb. Biol., 13: 174-234.
BODENSTEIN, D., 1938. Untersuchungen zum Metamorphoseproblem I. Roux Arch. f. Entiv.

mech., 137: 474-505.
BODENSTEIN, D., 1939a. Investigations on the problem of metamorphosis IV. Developmental

relations of interspecific organ transplants in Drosophila. Jour. Exp. Zool., 82: 1-30.
BODENSTEIN, D., 1939b. Investigations on the problem of metamorphosis V. Some factors

determining the facet number in the Drosophila mutant Bar. Genetics, 24: 494-508.
BODENSTEIN, D., 1939c. Investigations on the problem of metamorphosis VI. Further studies

on the pupal differentiation center. Jour. Exp. Zool., 82: 329-356.
BODENSTEIN, D., 1943a. Factors controlling growth and metamorphosis of the salivary gland

in Drosophila. Jour. Morph. (in press).
BODENSTEIN, D., 1943b. Hormone controlled processes in insect development. Cold Spring

Harbor Symp. on Quant. Biol. 10: (in press).
BURTT, E. T., 1938. On the corpora allata of dipterous insects II. Proc. Rov. Soc., London,

126: 210-223.
EPHRUSSI, B., 1943. Analysis of eye color differentiation in Drosophila. Cold Spring Harbor

Symp. on Quant. Biol., 10: (in press).
HADORN, E., 1937. An accelerating effect of normal "ring glands" on puparium formation in

lethal larvae of Drosophila melanogaster. Proc. Nat. Acad. Sci., 23: 478-84.
HADORN, E. AND J. NEEL, 1938. Der hormonelle Einfluss der Ringdruse (corpus allatum) auf

die Pupariumbildung bei Fliegen. Rmix Arch. f. Entw. mech., 138: 281-304.

THE LIFE-HISTORY OF PHYLLODISTOMUM SOLID UM RANKIN,
v 1937, WITH OBSERVATIONS ON THE MORPHOLOGY,
DEVELOPMENT AND TAXONOMY OF THE
GORGODERINAE (TREMATODA) l

CHAUNCEY G. GOODCHILD 2

(Department of Zoology, New York University)

INTRODUCTION

Digenetic trematodes are internal flatworm parasites which occur in all
classes of vertebrates. The life-cycles of these parasites involve two or more
hosts of which one,* the first intermediate, harbors the parasitic asexual stages
and eventually liberates a transfer stage which is typically a free-swimming
cercaria; the definitive host is usually a vertebrate which harbors the parasitic
sexual stages of the worm. The cyclic transfer from one host to another has
been variously modified by interpolations of additional intermediate hosts, and
by complications in the methods of infection of the first intermediate hosts.

Trematodes belonging to the family Gorgoderidae Looss, 1901 are character-
ized by having their bodies generally divided into two regions: a narrower,
mobile preacetabular part and a broader, sluggish postacetabular region. The
cuticula is usually smooth. The intestine may be simple to ramified. The
genital pore is median, preacetabular and behind the bifurcation of the gut caeca.
The testes, two to nine in number, are intercaecal or extracaecal, usually oblique,
rarely opposite. The ovary is usually pretesticular. The vitellaria are paired
and postacetabular. The uterus is much coiled postacetabularly. The eggs are
relatively small, numerous, without filaments, and usually fully embryonated.
Adults are found in the excretory ducts and urinary bladders of fishes, amphibians
and reptiles, and in the body and pericardial cavities of marine elasmobranchs.

The Gorgoderidae consists of two subfamilies with contrasting characters as
follows:

Gorgoderinae Looss, 1899 Anaporrhutinae Looss, 1901

1. No pharynx. 1. Well-developed muscular pharynx.

2. Laurer's canal present. 2. No Laurer's canal.

3. No seminal receptacle. 3. Prominent seminal receptacle.

4. Parasites in urinary bladders and ducts of 4. Parasites in pericardial and body cavities of
fishes, amphibians and reptiles. marine elasmobranchs and urinary bladders

of marine turtles.

The subfamily Gorgoderinae, at present, contains the following genera:
Gorgodera Looss, 1899; Gorgoderina Looss, 1902; Phyllodistomum Braun, 1899;
Xystretum Linton, 1910; Macia Travassos, 1922. The genus Phyllodistomum

1 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
at New York University.

2 Now located at State Teachers College, Springfield, Missouri.

60 CHAUNCEY G. GOODCHILD

has synonymy as follows: Spathidium Looss, 1899; Catoptroides Odhner, 1902;
Microlecithus Ozaki, 1926; Dendrorchis Travassos, 1926.

The subfamily Anaporrhutinae, according to Nagaty (1930), contains the
following genera: Anaporrhutum Ofenheim, 1900; Probolitrema Looss, 1901;
Plesiochorus Looss, 1901 ; Petalodistomum Johnston, 1912 ; Staphylorchis Travassos,
1920.

Generic diagnosis of Phyllodistomum Braun : Body usually spatulate. Ventral
sucker usually larger than oral sucker. Intestinal crura separate posteriorly.
Genital atrium usually present. Testes two, smooth to deeply lobed, usually
oblique, the one on ovarian side being more posterior. Anterior testis may be
cephalad of ovary. Vas deferens long, seminal vesicle conspicuous, pars prostatica
and ductus ejaculatorius generally short and inconspicuous. Ovary entire or
lobed, usually posterior or lateral, rarely anterior to vitellaria. Oviduct relatively
long, arising from dorsum of ovary. Fertilization space usually evident. Laurer's
canal usually paralleling vitelline duct on side opposite to ovary and opening to
exterior. Mehlis' gland present but indistinct. Metraterm large and distinct.
Vitellaria compact or lobed; common vitelline duct very short. Eggs in metra-
term embryonated. Excretory vesicle sac-like, elongate, its pore usually
subterminal.

Gorgoderid trematodes are usually found in the exretory systems of their
hosts; however, von Olsson (1876) described Distoma conostomum from the
esophagus and gills of Coregonus maraena. Nybelin (1926) stated that the worms
probably had emigrated from the bladder upon the death of the host, and that
he always found this species of trematode in the urinary bladder. Van Cleave
and Mueller (1934) have likewise reported ectoptic Phyllodistomum superbum
from the gut of Esox lucius, Percina caprodes zebra and Percopsis omnisco-maycus
from North America. Both Looss (1899) and Odhner (1902), upon the basis of
morphological similarity with other known phyllodistomes, agreed that von
Olsson 's Distoma conostomum is a species of Phyllodistomum.

There have been more than 40 seemingly valid species described in this genus.

According to Nybelin (1926) the first report of an adult phyllodistome is
probably that of Fabricus (1780) who described Fasciola umblae from the "kid-
ney" of Salmo alpinus (''in sanguine dorsali salmoni alpine"). Later Fabricus
(1794) redescribed and figured this same form. Rudolphi (1819) renamed it
Distoma seriate, expressing the opinion that "sanguine dorsali" is probably to
be interpreted as the kidney.

In the year 1816 v. Olfers described from Esox lucius a bladder fluke which
he called Distomum folium. Many of the distomes found by subsequent workers
in the bladders and urinary ducts of fishes and amphibians have been referred
to this species. This fact has created the extremely difficult taxonomic problem
of attempting, with meager data, the separation and identification of valid species.

Braun (1899) erected the genus Phyllodistomum with Dist. folium Olfers,
1816, as type, and included in the genus: Dist. cygnoides (Zed.) Looss, D. cymbi-
forme Rudolphi and D. patellare Sturges. Later in the same year Looss erected
the genus Spathidium with D. folium Olfers as type. By the laws of taxonomic
priority, Spathidium has been suppressed as a synonym of Phyllodistomum.

Zschokke (1884) found Distomum folium in Coitus gobio, Thymallus vulgaris,
Trutta variabilis and Salmo umbla, but not in Esox Indus. Braun (1892) found

PHYLLODISTOMUM SOL1DUM RANKIN, 1937 61

Distomum folium in Esox lucius at Konigsberg. His account indicates that
Zschokke had confused: the ovaries (2) with the vitellaria, the oviducts with the
vitelline ducts; he failed to see the ovary; the shell gland was confused with the
vitelline reservoir and the vitellaria were eggs in the uterus. Looss (1894)
reported Distomum folium from the urinary ducts of Acerina cernua. Sinitsin
(1905) studied fishes from Warsaw ponds and identified Phyllodistomum folium
in Carassius vulgaris, Barbus vulgaris, Gobio fluviatilis , Leuciscus rutilus, Scardinius
erythrophthalmus, Squalius cephalus, Idus melanotus, Aspius rapax, Abramis
brama and Blicca bjorkna. Finally, Zandt (1924) found Phyllodistomum folium
in Leuciscus leuciscus and Leuciscus rutilus from Lake Constance.

Looss (1901) described Phyllodistomum acceptum from Crenilabrus pavo and
C. griseus. Odhner (1902) described Phyllodistomum unicum from Serranus sp.,
Phyllodistomum linguale from Gymnarchus niloticus, P. spatula from Bagrus
docmac and B. bayad, and P. spatulaeforme from Malapterurus electricus. He
hesitated in placing the last two phyllodistomes in the genus because they had
symmetrically placed testes and sharply separated anterior and posterior body
regions. It was due to this hesitation that Odhner in Looss (1902) erected the
questionable genus Catoptroides with P. spatula Odhner as type.

Osborn (1903) described P. americanum from North American Amblystoma
tigrinum. Since that time, descriptions of phyllodistome species have been
generally complete enough to reduce taxonomic difficulties.

Two complete and several incomplete life-histories have been reported for
members of this subfamily. Incomplete cycles were determined by Sinitsin
(1905) for three species of Gorgodera and Gorgoderina vitelliloba. Krull (1935)
experimentally proved the life-cycle of Gorgodera amplicava. Rankin (1939)
determined the life-history of Gorgoderina attenuata, the first for any North
American species of that genus. Except for abstracts by Crawford (1939, 1940)
on the life-history of Phyllodistomum americanum, and Goodchild (1940) on the
life-history of P. solidum there have been no life-histories reported for North
American phyllodistomes.

Several European workers -have postulated, upon morphological similarity
and slight experimental evidence, certain relationships between cercariae or
metacercariae and sexually mature phyllodistomes. Nybelin (1926) summarized
and amplified these speculations concerning European phyllodistome life-
histories. Looss (1894), Liihe (1909) and Odhner (1911) proposed that Cercaria
duplicata v. Baer which develops in Anodontites cygnea, Anodontites anatina and
A. cygnea ventricosa is the larva of Phyllodistomum folium (Olfers). Nybelin
(1926), on the other hand, stated his belief that C. duplicata, as at present defined,
consists of three separate cercariae: (1) C. duplicata Wagener, 1851 (Nybelin
stated that this larva must be removed from the phyllodistome cercariae because
it possesses a pharynx and has a Y-shaped excretory bladder) ; (2) C. duplicata
Pagenstecher, 1857 (also accredited with a pharynx, which Nybelin regards as
an error in observation) — Nybelin was unable to link this cercaria with any
known adult phyllodistome; (3) C. duplicata Reuss, 1903 (= C. duplicata v. Baer
according to Nybelin). In extensive feeding experiments of metacercariae from
C. duplicata, using 16 different species of fish, several of which are natural hosts
for Phyllodistomum folium, Reuss was unable to obtain any sexually mature
distomes. He found metacercarial excystment only in two species, Cyprinus

62 CHAUNCEY G. GOODCHILD

carpio and Tinea vulgaris, but there was no progressive development; thus he
was unable to determine the species of the adult phyllodistome. Nybelin
postulated, upon the basis of the sucker ratio, the position of the genital pri-
mordium and the position of the genital pore, that C. duplicata of Reuss is the
larva of Phyllodistomum elongatum; this hypothesis seems untenable because
Reuss was unable to find metacercarial excystment in Abramis brama which
Nybelin listed as a normal host for the sexually mature P. elongatum.

Nybelin believed that the short-tailed cercaria described by Sinitsin (1901)
as the larva of Phyllodistomum folium is a true phyllodistome cercaria, but is not
that of P. folium because the cercaria had an acetabulum smaller than the oral
sucker; the smallest adult P. folium which Nybelin observed (0.45 mm. long),
had an acetabulum larger than the oral sucker. Nybelin stated that this short-
tailed cercaria may represent the larva of P. elongatum, the other known bladder
fluke of cyprinids. This assumption is inconsistent, however, because in the
same paper he had already proposed the C. duplicata of Reuss to be the larva of
P. elongatum, "Die einzige Larvenform fiir welche eine Vermutung der Artzu-
gehorigkeit einigermassen berechtigt erscheint, ist die von Reuss naher behandelte,
welche durch ihre etwa gleichgrossen Saugnapfen, durch die Anlage des vorderen
Hodens schrag hinter der Anlage des Germariums und vor alien durch die dicht
vor dem Bauchsaugnapf gelegene Anlage des Genitalporus sehr an Ph. elongatum
erinnert." This seeming contradiction Nybelin avoided by a second assumption
as to the fate of the cercaria described by Reuss, "Es ist also auch in diesem
Falle nicht moglich, etwas bestimmtes zu sagen; es ware sogar denkbar, dass
sich Reuss' Cercarie zu Ph. pseudofolium entwickeln konnte." However, the
validity of this species has been questioned; Lewis (1935) threw P. pseudofolium
into synonymy with P. folium.

Odhner (1911) stated that the stumpy-tailed cercaria of Sinitsin (1901) is
the larva of P. macrocotyle. However, Nvbelin (1926), Zandt (1924) and Lewis
(1935) agreed that P. macrocotyle is synonymous with P. folium. Odhner (1911)
believed that the marine " Rattenkonigcercaria " Cercaria clausii when eaten by
the fish Chrysophrys aurata develops into Phyllodistomum acceptum. This belief
was accepted by Steelman (1938) who accordingly suppressed P. acceptum as a
synonym of Phyllodistomum clausii? Nybelin summarized these life-history
studies and speculations in his statement, "Es ist also gegenwartig fiir keine
Phyllodistomum-Art moglich, die zugehorige Cercarienform sicher anzugeben;
die postembryonale Entwicklungsgeschichte der einzelnen Arten dieser Gattung
muss vielmehr ganz von neuem, und zwar am bestem auf experimentellem Wege
studiert werden." This is the only way reliable results are obtainable; and
furthermore, life-history studies based upon controlled experimentation will be
invaluable for clarification of the chaotic taxonomic status of the sexually mature
phyllodistomes.

The first phyllodistome life-history to be experimentally completed in the
laboratory was reported by Goodchild (1940) for Phyllodistomum solidum Rankin,
1937 which consisted of the following stages: sexually mature distomes in the
urinary bladder of Desmognathus fuscus fuscus, sporocysts and young cercariae
in Pisidium abditum, and metacercariae in naiads of several species of Odonata.

3 Cable [1942. Jour. Parasit. 28 (6 supp!.)] proposes that Cercaria clausii is the larva of a
fish trematode belonging to the family Lepocreadiidae or the family Gyliauchenidae.

PHYLLODISTOMUM SOLIDUM RANKIN, 1937 63

In the present paper, the various stages of the parasite are described and experi-
ments proving the life-cycle are cited.

The author wishes to acknowledge the encouragement and helpful criticism
given by Professor H. W. Stunkard during both the progress of the experimental
portions of the study and the preparation of the manuscript.

MATERIALS AND METHODS

A. Bivalves and asexual stages.

The fresh-water sphaeriid hosts of gorgoderid trematode asexual larval stages
are grouped into the three genera: Sphaerium, Musculium and Pisidium. Pisi-
dium abditum, host of C. Phyllodistomum solidum, were collected from Barrett
Pond near Cold Spring on Hudson, N. Y., in October and November 1937, and
during the summers of 1938, 1939 and 1940. The clams were collected by
careful examination of small quantities of bottom in regions where they were
abundant. In this way, two collectors were able to take about 300 clams in
one day. In the laboratory they were separated into lots of about 25 to 30 each
and placed with a small amount of pond debris into finger-bowls. They were
then set in running water to maintain a lower temperature. Every morning
each container was examined for cercariae with the aid of a 3X lens; this method
proved adequate for locating the cercariae, but as a final check, each bowl was
examined under a 17X binocular microscope.

When cercariae were found in any dish, the clams in that bowl were then
isolated to obtain the infected ones. All infected bivalves were placed in indi-
vidual containers; this separation reduced the death rate because infected clams
are less tolerant of conditions of crowding, increased temperature, starvation
and fungal infections than uninfected molluscs.

It was extremely difficult to keep the clams alive in the laboratory. The
water in which they lived was changed at least once a day. Culture water
containing various species of green algae and diatoms was introduced in small
amounts at frequent intervals. Nevertheless, the mortality during the period of
adjustment to laboratory conditions was discouragingly high. When kept in
running city water the results were more encouraging. Finally, an aquarium
was set up which approximated the conditions in nature as much as possible.
The pond debris which served as the bottom was sloped to one end of the tank.
The depth of water was approximately two inches in the deepest part. At the
other end, moss was introduced and a fine stream of water was allowed to trickle
over it. It was possible in this wray to have laboratory-raised specimens for
miracidial infection experiments.

Infected bivalves were teased apart to obtain mature living sporocysts for
study. Mature sporocysts were also fixed in warm (65°) Bouin's fixative and
stained with paracarmine and Delafield's haematoxylin.

B. Odonatan naiads and metacercariae.

The damsel-fly naiads which were used as the second intermediate hosts were
both grown from eggs in the laboratory, and collected from a small pond in
Van Cortlandt Park, New York City. This pond was free of Pisidium sp., so
the naiads were the equivalent of laboratory-raised specimens. As a control,

64 CHAUNCEY G. GOODCHILD

25 per cent of the damsel-flies were dissected under the binocular microscope;
they were all free of any helminth metacercarial infection. In addition, the
naiads which were to be used as hosts were isolated in the laboratory for two
weeks, after which time they were starved for periods of from three to seven days.
Starvation made them so transparent that examination by transmitted or
reflected light would disclose immediately any metacercariae. This period of
starvation served the additional function of causing the naiad to capture and to
devour eagerly any cercariae offered.

The cercariae were removed singly by micropipettes and introduced into a
finger-bowl with a starved damsel-fly naiad. As soon as the nymph had seized
a cercaria it was placed under the microscope and the penetration, wandering
and encystment of the cercaria were watched.

Encysted and mechanically excysted metacercariae were studied alive, and
also after fixation in Bouin's fluid and staining with Delafield's haematoxylin,
Ehrlich's haematoxylin and paracarmine. Serial sections of experimentally
infected odonatan naiads were also studied.

C. Desmognathus fuscus fuscus and adult worms.

The salamanders which were used in the experiments were captured as young
adults. They were kept in the laboratory for periods of from 12 to 17 months
before being used. During this time they were isolated at monthly intervals for
periods of from three to seven days in a small quantity of water in a finger-bowl.
The water in which they were living was examined about four times a day for
free-swimming miracidia. Since, in agreement with Rankin (1939), it was
found that miracidia will live under these conditions for as long as 24 hours,
any miracidia shed would be readily found. Salamanders kept under identical
conditions as controls, when dissected, were always found to be parasite free.

Adult worms were studied alive and after fixation. Bouin's fluid and satu-
rated mercuric chloride solution with about 5 per cent acetic acid added were
used as fixatives. Whole mounts were stained with paracarmine. Serial
sections were stained with Ehrlich's haematoxylin and Mallory's triple connective-
tissue stain.

EXPERIMENTS

1. Experiments with first intermediate hosts:

Infected Desmognathus fuscus fuscus were isolated in finger-bowls in small
quantities of water. The miracidia, which were given off in great numbers,
were picked up in a dropper and introduced into a finger-bowl containing actively
crawling laboratory-raised bivalves which were about 2 mm. long and approxi-
mately six months old. The miracidia moved about in more or less direct
courses. They were not attracted to the bivalves; they would swim within less
than 0.1 mm. of the clams and not change their courses. Miracidia were observed
being drawn into the mantle cavity of the bivalve by way of the incurrent mantle
cleft. The miracidium of Gorgoderina attenuate, according to Rankin (1939),
enters its bivalve host similarly.

A more successful method of infection of molluscs was to introduce an infected
Desmognathus into an aquarium with clams enclosed in small wire baskets.
The salamander and bivalves were then allowed to remain together for as long
as one week. It was possible, in this way, to obtain 50 to 70 per cent infections
in the clams.

PHYLLODISTOMUM SOLIDUM RANKIN, 1937 65

2. Experiments with second intermediate hosts:

Damsel-fly naiads were offered cercariae within four hours of emergence of the
latter from bivalves. The violent action of the cercariae attracted the attention
of the insect nymph. The labium of the naiad was projected out to grasp the
cercaria which was then pulled within reach of the mandibles. The enlarged
portion of the tail, because of its turgid spherical cells, prevented damage to the
distome in the chewing process. However, it was found that such protection is
not absolute, because about 15 per cent of the cercariae eaten failed to establish
themselves in the insect host. Some of the failure may be due to imperfect
cercariae, but in other cases, lacerated and partly digested cercariae have been
found in sections of the gut.

The insect nymph was able to engulf the cercaria completely in approximately
ten seconds. The tail collapsed and was digested, while the young distome
penetrated the intestinal wall. Usually only one minute elapsed from the time
the damsel-fly naiad took the cercaria until the young distome had penetrated
the crop wall. In one instance, the point of penetration was in the prothorax;
the worm migrated posteriad to the anterior edge of the metathorax, then turned
and wandered to the anterior edge of the mesothorax where encystment finally
took place. Usually the cercaria encysted, within four minutes of penetration,
in the segment in which it pierced the crop.

The act of penetration caused apparent discomfort to the insect. With one
cercaria the reactions were as follows: within 15 seconds after the act of swallow-
ing, the naiad rubbed the legs together and also rubbed the body with the legs;
then it started nervous wriggling movements, followed by short random dashes
through the water. If several cercariae were taken within ten seconds of each
other multiple penetrations occurred. The initial symptoms of the insect were
then intensified, but otherwise similar to those described above. However,
after about one minute the naiad lost its equilibrium and went into tetanus. A
damsel-fly thus treated would not take additional cercariae for at least 30 minutes.

Similar behavior of odonatan naiads during penetration of gorgoderid cercariae
has been reported by Sinitsin (1905) and Krull (1935).

The forming metacercarial cyst wall was recognizable as a delicate flexible
covering within 15 minutes after cercarial penetration. The anterior end of the
worm could easily push the wall out of shape. After cystogenous material,
which was emitted through the excretory pore, had been deposited in one region
of the forming cyst, the cercarial body was then thrown into a figure-8 shape
which brought the oral sucker into contact with the extruded cystogenous
material. The oral sucker and edge of the mouth opening then pushed the
material into a smooth layer on the inside of the growing cyst wall. Meanwhile,
the posterior end of the worm had been depositing cystogenous material elsewhere.
The formation of the complete cyst took approximately 18 minutes; the activity
then gradually diminished, and the young worm was practically quiescent one
hour after penetration. The metacercaria changed position in the cyst, as
indicated by drawings made over a period of several days.

Metacercariae were usually located in the thorax of the naiads; they have
been found as far posteriorly as the second abdominal segment, and as far an-
teriorly as the dorsum of the head.

Various odonatan species have been tried as second intermediate hosts:
Ischnura verticalis, and Argia sp., were satisfactory; Enallagma sp., and Libellula

66 CHAUNCEY G. GOODCHILD

sp., were less satisfactory. Ischnura verticalis was easiest to obtain in numbers,
and since it was the most transparent of the odonatans available, it was used
extensively as a metacercarial host.

3. Experiments with the definitive vertebrate host:

Metacercariae which had been watched during cyst formation were dissected
from the damsel-fly naiads and fed to Desmognathus fuscus fuscus. The sala-
manders became infected with phyllodistome bladder flukes. The metacercariae
excysted in the small intestine of the urodele; young worms were recovered from
the posterior portion of the large intestine, the cloaca, and the urinary bladder
within 24 hours after ingestion of the metacercariae.

In one experiment, a salamander was fed, at 5 P.M., one Ischnura naiad
containing five metacercariae; at 10 P.M., the same urodele was fed another
damsel-fly nymph containing two metacercariae. The next day, at 4 P.M., the
Desmognathus was sacrificed. Three small excysted phyllodistomes were re-
covered from it; one worm was found in the posterior portion of the large in-
testine, one in the cloaca, and one in the "urethra" migrating into the urinary
bladder. All these young worms still had concretions in their excretory bladders.
In routine examination of the digestive system the stomach was opened. The
damsel-fly which had been eaten at 10 P.M. was still being digested; one meta-
cercaria still encysted in it was found and studied. The stylet was attached
externally to a single-layered hyaline cyst wall. This fact seems to suggest the
presence of an acid-pepsin labile outer cystogenous layer. The three missing
young worms were not found, although the intestine, the Wolrfian ducts, the
mesonephroi and the coelom were examined carefully.

Worms, of increasing size, have been recovered from the urinary bladders of
the salamanders 1, 2, 3, 4, 5, 15, 21, 30, 66 and 123 days after being fed meta-
cercariae. See Figures 9-14 inclusive.

Various vertebrates have been used in experiments with these metacercariae:
Triturus viridescens viridescens, Rana pipiens, R. palustris, R. catesbeiana, R.
clamitans, Micropterus dolomieu, Eupomotis gibbosiis, Carassius auratus and
Cyprinus sp., all without success.

DESCRIPTION OF STAGES IN THE LIFE-CYCLE

1. Miracidium.

The ripe eggs in the metraterm of the uterus contain fully developed motile
miracidia. As many as 300 free-swimming miracidia have been recovered from
undiluted urine squeezed from the bladder of an infected salamander. When
the sexually mature distome is placed in water, eggs are passed through the
genital pore and hatch in 10 to 30 seconds. Ripe eggs of members of this sub-
family usually hatch immediately in water, although Crawford (1940), for
Phyllodistomum sp., reported a two-day interval between egg deposition and

PLATE I

FIGURE 1. Free-swimming miracidium.

FIGURE 2. Miracidium.

FIGURE 3. Miracidial epidermal plates.

FIGURE 4. Mother sporocyst (48 hours old).

FIGURE 5. Mother sporocyst.

PHYLLODISTOMUM SOLIDUM RANKIN, 1937

FIG. 5

PLATE 1

68 CHAUNCEY G. GOODCHILD

hatching. The free-swimming larva (Figure 1), 45 to 55 \L long by 40 to 47 /z
wide, is usually mucronate-cuneate in shape, but may change to pyriform (Figure
2), often with the narrower end anterior (Figure 3). These latter shapes (Figures
2 and 3) are usually assumed when the larva is fixed or imprisoned under a
cover glass.

Externally the miracidium is covered with cilia borne on 16 epidermal plates
(Figure 3) arranged in three transverse rows. There are six plates in the first
row, six in the second row, and four plates in the last row. Sinitsin (1905,
pi. Ill, Figures 56 and 57) likewise indicated three transverse rows of epidermal
plates in the miracidium of P. folium. Each cilium possesses a distinct basal
granule.

Internally the miracidium is provided with an anterior sac-like "gut" 15 /z
long by 24 iz wide, with a small apical opening. Situated laterally is one large
gland, 34 zz long by 15 /z wide, which opens beside the "mouth." This gland is
filled with refractive particles which stain deep maroon with neutral red. Rota-
tion upon the long axis is readily observed in swimming miracidia because the
asymmetrical gland appears as a rotating large, white mass. A much smaller
gland, which may represent the homologue of the larger gland, is located on the
opposite side of the miracidium and is often connected to the larger gland-cell
by a short narrow isthmus. The body of the larva is filled with many fat-like
droplets of various sizes. Similar droplets appear in all larval stages of this
bladder fluke. A group of small cells with large nuclei, which may represent the
germinal elements, is found in the posterior end of the miracidium. Two flame-
cells are present near the posterior part of the middle-third of the body. From
these flame-cells excretory ducts lead away; the ducts follow a tortuous course
anteriorly and laterally for a short distance and then they turn posteriad and
open separately by small lateral pores in the space anterior to the last row of
epidermal plates.

2. Mother sporocysts.

The miracidia after being drawn into the mantle cavity penetrate the gills of
the clam. The epidermal plates are sloughed off in one piece (Figure 4), exposing
the subepidermal layer of the miracidium, which then becomes the wall of the
mother sporocyst. The development of the larvae in the laboratory was slow.
Four weeks after miracidial penetration the mother sporocysts (Figure 6) are
small, 240 p. by 67 /z, and tubular, but well-organized germ-balls and developing
daughter sporocysts are already present. From six to ten daughter sporocysts
are produced by each mother sporocyst. Flame-cells in the young mother
sporocyst are located in the peripheral thickened region of the larva. The

PLATE 2

FIGURE 6. Young daughter sporocyst.

FIGURE 7. Mature daughter sporocyst.

FIGURE 8. Metacercaria, encysted (12 hours old).

FIGURE 9. Young adult (one day).

FIGURE 10. Young adult (five days).

FIGURE 11. Young adult (21 days).

PHYLLODISTOMUM SOLIDUM RANKIN, 1937

FIG. 8

FIG. II

PLATE 2

70 CHAUNCEY G. GOODCHILD

sporocyst wall is relatively thick (20 /*) and is composed of clear intercellular
fat-like droplets and cells with large nuclei. A six-weeks old mother sporocyst
measured 490 p. by 150 M- It had eight daughter sporocysts inside which ranged
in size from about 60 n by 50 ju for the smallest, to 102 n by 55 M for the largest.
These young daughter sporocysts already had a wall and a central cavity con-
taining an average of three large cell groups which may be germinal in nature
(Figure 5).

3. Daughter sporocysts.

The tubular, unbranched, sluggish, mature daughter sporocyst (Figure 7)
measures about 0.67-1.25 X 0.32-0.40 mm., and lies between the outermost gill
plates with its constricted, evertible anterior end embedded in the gill tissue,
and its posterior free end extending into the interlamellar gill space. The
sporocyst wall, 27 to 50 n in thickness, has a thin cuticula beneath which are
located very thin circular and longitudinal muscle fibers. The most conspicuous
elements of the wall are clear vesicular fat-droplets ranging from 5 to 17 n in
diameter. Dobrovolny (1939) reported similar structures in Plagioporus sinitsini
sporocysts. Definite cells of two main types also occur in the wall; the most
numerous are about 10 n in diameter with nuclei 6 to 7 n in diameter; from two
to six larger cells, 15 to 20 n in diameter, with conspicuous granular cytoplasm
are also present. Many opaque granules, about one n in diameter, occur in the
sporocyst wall. Flame-cells are similar in all respects to those described by
Thiry (1859), Looss (1894), Sinitsin (1905), Krull (1935) and Vickers (1940).

The interior of the sporocyst is filled with developing cercariae. The sporo-
cyst usually contains a number of germ-balls and a maximum of seven recognizable
cercariae. The large cercarial tails occupy most of the space in the sporocyst.
The developmental stages of the cercariae approximate those described by
Thiry (1859) for Cercaria macrocerca. A birth pore is located subterminally on
the constricted anterior region. Cercariae can be forced by pressure through
this pore; usually, however, the opening is indiscernible.

Between the sporocysts there are often numerous, opaque "yeast-like" cells
of unknown origin and function, which aid in determination of infected clams
in vivo by reflected light before cercariae are shed. The sporocysts are usually
grouped in the posterior to postero-dorsal gill regions. With reflected light
they appear lighter in color; with refracted light they appear as a darker mass.
Unfortunately, clams which are carrying young in the marsupium present much
the same appearance with both types of illumination; however, the developing
young clams are located in the center of the gill region, while the parasites are
located more posteriorly.

4. Cercaria.

A description of the cercaria of Phyllodistomum solidum has already been
published by Goodchild (1939a). In this same paper a discussion of the gorgo-
derid cercariae was given, and a review of the known species of gorgoderid
cercariae with their differences from Cercaria Phyllodistomum solidum ( = Cercaria
conica) was also included.

PHYLLODISTOMUM SOLIDUM RANKIN, 1937 71

KEY TO THE GORGODERID CERCARIAE

A. Cercariae without stylets (Parasites of Unionidae and Dreissenidae)

B. Tail shorter than body of distome C. Phyllodistomum folium Sinitsin, 1901

BB. Tail longer than body of distome

C. Tail with an anterior enlargement and terminal thread-like portion

C. mitocerca Miller, 1935
CC. Tail lacks anterior enlargement and terminal thread-like portion

D. European species C. duplicata v. Baer, 1827

DD. American species C. duplicata of Leidy, 1858

AA. Cercariae with stylets (Parasites of Sphaeriidae)
B. Tail inactive and very elongate

C. Suckers of distome provided with sensory bristles; tail 4.4-7.5 mm. long

C. Gorgodera amplicava Krull, 1935
CC. Suckers of distome lack sensory bristles; tail 8.8-9.6 mm. long

C. Gorgoderina attenuata Rankin, 1939
BB. Tail active and shorter

C. With anterior chamber containing the young distome
D. With anterior tail enlargement

E. Terminal filiform portion of tail shorter than anterior swelling plus anterior chamber

C. sphaerocerca Miller, 1935

EE. Terminal filiform portion of tail longer than anterior swelling plus anterior chamber
F. Cercarial chamber incorporated into anterior tail enlargement
G. Tail enlargement with lateral "wing-like" distentions

C. raicauda Steelman, 1939
GG. Tail enlargement without lateral "wing-like" distentions

C. Gorgoderina vitelliloba Sinitsin, 1905

FF. Cercarial chamber in front of and distinct from the tail enlargement
G. Anterior tail enlargement filled with spherical cells

H. Chamber containing distome ovoid; anterior swelling 1/10-1/13 of total tail

length C. macrocerca Thiry, 1859

HH. Chamber containing distome conical; anterior swelling 1/4-1/5 of total tail

length C. Phyllodistomum solidum Goodchild, 1939

GG. Anterior tail enlargement filled with polygonal cells

H. Filiform portion of tail with four equidistant compact longitudinal muscle

bands C. coelocerca Steelman, 1939

HH. Filiform portion of tail without compact longitudinal muscle bands

I. Distome with 9 pairs penetration glands; anlagen of testes in 5 parts

C. Gorgodera varsoviensis Sinitsin, 1905

II. Distome with 4 pairs penetration glands; anlagen of testes in 9 parts

C. macrocerca Wagener, 1857
according to Sinitsin (1905)
DD. Without anterior tail enlargement

E C. donecerca Goodchild, 1939

EE. Undescribed C. Phyllodistomum sp. Crawford, 1940

CC. Without anterior chamber C. macrocerca Wagener, 1857

according to Wagener (1857)

The presence of stylet-bearing and non-stylet-bearing cercariae in the same
trematode genus is unique. In the Gorgoderidae, moreover, certain morpho-
logical and physiological relationships always accompany the stylet condition:
non-stylet gorgoderid cercariae possess precociously differentiated testes, lack
both penetration glands and definitely organized cystogenous gland cells around
the excretory bladder, have a postacetabularly flared body, and parasitize
members of the family Unionidae and Dreissenidae; non-stylet cercariae lack
intermediate hosts according to Sinitsin (1901) and Reuss (1903); the adults of
these cercariae are parasites of fresh-water fish. Stylet bearing gorgoderid

72 CHAUNCEY G. GOODCHILD

cercariae, on the other hand, lack precociously developed testes [except C.
Gorgodera cygnoides, C. Gorgodera pagenstecheri, C. Gorgodera varsoviensis and
C. Gorgoderina vitelliloba described by Sinitsin (1905)], possess penetration and
cystogenous gland cells, usually lack a postacetabularly flared body, and parasitize
members of the family Sphaeriidae; stylet cercariae are eaten by second inter-
mediate vector hosts in which they encyst to form infective metacercariae;
adults from stylet gorgoderid cercariae are parasites of fishes and amphibians.

All sexually mature phyllodistomes have a similar morphological pattern.
Upon the basis of adult structures, it is impossible to determine the cercarial
type of any adult whose life-history is unknown. If subsequent investigation
proves that the adults, because of similar habitats, have converged to a common
morphological form from diverse ancestry (Text Figure 1) it will be a most

??7

(Catoptroldes) Phyllodlstomum Gorgoderlna Gorgodera

Cercariae without Cercariae with Cercariae with

stylets stylets; stylets;

motile non-motile

Unlonld and Drelas- Sphaerlld parasites.

enld parasites.

FIGURE 1. Assuming convergent evolution of adults from diverse cercarial ancestry.

striking case of convergent evolution ; if the present concept of close adult relation-
ships holds, however, which seems most likely, it must be assumed then that
cercarial structures in the gorgoderids, at least, are only caenogenetic features
which have been modified to fit the exigencies of their varied life-histories (Text
Figure 2).

5. Metacercaria.

One hundred and forty-four damsel-fly naiads were each experimentally
infected with from one to ten metacercariae. The latter are easily seen in vivo
with intense reflected light because the opaque concretions in the excretory
bladder appear pearly white. The metacercarial cysts (Figure 8) are spherical
to ellipsoidal in shape, with the relatively inactive distome occupying practically-
all the internal space. The very delicate hyaline cyst wall (one to four /j. thick)
of cercarial origin is evidently later, approximately five days, augmented inside
by a second cystogenous deposit of optically similar material. Vickers (1940)
found, in Cercaria macrocerca, a group of about one dozen large cells with granular
cytoplasm located antero-laterally to the penetration gland cells. He stated,
"they may well be the true cystogenous cells." These cells may be the source
of the secondary cystogenous material. Whether the cercaria of Phyllodistomum
solidum possesses such cellular elements is unknown, however, because Vicker's

PHYLLODISTOMUM SOLIDUM RANKIN, 1937

staining technique was not used. The host does not aid in the formation of the
cyst wall, but host cellular elements do adhere irregularly to the outer cyst
membrane.

The cercarial stylet which is discarded into the cavity of the metacercarial
cyst during the first 24 hours, is at first free-floating in the internal liquid; later
it attached to the internal surface of the cyst and is finally embedded between
primary and secondary cystogenous deposits.

The growth of the metacercaria is rapid: cysts after 24 hours measured
129 M X 124 M, after 15 days 162 M X 155 M, after 30 days 170/1 X 150 M, after
37 days 186 M X 150 M and after 61 days 209 M X 202 M-

"active stylet-bearing
gorgoderld cercarlae"
(e.g., C. macroeerca Fll.)

C. Gorgoderlna
vltellllo

C. Gorgoderlna attenuata

C. Gorgodera ampllcava

C. mltoeerca

C. dupllcata

C. Phyllodlatomum folium
Slnltsln, 1901.

Undiscovered
ancestor

With stylets Without stylets

FIGURE 2. Assuming divergent evolution of cercariae from a common cercarial ancestry.

The worms can be excysted mechanically without damage to the young
distome. The metacercaria retains the cercarial shape, but loses the oral and
acetabular sensory bristles. Unlike Gorgoderina attenuata, as reported by
Rankin (1939), the number and distribution of the sensory papillae remain
constant from cercaria to adult. An anterior cavity in the oral sucker indicates
the former position of the discarded stylet. The penetration glands with their
ducts disappear and, unlike some other phyllodistomes (e.g., P. singular e), there
are none visible in the adult. The excretory bladder and its contents are the
most conspicuous structures of the aging metacercaria. Peculiar "rosette-
shaped" refractive granules appear in the cavity of the bladder during the first
24 hours. These granules and the diameter of the bladder both slowly increase
in size with age. At all times, however, the granules are clustered in what
appears to be a cellular membrane. This membrane may be the outer wall of
a "cell" which concentrates the metacercarial metabolic wastes into insoluble

74 CHAUNCEY G. GOODCHILD

concretions. The excretory granules are composed of an insoluble carbonate
which gives a pink color reaction with mercuric chloride solution, and which
dissolves with the evolution of a gas when treated with an acid.

Little actual progressive development of organ systems takes place during
the metacercarial stage. The genital complex, the digestive system and the
nervous system all remain at the cercarial level of development.

A 24 hour metacercaria, the youngest infective larva, was 0.25 mm. long by
0.11 mm. wide; the oral sucker was 62 n in diameter and the acetabulum 64 /j.
in diameter; the club-shaped excretory bladder was 82 n long by 34 ^t at the
widest part. A 61 day old metacercaria was 0.33 mm. long by 0.12 mm. wide;
the oral sucker was 70 n and the acetabulum 79 n in diameter; the excretory
bladder was ovoid, 124 n long by 92 yu wide.

The length of time spent by trematode larvae in second intermediate hosts,
and the degree of differentiation attained in them, often give a clue to the funda-
mental importance of the host. Certain cercariae (e.g., microphallids) emerge
from the molluscan host in a state of relative immaturity and must remain long
enough in the succeeding intermediate host to complete their development.
Other cercariae (e.g., strigeids) may be apparently completely formed, but in the
intermediate host almost complete dedifferentiation occurs, followed by reorgan-
ization into new and different larval forms. Still other cercariae (e.g., am-
phistomes and psilostomes) normally encyst in the open, or within or outside
the host, but undergo few, if any, metacercarial changes.

In the gorgoderids we find what may be a partial recapitulation of such an
apparent larval phylogeny. Cercaria duplicata of Reuss, immediately upon
leaving its host, becomes a metacercaria which sinks to the bottom and lies
dormant until taken in by the next host. According to Sinitsin (1901), the
cercariae of Phyllodistomum folium encyst in the parent sporocyst which then
emerges from the bivalve and is eaten by the next host. These gorgoderids
have an apparent two-host cycle.

Typical three-host cycles also occur in this group of bladder worms. The
cercariae of Gorgodera amplicava, according to Krull (1935), and the cercariae of
Gorgoderina attenuata, according to Rankin (1939), are both passively ingested
with food by snails and amphibian larvae in which they encyst as metacercariae.
The cercariae of Phyllodistomum solidum, according to Goodchild (1939a, 1940),
and the cercariae of Phyllodistomum sp., according to Crawford (1939, 1940),
attract and are eaten by aquatic insect larvae in which they also encyst as
metacercariae. Sinitsin (1905), Lutz (1926) and Krull (1935) have also reported
finding gorgoderid metacercariae in aquatic insect larvae. Progenetic gorgoderid
metacercariae have been found by Wu (1938) in fresh-water shrimps. The
bladder flukes reported by Joyeux and Baer (1934), in the abdominal hypaxial
muscle of Rana esculenta, may also be precocious metacercariae. The inter-
mediate hosts in these three-host cycles, while necessary, are hardly more than
vectors which, by serving as food for the definitive host, enable easier completion
of the cycle.

6. Adult.

The time necessary for this bladder fluke to reach sexual maturity in the
final host is correlated with the length of time spent in the second intermediate

PHYLLODISTOiMUM SOLIDUM RANKIN, 1937 75

host; the size increase of a 61-day old metacercaria is comparable to a 15-day
old young adult worm developed from a four-day old metacercaria. The temper-
ature at which the urodele is kept may also influence the rate of development of
the worm, but as yet, no controlled experimental work has been done on this
aspect. These lungless plethodonids require a cool environment for satisfactory
maintenance in the laboratory which may partially explain the long period
required for sexual maturity of the worm (95 to 130 days).

Practically all development of the fluke takes place postacetabularly. This
region increases both in length and width at a greater relative rate than the
preacetabular body region.

The accompanying table of measurements (Table 1) shows clearly the increase
in size of the body organs while the series of camera lucida drawings (Figures 9
to 14 inclusive) shows relative sizes and growth rates, so that a discussion of
progressive development is unnecessary here.

The sexually mature distome (Figures 15, 16 and 17) can be characterized as
follows: Length 1.24 to 2.67 mm., width 0.76 to 1.27 mm. The body in fixed
specimens is usually not sharply divided into neck and discoidal regions. The
oral sucker is large, 0.26 to 0.47 mm. long by 0.27 to 0.48 mm. wide, terminal,
and cupshaped; the mouth opening is subterminal. There is no pharynx.
The short esophagus, 10 to 90 n long by 24 to 37 ^. wide, bifurcates into narrow
crura which immediately expand into the large digestive caeca, 105 fj. to 129 /j.
wide, the anterior lateral edges of which often extend forward to either side of
the oral sucker. Posteriorly the caeca reach approximately two-thirds of the
distance from the posterior testis to the end of the body. The acetabulum,
390 to 580 /u long by 435 to 560 p. wide, is located between the anterior and
middle body-thirds.

The ovary is large, 40 to 280 n long by 160 to 280 ^ wide, ovoid, usually
dextral rarely sinistral, located about midway between the acetabulum and the
posterior testis, and overlapping the respective gut caecum. The vitellaria
consist of a pair of smooth glands lying between the acetabulum and the ovary;
the right vitellarium is 78 to 199 n long by 106 to 163 /j. wide; the left vitellarium
is 65 to 177 // long by 110 to 140 /z wide. The vitellaria are connected by a
large common vitelline duct, the whole yolk-gland complex appearing dumbbell-
shaped. A Mehlis' gland lies at the middle of the common vitelline duct. The
oviduct leaves the dorsal side of the ovary and runs antero-mediad ; just before
it penetrates the shell-gland (Figure 18) it enlarges to form a fertilization space
filled with sperm cells; at this same point, Laurer's canal is given off dorsally;
it bends anteriorly and opens to the surface dorsally to the shell-gland. The
uterus loops over the vitelline duct and bends to the right, at the lateral edge of
the acetabulum it turns posteriad and runs to the level of the ovary where it
turns mediad between the ovary and posterior testis. The outlines of the uterus
are indistinct beyond this point, and the rest of the postacetabular body is
filled with developing miracidia. The uterus is visible again, running dorsad
and anteriad to the acetabulum, where it expands slightly to form the muscular
metraterm which opens into the genital sinus. The ovoid eggs are small, 30 to
31 fj, long by 22 to 24 fj, wide, and increase in size with the development of the
miracidia; the shells are thin and fragile.

The smoothly contoured testes are smaller than the ovary. The anterior

CHAUNCEY G. GOODCHILD

testis, 124 to 168 n long by 186 to 268 M wide, lies usually in the ovarian field;
often, however, its anterior edge may extend to or beyond a level with the
anterior edge of the vitellarium. The posterior testis, 88 to 118 fj. long by 270

TABLE I

Comparative measurements of Phyllodistomiim solidum at various ages and from different sources

All measurements in microns

One

day

Five
days

21
days

66
days

95
days

123
days

Mature
adult
no. 1

Mature
adult
no. 2

P. solidum
ex Rankin
(1937)

P. solidum
co-type of
Rankin
(1937)

Length

209

170

405

513

1102

1445

1460

1240

1820-2670

2120

Width

126

260

520

685

1000

1040

760-1270

1180

Oral sucker

Length

124

200

226

305

286

260

380-470

424

Width

130

179

243

305

286

273

380-480

453

Acetabulum

Length

155

186

325

378

430

390

440-580

459

Width

163

205

352

390

445

435

510-560

517

Ratio oral to acet.

sucker 1 :

1.25

1.12

1.25

1.03

1.44

1.26

1.54

1.55

—

1.12

Esophagus

Length

10-50

Width

—

Gut caeca

140

108

129

116

—

109

Posterior

109

108

105

—

Ovary

Length

140

160

112

40-280

212

Width

148

170

222

225

160-280

212

Vitellaria

Right

Half

Length

105

140

the

199

Width

132

121

163

size

106

Left

Length

108

132

the

177

Width

124

113

140

ovary

110

Testes

Length

110

124

127

168

Width

132

124

186

268

Posterior

Length

128

118

Width

112

102

270

325

300

Seminal vesicle

Length

116

108

Width

Eggs

28-34

30.4

30.7

X22-

X22

X22.4

X23.4

to 325 IJL wide, located on the ovarian side, is more attenuated transversely than
the anterior testis. The vas deferens is located to the left of the metraterm;
it expands near the anterior edge of the acetabulum into the conspicuous seminal

PHYLLODISTOMUM SOLIDUM RANKIN, 1937 77

vesicle, 93 to 108 M long by 47 to 50 M wide. The vesicle also leads into the
genital sinus which opens to the outside through the median genital pore.

DISCUSSION

Gorgoderid bladder flukes are usually found in the urinary bladders of their
hosts. In Phyllodistomum solidum, the young worms after excystment, emerge
from the anal opening and crawl about in the cloacal cavity. Eventually they
reach the urinary bladder where they reside permanently. The method of
orientation of the worm to the proper cloacal opening is unknown ; in Desmognathus
it may be the action of host cloacal cilia. The manner of infestation of fish
bladders by gorgoderid trematodes is still experimentally undetermined. Sinitsin
(1901), in feeding experiments with metacercariae of his P. folium, was able to
recover excysted specimens in the intestine of Carassius vulgaris and Abramis
brama two hours after feeding; 24 hours after feeding, he found fewer worms in
the urinary ducts. He suggested that young worms migrated from the anal to
the urogenital opening; this assumption remains as a distinct probability, and
the hazards of an external migration would explain the decrease in numbers of
flukes found in the urinary ducts.

The mesonephroi and mesonephric ducts of larger fishes and amphibians
have also been reported as additional sites for bladder flukes. Sinitsin (1901,
1905), Nybelin (1926), Lutz (1926), Odlaug (1937) and Rankin (1939) have
recovered gorgoderid trematodes from these structures in fishes, toads and frogs.
This fact is not surprising because the worms probably feed on the urinary
epithelium. In P. solidum, cells of an epithelial nature have been seen in sections
of gut caeca. The cercarial lytic penetration glands would undoubtedly aid in
loosening the epithelial cells which may explain the retention of these glands in
some gorgoderid adults. In fish, the "urinary bladder" is merely the dilation or
fusion of the posterior parts of the Wolffian ducts; the epithelium is continuous
and similar in both ducts and dilations.

Little can be said concerning host-specificity in the group. Phyllodistomum
folium has been reported from Carassius carassius, Barbus barbus, Gobio gobio.
Scardinius erythrophthalmus, Leuciscus rutilus, L. cephalus, L. idus, Aspius rapax,
Abramis brama and A. bjorkna, all of which are European Cyprinidae. P.
megalorchis has been found in Lota lota, Thymallus thymallus and Salmo trutta.
P. simile has been taken from Coitus gobio, C. poecilopus and Thymallus thymallus.
The remaining phyllodistomes are reported from only one or two host species.
P. solidum, as previously mentioned, was recovered in only one of a group of ten
vertebrate species used in feeding experiments. Rankin (1937) reviewed various
expressions concerning host-specificity among helminths and stated that the
North Carolina gorgoderids have an amphibian host-specificity.

The chaotic taxonomic status of the sexually mature phyllodistomes is due to
synonymy and homonymy. Mature worms have few distinctive qualitative
differences. Morphological structures which have been used taxonomically are:
size and shape; sucker ratio; position of ovary, testes and vitellaria; size of eggs
and contours of body organs. These anatomical features do not all remain
constant during the maturation of members of the same species. P. solidum,
as indicated by drawings (Figures 9 to 15 inclusive), passes from a " gorgoderina "
shape to a phyllodistome one; the oral-ventral sucker ratio varies greatly in

78 CHAUNCEY G. GOODCHILD

fixed specimens due to pressure differences during fixation (Table I). The eggs
likewise vary in length, from 24 to 31 fj, and in width, from 14 to 24 yu, depending
upon their age.

The synonymies proposed for members of this group should lead one to
exercise extreme caution in future gorgoderid taxonomy. Exact taxonomic
identification, without knowing the age of the worm, the physical conditions of
its host, and the normal size range of the parasite, is extremely difficult. Speci-
mens of P. solidum from New York State are usually smaller in all respects than
those taken by Rankin in North Carolina. This fact is explained by the longer
active period of the southern host, correlated with a probable one year cycle of
the mature worm. From observations made in the field, it appears that sala-
manders are infected with metacercariae in late summer and autumn; worms
develop to maturity during the remaining portions of those seasons, and during
the winter and early spring. Miracidia are shed in the early spring, infecting
clams which give off cercariae not before the first of July. This seasonal cycle
would be extended in the milder North Carolina weather, allowing time for the
worms to grow larger.

The caenogenetic organs of the cercariae offer more promise for positive
specific identification. Finally, life-cycle studies, using as many different hosts
as possible, may settle questionable synonymy.

Typical life-cycles were formerly believed to be family characteristics, similar
in their deep-seated significance to the structures and arrangements of adult
organs. In the Gorgoderinae, the life-histories so far reported, have varied
greatly both in larval structures and typical sequences. The non-stylet, stumpy-
tailed cercaria described by Sinitsin (1901), encysts in the sporocyst which then
emerges and being remarkably buoyant because of fat-filled columnar wall cells,
rises with sluggish wriggling movements to the surface, where it is eaten by fish.
Dobrovolny (1939) reported the same type of life-cycle for Plagioporus sinitsini,
a small allocread trematode from the gall-bladders of fresh-water fish. He also
commented on the loss or suppression of the free-swimming cercarial stage which
has been observed in other trematodes.

The non-stylet rhopalocercous gorgoderid Cercaria duplicate, of v. Baer (1827)
and Reuss (1903) emerges from its bivalve host and almost immediately the
anterior portion of the large cercarial tail distends anteriorly to enclose the
distome in a tail chamber. In the more typical gorgoderid cercariae, the same
enclosing mechanism is effected precociously in the sporocyst. The stage
represented by Reuss in Figure 4, if complete, does not represent an encysted
gorgoderid metacercaria; he shows the distome body free in the tail chamber
without any indication of a true enclosing cyst membrane. This fact may
explain Reuss' inability to get further development of the worm in feeding
experiments with any of 16 different species of fish.

Nybelin (1926) reported the encysted metacercariae of P. megalorchis from
the fish, Phoxinus sp. Identification of the trematode was circumstantially
based on the presence of only this one known species of sexually mature phyllo-
distome in the same waters. The stylet cercaria was not found.

Arthropods have also been reported as intermediate hosts of gorgoderid
trematodes by Sinitsin (1905), Liitz (1926), Krull (1935), Wti (1938), Crawford
(1939, 1940) and Goodchild (1939a, 1940).

PHYLLODISTOMUM SOLIDUM RANKIN, 1937

FIG. 13

FIG. 15

PLATE 3

FIGURE 12. Young adult (66 days).

FIGURE 13. Young adult (95 days).

FIGURE 14. Young adult (123 days).

FIGURE 15. Mature Phyllodistormtm solidiini.

CHAUNCEY G. GOODCHILD

FIG. 17

FIG. 16

PLATE 4

FIGURE 16. Co-type, P. solidum of Rankin.
FIGURE 17. Shapes of living P. solidum.
FIGURE 18. Genital complex of adult.

PHYLLODISTOMUM SOLIUUM RANKIN, 1937 81

Sinitsin (1905) described inclusive life-cycles for Gorgodera pagenstecheri and
Gorgoderina vitelliloba, while for Gorgodera cygnoides and Gorgodera varsoviensis,
he conducted incompletely controlled life-history studies. In all four of these
species odonatan naiads and beetle larvae served as second intermediate hosts.
Lutz (1926) reported from South America that macrocercous gorgoderid cercariac
when eaten by odonatan larvae penetrated the esophagus in the wall of which
they encysted. Wu (1938) reported the progenetic metacercariae of P. lesteri
from Chinese fresh-water shrimps, Palaemon asperuhis and P. nipponensis; he
was unable to determine the host of the adult worm. Crawford (1939, 1940)
found that odonatan larvae, caddice-fly larvae and diving-beetle larvae served
as the second intermediate hosts of P. americanum. Goodchild (1939a, 1940,
and in this paper) has reported odonatan nymphs as intermediate hosts of
P. solidum. Krull (1933, 1935) determined that the cercariae of Gorgodera
amplicava was ingested by the snail, Ilelisoma antrosa in which it became an
infective metacercariae. Krull (1935) reported that "frog bladder fluke cer-
cariae" were ingested by the nymphs of damsel-flies, Lestes sp., and formed
metacercariae in the body cavity.

Rankin (1939) determined that tadpoles of Rana pipiens, R. clamitans and
the snail, Pseudosuccinea columella serve as the intermediate hosts of Gorgoderina
attenuala.

Gorgoderids with two-host cycles and those with three-host cycles would
appear to be distantly related. It is possible to assume, however, that there is
present in this group, the fortunate retention of intermediate forms in a rather
complete evolutionary series. The phylogenetic modifications and specializations
of the life-cycle have not altered the fundamental morphologies of the adults or
the preference for their customary parasitic sites.

TRANSITIONS BETWEEN GENERA IN THE GORGODERINAE
1. Gorgodera and Gorgoderina.

These genera have been accepted as valid since Looss (1902) separated them
on the basis of the number of testes, of which the former had nine, the latter two.
Rankin (1939) showed that metacercariae of Gorgoderina attenuata have nine
testes (six on one side, three on the other) which gradually fuse to form two in
the maturing adult. In the same paper, he concluded that although these two
genera of worms are definitely separable, nevertheless, "the genera Gorgodera
and Gorgoderina are apparently very closely related, not only morphologically,
but also with respect to their life cycles and modes of development." Krull
(1935) reported that in one of the largest metacercariae of Gorgodera amplicava
a posteriorly extended string of cells showed enlargements which were the
primordia of testes. However, in young adults the, "dense testicular mass shows
little evidence of being subdivided into testes, and there is little separation into
right and left parts until maturity." This testicular condition again reveals the
closeness of these two genera. Odlaug (1937), reporting on young kidney stages
of Gorgodera amplicava, showed early separation into testicular masses. Although
Odlaug's interpretation of the number of these units was probably erroneous,
in that the ovary and vitellaria were interpreted as testes, nevertheless, the early
testicular segregation that he found is significant.

CHAUNCEY G. GOODCHILD

2. Gorgoderina and Phyllodistomum.

For opinions regarding the identity of these two genera see: Osborn (1903),
Sinitsin (1905), Cort (1912), Nybelin (1926), Ozaki (1926), Pande (1937) and
Byrd, Venard and Reiber (1940). Crawford (1939, 1940) described a bladder
fluke from Bufo boreas boreas and Amblystoma tigrinum which is intermediate
between the two genera. This worm has a phyllodistome-like postacetabularly
flared body, but also possesses prominent uterine coils between the vitelline
complex and the acetabulum (P. acceptum and P. marinum have similar morpho-
logical traits), a condition regularly found in the genus Gorgoderina. In its
early adult development this worm also passes through a typical Gorgoderina
shape.

Gorgoderina schistorchis Steelman, 1938 and Gorgoderina tenua Rankin, 1937
possess phyllodistome morphology and they should be included in the genus
Phyllodistomum .

Because of limited knowledge of life-histories in these genera it is unwise,
at present, to suppress dogmatically one of them as a synonym of the other.

3. Phyllodistomum and Catoptroides.

For a discussion of the history of these two genera consult: Lewis (1935) and
Byrd, Venard and Reiber (1940).

Nybelin (1926), Lewis (1935), Lynch (1936), Bhalerao (1937), Wu (1938)
and Steelman (1938) have advocated, upon morphological bases, the suppression
of Catoptroides as a synonym of Phyllodistomum. On the other hand, Loewen
(1929, 1935), and Arnold (1934) upheld generic separation and described new
species in the genus Catoptroides. Recently, Byrd, Venard and Reiber (1940),
upon the basis of the excretory system, have re-established the frequently
suppressed Catoptroides. The reasons for this action were: (1) differences in the
position of the main collecting duct bifurcation (anterior to the acetabulum in
the Gorgodera-Catoptroides group, and posterior to the acetabulum in the Phyllo-
distomum-Gorgoderina group), (2) the manner in which the collecting ducts
unite with the bladder, and (3) the way in which the accessory tubules arise
from the main collecting tubules.

Byrd et al (1940) found for Gorgodera amplicava, Catoptroides lacustri and
Phyllodistomum, lohrenzi, and Olsen (1937) found for Gorgoderina tanneri a
constant flame-cell pattern of 2 X 8 X 4 = 64 flame-cells. In their papers they
did not discuss the data which has been presented concerning the bifurcation of
the main collecting ducts in other species of Gorgoderinae.

The majority of gorgoderid species possess main collecting ducts which run
anterior to the acetabulum before bifurcation. Sinitsin (1905) reported such a
condition for the cercariae of Gorgodera varsoviensis, Gorgodera cygnoides, Gorgodera
pagenstecheri [Wesenberg-Lund (1934) figured this last cercaria and showed
bifurcation of the main collecting ducts at the level of the anterior-third of the
acetabulum], and Gorgoderina vitelliloba [Wesenberg-Lund (1934) figured post-
acetabular bifurcation in this species of cercaria, but wrote (p. 96), "two excretory
canals issue from the anterior part; near the middle of the ventral sucker they
seem to divide into an anterior and posterior branch."] The account given by
Vickers (1940) concerning the cercaria of Gorgoderina vitelliloba supports the
observations of Sinitsin.

PHYLLODISTOMUM SOLIDUM RANKIN, 1937 83

The following authors have also reported an anterior bifurcation of the main
collecting ducts: Reuss (1903) in Cercaria duplicata; Sturges (1897) in Phyllo-
distomum patellare; Krull (1935) in Gorgodera amplicava; Miller (1935) in Cercaria
sphaerocerca; Steen (1938) in Phyllodistomum brevicecum. Wu (1938) stated that
in Phyllodistomum lesteri, "the two tubules pass through the respective vitellaria
and go around the ventral sucker." Steelman (1939) remarked for Cercaria
coelocerca, "a pair of much-convoluted lateral collecting tubules extends anteriad
from front end of bladder to near level of brain before branching." Goodchild
(1939a, 1939b) found branching of the main collecting ducts anterior to the
acetabulum in the cercaria of Phyllodistomum solidiim and Cercaria donecerca.
Vickers (1940) in a very detailed morphological investigation of Cercaria macro-
cerca Fil. (•= C. Gorgoderina vitelliloba) described lateral excretory ducts which
proceeded anterior to the acetabulum before dividing. The excretory system as
described by Vickers for the cercaria is not complete, but the reported arrange-
ment does not fit any gorgoderid pattern so far determined. Fischthal (1942)
in describing Phyllodistomum semotili, P. notropidus and P. nocomis stated that
the right and left primary collecting ducts are seen, "extending anteriorly to
intestinal bifurcation, then looping posteriorly a short distance each receiving
two secondary collecting ducts."

Postacetabular bifurcation of main collecting ducts has also been reported in
a few gorgoderid species. Byrd et al (1940) have found this type in Phyllodis-
tomum lohrenzi. Rankin (1939) reported posterior bifurcation in Gorgoderina
attenuate; and Olsen (1937) found it in Gorgoderina tanneri. Walker (1937)
found in Gorgoderina vitelliloba a median excretory vesicle extending forward to
the anterior border of the posterior testis. He stated further, "here it bifurcates
into two lateral canals which diverge from one another and extend forwards
externally to the intestinal caeca until opposite the anterior testis, where each
canal divides to form two branches, one lying dorsally to the other. The ventral
branch extends forwards to the level of the acetabulum, while the dorsal extends
in front of the acetabulum."

A final type of bifurcation was described by Miller (1936) in Cercaria mitocerca.
Here, the main collecting ducts divide into an anterior and posterior branch at
the level of the middle of the acetabulum.

Because there is no uniformity in the literature concerning excretory patterns
of the same species (Gorgoderina vitelliloba), or members of the same genus, and
because definite transition forms (Cercaria mitocerca) occur between these two
different bifurcation types, it is evident that conclusions drawn now from such
data are premature and questionable.

CONCLUSIONS AND SUMMARY

1. The life-history of Phyllodistomum solidiim Rankin, 1937, a gorgoderid
trematode from the urinary bladder of the urodele, Desmognathus fuscus fuscus
(Raf.), has been completed experimentally in the laboratory.

2. Cercaria Phyllodistomum solidum (= Cercaria conica Goodchild, 1939)
from Pisidium abditum Haldeman by vigorous cercarial activity attracts and is
eaten by odonatan naiads (Ischnura verticalis, Argia sp., Enallagma sp., Libellnhi
sp.) which serve as metacercarial hosts.

84 CHAUNCEY G. GOODCHILD

3. Metacercariae encyst in the thoracic haemocoele of the insect within four
minutes of ingestion and are infective for the final host after 24 hours.

4. In the salamander, the metacercariae excyst in the small intestine and are
recoverable in the intestine, cloaca, "urethra" and urinary bladder within 24
hours after ingestion of the infective metacercariae.

5. In the laboratory these bladder flukes require three months time to attain
sexual maturity. The mature worms produce eggs which hatch immediately
liberating free-swimming miracidia.

6. The miracidia are drawn by water currents into the gills of the bivalve
where they transform into mother sporocysts. Mother sporocysts each produce
a single generation of daughter sporocysts which, in turn, give rise to the large-
tailed gorgoderid cercariae.

7. From miracidial penetration to cercarial production requires three months
time in the laboratory.

8. A key to the gorgoderid cercariae is included.

9. The phylogeny and life-cycles of non-stylet and stylet gorgoderid cercariae
are discussed.

10. Gorgoderina tenua Rankin, 1937 and Gorgoderina schistorchis Steelman,
1938 have been placed in the genus Phyllodistomum.

11. Comparisons between the genera: Gorgodera and Gorgoderina, Gorgoderina
and Phyllodistomum, and Phyllodistomum and Catoptroides are discussed.

12. Observations on the gorgoderid excretory system as a basis for taxonomy
are reviewed.

LITERATURE CITED

. \KNOLD, J. G., 1934. Some trematodes of the common bullhead Ameiunis nebulosus (Le Sueur).

Trans. Anier. Micr. Soc., 53: 267-276.
VON BAER, K. E., 1827. Beitrage zur Kenntniss der niederen Thiere. Nov. Act. Acad. Caes.

Leap. Carol., 13: 11.
BHALERAO, G. D., 1937. Studies on the helminths of India. Trematoda IV. Jour. Helminth.,

15: 97-124.

BRAUN, M., 1892. Ueber Distomum folium v. Olfers. Centr. Bakt. Parasit., 11: 461-463.
BRAUN, M., 1899. Uber Clinostomum Leidy. Zool. Anz., 22: 484-493.
BYRD, E. E., C. E. VENARD, AND R. J. REISER, 1940. The excretory system in Trematoda.

I. Studies on the excretory system in the trematode subfamily Gorgoderinae Looss,

1899. Jour. Parasit., 26: 407-420.

CORT, W. W., 1912. North American frog bladder flukes. Trans. Amer. Micr. Soc., 31: 151-166.
CRAWFORD, W. W., 1939. Studies on the life history of Colorado trematodes. Jour. Parasit.,

25 (6suppl.): 26.
CRAWFORD, W. W., 1940. The life history of a Gorgoderid trematode, presumably of the genus

Phyllodistomum. Jour. Parasit., 26 (6 suppl.): 38.
DOBROVOLNY, C. G., 1939. Life history of Plagioporus sinitsini Mueller and embryology of

new cotylocercous cercariae (Trematoda). Trans. Amer. Micr. Soc., 58: 121-155.
FABRICUS, O., 1780. Fauna groenlandica. Hafniae et Lipsiae.
FAHRICUS, O., 1794. Bidrag til Snylte-Ormenes Histoire. Skrivter af Naturhistorie-Selskabet.

Bd. Ill: H. 2. Ki0benharn. "
FISCHTHAL, J. H., 1942. Three new species of Phyllodistomum (Trematoda: Gorgoderidae)

from Michigan fishes. Jour. Parasit., 28: 269-276
GOODCHILD, C. G., 1939a. Cercaria conica n. sp. from the clam Pisidium abditum Haldeman.

Trans. Amer. Micr. Soc., 58: 179-184.
GOODCHILD, C. G., 1939b. Cercaria donecerca n. sp. (Gorgoderid Cercaria) from Musculium

partumeium (Say, 1822). Jour. Parasit., 25: 133-136.
GOODCHILD, C. G., 1940. The life history of Phyllodistomum solidum Rankin, 1937 (Trematoda:

Gorgoderidae). Jour. Parasit., 26 (6 suppl.): 36.

PHYLLODISTOMUM SOLIDUM RANKIN, 1937

JOYEUX, C. H., AND J. G. BAER, 1934. Note sur une nouvelle espece de Trematode, Gorgoderina

capsensis, n. sp. Rev. Suisse. Zool., 41: 197-201.

KRULL, W. H., 1933. Notes on the life history of a frog bladder fluke. Jour. Parasit., 20: 134.
KRULL, W. H., 1935. Studies on the life history of a frog bladder fluke, Gorgodera amplicava

Looss, 1899. Papers of the Mich. Acad. of Sci., Arts and Letters, 20: 697-710.
LEWIS, F. J., 1935. The trematode genus Phyllodistomum Braun. Trans. Amer. Micr. Soc.,

54: 103-117.
LOEWEN, S. L., 1929. A description of the trematode Catoptroides lacustri n. sp., with a review

of the known species of the genus. Parasitology, 21: 55-62.

LOEWEN, S. L., 1935. A new trematode of the family Gorgoderidae. Jour. Parasit., 21: 194-196.
Looss, A., 1894. Die Distomen unserer Fische un Frosche. Bibl. Zool., 16: 1-293.
Looss, A., 1899. Weitere Beitrage zur Kenntniss der Trematoden-Fauna Aegyptens. Zool.

Jahr. Abt.f. Syst., 12: 605.
Looss, A., 1901. Ueber einige Distomen der Labriden des Triester Hafens. Centr. Bakt. Parasit.,

29: 398-405.
Looss, A., 1902. Ueber neue und bekannte Trematoden aus Seeschildkroten. Zool. Jahr. Abt.

f. Syst., 16: 411-894.
LUHE, M., 1909. Parasitische Plattwiirmer. Trematodes. Die Susswasserfauna Deutschlands,

17: 100.
LUTZ, A., 1926. Trematodes et Oligochetes observes dans les canaux excreteurs du rein des

Batraciens de 1'Amerique Meridionale. Compt. Rend. Soc. Biol., 95: 1503.
LYNCH, J. E., 1936. Phyllodistomum singulare n. sp., a trematode from the urinary bladder of

Dicamptodon ensatus (Eschscholtz), with notes on related species. Jour. Parasit., 22:

42-47.
MILLER, E. L., 1935. Studies on North American Cercariae (Abstract). Jour. Parasit., 21:

244-254.

MILLER, E. L., 1936. Studies on North American Cercariae. Illinois Biol. Mono., 14: 7-125.
NAGATY, H. F., 1930. A new Anaporrhutine trematode genus and species Nagmia yorkei, with

a review of the classification of the sub-family. Ann. Trap. Med. Parasit., 24: 97-108.
NYBELIN, O., 1926. Zur Helminthenfauna der Siisswasserfische Schwedens. I. Phyllodistomen.

Goteborgs Kungl. Vetensk.-och Vitterhets-samhiilles Hand]., 31: 1-29.
ODHNER, T., 1902. Mitteilungen zur Kenntniss der Distomen I. Centr. Bakt. Parasit., 31:

58-69.
ODHNER, T., 1911. Zum natiirlichen System der digenen Trematoden IV. Zool. Anz., 38:

513-531.
ODLAUG, T. O., 1937. Notes on the development of Gorgodera amplicava in the final host. Biol.

Bull., 72: 80-87.
V. OLFERS, J., 1816. De vegetativis et animatis corporibus i corporibus animatis reperiundis

commentarius. Par. I. Berolini.
OLSEN, O. W., 1937. A new species of bladder fluke, Gorgoderina tanneri (Gorgoderidae: Trema-

toda), from Rana pretiosa. Jour. Parasit., 23: 499-503.
v. OLSSON, D., 1876. Bidrag till skandinaviens helminthfauna. K. Sven. Vet. Akad. Handl.,

14: 1-35.
OSBORN, H. L., 1903. On Phyllodistomum americanum (n. sp.); a new bladder distome from

Amblystoma piinctatum. Biol. Bull., 4: 252-258.
OZAKI, Y., 1926. On two new genera of frog trematodes, Cryptotrcma and Microlecithus, and a

new species of Pleurogenes. Jour. Fac. Sci. Tokyo Univ., 1: 33-44.
PANDE, B. P., 1937. On the morphology and s}'stematic position of a new bladder fluke from

an Indian frog. Annals and Mag. of Nat. Hist., 20: 250-256.
RANKIN, J. S., 1937. An ecological study of parasites of some North Carolina salamanders.

Ecol. Mono., 7: 169-269.
RANKIN, J. S., 1939. The life cycle of the frog bladder fluke, Gorgoderina attenuata Stafford,

1902 (Trematoda: Gorgoderidae). Am. Midi. Nat., 21: 476-488.
REUSS, H., 1903. Die Cercarie und Sporocyste des Distomum duplicatum Baer. Zeit. f. wiss.

Zool., 74: 458-477.
RUDOLPHI, C. A., 1819. Entozoorum synopsis cui accedunt mantissa duplex et indices locuple-

tissimi. x + 811 pp. Berolini.
SINITSIN, D. T., 1901. Einige Beobachtungen iiber die Entwicklungsgeschichte von Distomum

folium Olf. Zool. Anz., 24: 689-694.

*>6 CHAUNCEY G. GOODCHILD

SINITSIN, D. T., 1905. Data on the Natural History of Trematodes. Distomes of Fishes and

Frogs in the vicinity of Warsaw. Warsaw, 210 pp. (English translation from the

Russian.)

STEELMAN, G. M., 1938. A description of Cercaria raiacauda n. sp. Am. Midi. Nat., 20: 613-618.
STEELMAN, G. M., 1939. A new macrocercous cercaria. Trans. Amer. Micr. Soc., 58: 258-263.
STEEN, E. B., 1938. Two new species of Phyllodistomum (Trematoda: Gorgoderidae) from

Indiana fishes. Am. Midi. Nat., 20: 201-210.

STURGES, M. M., 1897. Preliminary notes on Distomiun patellare n. sp. Zool. Bull., 1: 57-69.
THIRY, L., 1859. Beitrage zur Kenntniss der Cercaria macrocerca Filippi. Zcit. f. wiss. Zool.,

10: 271-277.
VAN CLEAVE, H. J., AND J. F. MUELLER, 1934. Parasites of Oneida Lake fishes. Part III.

A biological and ecological survey of the worm parasites. Bull, of N. Y. State College

of Forestry at Syracuse Univ. Roosevelt Wild Life Annals, 3: 161-334.
YICKERS, G. G., 1940. On the anatomy of Cercaria macrocerca from Sphaeriuni corneum. Quart.

Jour. Micr. ScL, 82: 311-326.
WAGENER, G. R., 1857. Beitrage zur Entwicklungs-Geschichte der Eingeweidewurmer.

Natuurk. Verh. v. d. holl. maatsch. d. Wetensch. te Haarlem. II Vers. 13 Deel.

Haarlem.
WALKER, T., 1937. On the anatomy of Gorgoderina vitelliloba (Olss.), a trematode from the

urinary bladder of Rana tcinporaria. Proc. Zool. Soc. London, 107: 75-84.
WKSENBERG-LUND, G., 1934. Contributions to the development of the trematoda Digenea.

Part II. The biology of the freshwater cercariae in Danish freshwaters. Memoires de

1' Academic Royale des Sciences et des Lettres de Denmark Copenhague. Sec. des Sci.,

9 serie, 3: 1-223.
\Vu, K., 1938. Progenesis of Phyllodistomum lesteri sp. nov. (Trematoda: Gorgoderidae) in

freshwater shrimps. Parasitology, 30: 4-19.

ZANDT, F., 1924. Fischparasiten des Bodensees. Centr. Bakt. Parasit., 92: 225-271.
ZSCHOKKE, F., 1884. Recherches sur 1 'Organization et la Distribution Zoologique des vers

parasites des Poissons d'eau douce. Arch, de Biol., 5. Gand.

THE REPRODUCTIVE CYCLE OF THE VIVIPAROUS TELEOST,

NEOTOCA BILINEATA, A MEMBER OF THE FAMILY

GOODEIDAE. IV. THE GERMINAL TISSUE

GUILLERMO MENDOZA

(From the Department of Zoology, University College, Northwestern University, Chicago, Illinois]

INTRODUCTION'

The present article is the fourth of a series written by the author on the
reproductive cycle of the viviparous cyprinodont, Neotoca bilineata. The series
has been an attempt to present a more complete study than has been available
heretofore on the reproductive cycle of a viviparous teleost. Previous studies by
other investigators have been confined largely either to the breeding cycle or to
histological descriptions of the ovary, stressing particularly the adaptation of the
ovary to the retention of the young during development. Little or no attention
had been given to the actual changes in the ovarian soma during gestation until
the papers on Jenynsia (Fitzroyid) lineata (Hylton Scott, 1928; Siccardi, 1940),
Xiphophorus helleri (Bailey, 1933), Neotoca (Skiffia) bilineata (Turner, 1933),
and Cymatogaster aggregates (Turner, 1938b). Some of the above papers were
merely introductory descriptions; others were more complete. Furthermore,
with the exception of a study of the poeciliids by Turner in 1937, no reference
had been made even to the cyclic variation of the germ cells during gestation.
Consequently, the writer undertook this investigation with the purpose of
presenting in detail an analysis of the reproductive cycle stressing particularly
some of the phenomena generally omitted by previous investigators. Articles
by the writer on the reproductive cycle of Neotoca have described in detail
(1) the breeding cycle as observed in the laboratory (1939), (2) the marked
cyclic changes in the ovarian soma during gestation (1940), and (3) the variations
in the germ cell count during gestation (1941). The present article, the fourth
of the series, supplements brief preliminary descriptions (Turner, 1933; Mendoza,
1938) by considering in detail (1) the general description and growth of the
germ cells and their follicles, (2) fertilization, (3) the fate of the evacuated follicles,
(4) the nature and fate of the atretic follicles, and (5) the origin of germ cells
in the adult ovary.

MATERIALS

The present description of the germinal tissue is based on a study of over
30 ovaries in different stages of gestation. The greater part of the material was
fixed in Bouin's or Zenker's fluids although fixatives such as Flemming's fluid,
osmic acid, and corrosive sublimate were used for special techniques. The
principal stains used were iron hematoxylin and Delafield's hematoxylin followed
either by Eosin Y or Orange G. In addition, the following special stains were
used: Mallory's triple connective tissue stain, Van Geisen's stain, and Foot's
modification of Bieslchowsky's silver impregnation technique. The customary
alcohol-xylol dehydration series was used.

GUILLERMO MENDOZA

OVARY

The ovary of Neotoca is a single, spindle-shaped organ inserted in the median
sagittal line; it is attached dorsally to the pleuroperitoneal membrane and
ventrally to the mesogaster. The ovary is continuous caudad into a single
short gonoduct, a term preferred by Turner and others since, evidently, it is not
homologous to the true vertebrate oviduct. Neotoca is similar to most viviparous
teleosts since almost all possess the single median gonad; only in forms such as
Sebastodes rubrovinctus (Eigenmann, 1892) is the ovary double, and only in few
forms such as Dermogenys pusillus (Peters, 1865), Lucifuga subterraneous and
Stygicola dentata (Lane, 1903), is the ovary partially fused. So far as is known
to the writer, all viviparous teleosts have the single gonoduct even in forms that
have the double or partially fused gonads.

GERMINAL TISSUE
Ovigerous folds

The ovary of a teleost may be a solid or hollow organ. In the former case
the eggs merely rupture the wall of the gonad, fall into the coelom and then
escape to the outside through abdominal pores; in the latter type, the eggs never
reach the coelom but make their wray out directly from the ovary through the
gonoduct. In Neotoca the gonad is hollow and the germinal tissue is said to
occur internally; the same condition occurs in all other viviparous teleosts
known to the writer.

The oocytes are not scattered at random but are confined to "ovigerous" or
egg-bearing folds that are placed on either side of a median sagittal septum.

PLATE I

FIGURE 1. A cross-section of a typical non-gravid ovary (19. 5 x). Part of the ovarian wall
at the lower left-hand corner has been removed. In the center of the figure, in a nearly vertical
position is the median septum of the ovary. On either side of the septum lie the two ovigerous
folds attached by a narrow, constricted stalk to the ovarian wall. The attachment is particularly
clear on the left side. The arrow points to the lower edge of the ovigerous fold on the left side.
Prominent in the photograph are several developing oocytes.

FIGURE 2. A growing oocyte. The photograph shows clearly the nucleolus, the vesicular
nucleus, and the early follicle composed of squamous cells.

FIGURE 3. Two good examples of developing oocytes and their follicles. The medium size
oocyte in the upper portion of the figure shows a nucleus that has lost its vesicular character and
has become quite granular. The thickened follicle is the result of rapid mitoptic activity that does
not cease until the follicle cells become so abundant they form a compact layer of columnar cells
as in the egg at the bottom of the figure.

FIGURE 4. A figure that has a two-fold value. First, it shows an oocyte and its follicle in a
stage of development intermediate between that of the two eggs in the preceding figure. The
follicle cells now are cuboidal to low columnar and soon will attain the full columnar shape typical
of the full grown follicle. Secondly, the figure shows the vacuolated yolk-nucleus (arrow) in a
medium size oocyte. Figures 2 to 4 are intended to show the sequence of changes in the oocytes
and their follicles during growth.

FIGURE 5. A typical delle showing the funnel-like depression of the ovigerous fold epithelium
that reaches to the follicle of the egg at the lower left. The intimate contact between the ovigerous
fold epithelium and the follicle cells is clearly visible. Within the delle are two spermatozoa
(arrows). In the upper left-hand corner is a typical mitotic figure in a growing follicle.

FIGURE 6. A high-power photograph (283 x) of a follicle after the expulsion of the egg.
The position of the former delle is visible still at the upper left-hand corner, showing the place
through which the egg escaped from the follicle and the ovigerous fold. The follicle cells still
retain their peripheral position although degenerative changes already have started.

•

PLATE I !

1 All figures are contact prints of unretouched photomicrographs. Figure 1 was taken with
a Reflex-Korelle camera; all others were taken with a Voigtlander cut-film camera. With excep-
tion of Figures 1 and 6, all other photographs were taken with a fluorite, oil-immersion objective
and a Hyperplane ocular; the magnification of the oil-immersion photographs is approximately
650 x.

90 GUILLERMO MEXDOZA

Each fold is attached by a narrcnv strip along the dorso-lateral wall of the gonad ;
the two folds virtually fill the ovarian lumen (Figure 1). Internal ovigerous
folds by no means are characteristic of all viviparous teleosts since Xiphophorus
helleri, Anableps anableps, and Zoarces viviparus are devoid of special folds; the
ova merely develop in the walls of the ovary. On the other hand, forms such as
Cymatogaster aggregatus, Dermogenys pusillus, Jenynsia lineata, Sebastes marinus,
etc., resemble Neotoca and other members of the Goodeidae by the presence of
one type or another of ovigerous folds or septa. In fact, these folds are so
diagnostic in the Goodeidae that Hubbs and Turner (1939) used these same folds,
in part, as a basis for their recent taxonomic revision of the family.

Histologically, the folds consist of a loose, collagenous connective tissue that
in part shows a marked affinity for argyrophilic stains. A vascular network
ramifies throughout the folds and forms a very rich capillary plexus under the
epithelium of the folds. It has been shown (Mendoza, 1940) that during gesta-
tion, this internal epithelium covering the folds changes from a flattened, in-
different condition to a tall, highly secretory epithelium. Parallel with these
changes, the folds likewise become distended with fluid and are invaded by
numerous free cellular elements that contribute to the general tumescence of
the ovary during gestation. Within the ovigerous folds of Neotoca, the germ
cells are evenly distributed throughout the length of the gonad except at the
extreme anterior and posterior ends. The growing germ cells are not scattered
at random but are normally grouped into clusters of cells that range from minute
oocytes 7 to 10 /x in diameter to fully grown eggs approximately 200 n in diameter.
In addition, these clusters of cells normally occur at the surface of the folds,
retaining a very intimate connection with the epithelium (Figures 2, 10, 12), a
relationship that was stressed also for Anableps by Turner (1938a). At this
point of contact between the fully grown ovum and the epithelium, there appears
a deep, funnel-like depression of the ovigerous fold epithelium. The cells at the
base of the pocket are usually flattened strongly against the follicular cells
surrounding the ovum (Figures 5, 9). Normally this depression does not occur
at the outer surface of the folds but at the base of one of the many fissures that
crease the surface of the folds. This funnel-like depression has been described
in other viviparous teleosts and has been identified as a "tubular indentation,"
a "follicular pore," or a "clelle." The two-fold function of this structure will
be discussed elsewhere in this paper.

Germ cells and follicles

Germ cells are recognizable when they are but 7 /u in diameter and are under-
going early stages of maturation. Such early cells are grouped into small nests
and usually are attached closely to the ovigerous fold epithelium. In these
early cells the nucleus is vesicular and occupies fully two-thirds or more of the
diameter of the growing oocyte. The cytoplasm shows an affinity for acidophylic
stains and is heavily but evenly granular (Figure 2). The early follicle consists
of a few delicate, squamous cells flattened against the surface of the oocyte
(Figure 2) ; because of the manner of formation of the germ cell nests and the
structure of the follicle cells, it is likely that the latter are nothing more than
modified fibrocytes of the subepithelial connective tissue.

REPRODUCTIVE CYCLE OF NEOTOCA BILINEATA 91

In the growing oocyte, furthermore, many changes occur. The nucleus is
reduced to one-third of the diameter of the cell and the vesicular character is lost;
it becomes granular and even oxyphylic in nature (Figures 2 to 4). During
growth, the chromatin loses all affinity for stains although it later reappears
in the form of lampbrush chromosomes. Whereas in the early cells the nucleoli
may be numerous, less than a micron in diameter and strongly basic in reaction,
later they are but few in number, large, vacuolated, and even oxyphylic in their
reaction to stains. In general, however, their number, size, and appearance are
highly variable in the different cells and at different stages of development.
In the growing cells the cytoplasm also undergoes marked changes. The most
conspicuous characteristic of the oocytes is the complete absence of large masses
of yolk and the presence of numerous small droplets of oil scattered throughout
the granular cytoplasm. The yolk, such as it is, consists largely of a granular,
rlocculent mass evenly distributed throughout the cytoplasm and very difficult
to distinguish except with the use of differential stains and particularly in stages
immediately following fertilization. There is not the least similarity to forms
such as Zoarces in which the yolk appears as large spheres, nor to Jenynsia and
Xiphophorus in which the yolk appears as a large solid mass. Furthermore,
another goodeid, Lermichthys multiradiatus , shows a heavy yolk mass in the
developing embryos, a mass that, though small in size, is so heavy and compact,
it cannot in any way be compared to the "yolk" of Neotoca. It is regrettable
that lack of material prevented further testing or differentiating of fatty and
proteinaceous yolk in Neotoca although the use of Flemming's fixative on the ova
of another goodeid, Girardinichthys innominatus, showed beyond doubt that
there is a large number of droplets or spheres of fatty yolk concentrated around
the nucleus of the ovum. Another conspicuous feature of the fully-developed
ovum is a large, vacuolated structure that is very similar to or forms a yolk-
nucleus and pallial layer complex (Figure 4). The origin, development, and
fate of this yolk-nucleus complex is so interesting in these viviparous forms that
it will be discussed at length in a separate paper. It is interesting that, among
viviparous teleosts, Cymatogaster and Jenynsia have yolk nuclei equally as
prominent as that of Neotoca. The egg membranes of the enlarged oocytes
are not numerous; there is primarily one heavy vitelline membrane densely
perforated in part, if not in its entirety, by minute pores. Thus it is similar,
though not identical, to the thick, perforated, zona radiata described for Zoarces
by Stuhlmann and for Cymatogaster by Hubbard (1894). Internal to this heavy
egg membrane is a more delicate plasma membrane discernible only after
fertilization when the heavy outer membrane is separated somewhat from the egg.
During growth of the oocyte, the mitotic activity of the follicle cells not only
keeps pace with but actually surpasses the growth of the egg since the follicle
changes from a delicate layer of flattened cells to a single,. densely-packed row of
columnar cells (Figures 2 to 5), a follicle that more closely resembles that of
Xiphophorus and the early follicle of Anableps. In comparison, follicles of a
compound nature are found in forms such as Jenynsia, Stygicola, Lucifuga, and
Cymatogaster. The follicle in Neotoca is in turn invested by a thin layer of
condensed connective tissue fibers and fibrocytes comparable to the "theca"
described by Bailey for Xiphophorus. The connective tissue fibers of this
"theca" are intimately associated with those of the subepithelial network of the

GUILLERMO MENDOZA

ovigerous folds and, like reticular fibers, similarly show a marked affinity for
argyrophylic stains. Interspersed throughout the interstices of this network
there occurs a vascular network more or less prominent in the different follicles
and presumably of great importance in the physiological activity of the follicle
of the developing oocyte.

FATE OF THE GERM CELLS

After the ova have attained full growth they suffer one of two fates, either
they are fertilized and commence development or they undergo atresia. Both
possibilities are considered.

Fertilization

Among viviparous teleosts, fertilization may occur while the egg still is
retained within the follicle or after its extrusion into the ovarian lumen. In
forms such as Anableps and Xiphophorus, fertilization occurs within the follicle
and the embryo is not released until a short time preceding birth whereas in
Jenynsia, fertilization is similar but the embryo is discharged early in develop-
ment. On the other hand, in Cymatogaster, Sebastes marimis (Williamson,
1910), and Neotoca, fertilization and the expulsion of the egg must occur in
such rapid succession that it is difficult to separate the two phenomena. Finally,
in Zoarces, eggs are discharged first into the ovarian lumen and fertilized later.
Accompanying these varying conditions of fertilization, structural and functional
problems are presented by the different types, problems that are largely beyond
the scope of this paper.

Preceding fertilization in Neotoca, the different stages of meiosis can be
identified readily. Following synizesis which appears in oocytes 7 to 10 /x in
diameter, the chromatin temporarily loses all affinity for stains and later reappears
in the form of lampbrush chromosomes. Immediately preceding actual fertiliza-
tion the chromatin condenses markedly, resembling stages of diakinesis, while
the entire germinal vesicle migrates simultaneously toward the periphery of the
egg. This migration occurs normally in the direction of the delle, in preparation
for the ensuing meiotic divisions and fertilization. Evidently, the meiotic
divisions, fertilization, and the expulsion of the egg into the ovarian cavity must
occur simultaneously or in very rapid succession, for none of the three phenomena
actually have been seen although all stages immediately preceding and following
their occurrence have been identified. Unfertilized eggs always have been
identified within the follicles; on the other hand, fertilized eggs normally have
not been seen within the follicles but always free in the ovarian lumen. The
criterion used to distinguish fertilized eggs has been the very radical displacement
of cytoplasmic components within the egg and the ensuing segmentation. In
these eggs, the cytoplasm is concentrated as a thin peripheral layer, presumably
along the animal pole; small oil droplets occur evenly distributed throughout its
extent. Underneath the thin blastodisc, the central portion of the fertilized egg
is filled by a flocculent, albumen-like substance which apparently replaces the
heavy yolk of other viviparous eggs. Occasional spheres of actual yolk can be
found, but they are minute and extremely scarce. Although the meiotic divisions

REPRODUCTIVE CYCLE OF NEOTOCA BILINEATA 93

have not been seen, the polar bodies have been identified on the edge of the
heavy vitelline membrane. A unique phenomenon is that in the vicinity of the
polar bodies the vitelline membrane not only shows a strong affinity for basic
stains but also thickens conspicuously at time of fertilization to resemble the
typical zona radiata of teleost eggs. Since this thickening of the membrane
occurs only in evacuated eggs and only near the polar bodies, it is assumed that
it arises in response to the inciting action of the spermatozoa. Due largely to
the swelling of the vitelline membrane of these eggs, it can be distinguished from
the egg with ease.

It is at the time of fertilization that the delle formed by the ovigerous fold
epithelium plays an important role. Through the delle, the spermatozoa actually
can approach the egg very closely (Figures 5, 9). If the sperm do, in fact, enter
the egg while the latter still is within the follicle, the sperm then must penetrate
only through the single row of flattened cells at the base of the delle and the row
of columnar follicle cells. However, the writer never has identified an actual
pore within the follicular epithelium to permit the entry of the spermatozoa as
described for Xiphophorus by Bailey. Evidence is available from other vivip-
arous teleosts to show that fertilization within a follicle can and does occur.
In Xiphophorus, Anableps, Cymatogaster and Jenynsia, eggs are fertilized while
still enclosed within the follicle and later are discharged into the ovarian lumen
at different stages of development. The second and final function of the delle
is that it offers an attenuated and weakened place in the ovigerous fold epithelium
through which the egg escapes into the ovarian cavity. A similar role for the
delle has been reported for other viviparous teleosts in which it appears.

Upon evacuation of the egg, the follicular cells cease all mitotic activity and
suffer one of two or three fates. Sometimes the force of the expulsion of the
egg is so great that the follicle cells may be everted part way through the delle.
Normally, however, the follicle cells either merely collapse to form a cellular
mass of smaller diameter or the follicle cells may retain their peripheral position
around the reduced space previously occupied by the egg (Figure 6). Despite
these differences in evacuated follicles, the cells eventually lose their regularity
and become a disorganized mass of cells. The evacuated follicle does not assume
the "windswept appearance" of the Xiphophorus follicle (Bailey) nor does it
become hypertrophied as in Sebastes marinus and Anableps anableps. It is
impossible to compare the spent follicles of Neotoca with the mammalian corpora
lutea since in Neotoca there is no evidence of a pronounced physiological activity;
actually, the follicle cells are small and shrunken. Pycnotic figures of degenera-
tion make their appearance soon after the extrusion of the egg; vacuolization
and fatty degeneration have been found among these follicles (Figure 6). By
the time embryos are 1.5 mm. in length it is difficult to distinguish between spent
follicles and ordinary atretic follicles. It is to be expected, perhaps, that in
Neotoca the follicle should be passive and soon degenerate for, since the embryo
develops completely within the ovarian lumen, the follicle is relieved of all
responsibility of serving the developing embryo in a nutritive or respiratory
capacity. Thus the Neotoca follicle is not taxed with a physiological burden
similar to that of Anableps nor poeciliids like Xiphophorus in which the embryo
develops completely within the follicular sac.

94 GUILLERMO MENDOZA

Atresia of the eggs and follicles

Egg degeneration is found in all ovaries. It is evident that eggs degenerate
rapidly if not fertilized soon after they have attained full growth. As many as
25 eggs in various stages of atresia have been found in a single non-gravid ovary.
In typical cases of degeneration which occur during all stages of gestation, both
the follicle and the egg are involved. Normally, the egg and especially the
cytoplasm is the first to disintegrate; the alveolar structure is lost and frequently
the cytoplasmic residue coagulates upon fixation. Coexistent with these changes,
the follicle cells lose their regularity, mitosis ceases, the vitelline membrane is
thrown into folds, and the follicle cells are displaced toward the atretic egg.
Normally, however, the follicle cells retain their peripheral position for some time
while the cytoplasm of the egg breaks up. It is likely that the debris of the egg
is removed in part by absorption and in part by actual phagocytosis. With
the removal of most of the cytoplasmic residue, the follicle collapses completely
and forms a nearly solid group of cells surrounding a mass of debris (Figures 7, 8).
During this process, slight lymphocyte infiltration usually occurs and may be
instrumental in aiding the process of disintegration. In the follicle cells, the
nuclei are the first to undergo pycnotic degeneration; the breakdown occurs
gradually, cell by cell. The degeneration picture most definitely is not one of
complete breakdown of all or most cells at once. In a few isolated cases an
abnormally heavy lymphocyte infiltration may occur and in others the follicle
may precede the egg in degeneration. These, howrever, are infrequent in their
occurrence. Eventually, regardless of the method of atresia, the end result is
the removal of the degenerating mass of cells from the stroma of the ovary.

ORIGIN OF THE GERM CELLS

Although no study has been made of the origin of germ cells in the embryonic
gonad, the writer feels that there is available interesting evidence on the origin
of the cells in the adult gonad. The observation has been made repeatedly that

PLATE II

FIGURE 7. An atretic follicle showing the disorganized state of the follicle cells, the cellular
debris, some fatty degeneration, and several large vacuoles that have appeared between the cells.

FIGURE 8. A degenerating follicle that has been reduced to a small cellular mass. In the
center there occurs some cellular debris and one atretic cell (arrow).

FIGURE 9. This and the remaining figures on the plate have the single purpose of showing
the frequent occurrence of growing oocytes in the ovigerous fold epithelium and the possible origin
of some germ cell nests from the epithelium. The arrows indicate clearly an oocyte in the epi-
thelium that lines the delle and three spermatozoa within the delle proper. This and Figure 5
show the relation of the ovigerous fold epithelium of the delle to the egg and its follicle.

FIGURE 10. The arrows indicate first, a developing oocyte in the ovigerous fold epithelium
and second, a new germ cell nest apparently derived from the epithelium.

FIGURE 11. A single, distinct oocyte in the epithelium of the ovigerous folds. The sub-
epithelial connective tissue, showing black in the photograph, indicates clearly that the germ cell
is in the epithelium and not merely lying against it.

FIGURE 12. The opening that shows in the center of the photograph is a cross-section of a
delle. The arrows indicate two oocytes within the epithelium of the delle and other germ cells
that apparently have broken through the subepithelial connective tissue fibers to form a small
clump or nest of growing oocytes. The photograph shows clearly how the basement membrane has
been ruptured completely at this point, indicating a complete continuity between the growing
oocytes and the ovigerous fold epithelium.

REPRODUCTIVE CYCLE OF XEOTOCA D1LIXEATA

2 See footnote 1.

96 GUILLERMO MEXDOZA

cells normally occur in clusters at the surface of the ovigerous folds (Figures 2,
10, 12). This is true particularly at the interlobular fissures that occur in the
ovigerous folds. The attachment of the full grown oocytes to the ovarian delle
has been described in another part of this paper. Histologically, it is evident
that the subepithelial connective tissue fibers are in direct continuity with those
surrounding the large individual cells and clusters of small cells (Figure 10).
Furthermore, there are frequent examples of prominent invaginations of the
ovigerous fold epithelium to form small nests or clusters of epithelial cells.
These invaginated nests to all purposes appear like clusters of typical gonial
cells (Figures 10, 12). In addition, typical growing oocytes frequently appear
in these invaginated cell nests (Figure 12). The striking feature of many of the
nests is that the cells still are in direct continuity with the epithelial cells on the
surface of the ovigerous fold; in other words, the invaginated cluster of cells has
not been pinched off as yet from the superficial epithelium. With differential
stains, it is possible to determine that the invagination of the cells does not always
break through the underlying, subepithelial connective tissue fibers but rather
that the heavy fibers and the accompanying fibrocytes are carried along with the
invagination to form a thin connective tissue sheath around the nest of cells.
Thus, in these cases, not only the cells within the nests but also the connective
tissue fibers around the nests are continuous with the corresponding elements
at the surface of the ovigerous folds. These nests then are pinched off from the
surface. Lastly, the observation has been made frequently that oocytes may
occur within the epithelium proper of the ovigerous folds. They are much
larger than the adjacent epithelial cells and have the customary large vesicular
nucleus (Figures 9 to 12). A secondary migration of these oocytes into the
epithelium appears unlikely. Rather, it appears that these are examples of
epithelial cells differentiating in situ to form germ cells. In these cases, differ-
entiation occurs without the usual invagination. Therefore, in view of (1) the
intimate histological connection between the oocytes and the ovigerous fold
epithelium, (2) the actual invagination of the epithelial cells, and (3) the occur-
rence of typical oocytes in the epithelium proper, the writer is firmly convinced
that, in the adult gonad at least, some of the germ cells arise from the ovarian
epithelium on the ovigerous folds. It is interesting that Turner made similar
observations in the ovary- of Anableps anableps. He not only stressed the
subepithelial position of the germ cell nests and oocytes but also noted occasionally
single oocytes in the epithelium itself. However, he arrived at no conclusions
regarding the origin of the germ cells.

SUMMARY

1. Two ovigerous folds, one on either side of the median sagittal septum of
the ovary are described as bearing the germ cells.

. Oocytes normally occur in clusters at the surface of the ovigerous folds.
3. Follicular pores or clelles not only facilitate access of the spermatozoa to
the ovum but also provide a place for the escape of the fertilized egg.

The growing oocyte is characterized largely by the absence of large masses
of yolk and the presence of numerous oil droplets. During growth the nucleus
changes from a typical germinal vesicle to a granular, eosinophylic body in which
the chromatin exhibits only a weak affinity for stains.

REPRODUCTIVE CYCLE OF NEOTOCA BILINEATA 97

5. The follicle of Neotoca changes during growth from a tenuous layer of
scattered squamous cells to a thick, simple layer of columnar cells.

6. Fertilization, the completion of the meiotic divisions, and the escape of the
egg are described as occurring simultaneously or in extremely rapid succession.

7. Normally evacuated follicles cannot be compared in any way to the
mammalian corpus luteum.

8. In the atresia of the follicles the following phenomena are believed to
occur: some fatty degeneration, some liquefaction or vacuolization of cells, some
lymphocyte infiltration, some phagocytosis, and lastly, some absorption by the
surrounding cells.

9. Some, if not most, of the germ cells of the adult gonad of the female are
believed to arise from the epithelium of the ovigerous folds.

LITERATURE CITED

BAILEY, R. J., 1933. The ovarian cycle in the viviparous teleost Xiphophorus helleri. Biol.

Bull., 64: 206-225.
EIGENMANN, C. H., 1892. The fishes of San Diego, California. Proc. U. S. Nat. Mus., (1893),

15: 123-178.
FOOT, N. C. AND M. C. MENARD, 1927. A rapid method for the silver impregnation of reticulum.

Arch. Path, and Lab. Med., 4: 211-214.
HUBBARD, J. W., 1894. The yolk-nucleus in Cymatogaster aggregatus Gibbons. Proc. Amer.

Philos. Soc., 33: 74-83.
HUBBS, C. L. AND C. L. TURNER, 1939. Studies of the fishes of the order Cyprinodontes. XVI.

A revision of the Goodeidae. Univ. Mich. Zool. Misc. Pub., No. 42: 1-92.
HYLTON SCOTT, M. L, 1928 Sobre el desarrollo intraovarial de Fitzroyia lineata (Jen.) Berg.

Anal. Museo Hist. Nat. de Buenos Aires (Ictiologla, pub. mint. 13), 34: 361-424.
LANE, H. H., 1903. The ovarian structures of the viviparous blind fishes, Lucifuga and Stygicola.

Biol. Bull., 6: 38-54.
MENDOZA, G., 1938. El ciclo ovarico de la Neotoca bilineata. Rev. de Biol. y Med., num. 3:

20-25.
MENDOZA, G., 1939. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a

member of the family Goodeidae. I. The breeding cycle. Biol. Bull., 76: 359-370.
MENDOZA, G., 1940. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a

member of the family Goodeidae. II. The cyclic changes in the ovarian soma during

gestation. Biol. Bull., 78: 349-365.
MENDOZA, G., 1941. The reproductive cycle of the viviparous teleost, Neotoca bilineata, a

member of the family Goodeidae. III. The germ cell cycle. Biol. Bull., 81: 70-79.
PETERS, W. C., 1865. On viviparous fishes of the genus Hemirhamphus. Am. Mag. Nat. Hist.,

Ser. 3, 15: 500-501.
SICCARDI, E. M., 1940. La ovoviviparidad y viviparidad en los cyprinodontes argentinos. La

Prensa Medica Argentina, 27: 1-36.
STUHLMANN, F. L., 1887. Zur kenntnis des Ovariums der Aalmutter (Zoarces' viviparus Cuv.).

Abh. Naturw. Ver. Hamburg, 10: 1-48.
TURNER, C. L., 1933. Viviparity superimposed upon ovoviviparity in the Goodeidae, a family

of cyprinodont teleost fishes of the Mexican Plateau. Jour. Morph., 55: 207-251.
TURNER, C. L., 1937. Reproductive cycles and superfetation in poeciliid fishes. Biol. Bull., 72

145-164.
TURNER, C. L., 1938a. Adaptations for viviparity in embryos and ovary of Anableps anableps.

Jour. Morph., 62: 323-349.
TURNER, C. L., 1938b. Histological and cytological changes in the ovary of Cymatogaster

aggregatus during gestation. Jour. Morph., 62: 351-373.
WILLIAMSON, H. C., 1910. Report on the reproductive organs of Spams centrodontus, Delaroche;

Sparus cantharus, L.; Sebastes marinus (L.); and Sebastes dactylopterus (Delaroche);

and on the ripe eggs and larvae of Sparus centrodontus (?), and Sebastes marinus. Fish.

Scotland, Sci. Invest., (1910), no. 1 (Sept. 1911), 1-35.

THE REACTION OF CERTAIN CRUSTACEA TO DIRECT
AND TO DIFFUSE LIGHT

WILLIAM SCHALLEK

(From the Woods Hole Oceanographic Institution 1 and the Biological Laboratories,

Harvard University, Cambridge)

Many plankton organisms which move downward, away from daylight in
the sea, move toward a source of light in the laboratory. It was suggested in a
previous paper (Schallek, 1942) that this behavior is caused by the animals'
being exposed to diffuse light in nature, but to direct light in the laboratory.
It was reported that when a tall glass cylinder containing the copepod Acartia
tonsa was illuminated from above, the animals swam up toward the light. This
phototropic reaction seemed to depend on the fact that the light was shining
directly down the axis of the cylinder. But when the lamp was moved so as to
illuminate the container obliquely, the refraction of the light at the surface of the
water and its reflection from the curved inner wall of the cylinder seemed to
form a diffuse illumination. Under these circumstances the animals sank
downward, simulating their behavior in nature. The present paper provides a
further development of this hypothesis based on measurement of the light
distribution inside the cylinder.

APPARATUS

A glass cylinder 18 inches high and six inches in diameter was used for the
experiments. The cylinder was kept at 15° C. in a constant temperature bath
15 inches high and 15 X 18 inches in area. The bath had black walls to absorb
reflected light, and was kept in a photographic darkroom. Illumination was
from a 75-watt lamp with a parabolic, aluminum-coated reflector. The lamp
was placed in five different positions, each 12 inches from the top of the cylinder,
but separated by 22.5°. Position 1 was on a level with the top of the cylinder,
while position 5 was directly overhead.

Light measurements were made with a Westinghouse "Photox" cell connected
to a micro-ammeter, and calibrated in foot candles with a Macbeth illuminometer.
The cell was placed in a waterproof case, and covered with a cylindrical hood,
limiting the light received to an angular opening of 22.5°. The cell was held
six inches below the surface of the cylinder by clamps and rods attached to a
ringstand. As this apparatus would not fit inside the six-inch cylinder, an
11 -inch cylinder was used for the light measurements. This change is not
believed to be a significant source of error.

Twenty Acartia tonsa were placed in the cylinder at a time, while the lamp
was shifted from position 1 (oblique) to position 5 (overhead). Alternate runs
were made in the opposite direction. Two hours' exposure to each position was
allowed before counting to permit the animals to reach equilibrium. Results
are presented as the percentage of animals in the top third of the cylinder.

1 Contribution No. 323.

DIRECT AND DIFFUSE LIGHT

OBSERVATIONS

A. tonsa moves down when the illumination is oblique, and moves up when
the lamp is overhead (Table I). Measurements of the light distribution inside
the cylinder are given in Figure 1 .

TABLE I

Distribution of animals in cylinder
Average of three runs with 20 animals each

Light position

Elevation above horizontal

1
0

2
22.5

3
45

4
67.5

5
90 c

Acartia tonsa
Centropages typicus

Per cent animals in top third of cylinder

13 29 47 65 68

69 70 72 72 79

2 345

FIGURE 1. Light distribution inside the cylinder for each position of lamp. The small circle
indicates the location of the photometer, six inches below the surface of the water. The number
of each lamp position is given below the circle. The length of each line represents the light
intensity recorded when the photometer was pointed in that direction. Logarithmic scale: the
first crossbar = 100 foot-candles, the second = 1000 F. C. The hood limiting the incident light
to 22.5° made the cell insensitive to less than 20 F. C.

These measurements reveal several factors that may influence the behavior
of A . tonsa:

1. There is a 50-fold increase in the intensity of the maximum beam as the
lamp is raised over the cylinder. It may be that the animals swim up in a
bright light and sink down in a dim one. To test this factor, a slide-wire re-
sistance was connected in series with the lamp, permitting the light intensity
to be varied while the lamp was held in the overhead position. A 120-fold
increase in the intensity of the light had no effect on the distribution of A. tonsa
(Table 1 1 A).

2. The direction of the maximum beam is vertical in position 5, but is more
horizontal in the other positions. Perhaps the animals swim toward a vertical
light but sink downward in a horizontal one. This possibility was tested by

100

WILLIAM SCHALLEK

TABLE II

Effect of changing light intensity

A. Cylinder vertical, light overhead.

Average of three runs with 20 A. tonsa each

Light intensity

200

Per cent animals in top third of cylinder
Light intensity

1200 F. C.

56 52 54

B. Horizontal trough, illuminated from end.

10 20 200 1300 F. C.

Time to move 10 inches

Animal

130

150

125

130 seconds

placing the cylinder on its side. The light from position 5 (shining directly
down the axis of the cylinder) formerly was vertical, but now came in a horizontal
direction. When the lamp was in position 1 (oblique) the animals sank downward
(Figure 2A) ; when it was moved to position 5 (now horizontal) the animals
swam to the end of the cylinder nearest the light (Figure 2B). Hence the animals
swim toward the light when it is parallel to the axis of the cylinder, and sink
downward when it is oblique, regardless of whether the light comes from a hori-
zontal or a vertical direction.

A B

FIGURE 2. Arrangement of apparatus to test effect of light direction. A, lamp in position 1 ;
B, lamp in position 5. Position of animals shown by black dots. The cylinder is on its side in a
glass tank painted black (double line) on all sides except one through which the light enters.
A piece of tarpaper covers the top.

A further test was made with a horizontal tropism trough, which could be
illuminated at either end by a beam of light shining directly down the trough.
A. tonsa always moved toward the light under these conditions; by turning on
first the light at one end and then that at the other, it was possible to keep an
individual moving back and forth from end to end indefinitely. Here again the
reaction is independent of light intensity (Table IIB).

3. The distribution of the light changes as the lamp is moved. In position 5
the illumination is highly directional, with all the light coming from a single
direction. In position 1 the illumination is more diffuse, the light coming almost
equally from three directions. This angular distribution can be conveniently
measured by the ratio (sum of intensities in other directions) : (highest intensity).

DIRECT AND DIFFUSE LIGHT

101

The 22.5° hood permitted 16 readings to be taken by rotating the photometer
through 360°. For perfectly direct illumination, with all the light coming from
one direction, this diffusion ratio will be 0 : 1, or 0. For perfectly diffuse illumi-
nation, with the light coming equally from each of the 16 directions in which
measurements were made, it will be 15 : 1, or 15. The diffusion ratio for each
position of the lamp is given in Table III. The curve formed by plotting these
values against the percentage of animals in the top third of the cylinder is shown
in Figure 3.

0.2

o 0.4

0.6

0.8

1.0

ACARTIA TONSA

20 40 60

PERCENT TOP THIRD

8.0

FIGURE 3. Influence of distribution of light on behavior of A. tonsa. Ordinate, diffusion
ratio of light. Abscissa, per cent of animals in top third of cylinder.

The possibility remains that this relation is an artifact caused by the particular
apparatus or procedure used. This is not so, since another copepod, Centropages
typicus, is only slightly affected by the shift in lighting (Table I). It must
therefore be concluded that the behavior of A. tonsa depends upon the angular
distribution of the light:

1. A. tonsa moves toward a source of highly directional light, regardless of
the intensity of the light or the direction from which it comes. This is a typical
positive phototropism.

2. A. tonsa sinks downward in less directional (diffuse) light. This is a
positive geotropism and not a negative phototropism, since the animal does not
move along the beam of maximum light, but sinks passively downward.

102

WILLIAM SCHALLEK

Which of these types of illumination will animals encounter in nature?
Measurements were made of light distribution in air, while the distribution of
light in the sea was calculated from the data of Whitney (1941) (Figure 4,
Table III).

FIGURE 4. Light distribution three feet above roof of Marine Biological Laboratory.
August 2, 1942, 10 A.M. Arrangement as in Figure 1.

A. Clear sky, plane of sun.

B. Clear sky, plane perpendicular to sun.

It is evident that light in the sea becomes even more diffuse than any condi-
tions reached in the cylinder. A. tonsa sinks downward in diffuse light in the
laboratory, and this reaction apparently accounts for the fact that it leaves the
surface of the sea in the daytime (Esterly, 1928). Centropages typicus, which
moved down only slightly in the diffuse light in the cylinder, shows only a slight
downward movement in the sea (Clarke, 1934).

DISCUSSION

There are several reports in the literature of animals which react photo-
tropically in a horizontal tube illuminated from the end, but which sink downward
in a vertical tube illuminated obliquely (Table IV). Like A. tonsa, these animals
apparently have different responses to direct and to diffuse light. Most of them
show an upward movement in the dark.

The diversity of these animals, ranging from protozoans to arthropods and
echinoderms, suggests that a principle of general importance is involved. A

DIRECT AND DIFFUSE LIGHT

103

TABLE III

Summary of light measurements

Diffusion ratio =

sum of other intensities

highest intensity
Complete concentration = 0; complete diffusion = 15

Measurements in cylinder *
six inches below surface

Light position 1

2
3
4
5

Measurements in sea (Whitney, 1941)
5 met. below surface

Clear sky, plane of sun

perpendicular to sun
Diffuse sky, any plane
Measurements in air

3 feet above ground

Clear sky, plane of sun

perpendicular to sun

Diffuse sky, plane of sun

perpendicular to sun

Diffusion ratio

1.08

0.48

0.15

0.012

2.6

3.2
2.0

0.13

9.2

6.2

6.8

* As the hood limiting incident light to 22.5° made the photocell insensitive to less than
20 F. C., these values are probably somewhat lower than the true ones.

TABLE IV

Summary of geotropism experiments

Animal

Photo-
tropism in

Geotropism in

vertical tube

Author

horizontal
tube

In light

In dark

Corethra plumicornis larva

Harper, 1907

Cyclops albidus

Esterly, 1907

Branchipiis serratus

McGinnis, 1911

Daphnia pulex

Dice, 1914

Sagitta bipunctata

Esterly, 1919

Diadema setosum larva' Paramecium

Indif.

Fox, 1925

Holopedium gibbenim

Indif.

Kikuchi, 1938

Hemimysis lamornae

+ or -

Foxon, 1940

Acartia tonsa

Schallek, 1942

The nauplii of Balanus perforatus sink to the bottom of the aquarium when taken from the
dark into horizontal light, but swim up when the lamp is moved overhead (Ewald, 1912). Indif.
means indifferent.

possible explanation of this effect is provided by Clark (1933). The beetle
Dineutes moves toward the lamp if placed in a direct beam of light. If a piece
of white cardboard is held perpendicular to the beam 300 cm. behind the animal,
occasional circus movements appear. These become more frequent as the card-
board is brought closer to the animal, until at 10 cm. they become continuous.

104 WILLIAM SCHALLEK

This effect is attributed to the stimulation of additional ommatidia by the light
reflected from the cardboard. In concentrated light, the photoreceptor will be
stimulated from the front only, and the animal will then react in a typically
phototropic fashion. In diffuse light, however, the photoreceptor will be stimu-
lated from both front and side, and a different behavior appears. In the case of
A. tonsa this results in cessation of activity, since the animal may be observed to
sink passively in diffuse light.

Laboratory studies of the light reactions of animals have largely been con-
cerned with phototropic behavior in a direct beam of light. Measurement of the
light distribution in the sea shows that it is much more diffuse than in such
experimental conditions. The reaction of A . tonsa to diffuse light in the labora-
tory accords with its downward movement in the ocean during the day. Its
reaction in the direct light in which experiments on phototropism are usually
conducted has no bearing on its behavior in nature.

Such relations may not be confined to this particular copepod. Several
reports have been quoted suggesting similar behavior in other aquatic forms.
The measurements of light distribution in air show that it is generally diffuse.
Perhaps this will solve the riddle of why positively phototropic insects do not
fly up to the sun : they may move toward a direct light but behave differently in
diffuse light. Phototropism experiments in a direct beam of light need not
necessarily apply to the behavior of organisms in nature.

SUMMARY

When the copepod Acartia tonsa is placed in a tall glass cylinder illuminated
from above, the animal swims upward. When the cylinder is illuminated
obliquely, the animal sinks downward.

Measurement of the light distribution inside the cylinder shows that the
behavior of A. tonsa depends upon the angular distribution of the light. In
highly directional illumination, the animal reacts phototropically, and swims
toward the light. In less directional (diffuse) illumination, the animal stops
swimming and sinks passively downward.

Measurement of the light distribution in the air and in the sea shows that
it is generally more diffuse than the conditions in the cylinder. The reaction of
A. tonsa to diffuse light in the laboratory accords with its downward movement
in the ocean during the day. Its reaction in the direct light in which photo-
tropism experiments are usually performed has no bearing on its behavior in
nature.

ACKNOWLEDGMENTS

This work was made possible by a fellowship from the Woods Hole Oceano-
graphic Institution and aid from the Department of Biology of Harvard Uni-
versity. It was done under the general supervision of Dr. George L. Clarke.
I wish to thank him most sincerely for many helpful suggestions in the experi-
mental work and in the preparation of this report.

LITERATURE CITED

CLARK, L. B., 1933. Modification of circus movements in insects. Jour. Exper. ZooL, 66:
311-333.

DIRECT AND DIFFUSE LIGHT 105

CLARKE, G. L., 1934. Further observations on the diurnal migration of copepods in the Gulf of

Maine. Biol. Bull., 67: 432-455.
DICE, L. R., 1914. The factors determining the vertical movements of Daphnia. Jour. Anim.

Behav., 4: 229-265.
ESTERLY, C. O., 1907. The reactions of Cyclops to light and gravity. Amer. Jour. Physiol., 18:

47-57.
ESTERLY, C. O., 1919. Reactions of various plankton animals with reference to their diurnal

migrations. Univ. Calif. Publ. Zool., 19: 1-83.
ESTERLY, C. O., 1928. Periodic occurrence of Copepoda at La Jolla. Butt. Scripps InsL Oceanog.,

1: 247-345.
EWALD, W. F., 1912. On artificial modification of light reactions and the influence of electrolytes

on phototaxis. Jour. Exper. Zool., 13: 591-612.
Fox, H. M., 1925. The effect of light on the vertical movement of aquatic organisms. Biol.

Rev., 1: 219-224.
FOXON, G. H., 1940. The reactions of certain mysids to stimulation by light and gravity. Jour.

Mar. Biol. Ass'n, 24: 89-97.
HARPER, E. H., 1907. Behavior of the phantom larvae of Corethra plumicornis fabricus. Jour.

Comp. Neural., 17: 435-456.
KIKUCHI, K., 1938. Studies on the vertical distribution of the plankton Crustacea. II. The

reversal of phototropic and geotropic signs with reference to vertical movement. Records

Oceanog. Works Japan, 10: 17-42.
McGiNNis, M. O., 1911. Reactions of Branchipus serratus to light, heat and gravity. Jour.

Exper. Zool., 10: 227-240.
SCHALLEK, W., 1942. The vertical migration of the copepod Acartia tonsa under controlled

illumination. Biol. Bull., 82: 112-126.
WHITNEY, L. V., 1941. The angular distribution of characteristic diffuse light in natural waters.

Sears Found. Jour. Mar. Res., 4: 122-131.

STUDIES ON THE LIFE HISTORY OF THE MARINE ANNELID

NEREIS VEXILLOSA

MARTIN W. JOHNSON

(Scripps Institution of Oceanography, University of California, La Jolla, and the
University of Washington Oceanographic Laboratories, Seattle)

Contributions from the Scripps Institution of Oceanography, New Series, No. 191

INTRODUCTION

Nereis vexillosa Grube is the most abundant member of the larger annelids of
the Pacific northwest fauna. In Puget Sound it attains a size of about 30 cm.
but, as will be explained later, some heteronereid individuals may be only about
6 cm. long.

It is characteristically a cold water form occurring intertidally and in shallow
water from eastern Siberia, Alaska and southward along the northwest American
coast to Santa Barbara, California. Some reports extend its range as far south
as San Diego. In the southern range, however, it may have been confused with
Nereis mediator Chamberlain (Chamberlain, 1919) or with Neanthes succinea (Frey
and Leuckart) according to Hartman (personal communication). The apparent
absence of the specific egg masses, described below, from the southern range lends
support to the belief that TV. vexillosa may not occur in these waters.

The tiny eggs produced by this animal are about 0.2 mm. in diameter; they
are spawned in firm, irregular gelatinoid masses, somewhat translucent, and of
blue-green, greenish or brownish tints (Figure 1). These colors are most notice-
able in the freshly laid eggs owing to their greater compactness prior to absorption
of water by the capsular material. The firmness of the masses enables them to
withstand a good deal of handling or washing about on the beach by waves
without disintegrating. Hence they are often found in good condition on tidal
flats where bits of seaweed or other debris collects at the waters edge. They
appear never to be found in more than moderate quantities despite the abundance
of the species producing them.

In so far as known, there is no other nereid worm that deposits its eggs in
this manner. Commonly the eggs of other species of the genus, or of closely
related genera are deposited separately in the water or are only lightly aggluti-
nated. The size of masses varies considerably being about one to three inches
in diameter, apparently depending upon the size of the female depositing them.

The identity of the egg masses has long been a puzzle to biologists and others
alike, and, indeed, the present study was initiated in 1927 at Friday Harbor,
Washington, primarily for the purpose of identification. In the course of the
investigation, however, important aspects pertaining to the life history of the
species have come to light.

It is a pleasure to acknowledge the generous support given this investigation
by Director T. G. Thompson, Professor T. Kincaid, and other members of the
staff of the University of Washington Oceanographic Laboratories where facilities

106

LIFE HISTORY OF NEREIS VEXILLOSA

107

were provided for much of the work. My thanks are also due to Dr. Olga
Hartman of the Allen Hancock Foundation for identification of the heteronereis
worm and for helpful suggestions.

FIGURE 1. Nereis vexillosa. A small egg mass, about natural size.

CULTURE EXPERIMENTS AND DEVELOPMENT

The larvae resulting from culture of eggs collected in 1927 failed to survive
long beyond hatching and therefore served to establish only that they were
annelids of the nereid type. The failure of these to survive may be due in part
to inadequate food since they were fed only diatoms and algae. This diet brought
good results with Platynereis agasszi which was being reared at the same time
(see Guberlet, 1933), but subsequent experiments have indicated that Nereis
vexillosa thrives best in later life when animal food is provided.

In the summer of 1941 an opportunity was again presented to set up cultures
at Friday Harbor. Two egg masses were found in False Bay and rearing of the
larvae began on June 28. Some of the larvae were already hatching in the three
to five segmented stage (Figure 2). The larvae and young worms grew slowly
and were able to survive for about a week after hatching on the yolk content
which became concentrated in the digestive tract. Later they were fed sessile
diatoms (Navicula sp. and Biddulphia laevis) and powdered dry scallop muscle.

The rate of growth varied so greatly that on August 23 the most advanced
worms, now about 57 days old, had acquired 35 segments and were about 9 mm.
long, whereas the least advanced had but 15 segments and were about 5 mm. long.

108

MARTIN W. JOHNSON

FIGURES 2-7. Xereis vcxilhsa.

FIGURE 2. Five- segmented larva.

FIGURE 3. Head &f eight-segmented larva to show development of peristomial tentacles.
FIGURE 4. H-vi <yi fifteen-segmented larva to show further progress of head development.
FIGURES 5 and 6. Homogomph nototeta and heterogomph neuroseta from parapodium
shown in Figure '/.

FIGURE 7. Parapodium from posterior portion of body of eleven month old worm.
(Camera lucida drawings, Figures 2, 3, 4 same scale; Figures 5, 6, 7 enlarged scale.)

It was however, still not possible to establish identity of the worms from the
most advanced specimens, therefore, at the end of the summer session the animals
were m©yed alive to the Scripps Institution of Oceanography at La Jolla, Cali-
fornia. • This was accomplished by placing ten specimens of various sizes in
each q)f four vials with sea water and containing a wad of cotton among the
strands of which the young worms found protection against violent battering in
the vials while in transit. The vials were kept cool by being wrapped in wet,
frequently-changed paper towels.

Upon arrival at La Jolla the specimens were changed gradually to local sea
water (salinity about 3%<i above that at Friday Harbor) in small culture dishes
and it was found that 25 had survived the journey. Following some additional
mortality in the first month, 16 of various sizes lived for several months.

After setting up the cultures at La Jolla, about three-fourths of the water was
changed in each dish every two or three days and the animals were fed dried

LIFE HISTORY OF NEREIS VEXILLOSA 109

powdered pecten and sea mussel for a few weeks, but growth was so slow that
it was decided to try feeding finely chopped pieces of freshly killed Thoracophelia
mucronata, a polychaete worm found abundantly at La Jolla. This fresh food
was given in small quantities every second or third day. With it there was a
prompt response by more rapid growth. Later bits of fresh mussel were also
fed but the minced worms seemed to be preferred. Not only the killed and
minced worms were eaten but living specimens half the size of the young Nereis
were eaten. This rapacious habit was evident even in worms only 6 or 8 mm.
long that were seen to grasp and completely swallow in a few seconds others of
their kind that were fully half their own size. Diatoms were also added to the
dishes and these were readily eaten. The animals rejected partially decomposed
food and since the object of the experiment was to keep them growing as long as
possible no further experimenting with food was deemed advisable. Results
indicate that N. vexillosa is more or less omnivorous, utilizing mostly animal food
but that it is not a scavenger by preference. According to Copeland and Wieman
(1924) Nereis virens is also omnivorous though Gross (1921) found only evidence
of plant feeding.

No membranous tubes were constructed as in Platynereis agasszi, only flimsy
tubes of sand and debris were formed with the aid of secreted mucus, and after
the animals had reached a length of about 15 mm. they rarely left their tubes
completely to gather food. To facilitate study, pieces of glass tubing were
provided and these were readily accepted in most cases. Some animals refused
to accept new glass tubes that were provided to replace those outgrown and
deserted. In these instances the lack of security resulted in restlessness and
failure to feed normally.

The older specimens were kept in running (uncooled) sea water pumped in
from the sea. At the middle of July this water had reached a temperature of
20° C., which is about 6 to 8 degrees higher than might be expected in the natural
habits of N. vexillosa near Friday Harbor during the summer. This may well
have been a contributing factor in the failure of the worms to survive with the
advance of summer at La Jolla.

In Table I is given a summary of the condition of the worms that survived
ten or more months. Two of these were found dead (May 18 and May 29).
The others were killed and preserved only after they had deserted their tubes
and it seemed obvious that they would not survive much longer.

At the age of four and one-half months, when about 60 segments had been
formed, the worms reared from the 1941 egg masses had developed specific
characteristics of N. vexillosa. Especially characteristic are the elongate strap-
like dorsal ligules (Figure 7) of the parapodia of the posterior region of the body,
but the head structures and the setae of the posterior region are also distinctive
(Figures 5 and 6) in older specimens.

The process of cephalization is shown in Figures 2, 3, and 4. In this develop-
ment, the first setigerous segment of the early larva becomes modified to form
the peristomium. The anterior dorsal pair of peristomial tentacles appear first,
followed by the posterior dorsal pair which develop from the first larval para-
podium. A ventral pair of anterior peristomial tentacles then appear and finally
the posterior ventral pair of tentacles are in evidence when the worm has acquired
about 18 to 20 segments.

110

MARTIN W. JOHNSON

TABLE I

Nereis vexillosa. The seven oldest specimens from cultures set up at Friday Harbor, June 28, 1941

Date killed or found dead
1942

Number of
segments

Approximate length when
moderately relaxed

April 25

128

8 cm.

May 18

109

5 cm.

May 26

109

6.7 cm.

May 26

107

4.5 cm.

May 29

119

8.5 cm.

June 10

105

7 cm.

July 28

125

10 cm.

None of the specimens showed any indication of entering the heteronereis
phase. In this respect they differ from Platynereis agasszi, several of which
were found to enter this phase near the end of the first year.

SPAWNING HABITS

While culturing the larvae at Friday Harbor in 1941 a watch was kept for
spawning adults in the bay. Finally from isolated small, spawning heterjpereids
there was obtained several masses of eggs. These spawners proved to be Nereis

FIGURES 8-10. Nereis vexillosa.

FIGURE 8. Heteronereized female parapodium, middle portion of posterior body.
FIGURK 9. Heteronereized female parapodium, fourth from last segment.
FIGURE 10. Dorsal cirrus, heteronereized male parapodium.

(Figures 8, 9, 10 camera lucida drawings to same scale.)

LIFE HISTORY OF NEREIS VEXILLOSA HI

vexillosa Grube thus confirming the identification of the worms reared experi-
mentally from egg masses collected in the field. Heteronereized parapodia are
shown in Figures 8 and 9. Heteronereis males have 25 parapodial segments in
the anterior portion of the body while the females have 27 such segments (count
of 12 males and four females). In the males the dorsal cirrus of the heteronereized
segments bears a series of wart-like protuberances on the ventral surface (Figure
10) while in the females examined these cirri were smooth.

The spawning worms were obtained only at night while collecting with a
light at the end of the pier in front of the laboratories. They appeared only in
small numbers usually an hour or two before midnight and were mingled with
spawning swarms of the smaller species Platynereis (formerly Nereis} agasszi
which on all occasions was the first of the two to appear swimming at the surface.
It was not possible with these few observations to establish any correlation of
spawning with phases of the tide or moon as has been done with other marine
worms (cf. Woodworth, 1907; Lillie and Just, 1913; Guberlet, 1933).

Only small individuals of N. vexillosa were seen spawning and from what has
subsequently been learned through the above rearing experiments, it seems
certain that these were all spawning for the first time at the age of one year.
Not only were some of the experimentally reared worms as large at the age of
10 to 11 months as some of the spawning worms but there was also indication of
approach to sexual maturity. One of the worms reared at La Jolla was killed
April 25, 1942 after having deserted its tube. Upon examination it was found
to contain many eggs which were very small and not yet ripe, but their large
numbers might be interpreted to indicate that spawning would normally have
occurred in the coming summer. A second worm that died May 29 also showed
many eggs. However, it seems certain that in nature some heteronereis indi-
viduals must be older than one year at spawning since egg masses of much greater
size than those known to have been spawned by the smaller specimens have
been found. This is more fully discussed later.

In nature more males than females were observed swimming at the surface.
They are the first to come to the nuptial party where, as scattered individuals,
they suddenly appear from below and rise to the immediate surface, swimming
a few moments there in spirals and loops and then disappearing into the deeper
water or the darkness beyond the range of the collector's light. They continue
unabated in numbers and vigor as the females a little later appear to join the
dance, a dance which seemingly is a climax that marks the ends of their lives,
for none of the exhausted individuals kept in captivity was observed to live
more than a few days following the act of spawning.

Isolated small heteronereis females ripe with eggs were induced to spawn
almost instantly when a few drops of sperm laden water were added to the
water in which they were isolated. This is similar to the findings of Lillie and
Just (1913) for Nereis limbata and of Just (1929) for Platynereis dumerilii. In
large battery jars the act of spawning by the female Nereis vexillosa consists of
coming to or near the surface and suddenly exuding a mass of eggs which instantly
agglutinates (Figure 11). She then passively sinks to the bottom together with
the mass. A few moments later she frees herself from the mass which remains
demersal and which in a fewr hours swells to about three or four times its original
size through the absorption of water. The spawning of these demersal masses

112

MARTIN W. JOHNSON

in water just beyond the low tide limit may account for the finding of fewer egg
masses than are commensurate with the number of worms.

FIGURE 11. Nereis vexillosa. Method of egg deposition by small heteronereis. (Free hand
drawing.)

DISCUSSION

An answer has been found as to the identity of the egg masses, and certain
features of the life history of the species have been discerned. In the process of
interpreting the observations, however, another biologically important question
arises, namely, does Nereis vexillosa possess a diversified life history in which
there may be recognized several types of reproductive individuals, as indicated
for Platynereis dumerilii by Hempelmann? In the latter species at Naples,
Hempelmann (1911) distinguished (1) small sexually mature nereis (i.e., atokus)
individuals that gave rise to heavily yolked eggs producing characteristic larvae
which he called " nereidogene " ; (2) small heteronereis forms producing less

LIFE HISTORY OF NEREIS VEXILLOSA 113

heavily yolkcd eggs developing into pelagic larvae called " planktogene " ; (3) large
heteronereis forms with eggs as in the small form but whose larvae have not
been investigated. He found that after spawning, the small nereis form may in
experimental cultures be transformed into a small heteronereis form and produce
young for the second time and is therefore dissogenous. The nereis form may
also grow to a relatively large size and then transform to the large heteronereis
but the steps involved in arrival at the large form in this phase are uncertain.
It is believed that entrance into the heteronereis form and its spawning marks
the end of life for the individual.

More recently Just (1929) also worked on the Naples species and in so far
as his investigation was carried, the findings of Hempelmann were verified.

It has been noted by H. P. Johnson (1901) that the heteronereis form of
Nereis vexillosa occurs in individuals of 56 mm. and upward in length but the
maximum length is not given. The same author reports that sexual maturity is
frequently arrived at by the species without it becoming heteronereized. Ricketts
and Calvin (1939) report finding many large heteronereis of N. vexillosa but the
authors never found these to be free-swimming. The supposition is, however,
that they do spawn, and indeed the finding of egg masses in Puget Sound that
are much larger than those known to have been spawned by small heteronereis
forms of the species substantiates this.

What the destiny of the worms reared from the Friday Harbor material would
have been normally can be only a. matter of conjecture. In this connection it
may be significant to note that the number of segments in several large (14 to
21 cm. long) N. vexillosa collected in the field at Friday Harbor was 142 to 152
in the nereis phase, whereas the spawning heteronereids taken in the same region
had only 63 to 96 segments. The latter figure is less than the number occurring
in the seven most advanced specimens reared in cultures (Table I). These
specimens had 107 to 128 segments and the length of some was greater than the
spawning worms. This may mean that the reared worms were destined to reach
sexual maturity only in a more advanced nereis phase or in a large heteronereis
phase. Much additional study is needed to answer this question. The great
range in size that is possible in the heteronereis of N. vexillosa should make it
an ideal species for such a study if culture problems can be overcome.

SUMMARY

Nereis vexillosa deposits its eggs in firm irregular gelatinoid masses which
vary in size from about one to three inches in diameter.

Spawning of small (6 to 8 cm. long) heteronereids of this species was observed
to take place an hour or two before midnight. The eggs which are demersal are
apparently spawned in water at or just beyond the extreme low tide level and
this habit may account for the finding of fewer egg masses than seems commensu-
rate with the number of the species producing them. Isolated heteronereis
females were induced to discharge their eggs by the introduction of spermatozoa
into the water.

Worms cultured in the laboratory from egg masses collected on the beach
throve best on fresh animal food. A number of young worms transported from
Friday Harbor to La Jolla attained a maximum length of 10 cm. at the age of
13 months after hatching. All had acquired specific characteristics but none

114 MARTIN W. JOHNSON

became heteronereized. The transformations taking place during cephalization
are similar to that occurring in other nereid worms.

Since the above maximum size of the reared worms is comparable to that of
the small spawning heteronereis forms, it appears that the latter were spawning
at the age of about one year. Much larger nereis and also heteronereis individuals
are known to occur in this species but the time and steps involved in their de-
velopment are unknown.

LITERATURE CITED

CHAMBERLAIN, R. V., 1919. New polychaetous annelids from Laguna Beach. Pomona Jour.

Entom. and Zool., 11: 1-23.
COPELAND, M. AND H. L. WiEMAN, 1924. The chemical sense and feeding behavior of Nereis

virens Sars. Biol. Bull., 47: 231-238.
GROSS, A. O., 1921. The feeding habits and chemical sense of Nereis virens Sars. Jour. Exp.

Zool., 32:427-442.
GUBERLET, J. E., 1933. Observations on the spawning and development of some Pacific annelids.

Proc. Fifth Pac. Sci. Congr., 5: 4213-4220.
HEMPELMANN, P. F., 1911. Zur Naturgeschichte von Nereis dumerilii Aud. et Edw. Zoologica,

H. 62, 1-135.
JOHNSON, H. P., 1901. The Polychaeta of the Puget Sound region. Proc. Boston Soc. Nat. Hist.,

29: 381-437.

JUST, E. E., 1929. Breeding habits of Nereis dumerilii at Naples. Biol. Bull, 57: 307-310.
LILLIE, F. R. AND E. E. JUST, 1913. Breeding habits of the heteronereis form of Nereis limbata

at Woods Hole, Mass. Biol. Bull., 24: 147-160.
RICKETTS, E. F. AND J. CALVIN, 1939. Between Pacific Tides. 320 pp., Stanford Univ. Press,

1939.
WOODWORTH, W. McM., 1907. The Palolo worm, Eunice viridis (Gray). Bull. Harvard Mus.

Comp. Zool., 51: 3-21.

(LltRAfO

THE REPRODUCTIVE PROCESSES OF THE FISH,

ORYZIAS LATIPES

EDWIN J. ROBINSON ' AND ROBERTS RUGH

(Washington Square College of Arts and Science, New York University)

INTRODUCTION

The study of experimental vertebrate embryology has been limited to the
breeding seasons of the lower forms with the exception of the amphibia (Rugh,
1941) where ovulation can be induced at almost any time of the year. A study
of the reproductive processes of the fish Oryzias latipes, the Japanese medaka,
was planned in the hope that its eggs might also be made available for more
investigations in the experimental field.

MATERIALS AND METHOD

The fish in a sexually mature condition were obtained from a West Coast
importer. They were kept in ten-gallon aquaria in a south window, at room
temperature and in spring water which was oxygenated by Nitella. The food
consisted of freshly collected Tubifex which were cleaned in running water,
Daphnia, and some dried fish food. Artifical lighting was supplied only under
experimental conditions. This consisted of a 75-watt bulb held above each
tank, the light being increased with a metal reflector. When light was to be
eliminated this was accomplished by covering the entire tank with a light-tight
cardboard box which allowed sufficient circulation of room air and maintenance
at room temperature.

The female urino-genital system was studied from cleared whole mounts
and serial sections, as well as in the living condition. For a study of the ovulation
process females were anesthetized at the appropriate time in MS 222, one part
to 3000 of spring water, pinned in a permoplast dish and dissected.

OBSERVATIONS AND EXPERIMENTAL DATA

The female reproductive system.

The structure of the reproductive tract of the female Oryzias latipes (Figures
1-8) was studied as preliminary to observations and experiments on the sexual
cycle. The ovary and the oviduct are essentially like those previously described
for other poeciliids. The ovary is a median, unpaired sac-like organ (Figures 7
and 8) filling most of the body cavity behind the posterior edges of the liver.
It is composed of a large number of follicles in different stages of development,
all facing a central lumen which is somewhat occluded by the projecting ripe
follicles. These follicles are attached to the thin, muscular ovarian wall on all

1 Submitted in partial fulfillment of the requirements for the degree of Master of Science at
New York University.

115

116 E. J. ROBINSON AND ROBERTS RUGH

sides except the dorsal. Through this dorsal wall the lumen and the follicles
are visible even in the intact ovary.

The developing oocytes are practically identical with those of Fundulus
heteroclitus (Marza, Marza, and Guthrie, 1937 ; Solberg, 1938), except with respect
to size. A small oocyte with a large germinal vesicle becomes transformed into
a large egg containing a mass of yolk and oil drops surrounded by a thin layer of
cytoplasm. During this growth phase of maturation a thick chorion with long
fibers attached to its external surface is laid down around the ovum by the
follicle cells. This is a protective outer, non-living membrane comparable to the
jelly capsules of the amphibia.

The oviduct is a single thick-walled straight and non-glandular tube about
one millimeter in length (Figures 3-8), unpaired as in Xiphophorus helleri (Essen-
berg, 1923; Bailey, 1933; Regnier, 1938) in which species it is not the homologue
of the Mullerian duct (Essenberg, 1933). The walls consist largely of circular
muscle fibers (Figure 5). The lumen of the oviduct is lined with epithelium
which is columnar near the ovary and becomes squamous toward the external
opening. Numerous folds in the epithelium of the contracted oviduct allow
considerable expansion as the eggs pass rapidly through. There are no glands
present, as there are in the amphibian oviduct. The thick muscular walls of the
ovary and of the oviduct are histologically continuous, and show no appreciable
difference except in regard to total thickness. The duct extends from the pos-
terior end of the ovary to open behind the urino-genital papilla, between the
anus and the mesonephric duct (Figure 8). The mesonephric duct descends in
the muscle of the body wall posterior to the body cavity to run parallel to the
oviduct. Before it does this it bends anteriorly almost to the body cavity and
then sharply posteriorly (Figure 8). In the region near the external opening
there is no muscular wall, but one is soon organized from the surrounding tissue.
In the region of the two loops the lumen is large, the muscular wall is relatively
thin, and there are many villi in the mucosa.

The urino-genital papillae are a pair of protuberances from the ventral surface
of the female between the anus and oviduct opening (Figure 8). They extend
posteriorly and ventrally from their attachment to the body wall, covering the
opening of the oviduct, and are grown together in the mid-line for most of their
length. The papillae have a thick cortex of stratified epithelium and a highly
vascularized medulla.

PLATE I

FIGURE 1. Stained section through external opening of mesonephric duct.

FIGURE 2. Stained section anterior to Figure 1, through mesonephric duct.

FIGURE 3. Stained section anterior to Figure 2, level of mesonephric duct loop.

FIGURE 4. Stained section anterior to Figure 3.

FIGURE 5. Stained section anterior to Figure 4.

FIGURE 6. Stained section through posterior end of body cavity.

FIGURE 7. Stained section through posterior part of ovary.

FIGURE 8. Drawn from a cleared whole mount.

B — body wall; C — connective tissue; D— mesonephric duct; E — edge of rupture; F — fat;
I — intestine; L — loop of mesonephric duct; M — muscle; N — capillaries on follicle; O — oviduct;
P — body cavity; R — fin ray; S — scale; T — threads on egg; U — urino-genital papilla; Y — ovary;
X — overgrowth of body wall epithelium; Y — follicle wall; Z — constriction.

- ••'• ••

PLATE I

118

E. J. ROBIXSOX AND ROBERTS RUGH

PLATE II

FIGURE 9. Fixed ovary with the dorsal wall removed showing rupture and emergence of egg.
FIGURE 10. Profile view of living egg one-third emerged from follicle.
FIGURE 11. The same egg half emerged, transmitted light.
FIGURE 12. The same, reflected light.

REPRODUCTION IN ORYZIAS LATIPES 119

Ovulation and egg transport.

The ovulation process was observed in more than twenty ripe ovaries, either
removed from the fish at the appropriate time or observed in partially dissected
fish under anesthesia (Figures 9-12). Some of the observations were made
directly through the dorsal wall of the intact ovaries, others were made after
removal of this dorsal ovarian wall.

The rupture of the ripe follicle appears in the center of a vascular plexus on
the side toward the lumen (Figure 9). The hole is generally oval in shape at the
beginning of the process, and as it increases in size the blood vessels around it are
stretched but do not rupture. As the egg protrudes (Figure 10). the packed
threads attached to the chorion become unwound, and the edges of the hole
constrict the egg appreciably (Figures 11-12). There is no noticeable increase
in the rate of emergence after the egg is half extruded, probably because intra-
ovarian pressure from the muscular walls prevents any sudden eruption. The
process of rupture and emergence of the egg from its follicle is very similar to
that described for the frog (Rugh, 1935) except that in the frog the egg is released
into the body cavity. At the end of the process, from 20 to 30 distorted eggs
fill the lumen of the ovary, t

The exact duration of the normal process of ovulation cannot be stated since
the anesthetized and partially dissected fish are probably not physiologically
normal. However, it is possible to estimate approximate times. During these
observations most of the eggs in intact ovaries took from 15 to 45 minutes to be
extruded. The longer intervals were probably due to the ovary becoming mori-
bund. If intraovarian pressure is removed by tearing the dorsal wall of the
ovary, as was done frequently to obtain a better view of individual eggs, an egg
may emerge in ten minutes from the time of the first visible rupture. Examining
fish taken from an aquarium at intervals during the period of ovulation, it was
found that under normal conditions all the fish ovulate completely during a
period of about an hour and a half, thus setting that interval as an absolute
maximum. Since all fish do not begin to ovulate simultaneously, this estimate
is too high for any single specimen and it is presumed that an individual egg
normally takes about ten to 20 minutes to emerge from the follicle, and that a
single fish ovulates all of its eggs in less than one hour.

The forces causing the initial rupture of the follicle and the extrusion of the
egg are unknown. At no time were movements of the ovarian wall or of individual
follicles noticeable, although the presence of smooth muscle fibers in both implies
the possibility of such movements during ovulation. Furthermore, the whole
ovary often undulates remarkably when a fish is first dissected, the contortions
resembling euglenoid movement. It is interesting that these movements did
not cause rupture of the follicles. Until the forces causing ovulation are known
they may be presumed to be similar to those which act in other poikilothermous
forms (Rugh, 1935).

The eggs are probably forced into the oviduct by muscular movement of the
ovary similar to those observed under the stimulation of dissection. The
muscular oviduct and the muscles of the adjacent body wall may also aid in
moving the eggs quickly through the short oviduct.

120 E. J. ROBINSON AND ROBERTS RUGH

Fertilization.

The mature eggs are not stored in the ovary for more than a few hours, for
it was found that normally they are laid almost immediately after ovulation,
provided a sufficient number of males is present to supply the incentive. The
eggs are laid all at once by the female and are fertilized immediately, while they
remain attached to the female by chorionic threads. Egg-laying was not ob-
served, it being a very rapid process, but most of the courtship and fertilization
process was observed a number of times. Each time it lasted less than 60
seconds. Kami to (1928) stated that the entire process takes about 35 seconds,
and the present observations tend to confirm that statement. After fertilization
the eggs are accidentally brushed off on plants, during the day. They remain
attached to vegetation during early development.

The maturation cycle.

The maturation cycle of the meclaka is 24 hours long and is Very regular
since the eggs are almost invariably laid in the early morning, although Kamito
(1928) reported they may also be laid in the evening. Observation of more than
150 females over a period of three months showed only one possible exception
to the rule that the eggs are laid in the early morning. About 75 per cent of
all of the adult female fish in the aquarium produce eggs daily. The eggs are
laid almost immediately after ovulation. Dissection of numbers of stock fish
taken at random at different times of the day never showed ovulated eggs in the
ovary before about 1 A.M. or after about 9 A.M. On two occasions six and eight
fish were dissected about an hour and a half before the usual time of egg laying
and were found to be ovulating or about to ovulate. Thus the time at which
the eggs are laid indicates the approximate time of day they were ovulated.

One other important fact was discovered by daily observation of medakas in
stock aquaria, although not under experimental conditions. These fish were in
aquaria artificially instead of naturally aerated, but otherwise under the same
conditions as the other stock and experimental fish. There was no photo-
periodicity in these aquaria since lights were above them constantly. These
fish did not show the regularity of ovulation that characterizes the species.
Newly-laid eggs could be found during any period of the day, and the fish in an
aquarium did not lay eggs at approximately the same time, even though the
conditions for all were identical.

EXPERIMENTAL DATA

The knowledge that the eggs are normally ovulated and laid near dawn, and
that unusual lighting conditions upset the regular cycle, suggested that diurnal
light changes might be important in controlling the normal cycle. To test this
hypothesis experiments were performed as follows:

In the first, eight male and six female specimens of Oryzias latipes were put
in each of two five-gallon aquaria with food, water, and flora from stock aquaria.
One aquarium was covered with a light-tight box from 8:30 P.M. until 8:30 A.M.
(Tank I) and the other was covered from 8:30 A.M. until 8:30 P.M. (Tank II).

REPRODUCTION IN ORYZIAS LATIPES

121

For the remaining 12 hours each tank was lighted by a 75-watt bulb held directly
above in a metal reflector. After one week the lighting schedule for each tank
was reversed. The temperature varied with that of the room. The time the
eggs were laid was determined either by frequent observation or by noting the
stage of cleavage when the eggs were first seen and removed from the female.
The developmental rate of the Oryzias egg has been worked out for laboratory
temperatures (Rugh, 1941). The first division occurs over an hour after fertiliza-
tion, and the next two divisions at 45 minute intervals. Results are summarized
below (Table I).

TABLE I

Date Tank I (covered all night)'

Feb. 2 No eggs all day.

3 No eggs all day.

4 One laid eggs between 8:30 and 9:30

A.M. No others laid eggs.

5 One laid eggs between 8:30 and 9:30

A.M. No others laid eggs.

6 One laid eggs between 5 and 8:30 A.M.

One laid between 8:30 and 8:40 A.M.
No more laid eggs.

7 Two laid eggs between 6 and

One laid between 8:30 and
No more.

8 Two laid eggs between 8 and

One laid between 8:30 and
No more laid.

9 Schedule reversed. Both tanks

Tank II (covered all day)

No eggs all day.
No eggs all day.
One laid eggs between 8:30 and 9:30

P.M. No others laid eggs.
One laid eggs between 8:30 and 9:30

P.M. Others did not lay.
Two laid eggs between 5 and 8:30 P.M.

One laid eggs between 8:30 and 9 P.M.

8:30 A.M. One laid eggs at 9 P.M. One laid eggs
8:40 A.M. between 7 and 8:30 P.M. No more

laid.

8:30 A.M. Tank uncovered at 7:10 P.M. No eggs
9:30 A.M. up to 7:40 P.M.

uncovered until 2:30 P.M., then tank II was covered.

Tank I

10 One with eggs at 2:45 P.M. No other
eggs at 8:30 P.M. or at 10 P.M.

1 1 No eggs at 8:30 A.M., or at 8:30 P.M.

12 One laid eggs between 7:30 and 8:20

P.M. Two laid between 8:20 and
10 P.M. No others laid eggs.

13 No eggs at 8:30 P.M. Three laid be-

tween 8:30 and 9 P.M. No others
laid.

Tank II

No eggs at 8:30 A.M. One laid between
8:30 and 10:30 A.M., another between
10:30 and 11 A.M., another between 11
A.M. and 1 P.M.

No eggs up to 9 P.M.

No eggs at 8:30 A.M. One laid eggs be-
tween 8:30 and 9:50 A.M. No other
eggs all day.

No eggs at 8:30 A.M. One laid between
8:30 A.M. and 1 P.M. No other eggs
all day.

The same type of experiment was started with a new set of fish on March 23
(Table II). Twenty females which had been laying eggs between midnight and
8 A.M. without exception for several weeks were put in a ten-gallon aquarium
with 22 males, under conditions already described. The aquarium was covered
from 8 A.M. until 8 P.M. and lighted with a 75-watt bulb from 8 P.M. until
8 A.M. As controls, six females from the same lot were kept in a community
tank and these fish continued to lay eggs nearly every morning. The results
appear in the following table:

122 E. J. ROBINSON AND ROBERTS RUGH

TABLE II

Date Observations at 8 P.M.

March 23 No eggs; none laid all day.

24 No eggs; none laid all day.

25 Xo eggs; none laid all day.

26 Fifteen with eggs, mostly in the two-cell stage, some up to eight cells — i.e., one to

three hours old. None laid the rest of the day.

27 Ten with eggs, mostly in the two-cell stage, some up to eight. None laid the rest

of the day.

Eleven with eggs, mostly in the two-cell stage, some up to eight. None laid the
rest of the day.

29 Thirteen with eggs, mostly in the two-cell stage, some up to eight. None laid the

rest of the day.

30 Tank covered at 10:30 A.M. and kept covered until 8 P.M. March 31st. Not

examined for eggs at 8 P.M.

31 One with newly-laid eggs at 8:30 A.M. Three with eggs in late stages of cleavage

at 8 P.M., laid since 8:30 A.M.

April 1 No eggs at 8:40 A.M., nor at 8:40 P.M. Eleven with eggs at 9 P.M. None laid
later.

At the end of the experiment the fish were returned to a normal daylight schedule
and within two days as many as 75 per cent of the females were laying eggs
before 8 A.M. each day.

DISCUSSION

These experiments indicate that the ovulatory cycle of Oryzias latipes is in
some way correlated with diurnal photoperiodicity. With but four isolated
exceptions among the 32 experimental females, reversal of the photoperiodicity
changed the time of ovulation, as indicated by egg-laying, by approximately
12 hours. In the first experiment there was an appreciable change in addition
to the reversal during the experiment, as the fish had been laying eggs near the
middle of the day. In the second experiment two reversals occurred, including
the return to normal conditions. The two or three day lag between reversal of
the photoperiodicity and appearance of the first eggs is assumed to be caused
by an adjustment of the fish to the new lighting conditions.

The exceptional results are easily explained. The two fish in tank II on
February 10 undoubtedly were not yet adjusted to the sudden change in photo-
periodicity; the one in tank II on February 13 was in poor condition, having
been taken from slightly foul water. The exceptional fish on March 31 was
the result of the upset of photoperiodicity on the previous day.

These facts suggest the manner in which photoperiodicity may regulate the
ovulatory cycle in Oryzias latipes. The species is physiologically and genetically
adjusted to a daily cycle. This means that 20 to 30 relatively large eggs must
be matured, ovulated, and laid in the space of 24 hours, the bulk of this time
being utilized in the maturation process. Ovulation (i.e., rupture and emergence
of the egg from the ovary) must normally occur approximately at dawn because
these studies show that oviposition follows shortly thereafter and Oryzias latipes
has long been known to produce its eggs early in the morning. It is inconceivable
that the temperature of a 15-gallon tank of water would change as abruptly as
does the light factor early in the morning. Of the two variables, it seems most

REPRODUCTION IN ORYZIAS LATIPES 123

likely that since the temperature is relatively constant and the light factor is
extremely variable, that this latter factor is the trigger which sets off the ovulation
and oviposition reactions. Light in itself may not be necessary in the sense that
it acts through the sense organs to bring about breeding reactions for these fish
may ovulate on occasion and deposit their eggs even in total darkness, although
such reactions are not predictable. It is the thesis of this paper that the stimulus
of light brings about an increase in the metabolic activity of the fish and that
this activity in turn sets off the breeding reactions which have otherwise been
rather quiescent. By direct observation it can be shown that the periods of
light and dark do regulate the physical activity of the fish and those which have
been in darkness for a considerable period fail to respond normally to tactile
stimuli and generally swim very sluggishly. This has been previously reported
by Spencer (1929) who found the sunfish to be quiescent at night, and Shaw,
Escobar, and Baldwin (1938) who found the locomotor activity of goldfish to be
much reduced in reduced sunlight. The medaka is a very active fish during the
day so there may be a considerable difference in the amount of energy available
for the maturation process between periods of rest (in darkness) and of activity
(in light). There is no suggestion that radiant energy is thereby transformed
into metabolic energy but rather that light which penetrates the water stimulates
the fish to activity which activity in turn utilizes stored metabolic energy.
This energy might otherwise be utilized in the maturation process.

Such a thesis does not preclude the possibility of maturation (and ovulation)
in fish which are continuously active but that the reproductive cycle of such fish
should be longer and more irregular. These observations confirm such a proposi-
tion. But under the normal ration of light and darkness the fish will ovulate
near the end of the period of darkness no matter what time of day this happens
to be. Further confirmation of this thesis might be obtained by enucleation of
the females prior to a series of observations, but we would have to go further
and attempt to control the chromatophores as well. This would be extremely
difficult.

Other species studied have longer cycles, so no direct comparison is possible.
It is not impossible that light may also affect these longer cycles, particularly
if it can be proven that certain parts of the spectrum are more beneficial than
others, such as ultra-violet or the other extreme, infra-red. But this suggested
relationship is not offered as the explanation of the phenomena in Oryzias since
it produces eggs at almost all seasons. Barney and Anson (1921) and Turner
(1936, 1937, 1938) noted a direct relation between seasons and the reproductive
cycles in several poeciliids. But these authors associated the breeding reactions
more with seasonal temperature than light changes. Dildine (1936) did not
agree with Turner's (1937) conclusions after studying Lebistes reticulatus. Craig-
Bennett (1930) found no effect of variation of light on the cycle of Gasterosteus
aculeatus.

Matthews (1939) and Burger (1939b), both using the male Fundulus, state
that light is not essential and that it has no effect on the spermatogenetic cycle
but that temperature is the all-important environmental variable. Matthews
showed that low temperatures retarded spermatogenesis and Burger stated that
a low light ration of one and a half hours per day kept the fish sexually inactive
at 6-10° C., but that many spermatazoa were developed with the same light

124 E. J. ROBINSON AND ROBERTS RUGH

ration at 14-20° C. It is logical to assume that the low temperature would
retard any biological process and Burger admits that a basic light ration of one
and a half hours per day is needed to keep the fish sexually active.

Although other environmental factors may be involved, under normal
circumstances the time of ovulation (hence also the prior period of maturation)
in Oryzias latipes is related to the diurnal periodicity, probably through the
regulation of the states of activity during the daily cycle rather than through
any intrinsic value in light (energy) itself.

SUMMARY AND CONCLUSIONS

1. The female urino-genital tract of Oryzias latipes is, in general, similar to
that of other poeciliid fish. The ovary is an unpaired, hollow organ consisting
of a thin but strong wall, lined with developing follicles on all sides except the
dorsal. The development of the oocytes is similar to that in Fundulus hetero-
clitus. The oviduct is a short, muscular tube with no known function except
that of egg transport.

2. The ovulation process is not cataclysmic, but takes an average of about
30 minutes under the conditions stated. Contraction of the ovary as a whole
is not necessary, although this does not preclude the possibility of action of
smooth muscle in the walls of the follicles themselves. The force causing the
initial rupture of the follicle is unknown.

3. The eggs are laid almost immediately after ovulation, either just before
or during copulation. Copulation consists of a definite series of actions by both
fishes, and the sperm and eggs are shed simultaneously.

4. The fishes normally ovulate just before dawn, and inverting the periods of
light and darkness as compared to natural conditions causes ovulation to be
shifted from the time of the natural dawn to the time of the artificial dawn.
It was suggested that light governs the time of ovulation by regulating the
general metabolic activity of the female. The eggs are matured during a period
of quiescence and are released at the beginning of the period of activity, stimulated
by light. In the normal daily cycle light is probably the most important environ-
mental factor, acting as a stimulant to general activity and hence to ovulation
and oviposition in Oryzias latipes.

LITERATURE CITED

BAILEY, RALPH J., 1933. The ovarian cycle in the viviparous teleost Xiphophorus helleri. Biol.

Bull., 64: 206-225.

BARNEY, R. L., AND B. J. ANSON, 1921. Seasonal abundance of the mosquito-destroying top-
minnow, Gambusia affinis, especially in relation to male frequency. Ecology, 2: 53-69.
BURGER, J. \V., 1939a. Some preliminary experiments on the relation of the sexual cycle of

Fundulus heteroclitus to periods of increased and decreased daily illumination. Bull, of

the Mount Desert Island Biol. Lab., 39-40.
BURGER, J. W., 1939b. Some experiments on the relation of the external environment to the

spermatogenetic cycle of Fundulus heteroclitus. Biol. Bull., 77: 96 .

CRAIG-BENNETT, A., 1930. V. The reproductive cycle of the three-spined stickleback, Gas-

terosteus aculeatus, Linn. Phil. Trans, of the Roy. Soc. of London, B, 219: 197-280.
DILDINE, GLENN C., 1936. The effect of light and temperature on the gonads of Lebistes. Anal.

Rec., 67: supp., 61.
ESSENBERG, J. M., 1923. Sex-differentiation in the viviparous teleost Xiphophorus helleri Heckel.

Biol. Bull.. 45: 46-77.

REPRODUCTION IN ORYZIAS LATIPES 125

HOOVER, EARL E., AND HARRY E. HUBBARD, 1937. Modification of the sexual cycle in trout by

control of light. Copeia, 4: 206-210.
KAMITO, AKIRA, 1928. Early development of the Japanese killifish (Oryzias latipes), with notes

on its habits. Jour. Coll. Agric., Imp. Univ. Tokyo, 10: 21-38.
MARZA, V. D., EUGENIE V. MARZA AND MARY J. GUTHRIE, 1937. Histochemistry of the ovary of

Fundulus heteroclitus with special reference to the differentiating oocytes. Biol. Bull.,

73: 67-92.
MATTHEWS, S. A., 1939. The effects of light and temperature on the male sexual cycle in

Fundulus. Biol. Bull., 77: 92-95.
REGNIER, MARIE-THERESE, 1938. Contribution a 1'etude de la sexualite des Cyprinodontes

vivipares (Xiphophorus helleri, Lebistes reticulatus). Bull. Biol. de la France et de la

Belgique, 72: 385-493.
RUGH, ROBERTS, 1935. Ovulation in the frog. II. Follicular rupture to fertilization. Jour.

Exp. Zool., 71: 163-193.
RUGH, ROBERTS, 1941. Experimental embryology, a manual of techniques and procedures.

N. Y. U. Press.

SHAW, RALPH J., RAUL A. ESCOBAR AND FRANCIS M. BALDWIN, 1938. The influence of tempera-
ture and illumination on the locomotor activity of Carassius auratus. Ecology, 19:

343-346.
SOLBERG, ARCHIE NORMAN, 1938. The development of a bony fish. The Progressive Fish

Culturist, 40: 1-19.

SPENCER, WARREN P., 1929. Day and night periodicity in the activity of four species of fresh-
water fishes. Anal. Rec., 44: supp., 197.
TURNER, C. L., 1936. Reproductive cycles and superfoetation in the Poeciliidae, a family of

tropical and sub-tropical ovo-viviparous fishes. Anal. Rec., 67: supp., 9.
TURNER, C. L., 1937. Reproductive cycles and superfoetation in poeciliid fishes. Biol. Bull.,

72: 145-164.
TURNER, C. L., 1938. The reproductive cycle of Brachyraphis episcopi, an ovoviviparous

poeciliid fish, in the natural tropical habitat. Biol. Bull., 75: 56-65.

Vol. 84, No. 1, Supplement

February, 1943

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

SERIAL PUBLICATIONS

HELD BY

THE MARINE BIOLOGICAL LABORATORY LIBRARY

AND THE

WOODS HOLE OCEANOGRAPHIC INSTITUTION

EXPLANATION OF SYMBOLS

Titles recorded alphabetically with initial dates.

* ceased publication.
[ ] incomplete.

+ receive currently.

Abhandlungen aus der Abteilung fiir Phyto-
morphogenese des Timiriaseff-Instituts fiir
Biologic am Zentralen Exekutivkomitee der
U. d. S. S. R. 1933: Moscow. 1

Abhandlungen der Bayerischen Akademie der
Wissenschaften ; Mathematisch-Naturwis-
senschaftliche Abteilung (1871-1924 as
Mathematisch-Physikalische Abteilung)
1829: Munich. 25, nos. 6-10; 26, nos. 1-10;
28, no. 3; 30-32; N.F. 1-45; supplements 1-4;
10-15

Abhandlungen der Heidelberger Akademie
der Wissenschaften (Mathematisch-Natur-
wissenschaftliche Klasse) 1910: 1-23

Abhandlungen der Leopoldinisch-Carolini-
schen Deutschen Akademie der Natur-
forscher see Nova Acta Leopold ina

Abhandlungen der Naturforschenden Gesell-
schaft zu Gorlitz 1827: 18; 20-26

Abhandlungen der Naturhistorischen Gesell-
schaft zu Niirnberg 1852: 5-7; 10; 12-16;
18, no. 2; 20; 21, no. 4

Abhandlungen der Preussischen Akademie
der Wissenschaften; Mathematisch-Natur-
wissenschaftliche Klasse 1936: 15 +
*Abhandlungen der Preussischen Akademie
der Wissenschaften; Physikalisch-Mathe-
matische Klasse (1882-1900 as Abhand-
lungen der Koniglichen Akademie der Wis-

senschaften zu Berlin; 1901-07 as Abhand-
lungen der Koniglichen Preussischen Aka-
demie der Wissenschaften) 1804: 1882-1938;
also Verzeichnis der Abhandlungen der K.
Preussischen Akademie der Wissenschaften
von 1710-1870 in Alphabetischer Folge der
Verfasser

Abhandlungen (herausgegeben von) der
Senckenbergischen Naturforschenden Ge-
sellschaft 1854: Frankfurt a. M. 22; 33-35;
nos. 430-435; 440-442

Abhandlungen der Zoologisch-Botanischen

Gesellschaft in Wien 1901: 1 +
*Abhandlungen des Deutschen Seefischerei-
Vereins 1897: Berlin. 1; 4-5; 7; 8A; 9-13

Abhandlungen des Naturwissenschaftlichen
Vereins fiir Schwaben 1936: Augsburg. 1

Abhandlungen des Wissenschaftlichen Insti-
tutes fiir Fischereiwirtschaft see Trudy
Nauchnogo Instituta Rybnogo Khoziaistva

Abhandlungen des Wissenschaftlichen Zen-
tralinstitutes fur Fischereiwirtschaft see
Trudy Tsentral'nogo Nauchnogo Instituta
Rybnogo Khoziaistva

Abhandlungen herausgegeben vom Natur-
Wissenschaftlichen Verein zu Bremen (26+
subtitle Reihe B der Schriften der Bremer
Wissenschaftlichen Gesellschaft) 1868: 1-4;
6 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Abhandlungen zur Didaktik und Philosophie
der Naturwissenschaft; Sonderheft der
Zeitschrift fur den Physikalischen und
Chemischen Unterricht 1904: vols. 1-2; no.
16+

*Abhandlungen zur Physiologie der Sinne aus
dem Physiologischen Institut zu Freiburg
i. B. (Heft 1-4 as Abhandlungen zur Physio-
logie der Gesichtsempfindungen) 1897: 1-5

Abhandlungen zur Theoretischen Biologic
herausgegeben von Prof. Dr. Julius Schaxel ;
Vorstand der Anstalt fur Experimentelle
Biologic der Universitat Jena 1919: 1-30

Abhandlungen zur Theorie der Organischen
Entwicklung; Roux' Vortrage und Aufsatze
iiber Entwicklungsmechanik der Organis-
men, Neue Folge 1926: 1-6

Abridged Scientific Publications from the
Kodak Research Laboratories 1913: East-
man Kodak Company. 2 +

Abstract Bulletin of Lamp Development Lab-
oratory (1, no. 1 as Abstract-Bulletin of the
Physical Laboratory of the National Elec-
tric Lamp Association; 1, nos. 2-4 as
Abstract-Bulletin of Nela Research Labora-
tory) 1913: General Electric Company. 1 +

Abstracts from Rikwagaku-Kenkyu-jo Ib.6
see with Scientific Papers of the Institute of
Physical and Chemical Research
* Abstracts of Bacteriology 1917: 1-9

Abstracts of Chemical Papers issued by the
Bureau of Chemical Abstracts see Journal
of the Chemical Society; Journal of the
Society of Chemical Industry; British Chem-
ical and Physiological Abstracts, A and B
*Abstracts of Physical Papers from Foreign
Sources; Physical Society of London 1895:
1-3

Abstracts of the Works of the Zoological
Institute of the Moscow State University
see Sbornik Nauchno-Issledovatel'skogo In-
stituta Zoologii

Academy of Natural Sciences of Philadelphia;

Review 1932: 1932-35

*Academy of Natural Sciences of Philadelphia;
Yearbook (to 1920 in Proceedings; 1920-22
as Annual Reports) 1920: 1920-31

Acta Academiae Aboensis; Mathematica et
Physica 1922: Abo. 11 +

Acta Adriatica; Instituti (Biologico-) Oceano-
graphici Split; Jugoslavia 1932: 1 +

Acta Biologiae Experimentalis; Litterae So-
cietatis Physiologorum Polonorum 1928:
Institut Nencki, Warsaw. 1-12

Acta Biologica Latvica; Latvijas Biologijas
Biedribas Raksti (1-7 as Latvijas Biologijas
Biedribas Raksti; Acta Societatis Biologiae
Latviae, Bulletin de la Socie"te de Biologic
de Lettonie) 1929: 1 +

Acta Biotheoretica; Geschriften van de Prof.
Dr. Jan van der Hoeven Stichting voor
Theoretische Biologic van Dier en Mensch,
verbonden aan de Universiteit te Leiden;
Series A, 1935: 1+ Series D. Bibliotheca
Biotheoretica 1941: 1 +

Acta Botanica Fennica 1925: Societas pro
Fauna et Flora Fennica. 1 +

Acta Brevia Neerlandica de Physiologia, Phar-
macologia, Microbiologia e. a. 1931: Neder-
landsche Vereeniging voor Physiologie en
Pharmacologie, Amsterdam. 1 +

Acta Horti Bergiani 1890: Bergielund Botani-
ska Tradgard, Stockholm. 1 +

Acta Horti Botanici Universitatis Latviensis
1926; Riga. 1 +

Acta Instituti Botanici Academiae Sciencia-
rum, URSS see Trudy Botanicheskogo Insti-
tuta Akademii Nauk SSSR

Acta Instituti et Musei Zoologici Universitatis
Atheniensis (title given in Hebrew also)
1935: Zoological Institute and Museum,
University of Athens. 1, nos. 1-5

Acta Medica Scandinavica (1-51, 1869-1919
as Nordiskt Medicinskt Arkiv) 52, 1919:
Stockholm. 77, no. 4; 103 + ; Supplementum
1921: 17; 42; 56; 68; 71; 107 +

Acta Ornithologica ; Musei Zoologici Polonici
1933: Warsaw. 1-3, no. 3

Acta Pathologica et Microbiologica Scandina-
vica 1924: Copenhagen. 10, nos. 1-2; 16+

Acta Physicochimica U. R. S. S. 1934: 1 +

Acta Phytochimica 1922: Iwata Institute of
Plant Biochemistry, Tokyo. 1 +

Acta Radiologica 1921: Societies for Medical
Radiology in Denmark, Finland, Holland,
Norway, Sweden, Switzerland. 3, no. 6; 13 +

Acta Societatis Biologiae Latviae see Acta Bio-
logica Latvica

Acta Societatis Botanicorum Poloniae; Organ
Polskiego Towarzystwa Botanicznego (Pub-
lication de la Societe Botanique de Pologne)
1923: Warsaw. 1-15, no. 3; supplements to
9 and 11

Acta Societatis pro Fauna et Flora Fennica
1875: Helsingfors. 1 +

Acta Societatis Scientiarum Fennicae (after
50 as Nova Series A. Opera Physicomathe-
matica and B. Opera Biologica) 1842:
Helsingfors. 1 +

Acta Societatis Scientiarum Naturalium Mo-
ra vicae see Prace Moravske Prirodovedecke
Spolecnosti

Acta Universitatis Asiae Mediae see Trudy
Sredne-Aziatskogo Gosudarstvennogo Uni-
versiteta

Acta Universitatis Lundensis see Lunds Uni-
versitets Arsskrift

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Acta Universitatis Voronegiensis see Trudy
Voronezhskogo Gosudarstvennogo Univer-
siteta

Acta Zoologica; Internationell Tidskrift for
Zoologi 1920: Stockholm. 1 +

Acta Zoologica Fennica 1926: Societas pro
Fauna et Flora Fennica; Helsingfors. 1 +

Actes de la Societe Helvetique des Sciences
Naturelles see Verhandlungen der Schwei-
zerischen Naturforschenden Gesellschaft

Actes de la Societe Scientifique du Chili (fon-
dee par un groupe de Francais) 1891: San-
tiago. 1; 2, nos. 1, 4; 3-4

Actualites Scientifiques et Industrielles 1929:
Paris. 11; 32; 36; 38; 47-48; 64-66; 78; 96;
100; 102; 106; 108; 112-13; 119-20; 128;
133; 135-37; 170-71; 176; 178-180; 183;
187; 189; 207-09; 228; 238; 240; 242-44;
246; 254-55; 258-61; 264; 266-68; 272-73;
284; 300; 338; 350; 373; 401-03; 405; 408;
410; 413; 416; 466; 498; 583; 594-97

Administration Reports of the Government
Marine Biologist: Ceylon. 1910-1920;
1922 +

Advances in Colloid Science 1942: New
York. 1

Advances in Enzymology and Related Sub-
jects 1941: New York. 1 +

Advances in Modern Biology, Moscow see
Uspekhi Sovremennoi Biologii

Aeronautical Engineering Review 1942: Insti-
tute of the Aeronautical Sciences, Inc. 1 +

Aeronautical Review Section see Journal of the

Aeronautical Sciences

*Agricultural Journal of the Union of South
Africa 1911: Department of Agriculture.
1-8, no. 2

Algal Research Institute, Archangel see Trudy
Arkhangel'skogo Vodoroslevogo Nauchno-
Issledovatel'skogo Instituta Avnii

Allahabad University Studies 1925: 1 +
*Allgemeine Botanische Zeitschrift fur Syste-
matik, Floristik, Pflanzengeographie 1895:
Karlsruhe. 1-33

Allgemeine Zellforschung und Mikroskopische
Anatomie see Zeitschrift fur Zellforschung
und Mikroskopische Anatomie, Abt. A

American Agriculturist for Farm, Garden,
Household 1843: New York. 1-2; [20-52]

American Anatomical Memoirs (Memoirs of
the Wistar Institute of Anatomy and Biol-
ogy) 1911: Philadelphia. 1 +

American Association for the Advancement of
Science and Associated Societies; General
Program 1849: 22-23; 25; 36; 38; 40; 43-47;
51; 57; 60-63; 65-66; 68-69; 73; 76; 78-79;
83; 85 +

American Association for the Advancement of
Science Bulletin 1942: 1 +

American Botanist; a quarterly journal of
economic and ecological botany 1901 : 1 +

American Breeders' Magazine see Journal of

Heredity

* American Chemical Journal 1879: 1-50
*American Entomologist; an illustrated maga-
zine of popular and practical entomology
1868: 1-3

American Forests (8-14, no. 8 as Forestry and
Irrigation; 14, no. 9 — vol. 15 as Conserva-
tion; 16-29 as American Forestry) 1895:
American Forestry Association, Washing-
ton. [12-43]

American Heart Journal; a journal for the
study of the circulation 1925: American
Heart Association. 1 +

American Journal of Anatomy (preceded by
and including Proceedings of the Association
of American Anatomists 1888: 1-20) 1901:

1 +

American Journal of Botany; official publica-
tion of the Botanical Society of America
1914: 1 +

American Journal of Cancer (1-14 as Journal
of Cancer Research) 1916: American Asso-
ciation for Cancer Research. 1-40
*American Journal of Conchology 1865: Acad-
emy of Natural Sciences. 2-7

American Journal of Hygiene 1921: School of
Hygiene and Public Health of Johns Hop-
kins University. 1 + and Monographic
Series 1 +

*American Journal of Microscopy; a monthly
record of microscopical science 1871: Chi-
cago. 1, no. 1

*American Journal of Microscopy and Popular
Science 1875: New York City. 1-6

American Journal of Pathology 1925: Ameri-
can Association of Pathologists and Bac-
teriologists. 1 +

American Journal of Physical Anthropology;
organ of the Association of Physical Anthro-
pologists 1918: 1 +

*American Journal of Physiological Optics
1920: American Optical Company. 1-7

American Journal of Physiology 1898: 1 +

American Journal of Public Health and the
Nation's Health (1-17 as American Journal
of Public Health) 1911: American Public
Health Association! 2 +

American Journal of Roentgenology and Ra-
dium Therapy; official organ of the Ameri-
can Roentgen Ray Society and the Ameri-
can Radium Society (1-9 as American
Journal of Roentgenology) 1906: n.s. 2 +

American Journal of Science (1820-79 as
American Journal of Science and Arts.
Known also as Silliman's Journal of Science)
1818: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

American Journal of Surgery (1-17 as Ameri-
can Journal of Surgery and Gynecology)
1890: [17-37]; n.s. [4-10]; 19 +

American Journal of the Medical Sciences
1827: n.s. 99-106; [107-112]; 113; 115; 117;
119; [123-127]; 148; [149]; 150-51; [152];
153 +

American Journal of Tropical Medicine 1921:

I +

*American Meteorological Journal; a monthly
review of meteorology and allied branches
of study 1884: 1-9, no. 1; 10, nos. 5-6

American Midland Naturalist 1909: Univer-
sity of Notre Dame. 1 +

*American Monthly Microscopical Journal
1880: 1-20; 21, no. 10

American Museum Journal see Natural His-
tory

American Museum Novitates 1921: American
Museum of Natural History. 1 +

American Naturalist; a bi-monthly journal
devoted to the advancement of the biologi-
cal sciences, with special reference to the
factors of evolution 1867: 1 +

American Nautical Almanac: United States
Naval Observatory. 1934 +

American Philosophical Society; Yearbook
1937: 1937 +

American Public Health Association Yearbook
see in American Journal of Public Health
and the Nation's Health
*American Quarterly Microscopical Journal
1878: New York Microscopical Society. 1

American Scientist; the Sigma Xi Quarterly
(1-29 as Sigma XI Quarterly) 1913: [1-10];

II +

Anales de la Escuela Nacional de Ciencias
Biologicas 1938: Mexico. 1 +

Anales de la Sociedad Cientifica Argentina
1876: [2-98]; 99 +

Anales de la Sociedad Espanola de Fisica y
Quimica (10+ in two parts: Notas y Memo-
rias; and Actas, Revistas e Indices) 1903:
1 +

*Anales de la Sociedad Espanola de Historia
Natural 1872: Madrid. 1-30

Anales del Instituto de Biologia de la Universi-
dad Nacional de Mexico 1930: 1 +

Anales del Instituto Geologico de Mexico
1917: 1-9

Anales del Museo Argentino de Ciencias
Naturales "Bernardino Rivadavia" (3-20 as
Anales del Museo Nacional; 21-36 as Anales
del Museo Nacional de Historia Natural;
32+ with addition of "Bernardino Rivada-
via") 1864: 4 +

Anales del Museo de Historia Natural de
Montevideo (ser. 1, vols. 1-7 and ser. 2, vol.
1 as Anales del Museo Nacional de Monte-

video of which vols. 1-4 form Flora Uru-
guaya) 1894:* ser. 1, 1-7; ser. 2 (1904). 1 +

Anales del Museo de la Plata 1891: seccion
botanica, 1; seccion paleontologica, 5; ser.
2 (1907), 1-4, pt. 1

Anales Hidrograficos ; Ministerio de Marina,
Argentina 1918: 5-10

Analyst; the journal of the Society of Public
Analysts and other analytical chemists 1877:
Cambridge, England. 1 +

Anatomical Record 1906: 1 +

Anatomische Hefte, Abt. 1 ; Arbeiten aus
Anatomischen Instituten see Zeitschrift fur
Anatomie und Entwicklungsgeschichte

Anatomische Hefte, Abt. 2; Ergebnisse der
Anatomie und Entwicklungsgeschichte see
Ergebnisse der Anatomie und Entwick-
lungsgeschichte

*Anatomische und Entwicklungsgeschichtliche
Monographien herausgegeben von Prof.
Wilhelm Roux 1909: 1-3

Anatomischer Anzeiger; Centralblatt fur die
Gesamte Wissenschaftliche Anatomie; Amt-
liches Organ der Anatomischen Gesell-
schaft 1886: 1 +

Anatomischer Anzeiger; Erganzungsheft see
Verhandlungen der Anatomischen Gesell-
schaft

Anatomischer Bericht; Referierendes Organ
fur das Gesamtgebiet der Anatomie; im
Auftrage der Anatomischen Gesellschaft
1922: 1 +

*Anexos das Memorias do Instituto de Butan-
tan; Seccao de Ofiologia 1921: 1, fasc. 1

Angewandte Botanik; Zeitschrift der Vereini-
gung fur Angewandte Botanik (1-8 with sub-
title Zeitschrift fur Erforschung der Nutz-
pflanzen) 1919: 1 +

Angewandte Chemie; Zeitschrift und Eigen-
tum des Vereins Deutscher Chemiker (2-44
as Zeitschrift fur Angewandte Chemie) 1887:
2 +

Animal Kingdom (nos. 1-5 as News Bulletin)
(vol. 1, no. 6— vol. 44 as Bulletin) 1897:
New York Zoological Society. 1 +

Anleitungen der Deutschen Gesellschaft fur
Ziichtungskunde 1920: 16 +

Annale van die Transvaal Museum see Annals
of the Transvaal Museum

Annalen der Chemie und Pharmazie see Justus
Liebigs Annalen der Chemie

Annalen der Hydrographie und Maritimen
Meteorologie ; Zeitschrift fur Seefahrt- und
Meereskunde (1-2 as Hydrographische
Mittheilungen) (includes Jahresbericht iiber
die Tatigkeit der Deutschen Seewarte)
1873: 1 +

Annalen der Pharmazie see Justus Liebigs
Annalen der Chemie

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Annalen der Physik (1819-1824 as Anna'.en der
Physik und der Physikalischen Chemie;
1824-1899 05 Annalen der Physik und
Chemie) 1799: 1 +

Annalen der Physik; Beiblatter see Beiblatter

zu den Annalen der Physik
* Annalen der Physik; Erganzungsbande 1842:
1-8

Annalen des Naturhistorischen Museums in
Wien (1-32 as Annalen des K. K. Natur-
historischen Hof museums) 1886: 1 +

Annales; Station Oceanographique de Sal-
ammbo 1925: Tunis. 1-8

Annales Botanici Societatis Zoologicae-Botani-
cae Fennicae Vanamo see Suomalaisen
Elain-ja Kasvitieteellisen Seuran Vanamon;
Kasvitieteellisia Julkaisuja
*Annales de Biologic Lacustre 1906: Brussels.
1-15

Annales de Chimie Analytique et de Chimie
Appliquee et Revue de Chimie Analytique
Reunies 1896: ser. 2, 1 +

Annales de Chimie et de Physique (after ser.
8, divided into Annales de Chimie, and An-
nales de Physique) 1789: 1 +
*Annales de Cryptogamie Exotique 1928: Mu-
seum National d'Histoire Naturelle. 1-8
*Annales de 1'Association des Naturalistes de
Levallois-Perret 1895: 9-10; 12-19

Annales de 1'Institut Henri Poincare; Recueil
de Conferences et Memoires de Calcul des
Probabilites et Physique Theorique 1930:
4, no. 3

Annales de 1'Institut National Zootechnique
de Roumanie 1932: Bucarest. 1-3

Annales de 1'Institut Oceanographique; Mo-
naco 1909: 1 +

Annales de 1'Institut Pasteur 1887: 1 +

Annales de 1'Universite de Minsk see Trudy

Belorusskogo Gosudarstven. Universiteta
*Annales de la Societe Beige de Microscopic
(Memoires) 1875: 1-28

Annales de la Societe Entomologique de
Belgique see Bulletin et Annales de la
Societe Entomologique de Belgique

Annales de la Societe Linneenne de Lyon

1826: n.s. 76-80

*Annales de la Societe Royale des Sciences
Medicales et Naturelles de Bruxelles 1892:
10, no. 1; 15, no. 3; 19, no. 1

Annales de la Societe Royale Zoologique (et
Malacologique) de Belgique 1863: 1 +

Annales de la Societe Scientifique de Bruxelles
1875: 1-46; ser. B. Sciences Physiques et
Naturelles. 47+ *ser. C. Sciences Medicales.
47^8

*Annales de Micrographie ; specialment consa-
crees a la bacteriologie aux protophytes et
aux protozoaires 1888: 1-10

Annales de Parasitologie humaine et comparee

1923: 1 +
Annales de Physiologic et de Physicochimie

Biologique 1925: 1 +
Annales de Physique see Annales de Chimie

et de Physique
Annales de Protistologie ; recueil de travaux

originaux concernant la biologic et la sys-

tematique des protistes 1928: 1 +
Annales des Sciences Naturelles (after 30 in

two sections; Botanique; Zoologie) 1824: 1 +
Annales du Jardin Botanique de Buitenzorg

1876:41 +
Annales du Musee d'Histoire Naturelles de

Marseille 1883: 1 +
*Annales du Musee Royal d'Histoire Naturelle

de Belgique 1877: 1-14
*Annales du Museum d'Histoire Naturelle par

les Professeurs de cet Etablissement 1802:

1-20
Annales Entomologici Fennici see Suomen

Hyonteistieteellinen Aikakauskirja
Annales et Bulletin de la Societe Royale des

Sciences Medicales et Naturelles de

Bruxelles (1-69 as Bulletin de la Seance

de . . .) 1840: [57-77]; 78 +
Annales Musei Zoologici Polonici (1-6 as

Prace Zoologiczne Polskiego Panstwowego

Muzeum Przyrodniczego; 7-8 as Prace

Panstwowego Muzeum Zoologicznego) 192 1 :

1-12, no. 24
Annales Mycologici; Editi in Notitiam Scien-

tiae Mycologicae Universalis 1903: Berlin.

1 +
Annales Scientifiques de 1'Universite de Jassy

1902: 1-22; (partie 2) Sciences Naturelles.

23 +
Annales Societatis Chimicae Polonorum see

Roczniki Chemii
Annales Societatis Rebus Naturae Investi-

gandis in Universitate Tartuensi Constitu-

tae see Tartu Ulikooli juures oleva Loodu-

suurijate seltsi Aruanded
Annales Societatis Zoolog.-Botanicae Fenni-
cae Vanamo see Suomalaisen elain-ja Kas-
vitieteellisen Seuran Vanamon
Annales Zoologici Societatis Zoologicae-Bo-

tanicae Fennicae Vanamo see Suomalaisen

elain-ja Kasvitieteellisen Seuran Vanamon;

Elaintieteellisia Julkaisuja
Annaii d'Igiene (includes supplement, 1930-34

with no title, 1935 entitled "Igea," 1936+

entitled 1'Attualita Meclica, vol. 1+) 1889:

40+

Annaii di Botanica 1904: 1 +
Annaii di Chimica Applicata 1914: Associa-

zione Italianadi Chimica. 19, nos. 9, 12; 21 +
Annaii Idrografici; Raccolta di Document! e

Notizie circa 1'Idrografia e la Navigazione

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

1900: Istituto Idrografico della Regia
Marina. 1 +

Annals and Magazine of Natural History (ser.
1, vols. 1-5 as Annals of Natural History)
1838: London. 1 +

Annals of Applied Biology 1914: Cambridge,
England. 1 +

Annals of Botany 1887: Oxford. 1 +

Annals of Eugenics; a journal for the scientific
study of racial problems (subtitle 6+ jour-
nal devoted to the genetic study of human
populations) 1925: London. 1 +

Annals of Internal Medicine 1927: American
College of Physicians. 1 +

Annals of Natural History see Annals and
Magazine of Natural History

Annals of Science; a quarterly review of the
history of science since the Renaissance
1936: London. 1 +
* Annals of Scottish Natural History 1892: 1-20

Annals of the Association of American Geog-
raphers 1911: 1 +

Annals of the Astrophysical Observatory of

the Smithsonian Institution 1900: 1 +
*Annals of the Bolus Herbarium 1914: Uni-
versity of Cape Town. 1-4

Annals of the Carnegie Museum 1901: Pitts-
burgh. 1 +

Annals of the Durban Museum 1914: Natal.

1 +
Annals of the Entomological Society of

America 1908: 1 +
*Annals of the Institute of Jamaica 1897: 1,

no. 1
Annals of the Lyceum of Natural History of

New York see Annals of the New York

Academy of Sciences
Annals of the Missouri Botanical Garden

1914: 1 +
Annals of the Natal (Government) Museum

1906: Pietermaritzburg. 1 +
Annals of the New York Academy of Sciences

(ser. 1 as Annals of the Lyceum of Natural

History of New York) 1823: 1 +
*Annals of the Royal Botanic Gardens, Pera-

deniya; Ceylon Journal of Science, Section

A. Botany 1901: [1-11]
Annals of the South African Museum 1898:

1 +
Annals of the Transvaal Museum (Annale van

die Transvaal Museum) (Mededelingen

van het Transvaal Museum) 1908: [1-2];

3-9; [10]; 11 +
Annals of Tropical Medicine and Parasitol-

ogy; issued by the Liverpool School of

Tropical Medicine 1907: 1 +
1'Annee Biologique; comptes rendus des tra-

vaux de biologic generale (27-43 in two

parts: 1. Physiologic generale; 2. Morpholo-
gic et Biologic generales) 1895: 1 +

Annotation of the Oceanographical Research
1926: Imperial Fisheries Institute; Tokyo.
1-3, no. 2

Annotationes Zoologicae Japonenses 1897:
Zoological Society of Japan. 1 +

Annuaire de 1'Academie Royale de Belgique
(Jaarboek van de Koninklijke Belgische
Academie) (95-98 as Annuaire de 1'Acade-
mie Royale des Sciences des Lettres et des
Beaux-Arts de Belgique) 1835: 95 +

Annuaire de 1'Institut Oceanographique du
Royaume de Yougoslavie see Godisnjak
Oceanografskog Instituta Kraljevine Jugo-
slavije

Annuaire de la Societe des Sciences et des
Lettres de Varsovie see Rocznik Towar-
zystwa Naukowego Warszawskiego

Annuaire de la Societe Meteorologique de

France see Meteorologie

*Annuaire du Musee Zoologique; Academie
des Sciences de 1'URSS (Ezhegodnik
Zoologicheskogo Muzefa; Akademiya Nauk
SSSR) (1-18 as Ezhegodnik Zoologiches-
kago Muzeia; Imperatorskoi Akademii
Nauk) (Annuaire du Musee Zoologique de
1'Academie Imperiale des Sciences de St.-
Petersbourg) (19-20 as Ezhegodnik Zoologi-
cheskago Muzeia; Imperatorskoi Akademii
Nauk) (Annuaire du Musee Zoologique de
1'Academie ImpeYiale des Sciences de
Petrograd) (21-24 as Ezhegodnik Zoolo-
gicheskogo Muzeia; Rossiiskoi Akademii
Nauk) (Annuaire du Musee Zoologique de
1'Academie des Sciences de Russie) 1896:
1-32 (1932)

Annual Announcement; Marine Biological
Laboratory of Woods Hole, Massachusetts
1888: 1 +

Annual Announcement; Woods Hole Oceano-
graphic Institution 1931: 1 +

Annual Conference of the Universities of
Great Britain and Ireland; Report of Pro-
ceedings: 1925; 1928-29
*Annual of Scientific Discovery; or Year-book
of Facts in Science and Art 1850: Boston.
1-21

* Annual of the Universal Medical Sciences; a

yearly report of the progress of the general

sanitary sciences throughout the world

(Sajous) 1888: 5 (1891)

*Annual Record of Science and Industry 1871 :

New York. 1873

*Annual Report; American Breeders' Associa-
tion (1-2 as American Breeders' Association
Proceedings; 3-6 as American Breeders'
Association Report) 1903: Washington. 1-8

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Annual Report; Carnegie Foundation for the
Advancement of Teaching 1906: 1 +

Annual Report; Fouad I University; Faculty
of Science 1926: Cairo. 1934 +

Annual Report; General Education Board
(1914-17 as Report of the Secretary) 1914:
1914 +

Annual Report; Geological and Natural His-
tory Survey of Minnesota 1872: 1 ; 7; 12-13;
15-16; 18

Annual Report; Indian Association for the
Cultivation of Science (1925, 1929-34 see
in Indian Journal of Physics): 1935 +

Annual Report; International Hydrographic
Bureau 1921: Monaco. 1 +

Annual Report; John Innes Horticultural In-
stitution 1909: 28 +

Annual Report; Liverpool Observatory and
Tidal Institute 1920: University of Liver-
pool. 1921 +

* Annual Report; Missouri Botanical Garden
1889: 1-23

Annual Report; Nantucket Maria Mitchell
Association 1902: 1; 4-5; 10; 12-14; 17-18;
20 +

Annual Report; National Foundation for In-
fantile Paralysis, Inc. 1939: 1939 +

Annual Report; New York Academy of Medi-
cine see Bulletin of the New York Academy
of Medicine

Annual Report; New York Zoological Society
(1-28 as Annual Report of the New York
Zoological Society) 1896: 1 +

Annual Report; Oceanographical Investigation
(through no. 43 as Quarterly Report) 1913:
Imperial Fisheries Experimental Station,
Tokyo. 21-31; 33-44; 51 +
*Annual Report; Peabody Academy of Sciences
(1-6 as Annual Report of the Trustees of
the Peabody Academy of Science) 1867:
1-6; 17-18

Annual Report; Rockefeller Foundation 1913:
1 +

Annual Report; Rockefeller Foundation; In-
ternational Health Division (through 1933
see in Annual Report; Rockefeller Founda-
tion) 1913: 1934 +

Annual Report; Scottish Marine Biological
Association (1894-1901 as Millport Marine
Biological Station; 1901-13 as Marine Bio-
logical Association of the West of Scotland)
1897: 1898-99; 1902-05; 1913-18; 1924 +

Annual Report; Scripps Institution of Ocean-
ography: La Jolla. 1938 +

Annual Report; Texas Agricultural Experi-
ment Station 1887: 32 +

Annual Report; United Fruit Company; Medi-
cal Department 1912: 1; 3; 5-20

Annual Report about the activity of the Breed-

ing and Biological Section of the Zootechni-
cal Research Institute hi Brno see Zprava
o Cinnosti sekce pro plemenarskou Biologii
Moravskeho Zemskeho Vyzkumneho Us-
tavu Zootechnickeho v Brne
*Annual Report and Proceedings of the Botani-
cal Society; Edinburgh 1836: 1-10

Annual Report and Transactions; Manchester
Microscopical Society 1880: 1886; 1888;
1890-96; 1900; 1907-13; 1916; 1919

Annual Report of Fishery Research Labora-
tory (Institute); Department of Natural
Resources; Division of Fishery Research:
St. Johns, Newfoundland. 1931 +

Annual Report of Hydrographical Observa-
tions 1926: Fishery Experiment Station;
Fusan. 1 +

Annual Report of the Adelaide Philosophical
Society see Transactions of the Royal So-
ciety of South Australia

Annual Report of the Agricultural Experiment
Station; University of Puerto Rico (formerly
Insular Experiment Station of the Depart-
ment of Agriculture and Labor) (title
varies): 1922 +

Annual Report of the American Museum of
Natural History 1869: 1 +

Annual Report of the Auckland Institute and
Museum: New Zealand. 1940 +

Annual Report of the Biological Board of
Canada see Annual Report of the Fisheries
Research Board of Canada

Annual Report of the Biological Laboratory;
Long Island Biological Association 1889:
35 +

Annual Report of the Biological Station to the
Governor of North Dakota 1909: 1909-12;
1915-16

Annual Report of the Board of Directors of
the Zoological Society of Philadelphia 1873:
13-16; 18; 20-23; 25-28; 30-33; 35-38; 40

Annual Report of the Board of Regents of the
Smithsonian Institution 1846: 1847; 1849 +

Annual Report of the Brooklyn Botanic Gar-
den see in Brooklyn Botanic Garden Record

Annual Report of the Bureau of American
Ethnology; Smithsonian Institution 1879:
1885-89; 1892-96; 1899-1905

Annual Report of the Bureau of Science ; Phil-
ippine Islands (no. 2 as Report of the Super-
intendent of Government Laboratories; no.
3 as Annual Report of the Superintendent of
the Bureau of Government Laboratories;
5-18 as Annual Report of the Director of the
Bureau of Science) 1901: Manila. 2-3; 5-7;
18; 26-31; 33

Annual Report of the Carnegie Museum ; Car-
negie Institute 1896: Pittsburgh. 7-27; 30 +

Annual Report of the Chairman ; National Re-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

search Council; Division of Biology and
Agriculture see National Research Council;
Annual Report of the Chairman of the Divi-
sion of Biology and Agriculture

Annual Report of the Chief Signal Officer of
the Army to the Secretary of War: 1889

Annual Report of the Colorado Agricultural
Experiment Station 1886: Fort Collins. 37;
39 +

*Annual Report of the Commissioners of Inland
Fisheries ; State of Rhode Island and Provi-
dence Plantations (no. 1 forms report of the
joint special committee of the general as-
sembly of Rhode Island appointed to exam-
ine into the fisheries of Narragansett Bay,
1870) 1870: 1-5; 7-8; 10-13; 18-65
*Annual Report of the Commissioners of Shell
Fisheries, made to the General Assembly;
State of Rhode Island and Providence Plan-
tations 1890: 1890-91; 1893-94; 1905-07;
1911-12; 1914-15; 1917; 1919-25; 1928-30

Annual Report of the Commissioners on Fish-
eries and Game ; Massachusetts (28-29 and
33 as Report of the Commissioners on In-
land Fisheries and Game; 40-49 as Report
of the Commissioners on Fisheries and
Game) 1865: 28-29; 33; 40-49; 51

Annual Report of the Conservation Depart-
ment of the State of Maryland 1923: 1-3;
6+

Annual Report of the Council for Scientific and
Industrial Research 1926: Melbourne. 1 +

Annual Report of the Department of Agricul-
ture and Conservation of the State of Rhode
Island and Providence Plantations 1935: 1

Annual Report of the Department of Fisheries;
Dominion of Canada (1868-83 issued as
Supplements to the Annual Report of the
Department of Marine and Fisheries; 1875-
83 as Report of the Commissioner of Fish-
eries and variations of this title; 34-47,
1901-14 as Annual Report of the Depart-
ment of Marine and Fisheries; 48-53, 1914-
19 as Annual Report of the Fisheries Branch ;
Department of the Naval Service; 56-63,
1922-30 as Annual Report of the Fisheries
Branch; Department of Marine and Fish-
eries) 1868: [4-53]; 56+ Supplements: to
vols. [23-51]

Annual Report of the Department of Fisheries,
Bengal, Bihar, and Orissa 1913: 1916-19;
1920-22

Annual Report of the Department of Public
Health; Massachusetts 1913: 15 +

Annual Report of the Department of Water
Supplies and Sewage Disposal ; New Jersey
Agricultural Experiment Station (1923-26 as
Report of the Sewage Sub-station) 1921:
1922-26; 1929+

Annual Report of the Director; United States
Coast and Geodetic Survey (1834-50 as Re-
port (Letter) from the Secretary of the
Treasury; 1851-191 1 as Report of the Super-
intendent; 1911-19 as Annual Report of the
Superintendent; 1935+ see in Annual Re-
port of the Secretary of Commerce) 1832:
1834; 1836-41; 1843 +

Annual Report of the Director of the Museum
of Comparative Zoology at Harvard College
1861: 1861 +

Annual Report of the Entomological Society of
Ontario 1870: [3-66]

Annual Report of the Essex Institute 1898:
Salem. 1903-06; 1912-13; 1917-18

Annual Report of the Fan Memorial Institute
of Biology 1928: Peiping. 1 +

Annual Report of the Fisheries Research
Board of Canada (1930-33 as Annual Report
on the work of the Biological Board of
Canada; 1934-37 as Annual Report of the
Biological Board of Canada): 1930 +

Annual Report of the Fishery Board for Scot-
land 1881: 40; 47-49; 51-52

Annual Report of the Imperial Cancer Research

Fund 1902: London. 1 +

*Annual Report of the Indian Museum, Zoolog-
ical and Anthropological: 1909-16

Annual Report of the Laguna Marine Labora-
tory 1912: Laguna Beach, Orange County,
California. 1

Annual Report of the Librarian of Congress
(1910-37 as Report) 1800: 1902; 1906 +

Annual Report of the Liverpool Marine Bio-
logical Station on Puffin Island see Report
of the Marine Biological Station at Port
Erin, Isle of Man

Annual Report of the Maine Agricultural Ex-
periment Station (includes Bulletins and Of-
ficial Inspections) 1884: 1889-91; 1893-94;
1898 +

Annual Report of the Marine Biological Sta-
tion at Port Erin see Report of the Marine
Biological Station at Port Erin, Isle of Man

*Annual Report of the Michigan Academy of
Science 1894: 1894-1920

Annual Report of the (Metropolitan) National
Library of Peiping 1926: 1927-33; 1935-38

Annual Report of the National Research Coun-
cil; Dominion of Canada 1916: 17-20; 22

* Annual Report of the New Jersey State Mu-

seum 1905: 1905-11

* Annual Report of the Newfoundland Fisheries

Commission 1889: 1889-93
*Annual Report of the Ohio (State) Academy
of Science (12-47 see in Proceedings of the
Ohio Academy of Sciences and in Ohio Jour-
nal of Science) 1892: 1-11

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Annual Report of the Secretary of Commerce
1912:23 +

Annual Report of the Secretary of the Interior;
Philippine Islands 1901: 1901-02; 1910-13

Annual Report of the Smithsonian Institution
see Annual Report of the Board of Regents
of the Smithsonian Institution

Annual Report of the Tortugas Laboratory;
Carnegie Institution of Washington see Car-
negie Institution of Washington Yearbook

Annual Report on the Geology of the State of
Maine 1837: 1-3

Annual Report on the Geology of the State of
New Hampshire 1841: 1

Annual Report on the Marine Biological Sta-
tion at Ghardaqa, Red Sea: Fouad I Uni-
versity; Cairo. 1932-38

Annual Report on the Progress of Chemistry
1904: Chemical Society, London. 1 +

Annual Report on the Work of the Biological
Board of Canada see Annual Report of the
Fisheries Research Board of Canada

Annual Review of Biochemistry 1932: 1 +

Annual Review of Physiology 1939: American

Physiological Society. 1 +
*Annual Survey of American Chemistry 1925:
National Research Council. 1-10

Annuario del Museo Zoologico della R. Uni-
versita di Napoli 1861: n.s. 1 +

Annuario della Reale Accademia d'ltalia 1929:
1-9

Annuario della Pontificia Accademia delle

Scienze 1936: 1

*Anuari; Junta de Ciencies Naturals de Bar-
celona 1916: 1-3

Anuario da Escola Medico-Cinirgica de Nova
Goa 1915: 2-4; 6

Anzeiger der K. Akademie der Wissenschaf-
ten, Wien; Mathematisch-Naturwissen-
schaftliche Klasse 1864: 4; [5-6]; 8-9; 12 +

*Applied Photography 1931: Eastman Kodak
Company. 1-14

*Apteryx; a New England quarterly of natural
history 1905: Providence, R. I. 1, nos. 1-2

Aquarium 1932: Philadelphia. [1-4]

*Aquarium; a quarterly magazine for students
and lovers of nature, education, and recrea-
tion 1892: [3-4]

* Aquarium; Aquarium Societies of New York,
Brooklyn, Chicago, Philadelphia, etc. 1912:
[1-2]

Aquarium Digest: Toledo Aquarium Society.
4 +

Aquatic Life 1915: Philadelphia. [1-2]

Aquila; journal of ornithology 1894: Budapest.
11-18

Arbeiten aus Anatomischen Instituten see Zeit-
schrift fiir Anatomie und Entwicklungsge-
schichte

*Arbeiten aus dem Gebiet der Experimentellen
Biologie 1921: 1-3

Arbeiten aus dem Staatlichen Institut fur Ex-
perimentelle Therapie und dem Forschungs-
institut fiir Chemotherapie zu Frankfurt
a. M. (17-35 as Arbeiten aus dem Staats-
institut fiir Experimentelle Therapie und
dem Georg Speyer-Hause zu Frankfurt
a. M.) 1905: 17; 19-20; 22 +
*Arbeiten aus dem Zoologischen Institut zu

Graz 1886: 1-9

*Arbeiten aus den Zoologischen Instituten der
Universitat Wien und der Zoologischen Sta-
tion in Triest 1878: 1-20

Arbeiten aus der Biologischen Meeresstation
am Schwarzen Meer in Varna, Bulgarien
see Trudove na Chernomorskata Biologichna
Stantsiia v Varna

Arbeiten aus der Dritten Abteilung des Anato-
mischen Institutes der Kaiserlichen Uni-
versitat Kyoto; Ausserseriale Monographic
1932: 1

Arbeiten aus der Dritten Abteilung des Anato-
mischen Institutes der Kaiserlichen Uni-
versitat Kyoto; Serie A. Untersuchungen
iiber das Periphere Nervensystem 1930:
1 + ; Supplementheft 1

Arbeiten aus der Dritten Abteilung des Anato-
mischen Institutes der Kaiserlichen Uni-
versitat Kyoto; Serie C. Experimentelle
Tuberkuloseforschung 1930: 1 + ; Supple-
mentheft 2

Arbeiten aus der Dritten Abteilung des Anato-
mischen Institutes der Kaiserlichen Uni-
versitat Kyoto ; Serie D. Lymphopathologie
(1930-35 as Untersuchungen iiber die
Physiologic der Lymphbewegung) 1930: 1 +
*Arbeiten aus der Physiologischen Anstalt zu
Leipzig 1866: 1-11

Arbeiten der Biologischen Noworossijsk-
Station see Raboty Novorossiiskoi Biologi-
cheskoi Stantsii

Arbeiten der Biologischen Station zu Kossino
(bei Moskau) see Trudy Limnologicheskoi
Stantsii v Kosine

Arbeiten der Biologischen Sungari-Station see
Trudy Sungariiskoi Rechnoi Biologicheskoi

Arbeiten der Biologischen Wolga-Station
(Raboty Volzhskoi Biologicheskoi Stantsii)
1900: Russia. 1-10

Arbeiten der Hydrophysiologischen Station
des Instituts fiir Experimentelle Biologie see
Trudy Zvenigorodskoi Gidrofiziologicheskoi
Stantsii Instituta Eksperimental'noi Biologii
ginz'a Moskva

Arbeiten der Limnologischen Station zu Kos-
sino der Hydrometeorologischen Adminis-
tration der USSR see Trudy Limnologiches-
koi Stantsii v Kosine

SERIAL PUBLICATIONS, A1ARINE BIOLOGICAL LABORATORY

Arbeiten des Ichthyologischen Laboratoriums
der Kaspi-Wolgaschen Fischerei-Verwal-
tung in Astrachan see Report (s) of the
(Astrakhan) Scientific Station of Fisheries
of Volga and Caspian Sea

Arbeiten des Instituts fur Experimentelle
Morphogenese, Moskau see Trudy Nauchno-
Issledovatel'skogo Instituta Eksperimen-
tal'nogo Morfogeneza: Moskovskogo Go-
sudarstvennogo Universiteta
*Arbeiten des Naturforscher-Vereins zu Riga
1847: n.f. 2-22

Arbeiten des Ungarischen Biologischen For-
schungsinstitutes see Magyar Biologiai
Kutatointezet Munkai

Arbeiten des Zoologischen Forschungsinsti-
tutes see Trudy Nauchno-Issledovatel'skogo
Instituta Zoologii

Arbeitsphysiologie ; Zeitschrif t fur die Physio-
logic des Menschen bei Arbeit und Sport
1928: 1 +

Arbok; Norske Videnskaps-Akademi i Oslo
1925: 1925 +

(Aus dem) Archiv der Deutschen Seewarte
und des Marineobservatorium (1-57, no. 3
as Aus dem Archiv der Deutschen Seewarte)
1878: 1 +

Archiv der Julius Klaus-Stiftung fur Verer-
bungsforschung, Sozialanthropologie und
Rassenhygiene 1925: Zurich. 1+ Ergan-
zungsband 7

Archiv fur Pharmazie und Berichte der Deut-
schen Pharmazeutischen Gesellschaft 1822:
Archiv 271+ Berichte 43 +

Archiv des Vereins der Freunde der Naturge-
schichte in Mecklenburg (78+ also as n.s.
1+) 1847: 75; N.F. 1-13

Archiv for Mathematik og Naturvidenskab

1876: Christiania. 4-8, no. 2; 11, nos. 1-2
*Archiv fur Anatomie, Physiologic und Wissen-

schaftliche Medicin 1834: 43, Heft 1-2
*Archiv fur Anatomie und Physiologic ; Anato-
mische Abt. ; Archiv fiir Anatomie (1877-
1913 as Archiv fiir Anatomie und Entwick-
lungsgeschichte) 1877: 1-43
*Archiv fiir Anatomie und Physiologic; Ana-
tomische Abt.; Supplement 1877: 1889-90;
1895; 1897; 1902; 1905-07; 1909; 1912-13;
1915

*Archiv fiir Anatomie und Physiologic ; Physio-
logische Abt.; Archiv fiir Physiologic (con-
tains Verhandlungen der Physiologischen
Gesellschaft zu Berlin 1-31) 1877: 1-43

*Archiv fiir Anatomie und Physiologic ; Physio-
logische Abt.; Supplement 1877: 1879-80;
1882-87; 1889-90; 1892-93; 1899-1908;
1910; 1912

Archiv fur Biontologie; Gesellschaft Natur-

forschender Freunde zu Berlin (suspended
1909-12, 1921) 1906: 1-4

Archiv fiir die Gesamte Physiologie see: Pflii-
gers Archiv fiir die Gesamte Physiologie der
Menschen und der Tiere

Archiv fiir die Naturkunde Estlands (Eesti
Loodusteaduse Arhiiv) (1854-1905 as Archiv
fiir die Naturkunde Liv- Ehst- und Kur-
lands; 1920-23 as Archiv fiir die Naturkunde
des Ostbaltikums) (suspended 1906-19)
1854: Naturforscher-Gesellschaft, Universi-
tat Tartu (Dorpat). Erste Serie. Geologica,
Chemica et Physica (1-9 pt. 5 as Minera-
logische Wissenschaften, nebst Chemie,
Physik, etc.) 1 + ; Zweite Serie. Biologische
Naturkunde 1 +

*Archiv fiir die Physiologie 1795: Halle. 1-12

Archiv fiir Entwicklungsmechanik der Or-
ganismus see Wilhelm Roux' Archiv fiir
Entwicklungsmechanik der Organismen

Archiv fiir Experimentelle Pathologic und
Pharmakologie see Naunyn-Schmiedebergs
Archiv fiir Experimentelle Pathologie und
Pharmakologie

Archiv fiir Experimentelle Zellforschung, be-
sonders Gewebeziichtung (Explantation)
1925: 1 +

Archiv fiir Hydrobiologie ; Organ der Inter-
nationalen Vereinigung fiir Theoretische
und Angewandte Limnologie (1-11 as
Archiv fur Hydrobiologie und Plankton-
kunde) 1906: 1 + ; Supplement 1 + ; Literary
Supplement 1-3

Archiv fiir Hygiene 1883: 97-100

Archiv fiir Mikrobiologie ; Zeitschrift fiir die
Erforschung der Pflanzlichen Mikroorganis-
men 1930: 1 +

*Archiv fiir Mikroskopische Anatomie (98-104
as Archiv fiir Mikroskopische Anatomie und
Entwicklungsmechanik) 1865: 1-104; Sup-
plement to vol. 5

*Archiv fiir Naturgeschichte 1835: 1-77
*Archivfiir Naturgeschichte Abt. A. 1912: 78-92

Archiv fiir Naturgeschichte; Zeitschrift fiir
Wissenschaftliche Zoologie Abt. B, Zeit-
schrift fiir Systematische Zoologie (78, 1912
to, not including, n.f. 1, 1932 as Archiv fiir
Naturgeschichte Abt. B) 1912: 78 +

Archiv fiir Pathologische Anatomie und
Physiologie see Virchow's Archiv fiir Patho-
logische Anatomie und Physiologie und fiir
Klinische Medizin

Archiv fiir Physiologie see Archiv fiir Anatomie

und Physiologie; Physiologische Abtheilung

*Archiv fiir Physiologische und Pathologische

Chemie und Mikroskopie in ihrer Anwen-

dung auf die Praktische Medizin 1844: 1-4

Archiv fiir Protistenkunde 1902: 1 +

Archiv fiir Rassen- und Gesellschaftsbiologie ;

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Einschliesslich Rassen- und Gesellschafts-
hygiene 1904: 1 +

Archiv fur Schiffs- und Tropenhygiene ; Patho-
logie und Therapie Exotischer Krankheiten
see Deutsche Tropenmedizinische Zeitschrift

*Archiv fur Vergleichende Ophthalmologie

1910: 1-4, Heft 3

Archiv fiir Wissenschaftliche Botanik see
Planta; Archiv fiir Wissenschaftliche Bo-
tanik

*Archiv fiir Zellforschung 1908: 1-17

* Archiv Mecklenburgischer Naturforscher 1923 :

Verein der Freunde der Naturgeschichte in

Mecklenburg 1923: 1, Heft 2
Archives d'Anatomie, d'Histologie et d'Em-

bryologie 1922: 1 +

Archives d'Anatomie Microscopique 1897: 1 +
Archives d'Hydrobiologie et d'Ichthyologie see

Archiwum Hydrobiologji i Rybactwa
Archives de Biologie 1880: Paris and Liege.

1 +
Archives de Biologie de la Societe des Sciences

et des Lettres de Varsovie see Archiwum

Nauk Biologicznych
*Archives de 1'Electricite, supplement a la Bib-

liotheque Universelle de Geneve 1841: 1-5
Archives de 1'Institut de Botanique de 1'Uni-

versite de Liege 1897: 1 +
Archives de 1'Institut Pasteur d'Algerie 1923:

1 +
Archives de 1'Institut Pasteur de Tunis 1906:

15, no. 3; 22, nos. 3-4
Archives de la Societe des Sciences Medicales

et Biologiques de Montpellier et du Langue-

doc Mediterranean (1-8 as Bulletin) 1919:

9+
*Archives de Morphologie Generale et Experi-

mentale 1921: 1-29

* Archives de Parasitologie 1898: 1-16
Archives de Pharmacodynamie see Archives

Internationales de Pharmacodynamie et de
Therapie

*Archives de Physiologic Normale et Patho-
logique 1868: 1— ser. 5, vol. 10

Archives de Physique Biologique et de Chimie-
Physique des Corps Organises (1-7 as
Archives de Physique Biologique) 1921: 1 +

Archives de Zoologie Experimentale et Gene-
rale; histoire naturelle, morphologic, histo-
logie, evolution des animaux (1907+ in-
cludes Biospeologica) 1872: 1 +

Archives des Sciences Biologiques see Arkhiv
Biologicheskikh Nauk

Archives des Sciences Physiques et Naturelles
(Bibliotheque Universelle, 1848-1918) 1846:
1+

Archives du Musee Zoologique de 1'Universite
de Moscou see Sbornik Trudov Gosudarst-
vennogo Zoologicheskogo Muzeya

Archives du Museum d'Histoire Naturelle de
Lyon 1872: 1-13

Archives Internationales de Medecine Experi-
mentale 1924: 1 +

Archives Internationales de Pharmacodyna-
mie et de Therapie (1-3 as Archives de
Pharmacodynamie) 1894: 1 +

Archives Internationales de Physiologic 1904:

*Archives Italiennes de Biologie; Revues, Re-
sumes, Reproductions des Travaux Scien-
tifiques Italiens 1882: 1-6; 10-11; 13-94

Archives Neerlandaises de Phonetique Ex-
perimentale 1926: Societe Hollandaise des
Sciences a Harlem. 1 +

Archives Neerlandaises de Physiologie de
1'Homme et des Animaux (forms series 3C of
Archives Neerlandaises des Sciences Exactes
et Naturelles) 1916: Societe Hollandaise des
Sciences a Harlem. 1 +

Archives Neerlandaises de Zoologie 1934:

Societe Neerlandaise de Zoologie. 1 +
*Archives Neerlandaises des Sciences Exactes
et Naturelles 1866: Societe Hollandaise des
Sciences a Harlem. 1— ser. 2, 15
*Archives Neerlandaises des Sciences Exactes
et Naturelles 1911: ser. 3A, 1-14; ser. 3B,
1-5; ser. 3C, see Archives Neerlandaises de
Physiologie de l'Homme et des Animaux

Archives Neerlandaises des Sciences Exactes
et Naturelles, 4A, see Physica

Archives of Biochemistry 1942: 1 +

Archives of Internal Medicine 1908: American
Medical Association. 1 +

Archives of Neurology and Psychiatry 1919:
ibid. 1 +

Archives of Pathology (1-5, no. 2 as Archives
of Pathology and Laboratory Medicine)
1926: ibid. 1 +

Archives Portugaises des Sciences Biologiques
1921: Sociedade Portuguesa de Sciencias
Naturais. 1-2

Archives Roumaines de Pathologic Experi-
mentale et de Microbiologie 1928: Paris. 1 +

Archives Russes d'Anatomie, d'Histologie et
d'Embryologie see Arkhiv Anatomii, Gisto-
logii i Embriologii

Archives Russes de Protistologie ; publiees
sous la direction de G. Epstein et L.
Kourssanow see Russkii Arkhiv Protistologii

Archivio di Farmacologia Sperimentale e
Scienze Affini 1902: 1 +

Archivio di Fisiologia 1904: 1 +

Archivio di Scienze Biologiche; Fisiologia,
Farmacologia, Patologia Sperimentale 1919:
Societa Italiana di Biologia Sperimentale.
1 +

Archivio Italiano di Anatomia e di Embriologia
1902: 1-9; [10-12]; 13; [14]; 15 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Archivio Italiano di Medicina Sperimentale

see Medicina Sperimentale Archivio Italiano
Archivio Triennale del Laboratorio di Botanica

Crittogamica see Atti dell'Istituto Botanico

"Giovanni Briosi"
Archivio Zoologico Italiano (1-4 as Archivio

Zoologico) 1902: 1 + ; Supplemento; Attua-

lita Zoologiche 1933: 1 +
Archives da Escola Superior de Agricultura e

Medicina Veterinaria 1917: Rio de Janeiro.

1-4, no. 1 ; 6-8
Archives de Neurobiologia (1-8 as Archives de

Neurobiologia, Psicologia, Fisiologia, His-

tologia, Neurologia y Psiquiatria) 1920:

Madrid. 1-8; [9-15]

*Archivos do Institute Vital Brazil 1923: 1-4
Archives do Museo Nacional (do Rio de

Janeiro) (9 as Revista) 1876: 1 +
Archivum Balatonicum see Magyar Biologiai

Kutatointezet Munkai
Archiwum Hydrobiologji i Rybactwa (Archives

d'Hydrobiologie et d'Ichthyologie) 1926: .

Stacja Hydrobiologiczna na Wigrach Su-

walki. 1-11, no. 2
Archiwum Nauk Biologicznych Towarzystwa

Naukowego Warszawskiego (Archives de

Biologic de la Societe des Sciences et des

Lettres de Varsovie) 1921: 1-8, no. 2
Arkhiv Anatomii, Gistologii i Embriologii

(Archives Russes d'Anatomie, d'Histologie

et d'Embryologie) 1916: 1 +
Arkhiv Biologicheskikh Nauk; Institutom Eks-

perimental'noi Meditsin' i (Archives des

Sciences Biologiques) 1892: 1 +
Arkiv for Botanik 1903: K. Svenska Vetens-

kapsakademie. 1 +
Arkiv for Matematik, Astronomi och Fysik

1903: ibid. 1 +

Arkiv for Zoologi 1903: ibid. 1 +
Arquivo de Anatomia e Antropologia 1912:

Lisbon. 1 +
Arquivos da Escola Medico-Cirurgica de Nova

Goa 1927: ser. A, 1-12; ser. B, 1-7
Arquivos de Zoologia do Estado de Sao Paulo

1940: 1 +

Arquivos do Institute Biologico (1-4 as Archi-
ves do Institute Biologico de Defesa Agri-
cola e Animal; 5-8 as Archives do Institute

Biologico) 1928: Brazil. 1 +
Arquivos do Institute de Biologia Vegetal

1934: Ministerio da Agricultura, Brazil. 1,

nos. 1-2
*Arquivos Indo-Portugueses de Medicina e

Historia Natural; Orgao do Institute Bac-

teriologico de Nova Goa 1921: 1-4
Arsbok Vuosikirja 1922: Societas Scientiarum

Fennica. 1 +
Asher-Spiro; Ergebnisse der Physiologic see

Ergebnisse der Physiologie, Biologischen
Chemie und Experimentellen Pharmakologie

Astrophysical Journal; an international review
of spectroscopy and astronomical physics
1895: 1 +

Atti del Congresso Nazionale di Botanica
Crittogamica in Parma 1887: Societa Critto-
gamalogica Italiana Varese. 1-2

Atti del Congresso Nazionale di Microbiologia
1930: Societa Internazionale di Microbio-
logia, Sezione Italiana. 3-4

Atti dell'Istituto Botanico "Giovanni Briosi" e
Laboratorio Crittogamico Italiano della R.
Universita di Pavia (1-5 as Archivio Trien-
nale del Laboratorio di Botanica Crittoga-
mica; ser. 2-3 as Atti dell'Istituto Botanico
dell' Universita di Pavia) 1874: 1 +

Atti della Accademia Gioenia di Scienze Natu-
rali in Catania 1825: 1 +

Atti della Reale Accademia d'ltalia; Rendi-
conti della Classe di Scienze Fisiche, Mate-
matiche e Naturali (ser. 1 — ser. 2, vol. 3,
pt. 1 as Atti della Reale Accademia dei
Lincei; ser. 2, vol. 3, pt. 2, 1876 — ser. 6,
1939 as Atti della Reale Accademia Nazio-
nale di Lincei; Memorie della Classe di
Scienze Fisiche, Matematiche e Naturali; to
1920 as Atti della Reale Accademia dei
Lincei) 1847: 24-26; ser. 2-f

Atti della Reale Accademia delle Scienze di
Torino; Classe di Scienze Fisiche, Mate-
matiche e Naturali 1865: [27-51]

Atti della Reale Accademia Nazionale dei
Lincei; Memorie; Classe di Scienze Fisiche,
Matematiche e Naturali (to 1920 as Atti
della Reale Accademia dei Lincei) see Atti
della Reale Accademia d'ltalia; Rendiconti
della Classe di Scienze Fisiche, Matematiche
e Naturali

*Atti della Reale Accademia Nazionale dei
Lincei; Rendiconti 1884: ser. 4, 1-7

Atti della Reale Accademia Nazionale dei
Lincei (to 1920 as Atti della Reale Acca-
demia dei Lincei); Rendiconti; Classe di
Scienze Fisiche, Matematiche e Naturali
1895: ser. 5 +

Atti della Reale Accademia Nazionale dei
Lincei; Rendiconto dell'adunanze solenne
(1 as Rendiconti delle sedute solenni) 1892:

1 +
*Atti della Reale Accademia Nazionale dei

Lincei; Transunti 1876: 1-8
Atti della Societa Italiana di Genetica ed

Eugenica 1920: 1
Atti della Societa Italiana di Scienze Naturali

e del Museo Civico di Storia Naturale in

Milano 1855: [18]; [41-42]; 63 +
Atti della Societa Toscana di Scienze Naturali

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

residente in Pisa; Process! Verbali 1878:
[14]; 15-26; [27-29]; Memorie 1875: 15 +

Atti e Memorie dell'Accademia d'Agricoltura
Scienze Lettere Arti e Commercio di Verona
(1-75 as Memorie della Accademia di Ve-
rona; Agricoltura, Scienze, Lettere, Arti e
Commercio) 1807: 1; 17; ser. 3. [64-75]; ser.
4. [1-6]; 10-11

Attualita. Medica see Annali d'Igiene

Attualita Zoologiche see Archivio Zoologico
Italiano Supplement

Auk; a quarterly journal of ornithology 1876:
ser. 2 (1884), 1 +

Australian Journal of Experimental Biology
and Medical Science 1924: 14-

Australian Journal of Science (includes Aus-
tralian Science Abstracts 174") 1938: Aus-
tralian National Research Council. 14-

Australian Science Abstracts 1922: 1-16

Australian Zoologist 1914: Royal Zoological
Society of New South Wales. 1 4-

Avhandlinger utgitt av det Norske Videnskaps-
Akademi i Oslo. I. Matematisknaturvi-
denskapelig Klasse 1925: 19254-

Avicultural Magazine; journal of the Avicul-
tural Society 1894: n.s. [4-7]

Bacteriological Reviews 1937: 1 +

(Der) Balneologe; Zeitschrift fur die Gesamte
Physikalische und Diatetische Therapie;
Organ der Deutschen Gesellschaft fur
Bader- und Klimaheilkunde 1934: 1-6

"Baumgarten's Jahresbericht" see Jahresbe-
richt iiber die Fortschritte in der Lehre von
den Pathogenen Mikroorganismen, um-
fassend Bacterien, Pilze und Protozoen
*Behavior Monographs 1911: 1-4, no. 5
*Beiblatter zu den Annalen der Physik (1-23 as
Beiblatter zu den Annalen der Physik und
Chemie) 1877: 1-43

Beihefte zum Archiv fiir Schiffs- und Tropen-
hygiene; Pathologic und Therapie Exoti-
scher Krankheiten see Deutsche Tropen-
medizinische Zeitschrift

Beihefte zum Botanischen Centralblatt (18,
19054- as Abt. A or 1 and B or 2) 1891:
Abt. A, 1 + ; Abt. B, 274-

Beitrage zur Anatomic Funktioneller Systeme
1930: 1, no. 1

Beitrage zur Angewandten Geophysik (1-3,
no. 3 as Gerlands Beitrage zur Geophysik;
Erganzungshefte fiir Angewandte Geo-
physik) 1931: 1 +

Beitrage zur Biologic der Pflanzen 1875: 14-
*Beitrage zur Chemischen Physiologic und
Pathologic; Zeitschrift fiir die Gesamte Bio-
chemie (Franz Hofmeister) 1901: 1-11

Beitrage zur Geophysik see Gerlands Beitrage
zur Geophysik

*Beitrage zur Lehre von den Geschlechts-
Unterschieden, von Dr. P. J. Mb'bius in
Leipzig 1903: 1-8

*Beitrage zur Morphologic und Morphogenie;
Untersuchungen aus dem Anatomischen
Institut zu Erlangen 1883: 1
*Beitrage zur Morphologic und Physiologic der
Pflanzenzelle 1890: Tubingen. 2, heft 1

Beitrage zur Pathologischen Anatomic und zur
Allgemeinen Pathologic (Ziegler's) (1-2 as
Beitrage zur Pathologischen Anatomie und
Physiologie) 1886: 1 +

Beitrage zur Physik der Freien Atmosphare;
Zeitschrift fiir die Erforschung der Hoheren
Luftschichten und der Stromungserschei-
nungen in der Atmosphare (1-12 subtitle as
Zeitschrift fiir die Erforschung der Hoheren
Luftschichten) 1904: 1 +
*Beitrage zur Physiologie; herausgegeben von
Max Cremer 1920: 1-4

Bell System Technical Journal; a journal de-
voted to the scientific and engineering as-
pects of electrical communication 1922:
American Telephone and Telegraph Com-
pany. 1 4-

Beretninger fra Chr. Michelsens Institutt for
Videnskap og Andsfrihet 1931: Bergen. 1 +

Bergens Museum, Arsberetning 1885: 18854-
*Bergens Museum, Meeresfauna von Bergen
1901: 1-3

Bergens Museums Arbok (1885-91 as Arsbe-
retning) 1883: 18854-

Bergens Museums Skrifter 1878: 14-

Bericht; Zoologisches Staatsinstitut und Zoolo-
gisches Museum in Hamburg (1913-15 see in
Mitteilungen aus dem Hamburgischen
Zoologischen Museum und Institut) 1882:
1916-29

*Bericht(e) aus der Kgl. Bayerischen Biologi-
schen Versuchsstation in Miinchen ; heraus-
gegeben von Prof. Dr. Bruno Hofer 1908:
1-2

Bericht(e) der Akademischen Biologischen
Siisswasser-Station Borodin see Trudy Bo-
rodinskoi Biologicheskoi Stantsii v Karelii

Bericht(e) der Biologischen Borodin Station
see ibid

Bericht(e) der Biologischen Siisswassersta-
tion der Kaiserlichen Naturforscher-Gesell-
schaft zu St. Petersburg see ibid

Bericht(e) der Deutschen Botanischen Gesell-
schaft 1883: 1 +

Bericht(e) der Deutschen Chemischen Gesell-
schaft 1868: 14-

Bericht(e) der Deutschen Pharmazeutischen
Gesellschaft see Archiv der Pharmazie und
Berichte der Deutschen Pharmazeutischen
Gesellschaft

Bericht(e) der Deutschen Wissenschaftlichen

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Kommission fur Meeresforschung N.F.
(1925) (1-5 as Beteiligung Deutschlands an
der Internationalen Meeresforschung) 1902:
N.F. 1 +

Bericht(e) der Gesellschaft Russischer Physio-
logen (Trudy O-va Rossiiskikh Fiziologov)
1928: 1-3

*Bericht der Kommission zur Wissenschaft-
lichen Untersuchung der Deutschen Meere
in Kiel (1-3 as Jahresbericht der Kommis-
sion zur Wissenschaftlichen Untersuchung
der Deutschen Meere in Kiel) 1871: 1-6
(Jahrgang 1-21, 1871-91; Neue Folge see
Wissenschaftliche Meeresuntersuchungen
*Bericht der Meteorologischen Commission des
Naturforschenden Vereines in Briinn; Er-
gebnisse der Meteorologischen Beobach-
tungen 1882: 17-27

Bericht der Naturforschenden Gesellschaft in
Bamberg 1852: 6-7; 9-14; 21

Bericht (e) der Naturforschenden Gesellschaft
zu Freiburg i. Br. 1886: 1 +

Bericht der Notgemeinschaft der Deutschen

Wissenschaft 1921: Berlin. 1-9
*Bericht der Oberhessischen Gesellschaft fur
Natur- und Heilkunde; Giessen 1847: 7; 9;
14-34

Bericht der Oberhessischen Gesellschaft fur
Natur- und Heilkunde zu Giessen; N.F.
Medizinische Abt. 1906: 1-14; N.F. Natur-
wissenschaftliche Abt. 1904: 1 +

Bericht(e) der Physikalisch-Medizinischen
Gesellschaft zu Wiirzburg (1868-1935 as
Verhandlungen der Physikalisch-Medizini-
schen Gesellschaft zu Wiirzburg) (after 1923
includes Sitzungsberichte) 1850: n.s. 16; 21-
22; 29 +

Bericht der Senckenbergischen Naturfor-
schenden Gesellschaft in Frankfurt am Main
see Natur und Yolk

Bericht (e) der Tomsker Staats-Universitat see
Transactions of Tomsk State University

Bericht(e) des Naturwissenschaftlich-Medi-
zinischen Vereines in Innsbruck 1870: 1-10;
14-44

*Bericht des Naturwissenschaftlichen Vereins
fur Schwaben und Neuburg (E. V.) (friiher
Naturhistorischen Vereins in Augsburg)
1848: 30-32; 34-39; 41; 43-50

Bericht (e) des Naturwissenschaftlichen (frii-
her Zoologisch-Mineralogischen) Vereines
zu Regensburg 1886: 7-9

Bericht (e) des Ohara Instituts fur Landwirt-
schaftliche Forschungen in Kurashiki Pro-
vinz Okayama, Japan 1916: 3 +

Bericht des Westpreussischen Botanisch-
Zoologischen Vereins (1-25 as Bericht iiber
die Versammlungen) 1878: 1-2; 4-6; 8-10;
15-16; 19; 22; 29 +

*Bericht(e) des Wissenschaftlichen Meeresin-
stituts (Trudy, Plovuchego, Morskogo
Nauchnogo Instituta) (1, lief. 2-5 as Rus-
sische Hydrobiologische Zeitschrift, by A.
L. Behning) 1923: 1-4

Bericht iiber das Zoologische Museum (der
Universitat) in Berlin 1896: 1897-1909;
1911-26

*Bericht iiber die Fortschritte der Anatomic
und Physiologic; als Besondere Abtheilung
der Zeitschrift fur Rationelle Medicin 1856:
1-16

Bericht (e) iiber die Gesamte Biologic, Ab-
teilung A see Berichte iiber die Wissen-
schaftliche Biologie

Bericht(e) iiber die Gesamte Biologie, Ab-
teilung B see Berichte iiber die Gesamte
Physiologic und Experimentelle Pharmako-
logie

Bericht (e) iiber die Gesamte Physiologic und
Experimentelle Pharmakologie ; (since vol.
35, 1926) Berichte iiber die Gesamte Biologie
Abt. B (1-2 as Berichte iiber die Gesamte
Physiologic, Neue Folge des Zentralblattes
fur Biochemie und Biophysik; until vol. 35,
1926) 1920: Deutsche Physiologische Gesell-
schaft und Deutsche Pharmakologische
Gesellschaft. 1 +

Bericht iiber die Tagung der Deutschen Ver-
einigung fur Mikrobiologie 1906+ see in
Zentralblatt fiir Bakteriologie, Parasiten-
kunde und Infektionskrankheiten
*Bericht(e) iiber die Verhandlungen der Natur-
forschenden Gesellschaft zu Freiburg i. B.
1858: 4-5

Bericht u'ber die Versammlung des West-
preussischen Botanisch-Zoologischen Ver-
eins zu Danzig see Bericht des Westpreus-
sichen Botanisch-Zoologischen Vereins

Bericht(e) iiber die Wissenschaftliche Biologie
(Berichte iiber die Gesamte Biologie, Abt.
A) 1926: 1 +

*Berliner Entomologische Zeitschrift (19-24 as
Deutsche Entomologische Zeitschrift) 1857:
Entomologischen Vereine in Berlin. 11-24;
26-27, Heft 1; 28; 29, Heft 1; 30-33; 35-40;
46, Heft 2; 49-58

Bermuda Biological Station for Research ; Re-
ports of Officers 1926: 1 +

Bermuda Meteorological Office; Summary of

Observations: 1936

*Beteiligung Deutschlands an der Interna-
tionalen Meeresforschung; (Jahres)Bericht
1902: 1-5

*Bibliographia Chimica; International Litera-
tur-Anzeiger fiir Chemie, Chemische Tech-
nologie und alle Grenzgebiete 1922: 1-5

Bibliographia Eugenica see Eugenical News;
Supplement

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Bibliographia Genetica 1925: 1 +

Bibliographia Oceanographica (also for the year
1928, Essai d'une Bibliographic Generate
des Sciences de la Mer) 1929: 1 +
*Bibliographia Physiologica, adhuc diario "Zen-
tralblatt fiir Physiologie" adnexa, edidit
Concilium Bibliographicum (ser. 3) Turici
constitutum ab H. H. Field (ser. 1 as Bi-
bliographia Physiologica; ser. 2 as Biblio-
graphia Universalis quae auspiciis Instituti
Bibliographici Internationalis Bruxellensis)
1897: ser. 1-4

*Bibliographia Zoologica, antea diario "Zoolo-
gischer Anzeiger" adnexa, condita ab J.
Victor Carus; edidit Concilium Bibliogra-
phicum Turici constitutum ab H. H. Field
1896: 1-43

Bibliographic Service see Wistar Institute

Bibliographic Service

*Bibliographical Contributions from the Lloyd
Library (nos. 1-13 also as vol. 1; 14-25 as
vol. 2; 26-32 as vol. 3) 1911: 1-32
*Bibliographie Anatomique; Revue des Tra-
vaux en Langue Francaise; Anatomic, His-
tologie, Embryologie, Anthropologie 1893:
1-25

Bibliography and Index of Geology Exclusive
of North America; Geological Society of
America 1933: 1 +

Bibliography of Agriculture; Section D.
Plant Science 1942: U. S. Department of
Agriculture Library. 1 +

Bibliography of Helminthology see Helmin-
thological Abstracts

Bibliography of Meteorological Literature see
Quarterly Journal of the Royal Meteoro-
logical Society; Supplement

Biblioteka Centralnog Higijenskog Zavoda:
Belgrade. 2 +

Bibliotheca Biotheoretica; Series D see Acta
Biotheoretica which forms A of this Series

Bibliotheca Genetica herausgegeben von Pro-
fessor Dr. E. Baur 1917: Leipzig. 1 +

Bibliotheca Universitatis Liberae Poloniae
1922: A, 1-23; B, 4

Bibliotheca Zoologica see Zoologica; Originate
Abhandlungen aus dem Gesamtgebiete der
Zoologie

Bibliotheque Universelle de Geneve; Supple-
ments see Archives de 1'Electricite; and
Archives des Sciences Physiques et Natu-
relles

Bidrag till Kannedom af Finlands Natur och
Folk 1858: Finska Vetenskaps-Societetet.
[63-831

*Bihang till Kongl. Svenska Vetenskapsakade-
miens Handlingar 1872: [6]; [8]; [10]; Afd.
1. Mathematik, Astronomi, Mekanik, Fysik,
Meteorologi och Beslagtade Amnen 1886:

[12-14]; Afd. 2. Kemi, Mineralogi, Geog-
nosi, Fysisk geografi och Beslagtade Amnen
1886: 14; Afd. 3. Botanik 1886: 14; 19-28;
Afd. 4. Zoologi 1886: [13-14]; 21-28

Bijdragen tot de Dierkunde (K. Zoologische
Genootschap "Natura artis magistra")
1848: Amsterdam. 1 +

*Biochemical Bulletin 1911: Columbia Uni-
versity Biochemical Association. 1-5, no. 21

Biochemical Journal 1906: Biochemical So-
ciety, England. 1 +

Biochemische Zeitschrift; unter Mitwirkung
Zahlreicher Fachgenossen (12-134 subtitle
as Beitrage zur Chemischen Physiologie und
Pathologie) 1906: 1 +

Biochemisches Centralblatt see Zentralblatt
fiir Biochemie und Biophysik

Biodynamica; a (scientific) journal for the
(elaboration and the experimental) study of
(working hypotheses on) the nature of life;
structure and dynamics of living matter
1934: Normandy, Mo. 1 +

Biographical Memoirs; National Academy of
Sciences 1877: 1 +

Biokhimiia (Biochimia) 1936: Izdatel'stvo
Akademii Nauk SSSR. 1; 2, nos. 2-6; 3 +

(Der) Biologe; Monatsschrift des Reichs-
bundes fiir Biologie und der Unterabteilung
Lebens- und Rassenkunde des N.S.L.B.
1931: [1-2]; 3-7; 8, nos. 1-11; 9

Biologia Generalis ; Archiv fiir die allgemeinen
Fragen der Lebensforschung; begriindet
von L. Lohner, R. Pearl, V. Riizicka (1-13,
1937 has subtitle "Internationales Archiv")
1925: 1 +

Biological Abstracts 1926: a comprehensive
abstracting and indexing journal of the
world's literature in theoretical and applied
biology, exclusive of clinical medicine. 1 +

Biological Bulletin 1899: Marine Biological

Laboratory. 1-f-

*Biological Laboratory 1929: Long Island Bio-
logical Association. 1-5, no. 1
*Biological Lectures delivered at the Marine
Biological Laboratory, Woods Hole, Mass.
1890: 1-7

Biological Reviews of the Cambridge Philo-
sophical Society (1 as Proceedings of the
Cambridge Philosophical Society, Biologi-
cal Sciences; 2-10 as Biological Reviews and
Biological Proceedings of the Cambridge
Philosophical Society) 1923: 1 +

Biological Symposia; a series of volumes de-
voted to current symposia in the field of
biology; edited by Jaques Cattell 1940: 1 +

Biologicheskii Zhurnal ; Otvetstvennyi Redak-
tor, N. K. Kol'tsov (1-4 also as Journal de
Biologie, or Zeitschrift fiir Biologie) 1932:
Moscow. 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Biologicke Spisy Vysoke Skoly Zverolekarske ;
Brno, Ceskoslovenska Republika (Publica-
tions Biologiques de 1'Ecole des Hautes
Etudes Vete"rinaires) 1922: 1-15

Biologisches Zentralblatt 1881: 1 +

Biologiske Meddelelser; Det Kgl. Danske
Videnskabernes Selskab 1917: 1 +

Biologiske Skrifter; Det Kgl. Danske Viden-
skabernes Selskab 1939: 1 +

Biologist; official publication of the Phi Sigma
Society 1916: 1-20, no. 1

Biometrika; a journal for the statistical study
of biological problems 1901: Cambridge,
England. 1 +

*Biophysikalisches Centralblatt ; Centralblatt
fur die Gesamte Biologic, Abt. 2; Vollstan-
diges Sammelorgan fiir Biologic, Physio-
logic und Pathologic mit Ausschluss der
Biochemie 1905: 1-4 (1910)

Bios 1930: Beta Beta Beta Biological Frater-
nity at Mount Vernon, Iowa. 1 +
*"Bios"; Rivista di Biologia Sperimentale e
Gen°rale; Direttore, Paola Enriques, Isti-
tuto Zoologico (Bologna) 1913: 1-2

Biospeologica see Archives de Zoologie ExpeYi-
mentale et Ge'ne'rale

Bird-Banding; a journal of ornithological in-
vestigation 1930: 8+

Bird Lore; an illustrated bi-monthly magazine
devoted to the study and protection of birds
1899: 1-14; [15]; 16; [17]; 18-20, no. 1

Biuletyn Stacji Morskiej w Helu (Bulletin de
la Station Maritime de Hel): Warsaw. 2,
no. 3 (1938)

Biuletyn Towarzystwa Geofizykow w Warsza-
wie (Bulletin de la Societe Geophysique de
Varsovie) 1931: 1 +

Boilleten Arkticheskogo Instituta (Bulletin of
the Arctic Institute of the USSR) 1931 : Chief
Administration of Northern Sea Route,
Leningrad. 1931-36, no. 7

B/Clleten Gosudarstvennogo Okeanografiches-
kogo Instituta (Bulletin of the State Oceano-
graphical Institute) 1931: Gidrometeoro-
logicheskii Komitet SSSR. 1-18

Bailleten Moskovskogo Obshchestva Ispyta-
telei Prirody see Bulletin de la Societ£ des
Naturalistes de Moscou

Bmlleten Nauchno-Issledovatel'skogo Insti-
tuta Zoologii see Sbornik Nauchno-Issledo-
vatel'skogo Instituta Zoologii

Bfulleten Obshchestva Estestvoispytatelei pri
Voronezhskom Gosudarstvennom Universi-
tete see Bulletin de la Socie'te' des Naturalis-
tes de Voroneje

Biulleten Sredne-Aziatskogo Gosudarstven-
nogo Universiteta (Bulletin de 1'Universite
de 1'Asie Centrale; Tachkent) 1923: 11-13;
15-16

B'ack Rock Forest Bulletin 1930: Cornwall-
on-the-Hudson. 1-6

Black Rock Forest Papers 1935: Cornwall-
on-the-Hudson. 1, nos. 1-11

Blue Hill Meteorological Observatory of Har-
vard University see Harvard Meteorological
Studies

Blumea; Gijdschrift voor de Systematiek en
de Geografie der Planten (a journal of plant
taxonomy and plant geography) 1934:
Rijksherbarium. 1 +

Boletim; Institute de Higiene de Sao Paulo
1919: 1-2; 4-6; 15; 23-27; 29-45; 47; 53

Boletim; Secretaria da Agricultura, Commer-
cio e Obras Publicas do Estado de Sao
Paulo; Servicio Meteorologico (1-21 as
Boletim da Commissao Geographica e Geo-
logica do Estado de S. Paulo) 1889: 4-21;
ser. 2, 6; 8-11; 25-28 (1916)

Boletim Biologico; Orgao do Clube Zoologico
do Brasil 1926: 1-21; n.s. 1-3, no. 5

Boletim da Commissao Geographica e Geolo-
gica do Estado de S. Paulo see Boletim;
Secretaria da Agricultura, Commercio e
Obras Publicas do Estado de Sao Paulo;
Servicio Meteorologico

Boletim da Inspetoria Federal de Obras Con-
tra as Secas 1934: Brazil. [1-9]; 10+

Boletim do Institute Brasileiro de Sciencias
1925: 1, no. 1; 2, no. 8

Boletim do Institute Vital Brazil 1927: 1-17;

19-23

*Boletim do Museu Goeldi (Museu Paraense)
de Historia Natural e Ethnographia 1894:
Brazil. [1-2]; 3-7

Boletim do Museu Nacional ; Universidade do
Brasil 1923: 1 +

Boletm; Departamento de Agricultura y Tra-
bajo see Bulletin; Agricultural Experiment
Station; Puerto Rico

Boletin(s) da Faculdade de Filosofia, Ciencias
e Letras; Universidade de Sao Paulo; Bio-
logia Geral 1937: 1 + ; Botanica 1937: 1 + ;
Fisica 1938: 1 + ; Zoologia 1937: 1 +

Boletin de la Compania Administradora del

Guano: Lima. 16, nos. 8-11; 17, nos. 9-12
*Boletin de la Direction de Estudios Biologicos
1915: Mexico. 1-3

Boletm de la Sociedad de Biologia de Concep-
tion (Chile) 1927: Universidad de Concep-
tion. 1 +

Boletin de la Sociedad Espanola de Biologia
1911: 1-7; [10-13]

Boletm de la (Real) Sociedad Espanola de
Historia Natural 1901: 1-37, no. 6

*Boletin de la Sociedad Geologica Mexicana
1904: 5-7

Boletin del Hospital Sanatorio "El Feral";

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Enfermedades Broncopulmonares y Tuber-
culosis 1941: Santiago, Chile. 1, no. 1

Boletin del Institute Geologicos de Mexico
1895: 17-32; 34; [35]; 36

Boletin del Ministerio de Agricultura de la
Nacion (32-35 as Boletin Mensual del
Ministerio de Agricultura de la Nacion)
1904: Argentina. 28-37, no. 4

Boletin del Museo Nacional de Chile 1908:
[1-2]; 3

Boletin del Servicio Oceanografico y de Pesca;
Ministerio de Defensa Nacional inspeccion
general de marina 1938: Montevideo. 1,
no. 1

Boletin Estadistica Agropecuaria (1-37 as
Boletin Mensual de Estadistica Agropecua-
ria) 1900: Direccion de Economia Rural y
Estadistica, Argentina. 35 +
*Boletin oficial de la Secretaria de Agricultura
y Fomento (1-2 as Boletin oficial de la
Secretaria de Fomento, Colonizacion e In-
dustria) 1916: [1-7]

*Bollettino Bimestrale; R. Comitato Talasso-
grafico Italiano (nos. 1-86 also as vols. 1-13)
1909: 1-23; 25-86

Bollettino dei Musei di Zoologia e di Anatomia
Comparata della R. Universita di Torino
1886: 1; [2]; 3-12; [13]; 14-15; [16]; 17 +

Bollettino dei Musei e Laboratorii di Zoologia
e Anatomia Comparata della R. Universita
di Geneva (2-13 as Bollettino dei Musei di
Zoologia, etc.) 1892: 2 +

Bollettino del Laboratorio di Zoologia Agraria
e Bachicoltura del R. Istituto Superiore
Agrario di Milano see Bollettino di Zoologia
e Bachicoltura

Bollettino del Laboratorio di Zoologia Gene-
rale e Agraria del R. Istituto Superiore
Agrario in Portici 1907: 1 +

Bollettino del R. Laboratorio di Entomologia
Agraria di Portici 1937: 1 +

Bollettino del R. Osservatorio Fitopatolo^ico
di Milano Sezione Entomologica see Bollet-
tino di Zoologia Agraria e Bachicoltura

Bollettino dell'Istituto di Entomologia della R.
Universita degli Studi di Bologna (1-7 as
Bollettino del Laboratorio di Entomologia
del R. Istituto Superiore Agrario di Bo-
logna) 1928: 1 +

Bollettino dell'Istituto Sieroterapico Milanese
(pubblicazione Italiana di Batteriologia ed
Immunologia) 1917: 8+

*Bollettino dell'Istituto Zoologico della R. Uni-
versita di Roma 1923: 1-8

Bollettino della Sezione Italiana; Societa In-
ternazionale di Microbiologia 1929: 1-7

Bollettino della Societa Adriatica di Scienze
Naturali in Trieste 1874: 1-5, no. 1; 8, no.
1; 9-15; 17-18; 20+

Bollettino della Societa dei Naturalisti in
Napoli (21+ also numbered ser. 2, vol. 1+)
1887: 1-7; 8, no. 1; 10-12; 14; 16+

Bollettino della Societa Italiana di Biologia
Sperimentale 1926: 1 +

Bollettino delle Sedute della Accademia
Gioenia di Scienze Naturali in Catania (1-31
as Bullettino Mensile) 1888: 26-28; 36^45;
47^9; 51-56; 59-71; 73-82; 84-87; ser. 2,
36; 53; 57-69; ser. 3, 1 +

Bollettino di Pesca, di Piscicoltura et di Idro-
biologia 1925: Ministero dell'Agricoltura e
delle Foreste; R. Laboratorio Centrale di
Idrobiologia applicata alia Pesca (1-5,
Ministero dell'Economia Nazionale; Dire-
zione generale dell'Industria e delle Miniere)

1 +

Bollettino di Pesca, di Piscicoltura et di Idro-
biologia, Supplemento see Memorie Scien-
tifiche; Memorie Storico-Giuridiche

Bollettino di Zoologia; pubblicato dall'Unione
Zoologica Italiana 1930: 1 +

Bollettino di Zoologia Agraria e Bachicoltura
della R. Universita di Milano (1-4, 6 as
Bollettino del Laboratorio di Zoologia Agra-
ria e Bachicoltura del R. Istituto Superiore
Agrario di Milano; 5 as Bollettino del R.
Osservatorio Fitopatologico di Milano Se-
zione Entomologica) 1928: 1 +
Bollettino Scientifico della Facolta di Chimica
Industriale. Bologna (1-4 as Giornale di
Biologia Applicata alia Industria Chimica,
5-7, Industriale, Agraria ed Alimentare, or-
gano ufficiale della Societa di Radiobiologia-
Centro di Bologna; 15-16, no. 5 merged with
Zymologica e Chimica dei Colloidi, using
volume numbering of Zymologica) 1931: 1 +

*Boston Journal of Natural History; contain-
ing papers and communications read to the
Boston Society of Natural History 1834: 1-7

*Botanical Abstracts; a monthly serial furnish-
ing abstracts and citations of publications
in the international field of botany in its
broadest sense 1918: 1-15
Botanical Gazette (1 as Botanical Bulletin)
1875: 1 +

*Botanical Gazette; a journal of the progress of
British botany and the contemporary litera-
ture of the science 1849: 1-3
Botanical Magazine 1887: Botanical Society of

Japan. [16-36]; 37 +

Botanical Review; Interpreting Botanical
Progress 1935: New York Botanical Garden.
1 +

*Botanicheskie Materialy Instituta Sporovykh
Rastenii Glavnogo Botanicheskogo Sada
(Notulae Systematicae ex Institute Crypto-
gamico Horti Botanici Petropolitani) 1922:
Leningrad. 1-4, no. 7

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Botanische Jahrbiicher fur Systematik, Pflan-
zengeschichte und Pflanzengeographie be-
griindet von A. Engler 1881: 1 +
*Botanische Zeitung 1843: 1-68

Botanisches Archiv; Zeitschrift fur die ge-
samte Botanik, begriindet ^von'Carl Mez
1922: 1 +

Botanisches Zentralblatt ; Referierendes Or-
gan fiir das Gesamtgebiet der Botanik im
Auftrage der Deutschen Botanischen Ge-
sellschaft (in two parts, Referate and Lite-
ratur) 1880: 1 +

Botanisk Tidsskrift udgivet afjDansk Botanisk
Forening 1866: 1+ « 41 •+ * '•-* ^ •***•!

Botaniska Notiser utgivna av Lunds Botaniska
Forening 1839: 1922+ **•«>!<<

(Le) Botaniste 1889: 1 +

*Botany; current literature; additions to the
botanical catalogue of the Bureau of Plant
Industry, U. S. Department of Agriculture:
[10]; 11; [12]; 13-16

Botany and Zoology, Theoretical and Applied

(text in Japanese): 3 +

*Bowdoin Scientific Review; a fortnightly jour-
nal 1870: 1-2

Brain; a journal of neurology 1878: 1 +

British Chemical and Physiological Abstracts.
A (1924-25 as Abstracts of Chemical Papers,
A; 1926-37 as British Chemical Abstracts,
A; in 1938 absorbed Physiological Abstracts)
(in three sections: I. General, Physical and
Inorganic Chemistry; II. Organic Chemis-
try; III. Physiology and Biochemistry) 1924:
1924+

British Chemical and Physiological Abstracts.
B (1924-25 as Abstracts issued by Bureau
of Chemical Abstracts; 1926-37 as British
Chemical Abstracts. B) (in three sections: I.
General and Inorganic Industrial Chemis-
try; II. Industrial Organic Chemistry; III.
Agriculture, Foods, Sanitation, etc.) 1924:
1924+

British Journal of Experimental Biology see
Journal of Experimental Biology

British Journal of Experimental Pathology
1920: 1 +

British Journal of Radiology 1896: n.s. 1 +

British Medical Journal; the journal of the
British Medical Association 1857: 1923 +

British Medical Research Council Special Re-
port see Special Report Series; Privy Coun-
cil; Medical Research Council

British Meteorological and Magnetic Year
Book see Reseau Mondial; monthly and an-
nual summaries of pressure, temperature,
and precipitation

Brooklyn Botanic Garden Memoirs 1918: 1 +

Brooklyn Botanic Garden Record 1912: 1 +

Broteria ; Revista Luso Brazileira (8-9 Revista

de Sciencias Naturaes) serie Botanica 1907:
8-21, no. 1

Bryologist; journal of the Sullivant Moss So-
ciety 1898: 1 +

Buletinul Societatei de Medici si Naturalisti

' » i

din lasi see Revue Medico-Chirurgicale di
Jassy

Bulletin; Agricultural Experiment Station;
Puerto Rico (32-39 as Boletin; Departa-
mento de Agricultura y Trabajo): 15; 32 +

Bulletin ; Association Francaise pour 1'Avance-
ment des Sciences (through 63 as Bulletin
Mensuel) 1896: 63, no. 119+

Bulletin; Bernice P. Bishop Museum of Poly-
nesian Ethnology and Natural History 1922:

1 +

Bulletin; Bureau of Meteorology 1908: Minis-
ter of the Interior, Australia. 1-12; 14 +

Bulletin; Carnegie Foundation for the Ad-
vancement of Teaching 1907: 1 +

^Bulletin; Coastguards and Fisheries Service;
Fisheries Research Section 1930: Ministry
of Finance, Egypt. 1

Bulletin; Department of Agriculture; Union
of South Africa 1911: [1916-30]

Bulletin; Department of Fisheries, Bengal
(Bihar and Orissa) 1913:8; 11; 14-17; 19-20

Bulletin ; Fisheries Research Board of Canada
(1-55 as Bulletin; Biological Board of
Canada) 1918: 1 +

*Bulletin ; Gulf Biologic Station 1902: Cameron,
Louisiana. 2; 4-11; 13-15

Bulletin; New Jersey Agricultural Experiment
Station: [103-669]

Bulletin; Societe Neuchateloise des Sciences
Naturelles (1-25 as Bulletin de la Societe
des Sciences Naturelles cle Neuchatel; 26 as
Bulletin de la Societe Neuchateloise des
Sciences Naturelles) (52+ also numbered as
n.s. 1 + ) 1843: 1 +

Bulletin; Station Oceanographique de Sa-
lammbo 1924: Tunis. 1-37

Bulletin; Storrs Agricultural Experiment Sta-
tion; Connecticut Agricultural College:
[23-177]

Bulletin; Texas Agricultural Experiment Sta-
tion: 18; 65; 69; 119; 180; 241-267; 269-317;
319-445; 447-448; 450 +

Bulletin; University of Wyoming Agricultural
Experiment Station : [49-138]

Bulletin; Wisconsin Geological and Natural
History Survey 1898: [1-70]

Bulletin Biologique de la France et de la Bel-
gique (19-50 as Bulletin Scientifique de la
France et de la Belgique; 1-18 under various
titles beginning Bulletin Scientifique. . . .)
1869: 1+ Supplements 1919: 1 +

Bulletin d'Histoire Naturelle de la Societe
Linneenne de Bordeaux 1826: 2-3

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Bulletin d'Histologie appliquee a la Physio-
logic et a la Pathologic et de Technique
Microscopique 1924: 1 +

Bulletin de Biologic et de Medecine Experi-
mental de 1'URSS 1936: 1 +

Bulletin de 1'Academie des Sciences de 1'Union
des Republiques Sovietiques Socialistes (Iz-
vestiia Akademii Nauk Soyousa Sovetskic
Socialisticheskic Respublik) (formerly Bul-
letin de 1' Academic Imperiale des Sciences
de St. Petersbourg) 1836: ser. 3, 1-32; ser.
4, 1, no. 1; 4, nos. 1-2; ser. 5, 1-25; ser. 6..
1-21; Classe des Sciences Physico-mathe-
matiques, ser. 7, 1928, 1-8 (1935)

Bulletin de 1'Academie des Sciences de 1'Union
des Republiques Sovietiques Socialistes;
Classe des Sciences Mathematiques et Na-
turelles 1936: Serie Biologique: 1936+ ;
Classe des Sciences Chimiques (1936-38 as
Serie Chimique; 1939 as Journal de Chimie
Generate, vol. 9) 1936: 1936+

Bulletin de 1'Academie des Sciences Mathe-
matiques et Naturelles; B. Sciences Natu-
relles 1933: Academic Royale Serbe, Bel-
grade. 1 +

Bulletin de 1'Academie Royale des Sciences,
des Lettres et des Beaux-Arts de Belgique
see Bulletin (s) de la Classe des Sciences;
Academie Royale de Belgique

Bulletin de 1'Academie Royale des Sciences et
des Lettres de Danemark, Copenhague
(Oversigt over det Kongelige Danske
Videnskabernes Selskabs Forhandlinger)
1814: [1874-1908]; 1915-31

Bulletin de 1'Association des Anatomistes; Re-
union de 1'Association 1926: [1-21]; 25 +

Bulletin de 1'Association des Psychiatres Rou-
mains see Bulletin de la Societe Roumaine
de Neurologie, Psychiatrie, Psychologic et
Endocrinologie

Bulletin de 1'Association Francaise pour
1'Avancement des Sciences see Comptes
Rendus; Association Francaise pour 1'Avan-
cement des Sciences

Bulletin de 1'Association Russe pour les Re-
cherches Scientifiques a Prague (Rozpravy
Vedecke Spolecnosti Badatelske Pfi Ruske
Svobodne Universite v Praze) ; Section des
Sciences Naturelles et Mathematiques: 19;
27; 34; 37

Bulletin de 1'Institut des Recherches Biologi-
ques de Perm see Izvestiia Permskogo Biolo-
gicheskogo Nauchno-Issledovatel'skogo Ins-
tituta

Bulletin de 1'Institut et du Jardin Botaniques
de 1'Universite de Beograde (Glasnik Bo-
tanichkog Zavoda i Bashte Univerziteta
u Beogradu) 1928: 1 +

Bulletin de 1'Institut Hydrologique ; Comite
Hydrometeorologique d'U.R.S.S.; Institut
Hydrologique see Izvestiia Gosudarstven-
nogo Gidrologicheskogo Instituta

Bulletin de 1'Institut Metchnikoff 1936: Kra-
kov. 1

Bulletin de 1'Institut National (1'Ecole Supe-
rieure) Agronomique, Brno, R C S see Sbor-
nik Vysoke Skoly Zemedelske v Brne C S R

Bulletin de 1'Institut Oceanographique; Fon-
dation Albert I; Prince de Monaco (1-278
as Bulletin du Musee Oceanographique de
Monaco) 1904: 1 +

Bulletin de 1'Institut Pasteur; revues et analy-
ses des travaux de bacteriologie, et de mede-
cine, biologie generate, physiologic, chemie
biologique, dans leur rapports avec la micro-
biologie 1903: 1 +

Bulletin de 1'Universite de 1'Asie Centrale see
Biulleten Sredne-Aziatskogo Gosudarstven-
nogo Universiteta

Bulletin (s) de la Classe des Sciences; Acade-
mie Royale de Belgique (Mededeelingen
van de Afdeeling Wetenschappen; Kon-
inklijke Belgische Academie) (series 1-3 as
Bulletin de 1'Academie Royale des Sciences,
des Lettres et des Beaux-Arts de Belgique)
1832: ser. 3, 32; 33; 35; ser. 4, 1 +

Bulletin de la Commission Internationale pour
1'Exploration Scientifique de la Mer Medi-
terranee 1920: Monaco. 1-10
*Bulletin de la Federation des Industries Chi-
miques de Belgiques 1921: 1-8

Bulletin de la Seance de la Societe Royale des
Sciences Medicales et Naturelles de Bru-
xelles see Annales et Bulletin de la Societe
Royale des Sciences Medicales et Naturelles

Bulletin de la Section Scientifique; Academie
Roumaine 1912: Bucharest. [1-22]

Bulletin de la Societe Botanique de France
1854: ser. 2, 5-7; ser. 3, 1-3; ser. 4, 75 +

Bulletin de la Societe Chimique de Belgique
1887: [22-30]; 31-33; [34]; 35; [36]; 38 +

Bulletin de la Societe Chimique de France
(1 — ser. 3, 36 as Bulletin de la Societe
Chimique de Paris) (1934+ in two parts,
Memoires and Documentation) 1858: 1 +

Bulletin de la Societe d'Etude des Sciences
Naturelles et du Musee d'Histoire Natu-
relle d'Elbeuf 1881: 23-40

Bulletin de la Societe d'Histoire Naturelle de
Colmar see Mitteilungen der Naturhisto-
rischen Gesellschaft in Colmar

Bulletin de la Societe d'Oceanographie de
France 1921: 1 +

Bulletin de la Societe de Biologie de Lettonie
see Acta Biologica Latvica

Bulletin de la Societe de Chimie Biologique
1914: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Bulletin(s) de la Societe de Pathologic Exo-
tique et de ses Filiales (de 1'Ouest Africain
et de Madagascar) 1908: 29 +

Bulletin de la Societe de Psychiatric de Buca-
rest 1936: 1 +

Bulletin de la Societe des Amis de 1'Institut
Oceanographique du Havre: 14, no. 42 +

Bulletin de la Societe des Etudes Oceaniennes
1917: Tahiti. 1 +

Bulletin de la Societe des Medecins et des
Naturalistes see Revue Medico-Chirurgicale
de Jassy

Bulletin de la Societe des Naturalistes de
Moscou (Biulleten Moskovskogo Obsh-
chestva Ispytatelei Prirody) (1-62 and n.s.
1-30 as Bulletin de la Societe Imperiale des
Naturalistes de Moscou) (includes Meteoro-
logische Beobachtungen 1882-94) (n.s. 31 +
in sections) 1829: 1; 2, no. 2; 3-5; 9, pp.
1-115; 10-15; 16, nos. 2-4; 17-23; 24, no. 3;
25, nos. 1, 3-4; 26-62; n.s. 1-30; Section
Biologique: 31 + ; Section de la Biologic Ex-
perimentale: 32, no. 3 +

*Bulletin de la Societe des Naturalistes de
Voroneje (Biulleten Obshchestva Estest-
voispytatelei pri Voronezhskom Gosudarst-
vennom Universitete) 1925: 1-2, no. 4

Bulletin de la Societe des Sciences Medicales
et Biologiques de Montpellier et du Langue-
doc Mediterranean see Archives de la So-
ciete des Sciences Medicales et Biologiques
de Montpellier et du Languedoc Mediter-
raneen

Bulletin de la Societe des Sciences Naturelles
de 1'Ouest de la France 1891: Secretariat
au Museum d'Histoire Naturelles de Nantes.
ser. 2, 2, nos. 3-4; 3, nos. 1-2; 4-5; 8, nos.
1-2; ser. 3 +

Bulletin de la Societe des Sciences Naturelles
de Neuchatel see Bulletin; Societe Neucha-
teloise des Sciences Naturelles

Bulletin de la Societe des Sciences Naturelles
du Maroc; Empire Cherifien; archives sci-
entifiques du protectorat frangais 1921: 1 +

Bulletin de la Societe Fouad I d'Entomologie
(1-6 as Bulletin de la Societe Entomologique
d'Egypte; 7-21 as Bulletin de la Societe
Royale Entomologique d'Egypte) 1908: 1 +

Bulletin de la Societe Francaise de Micro-
scopic 1932: 1 +

Bulletin de la Societe Geophysique de Varso-
vie see Biuletyn Towarzystwa Geofizykow w
Warszawie

Bulletin de la Societe Linneenne de Norman-
die 1855: Caen. 1 +

Bulletin de la Societe Polonaise d' Anatomic et
de Zoologie see Folia Morphologica

Bulletin de la Societe Portugaise des Sciences
Naturelles 1907: [1-7]; 8-11, no. 7

Bulletin de la Societe Roumaine de Neurol-
ogic, Psychiatric, Psychologic et Endocrin-
ologie; (1-3 as Bulletins et Memoires de la
Societe" de Neurologie, Psychiatrie et Psy-
chologie de Jassy; 4-5 as Bulletin de
1'Association des Psychiatres Roumains)
1919: 1 +

Bulletin de la Societe Royale de Botanique de
Belgique (18-42 in two parts; Memoires and
Comptes-Rendus; 51+ also as ser. 2, 1+)
1862: 1 +

Bulletin de la Societe Royale des Sciences de
Liege 1832: 1 +

Bulletin de la Societe Royale Entomologique
d'Egypte see Bulletin de la Societe Fouad I
d'Entomologie

Bulletin de la Societe Scientifique de Bretagne;
Sciences mathematiques, physiques et na-
turelles 1924: Rennes. 1 +

Bulletin de la Societe Vaudoise des Sciences
Naturelles 1842: Lausanne, ser. 5, 42 +

Bulletin de la Societe Zoologique de France
1876: 1 +

Bulletin de la Station Biologique d'Arcachon;
Travaux des Laboratoires ; Universite de
Bordeaux et Societe Scientifique d'Arca-
chon; Station Biologique 1895: 1 +

Bull etui de la Station Maritime de Hel see
Biuletyn Stacji Morskiej w Helu

Bulletin de Pharmacie et des Sciences Ac-
cessoires see Journal de Pharmacie et de
Chimie

Bulletin decadaire (mensuell) Meteorologique
et Glacial du Service des Previsions du
Temps de 1'Administration Centrale des
Voies Maritimes du Nord see Dekadnyi i
Ezhemesiachnyi Biulleten Sluzhby Pogody
i Ledovoi Informatsii

Bulletin des Institutions Royales d'Histoire
Naturelle a Sophia see Izvestiia. na Tsarskitye
Prirodonauchni Instituti v Sofiia
*Bulletin des Seances de la Societe Beige de
Microscopic (Proces-Verbaux) 1875: 1-25
(bound in with Annales de la Societe)

Bulletin des Travaux publics par la Station
d'Aquiculture et de Peche de Castiglione
1926: [1928-33]

Bulletin du Jardin Botanique de Buitenzorg
see Bulletin of the Botanic Gardens; Buiten-
zorg

Bulletin du Jardin Botanique de Kyiv see Vis-
nik Kiivs'kogo Botanichnogo Sadu

Bulletin du Jardin Botanique de 1'Academie
des Sciences de 1'URSS (Principal de
1'URSS) see Izvestiia Botanicheskogo^Sada

Bulletin du Jardin Botanique de 1'Etat a
Bruxelles; Ministere de 1' Agriculture 1902:
1, fasc. 4+

Bulletin du Laboratoire et de la Societe Inter-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

nationals de Plasmogenie (1-88 as Bulle-
tin du Laboratoire de Plasmogenie) 1932:
Mexico. 1 +

Bulletin du Laboratoire Maritime de Dinard
(1-3 as Laboratoire Maritime du Museum
National d'Histoire Naturelles a 1'Arsenal
de Saint-Servan ; 4-10 as Bulletin du Labo-
ratoire Maritime du Museum (National)
d'Histoire Naturelles a Saint-Servan; 11-14
as Bulletin du Laboratoire Maritime de
Saint-Servan) 1928: 1 +

Bulletin du Musee Oceanographique de Mo-
naco see Bulletin de 1'Institut Oceanogra-
phique

Bulletin du Musee Royal d'Histoire Naturelle
de Belgique 1882: 1 +

Bulletin du Museum National d'Histoire
Naturelle; Paris 1895: 1 +

Bulletin du Service Hydrographique see Wia-
domos'ci Sluzby Hydrograficznej

Bulletin et Annales de la Societe Entomolo-
gique de Belgique (1-64 as Annales de la So-
ciete Entomologique de Belgique) 1857: 53-
54; 56 +

Bulletin(s) et Memoires de la Societe de
Neurologic, Psychiatric et Psychologic de
Jassy see Bulletin de la Societe Roumaine
de Neurologic, Psychiatric, Psychologic et
Endocrinologie

*Bulletin for Scientific Relations ; International
Institute of Intellectual Co-operation ; League
of Nations 1926: Paris. 1-3

Bulletin from the Laboratories of Natural His-
tory of the State University of Iowa see
University of Iowa Studies in Natural
History

Bulletin Hydrographique 1908: Conseil Per-
manent International pour 1'Exploration de
la Mer. 1908 +

Bulletin International; Resumes des Travaux
Presentes; Classe des Sciences Mathe-
matiques, Naturelles et de la Medecine'.
Academic Tcheque des Sciences (Academic
des Sciences de 1'Empereur Francois
Joseph 1895-1916) 1895: [6-9]; 10-13;
15-17; 19+

*Bulletin International de I'Academie Polonaise
des Sciences et des Lettres (1890-1919 as
Bulletin International de I1 Academic des
Sciences de Cracovie) 1890: 1890-1909;
Classe des Sciences Mathematiques et Na-
turelles ser. A and B. 1910: 1910-38

Bulletin Mensuel; Association Francaise pour
1'Avancement des Sciences see Bulletin; As-
sociation Francaise pour 1'Avancement des
Sciences

Bulletin Mensuel de la Societe Linneenne de
Lyon 1932: [1-10]

Bulletin Meteorologique et Hydrographique,
Warsaw see Wiadomos'ci Meteorologiczne i
Hydrograficzne

Bulletin of Applied Botany, Genetics and
Plant-Breeding see Trudy po Prikladnoi
Botanike, Genetike i Selektsii
*Bulletin of Basic Science Research 1926: Basic
Science Research Laboratory, University of
Cincinnati. 1-5

Bulletin of the Chekiang Provincial Fisheries
Experiment Station 1935: China. [1-3]

Bulletin of Entomological Research 1910: Im-
perial Bureau of Entomology, London. 1 +

Bulletin (s) of Ichthyological Laboratory of
Baku see Izvestifa Bakinskoi Ikhtiologiches-
koi Laboratorii

Bulletin of Mathematical Biophysics (1 pub-
lished as supplement to Psychometrika)
1939: 1 +

Bulletin of Plant Protection (Trudy po Zash-
chite Rastenii) ; Lenin Academy of Agricul-
tural Sciences in U.S.S.R. ; Institute for
Plant Protection 1930: series 1. Entomol-
ogy: vol. 1, nos. 1-2 (1930-31); vol. 3, no. 1
(1931); vol. 4, no. 1 (1931), nos. 3 (1932), 4
(1932), 6 (1932); series 2. Phytopathology:
no. 3 (1933)

Bulletin of War Medicine; Medical Research
Council 1940: edited by the staff of the
Bureau of Hygiene and Tropical Diseases.
2, no. 3 +

Bulletin of the Academy of Sciences of the
United Provinces of Agra and Oudh Allaha-
bad see Proceedings of the National Acad-
emy of Sciences; India

Bulletin of the American Association of Uni-
versity Professors 1915: 1 +

Bulletin of the American Meteorological So-
ciety 1920: 1 +

Bulletin of the American Museum of Natural

History 1881: 1-19; 23 +

*Bulletin of the Antivenin Institute of America
1927: 1-5, no. 3

Bulletin of the Arctic Institute of the USSR
see Biulleten Arkticheskogo Instituta

Bulletin of the Association of American
Medical Colleges see Journal of the Asso-
ciation of American Medical Colleges

Bulletin of the Auckland Institute and Mu-
seum 1941: New Zealand. 1 +

Bulletin of the Bingham Oceanographic Col-
lection 1927: Peabody Museum of Natural
History, Yale University. 1 +

Bulletin of the Biological Department; Science
College; Sun Yat-Sen University 1929: 1,
no. 1

Bulletin of the Boston Society of Natural
History see New England Naturalist

Bulletin of the Botanic Gardens; 's Lands

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Plantentuin; Botanic Gardens, Buitenzorg
1911: ser. 3, 1+ Suppl. 1-3, no. 2
Bulletin of the Bowdoin Scientific Station (in-
cludes Annual Report) 1935: Kent's Island,
New Brunswick. 3-4; 6

*Bulletin of the Brookville Society of Natural
History; contributions to the natural history
of Franklin County, Indiana 1885: 1-2
*Bulletin of the Buffalo Naturalists' Field Club

1883: 1
Bulletin of the Buffalo Society of Natural

Sciences 1873: 1 +

Bulletin of the Bureau of Agricultural Micro-
biology, Leningrad see Trudy Vsesoiuznogo
Instituta sel'skokhozfaistvennoi Mikrobio-
logii
Bulletin of the Bureau of Eugenics see Trudy

Instituta Genetiki

Bulletin of the Bureau of Standards see Scien-
tific Papers of the Bureau of Standards
Bulletin of the Bussey Institution; Harvard

University 1874: 1-3, pt. 4
*Bulletin of the California Academy of Sciences

1884: 1-2
Bulletin (s) of the Ceylon Fisheries see Ceylon

Journal of Science; Section C. Fisheries
Bulletin of the Chemical Society of Japan

1926: 1 +
Bulletin of the Chicago Academy of Science

1883: 1; [2]; 3; [4-5]
*Bulletin of the College of Agriculture, Tokyo

Imperial University 1887: 5-6
*Bulletin of the Department of Biology of

Yenching University 1930: Peiping. 1
Bulletin of the Faculty of Science ; The Egyp-
tian University 1934: 1-3; 6+
Bulletin of the Fan Memorial Institute of
Biology (5+ in two sections; Botany and
Zoology) 1929: 1 +

Bulletin of the Fishery Experiment Station,
Canton, China 1929: 1929-30 (text in
Chinese)

Bulletin of the Fishery Experiment Station of
the Government-General of Chosen 1925:
Fusan. 1 +

Bulletin of the Geographical Society of Phila-
delphia 1893: 3, no. 4 (1902)
Bulletin of the Geological Institution of the
University of Upsala 1892: 1-5; 8; 10-11;
[13]; 14-16; 18
Bulletin of the Geological Society of America

1888: 52 +

Bulletin of the Harpswell Laboratory see Bul-
letin of the Mount Desert Island Biological
Laboratory

Bulletin of the History of Medicine; organ of
the American Association of the History of
Medicine and the Johns Hopkins Institute
of the History of Medicine (1-6 as Bulletin

of the Institute of the History of Medicine;
1-2 a supplement to Bulletin of the Johns
Hopkins Hospital) 1933: 1 +

Bulletin of the Hydrographic Department, Im-
perial Japanese Navy 1917: 1 +

Bulletin of the Hygienic Laboratory see Na-
tional Institute of Health Bulletin
*Bulletin of the Illinois State Museum of
Natural History 1876: 1; 3-10

Bulletin of the Illinois State Natural History
Survey see Illinois Natural History Survey
Bulletin

Bulletin of the Independent Biological Labora-
tories 1932: Tel-Aviv, Palestine. [1-2] +

Bulletin of the Institute of Fresh-water (Ich-
thyology) Fisheries (formerly Bulletin of the
Bureau of Applied Ichthyology) (Izvestiia
Vsesoiuznogo Nauchno-Issledovatel'skogo
Instituta Ozernogo i Rechnogo Rybnogo
Khoziaistva) 1918: Leningrad. [11]; 12 +

Bulletin(s) of the Institute of Genetics see
Trudy Instituta Genetiki

Bulletin of the Institute of Jamaica; Science
Series 1940: 1 +

Bulletin of the Institute of Physical and Chem-
ical Research (Rikwagaku-Kenkyujo Iho)
see with Scientific Papers of the Institute of
Physical and Chemical Research, Tokyo

Bulletin of the Japanese Society of Scientific
Fisheries 1932: 1 +

Bulletin of the Johns Hopkins Hospital 1889:
[1-3]; 4+

Bulletin (s) of the Laboratory of Genetics
(Trudy Laboratorii Genetiki) see Trudy
Instituta Genetiki

Bulletin of the Lloyd Library of Botany,
Pharmacy and Materia Medica 1900: 1 +

Bulletin of the Madras Government Museum

1894: n.s. Natural History Section 1927: 1 +

*Bulletin of the Michigan Fish Commission

1890: 1-8

*Bulletin of the Minnesota Academy of Natural
Sciences 1873: [2-4]

Bulletin of the Mount Desert Island Biological
Laboratory (1898-1923 as Bulletin of the
Harpswell Laboratory) : 9 ; 24; 26-27; 29-32;
34+

Bulletin of the Museum of Comparative Zool-
ogy at Harvard College in Cambridge 1863:
1-2; [3]; 4-33; 35 +

Bulletin of the National Research Council

(nos. 1-57 also as vols. 1-11) 1919: 1 +
*Bulletin of the Natural History Society of
British Columbia 1893: 1

Bulletin of the Natural History Society of New
Brunswick 1882: 1-2; 4-9; 11-18; 20-21

Bulletin of the Natural History Survey; Chi-
cago Academy of Sciences 1896: 3; 5-6; 7,
no. 2; 8

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

*Bulletin of the Neurological Institute of New
York 1931: 1-7

Bulletin of the New England Museum of
Natural History see New England Naturalist

Bulletin of the New York Academy of Medi-
cine (includes Annual Report 1924 + ) 1871:
sen 2, 1 +

^Bulletin of the New York Botanical Garden
1896: 1-14

Bulletin of the New York State Museum see
New York State Museum Bulletin

Bulletin (s) of the Pacific Scientific Institute of
Fisheries and Oceanography (Izvestiia
Tikhookeanskogo Nauchnogo Instituta Rib-
nogo Khozaistya i Okeanografii) (1-4, no. 1
as Bulletins of the Pacific Scientific Fishery
Research Station) (Izvestiia Tikhookeanskoi
Nauchno-Promyslovoi Stantsii) 1928: 1-6;
8-10; 12 +

*Bulletin of the Philosophical Society of Wash-
ington 1871: 1-15

Bulletin of the Scientific Laboratories of
Denison University 1885: 1-12; [13]; 14-17

Bulletin of the Scripps Institution of Ocean-
ography (1-11 as Bulletin of the Scripps
Institution for Biological Research); Non-
technical series 1916: 1 + ; technical series
1927: 1 +

Bulletin of the Shanghai Science Institute

1929: 1 +

*Bulletin of the South African Biological So-
ciety 1918: Pretoria, ser. 1, no. 2

Bulletin of the State Oceanographical Institute
see Biulleten Gosudarstvennogo Okeano-
graficheskogo Instituta

Bulletin of the Torrey Botanical Club 1870:

1 +

Bulletin of the USSR Institute of Agricultural
Microbiology see Trudy Vsesoiuznogo Insti-
tuta sel'skokhoziaistvennoi Mikrobiologii

Bulletin of the United States Bureau of
Fisheries (1-23 as Bulletin of the United
States Fish Commission) 1881: 1 +

Bulletin of the United States Coast Guard:

2 (1913)— 3; 5 +

Bulletin of the United States National Mu-
seum 1875: 1 +

Bulletin of the University of Kansas see Kansas
University Science Bulletin

Bulletin of the University of Texas see Uni-
versity of Texas Bulletin
*Bulletin of the University of Wisconsin;
Science series 1894: 1— i

Bulletin of the Vanderbilt Marine Museum (1,

art. 1 as Bulletin of the Vanderbilt Oceano-

graphic Museum) 1928: Huntington, Long

Island. 1 +

*Bulletin of the Washburn Laboratory of

Natural History 1884: Washburn College.
1, nos. 1, 2, 4

*Bulletin of the Wisconsin Natural History
Society 1900: n.s. 1-10

Bulletin of the Wistar Institute of Anatomy
and Biology 1905: 1-8

Bulletin (s) of the Zoological Society of San

Diego 1924: 1-2; 4-6; 9; 11 +
*Bulletin Planktonique 1908: Conseil Perma-
nent International pour 1'Exploration de la
Mer. 1908-12

Bulletin Scientifique; Recueil Biologique,
Chimique, Phys.-Mathematique see Nau-
kovi Zapiski; Kiivs'kii Derzhavnii Uni-
versitet

Bulletin Scientifique de la France et de la
Belgique see Bulletin Biologique de la
France et de la Belgique

Bulletin Statistique des Peches Maritimes des
Pays du Nord de 1'Ouest de 1'Europe 1903:
Conseil Permanent International pour 1'Ex-
ploration de la Mer. 2 +

*Bulletin Trimestriel des Resultats acquis pen-
dant les Croisieres periodiques et dans les
periodes intermediaires (1902-05 as Bulle-
tin des Resultats acquis pendant les Courses
periodiques) 1902: Conseil Permanent Inter-
national pour 1'Exploration de la Mer.
1902-08

*Bulletin van het Koloniaal Museum te Haar-
lem 1892: 4-23; 25; 30; 33-34; 39-41; 45-50

Bullettino dell'Instituto Zoologico della R.
Universita di Palermo 1918: 1-2, no. 11

Bullettino della Societa Botanica Italiana 1892:
Florence. [1910]; [1919]; 1923

Bullettino Mensile della Accademia Gioenia di
Scienze Naturali in Catania see Bollettino
delle Sedute della Accademia Gioenia di
Scienze Naturali in Catania

Bulteno Scienca de la Fakultato Terkultura;
Kyusyu (Kjusu) Imperia Universitato ; Hu-
kuoka (Fukuoka), Japanujo 1924: 1 +

Bureau of Standards Journal of Research see
Journal of Research of the National Bureau
of Standards

*Calcutta Journal of Natural History; con-
ducted by John M'Clelland, Bengal Medi-
cal Service 1840: 1, no. 1

California Fish and Game 1914: State of Cali-
fornia; Division of Fish and Game. [1]; [8];
9-10; [11-12]; 13 +

Canadian Entomologist 1 868 : 1 +

Canadian Field- Naturalist (subtitle 1-33 as
Transactions of the Ottawa Field-Natu-
ralists' Club 3-35) (1-32 as Ottawa Natu-
ralist) (2-7 contains Flora Ottawaensis)
1887: 1 +

Canadian Journal of Research (13+ in sec-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

tions: A. Physical Sciences; B. Chemical
Sciences; C. Botanical Sciences; D. Zoologi-
cal Sciences) 1929: National Research Coun-
cil of Canada, Ottawa. 1 +
Canadian Medical Association Journal 1911:

1 +

Canadian Meteorological Memoirs 1935: Di-
vision of Meteorological Services, Canada.

1 +

Cancer Research; a monthly journal of arti-
cles and abstracts reporting cancer research
1941: American Association for Cancer Re-
search, Inc. 1 +

*Cancer Review; a journal of abstracts; British
Empire Cancer Campaign 1926: 1-7

Carnegie Corporation of New York; Report of

the President and of the Treasurer: 1934 +
*Carnegie Institution of Washington; News
Service Bulletin, School Edition 1926: 1-4

Carnegie Institution of Washington; Publica-
tion 1902: [4-535]+; Classified List 1919 +

Carnegie Institution of Washington; Year

Book 1902: 1 +

*Catalogue of Scientific Papers; compiled by
the Royal Society of London 1800-1900:
series 1-4

Catalogue of United States Public Documents
see Monthly Catalogue; United States Gov-
ernment Publications

*Causeries Scientifiques de la Societe Zoologi-
que de France 1900: 1-10 (1906)

(La) Cellule ; Recueil de Cytologie et d'Histo-
logie generate; fonde par J. B. Carnoy 1884:
Belgium. 1 +

Centralblatt see Zentralblatt

Ceylon Journal of Science; Section A, see An-
nals of the Royal Botanic Gardens, Pera-
deniya; Section B, see Spolia Zeylanica

Ceylon Journal of Science; Section C. Fish-
eries (1-4 as Bulletins of the Ceylon Fish-
eries) 1922: 1 +

*Ceylon Marine Biological Reports 1904: vol. 1,
pt. 6, nos. 20-22 (1912)

Chemical Abstracts; American Chemical So-
ciety 1907: 1 +

Chemical and Engineering News (1-17 as In-
dustrial and Engineering Chemistry; News
Edition) (18-19 as News Edition; American
Chemical Society) 1923: 1 +

Chemical Reviews 1924: American Chemical

Society. 1 +

*Chemie der Zelle und Gewebe; Zeitschrift fur
die Probleme der Garung, Atmung und Vi-
taminforschung (1-6 as Zeitschrift fur Ga-
rungsphysiologie; 7-11 as Zeitschrift fur
Technische Biologic) 1912: 1-13, no. 2

Chemisches Zentralblatt (1-20 as Pharmaceu-
tisches Central-Blatt; 21-26 as Chemisch-
Pharmaceutisches Central-Blatt; 27-40 also

as n.f. 1-14; 41-59 as ser. 3, 1-19; 60-67 as
ser. 4, 1-8; 68-89 as ser. 5, 1-22; 90-95 as
ser. 6, 1-6; 96 as ser. 7, 7) 1830: Deutsche
Chemische Gesellschaft. 1-104 (1933)

Chemistry and Industry see Journal of the So-
ciety of Chemical Industry

China Journal (1-5 as China Journal of Science
and Arts) 1923: 1-12

Chinese Journal of Physics 1933: 4, no. 1

Chinese Journal of Physiology 1927: Chinese

Physiological Society. 1 +
*Chinese Journal of Physiology; Report Series;
Metabolism 1928: Chinese Physiological
Society. 1

Chinese Journal of Zoology 1935: Zoological
Society of China. 1

Chinese Medical Journal (21-45 as China
Medical Journal which, with 46, amalga-
mated with National Medical Journal of
China to be published as Chinese Medical
Journal) 1887: Chinese Medical Associa-
tion. 30-34; [35]; 36+

Chromosoma; Zeitschrift fiir Zellkern- und
Chromosomenforschung; Abt. B der Zeit-
schrift fiir Zellforschung und Mikroskopische
Anatomic 1939: 1 +

Chronica Botanica 1935: Chronica Botanica
Co. 1 +

Chronik der Ukrainischen Sevcenko-Gesell-
schaft der Wissenschaften in Lemberg 1900:
1-2; 6-12; 15-16; 19; 27-30; 35-37; 41-50

Ciencia; revista hispano-americana de ciencias
puras y aplicadas 1940: Mexico. 1 +

Circular; Texas Agricultural Experiment Sta-
tion: 4; 19-20; 22-34; 37-50; 52 +

Circular of the National Bureau of Standards
1901: United States Department of Com-
merce. [1-425]
*Cleveland Clinic Bulletin 1931: 1, no. 1

Cleveland Clinic Quarterly 1932: 1 +

Clinical Science, incorporating Heart 1933:

London. 1 +

*Cold Spring Harbor Monographs 1903: Brook-
lyn Institute of Arts and Sciences. 1-10

Cold Spring Harbor Symposia on Quantitative

Biology 1933: 1 +
*Colecgao "Natura"; Sociedade Portuguesa de

Sciencias Naturals. 5-10

*Collectanea dos Trabalhos do Institute de
Butantan (1901-17 as Collectanea de Tra-
balhos) 1901: Sao Paulo. 1-2

Collecting Net 1926: Woods Hole. 1 +

Collection of Czech Chemical Communications
(Tschechischer Chemischer Forschungsar-
beiten) 1929: Societas Scientiarum Bohe-
mica, Prague. 1-11

*Colloid Symposium Monographs (9+ pub-
lished in part in the Journal of Physical
Chemistry) 1923: 1-8

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

"Colorado College Publication (1-10 as Colo-
rado College Studies) 1890: 10; Science
series 1904: [11-13]

Colorado Scientific Society Proceedings (1-10
as Proceedings of the Colorado Scientific
Society) 1883: 1 +

(R.) Comitato Talassografico Italiano; Mono-
grafia: 2, parts 1-3 (1928)

Commentationes Biologicae; Societas Scien-
tiarum Fennica 1922: 1 +

Commentationes Physico-mathematicae ; So-
cietas Scientiarum Fennica 1922: 1 +

Comparative Psychology Monographs 1922:

1 +

Compte-Rendu Annuel de la Societe Royale
des Lettres et des Sciences de Boheme see
Vyrocni Zprava Kralovske Ceske Spolec-
nosti Nauk

Comptes Rendus; Association Franc, aise pour
1'Avancement des Sciences (1887, fusion-
nee avec 1' Association Scientifique de France)
1872: 1 + ; Bulletin: nos. 119-136 (1934-36)
*Comptes Rendus de 1'Academie des Sciences
de 1'URSS; Series A (Doklady Akademii
Nauk) 1922: 1922-33

Comptes Rendus (Doklady) de 1'Academie des
Sciences de 1'URSS n.s. 1933: 1 +

Comptes Rendus de 1'Association des Anato-
mistes 1899: 1 +

Comptes Rendus de (1'Association Libre) la
Societe des Physiologistes Suisses see Ver-
handlungen des Vereins der Schweizer
Physiologen

Comptes Rendus de la Station Hydrobiologi-
que du Lac de Wigry see Sprawozdania
Stacji Hydrobiologicznej na Wigrach

Compte Rendu des Seances de la Societe de
Physique et d'Histoire Naturelle de Geneve
(issued first in Archives des Sciences Phy-
siques et Naturelles, then independently as a
Supplement) 1883: 1 +

Comptes Rendus des Seances de la Societe
des Sciences et des Lettres de Varsovie see
Sprawozdania z Posiedzen Towarzystwa
Naukowego Warszawskiego

Comptes Rendus des Seances de la Societe
Meteorologique de France see in Meteoro-
logie; Revue de Meteorologique et de Phy-
sique du Globe

Comptes Rendus des Travaux du Laboratoire
Carlsberg (also have 1-4 as Meddelelser fra
Carlsberg Laboratoriet) 1878: 1-20; seYie
physiologique 21 + ; serie chimique 21 +
*Compte Rendu du Congres des Botanistes
Slaves a Varsovie (Sprawozdanie z zjazdu-
botanikow Slowianskich w Warszawie):
Polskie Towarzystwo Botaniczne, Warsaw.
3rd (1931)

Comptes Rendus hebdomadaires des Seances
de 1'Academie des Sciences 1935: Paris. 1 +

Comptes Rendus hebdomadaires des Seances
(et Memoires) de la Societe de Biologie et
de ses filiales 1849: (includes Societe de
Biologie d'Alger, Bordeaux, Lille, Lyon,
Marseille, Nancy, Strasbourg, Athenes, Bar-
celone, Belgrade, Montevideo, Montreal,
'Argentine, Beige, Bresilienne, Chilienne,
Danoise, Mexicane, Polonaise, Portugaise,
Roumaine, Tchecoslovaque, Suede, Letto-
nie, Franco-Japonaise) Paris. 1 +

Comptes Rendus Mensuels des Seances de la
Classe de Medecine; Academic Polonaise
des Sciences et des Lettres 1930: [1930-31]

Comptes Rendus Mensuels des Seances de la
Classe des Sciences Mathematiques et Na-
turelles; Academic Polonaise des Sciences
et des Lettres 1929: 1929-39, no. 3

Compte Rendu Sommaire des Seances; So-
ciete de Biogeographie 1924: Paris. 1-10;
[11-14]

Condor; a magazine of western ornithology
(1 as Bulletin of the Cooper Ornithological
Club) 1899: [1-3]; 6-11; [12-13]; 14-18;
[19-20]; 21-23; [24]; 25-26; [29]; 30; [31];
32-37, no. 5

Conference Internationale pour 1'Exploration
de la Mer: Stockholm, 1899

Congreso Internacional de Oceanografia, Hi-
drografia Marina e Hidrologia continental
see International Congress of Oceanography,
Marine Hydrography and Continenta Hy-
drology

Connecticut State Geological and Natural His-
tory Survey Bulletin 1903: 1 +

Conseil International des Unions Scientifiques
(formerly Conseil International de Recher-
ches) see Union Geodesique et Geophysique
Internationale

Conseil Permanent International pour 1'Ex-
ploration de la Mer; Index to Publications.
Copenhagen. 1899 +

Conseil Permanent International pour 1'Explo-
ration de la Mer (Copenhagen) see also
Bulletin Hydrographique; Bulletin Plank-
tonique; Bulletin Statistique des Peches
Maritimes des Pays du Nord et de 1'Europe;
Bulletin Trimestriel des Resultats acquis
pendant les Croisieres Periodiques etc.;
Journal du Conseil; Publications de Cir-
constance

Contributiones pro Fauna et Flora URPSS see
Materialy k Poznaniiu Fauny i Flory SSSR

Contributions ; Chesapeake Biological Labora-
tory: Solomons Island, Maryland. 12; 15-
20; 23-24; 26-29; 31 +

Contributions; Ohio State University; The
Franz Theodore Stone Laboratory 1928: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Contributions a la Connaissance de la Faune
et la Flore de 1'URSS see Materialy k Poz-
nanim Fauny i Flory SSSR

Contributions a la Flore de la Pologne et des
Pays limitrophes see Planta Polonica; Ma-
terijaly do Flory Polski i Krajow Sasiednich

Contributions de 1'Institut Botanique de 1'Uni-
versite de Montreal (1-30 as Contributions
du Laboratoire de Botanique de 1'Univer-
site de Montreal) 1922: 1 +

Contributions de 1'Institut de Biologie (Zoolo-
gie) de 1'Universite de Montreal 1937: 1-9

Contributions de la Station Biologique du St-
Laurent P. Q., Canada 1932: Universitd La-
val, Quebec. 1 +

Contributions from Boyce Thompson Institute
(for Plant Research) 1925: Yonkers, New
York. 1 +

Contributions from the Biological Laboratory
of the Science Society of China; supported
by the China Foundation for the Promotion
of Education and Culture and affiliated with
the Fan Memorial Institute of Biology (64-
issued in two sections: Botany and Zoology)
1925: 1 +

Contributions from the Botanical Laboratory
of the University of Pennsylvania (2, no. 2
as Transactions and Proceedings of the Bo-
tanical Society of Pennsylvania) 1892: [1-5]

Contributions from the Cushman Laboratory
for Foraminiferal Research 1925: Sharon,
Mass. 1 +

Contributions from the Department of Geology
of Stanford University 1930: 1 +

Contributions from the Department of Tropi-
cal Medicine and the Institute for Tropical
Biology and Medicine (1-4 as Contributions
from the Harvard Institute for Tropical Bi-
ology and Medicine) 1925: 1-6

Contributions from the Dudley Herbarium of
Stanford University 1927: 14-

Contributions from the Institute of Physiol-
ogy; National Academy of Peiping 1934:
1-4, no. 4

Contributions from the Institute of Zoology;
National Academy of Peiping 1932: 1-3,
no. 4

*Contributions from the Laboratory of the Ma-
rine Biological Association, San Diego 1904:
1—33 (included in the University of Cali-
fornia Publications in Zoology)

Contributions from the Laboratory of Verte-
brate Genetics ; University of Michigan 1936 :
1 +

Contributions from the United States National
Herbarium (1-8 as United States Depart-
ment of Agriculture; Division of Botany)
1890: 1 +

Contributions from the Zoological Laboratory;

University of Pennsylvania 1893: 1+ (filed
with reprints)

Contributions to Canadian Biology and Fish-
eries; being Studies from the Biological Sta-
tion of Canada 1901: 1901-21; n.s. 1-8
*Contributions to Canadian Paleontology; Geo-
logical and Natural History Survey of Can-
ada 1885: 1, pts. 2-3 (1891)

Contributions to Embryology see Carnegie In-
stitution of Washington Publication
*Contributions to the Micro-Palaeontology of
the Cambro-Silurian Rocks of Canada 1883:
Geological and Natural History Survey of
Canada. 1-4 (1892)

Copeia; a journal of cold blooded vertebrates
1913: American Society of Ichthyologists
and Herpetologists. 14-

Crop Protection Digest Bulletin Series 1921:
Crop Protection Institute, Washington,
D. C. 1-2; 4

Cumulative Book Index 1898: H. W. Wilson
Co. 244-

Current List of Medical Literature 1941:
Friends of the Army Medical Library,
Washington, D. C. and Medical Library
Association Inc. 1 4-

Current Science; Science in the Making 1932:
Indian Institute of Science. 1 +

Current Tables; Atlantic Coast; North Amer-
ica; United States Coast and Geodetic Sur-
vey: 1926; 1930+

Current Titles from Biological Journals; a
monthly register of selected tables of con-
tents 1937: L. R. Kuhn, University of Chi-
cago. 1, nos. 1-3

Cytologia; International Journal of Cytology
1929: Tokyo. 1 +

Dados Climatologicos ; Servico Meteorologico
1887: 1894; 1898; 1902 (1889 see Boletim;
Secretaria da Agricultura, Commercio e
Obras Publicas do Estado de S. Paulo ; Ser-
vico Meteorologico no. 6, 1890; 1890 ibid
no. 8, 1891; 1903 ibid no. 17, 1906; 1904
ibid ser. 2, no. 6, 1909; 1906 ibid nos. 18-21,
1906; 1908 ibid nos. 8-11, 1910; 1912 ibid
nos. 25-28, 1916)

Dansk Botanisk Arkiv udgivet af Dansk Bo-

tanisk Forening 1913: 1 +
*Danske Videnskabernes Selskabs Skrifter;
Naturvidenskabelig og Mathematisk Afde-
ling (Memoires de 1'Academie Royale des
Sciences et des Lettres de Danemark, sec-
tion des sciences) (ser. 4 as Danske Vi-
denskabernes Selskabs Naturvidenskabelige
og Mathematiske Afhandlinger) 1824: ser.
4-9

Dekadnyi i Ezhemesiachnyi Bmlleten ; Sluzhby
Pogody i Ledovoi Informatsii (Bulletin de-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

cadaire meteorologique et glacial du service
des previsions du temps de 1'Administration
Centrale des Voies Maritimes du Nord) :
1935-36

De Lamar Lectures; Johns Hopkins Univer-
sity School of Hygiene and Public Health
1925: 1925-27

Denkschriften der K. Akademie der Wis-
senschaften ; Mathematisch-Naturwissen-
schaftliche Classe 1850: Wien. 1-19; 36-
38; 41; 52; 59 +

Denkschriften der Kgl. Botanischen Gesell-
schaft in Regensburg 1815: 7-9

Denkschriften der Schweizerischen Natur-
forschenden Gesellschaft (Memoires de la
Societe Helvetique des Sciences Naturelles)
1837: 56+

Denkschriften des Naturhistorischen Mu-
seums in Wien; Geologisch-Palaeontolo-
gische Reihe (1 as Denkschriften des K. K.
Naturhistorischen Hofmuseums) 1917: 1 +
*Deutsche Entomologische National-Biblio-
thek; Deutsches Entomologisches Na-
tional-Museum zu Berlin 1910: 1-2

Deutsche Entomologische Zeitschrift (1875-
80 as vols. 19—24 of Berliner Entomologische
Zeitschrift) 1881: 1882; [1883]; 1884-86;
[1887]; 1888-94; [1906]; 1907-14; [1915-
16]; 1929-38

Deutsche Entomologische Zeitschrift "Iris"
(2-14 as Deutsche Entomologische Zeit-
schrift, Lepidopterologische Hefte) 1884:
Entomologischer Verein, Iris, zu Dresden.
2-16; [17-20]

Deutsche Fischwirtschaft; Zeitschrift des
Reichsnahrstandes fiir die Deutsche Fisch-
wirtschaft 1934: 1-5, no. 5

Deutsche Forschung; aus der Arbeit der Not-
gemeinschaft der Deutschen Wissenschaft
1928: Berlin. 1-27

Deutsche Mechaniker-Zeitung see Zeitschrift
fiir Instrumentenkunde; Beiblatt ; Zeitschrift
der Deutschen Gesellschaft fiir Mechanik
und Optik

Deutsche Medizinische Wochenschrif t ; Organ
der Berliner Medizinischen Gesellschaft
und Anderer Vereinigungen 1875: 56 +

Deutsche Tropenmedizinische Zeitschrift (1-
44, 1940 as Archiv fiir Schiffs- und Tropen-
hygiene ; Pathologie und Therapie Exotischer
Krankheiten) 1897: 1 + ; Beihefte 1907: 1 +

Deutsches Archiv fiir Klinische Medizin 1865:
175 +

Deutsches Reich ; Reichsamt fiir Wetterdienst ;
Wissenschaftliche Abhandlungen 1935: 1 +

Deutsches Tuberkulose-Blatt; Sonderbeilage
zur Deutschen Medizinischen Wochen-
schrift (1-7 as Praktische Tuberkulose-
Blatter) 1929: 8 +

Dobutugaku Zassi (43-47 as Dobutsugaku
Zasshi; 48-50 as Zoological Magazine) 1888:
Zoological Society of Japan. 43 +

Doklady Akademii Nauk SSSR see Comptes
Rendus de 1'Academie des Sciences de
1'URSS

Doklady Gosudarstevnnogo Okeanografiches-
kogo Instituta see Reports of the State
Oceanographical Institute

Dopovidi Akademii Nauk URSR (Reports of
the Academy of Sciences of the Ukrainian
SSR): Kief. [1939-40]

*D6rfleria; Internationale Zeitschrift fiir For-
derung Praktischer Interessen der Botani-
ker und der Botanik 1909: Vienna. 1, no. 1
*Drapers' Company Research Memoirs; Bio-
metric series, Department of Applied Statis-
tics, University College, University of Lon-
don 1904: 1-12

Ecological Monographs; Ecological Society of
America 1931: 1 +

Ecology; all forms of life in relation to environ-
ment; Ecological Society of America 1920:
1 +

Economic Proceedings of the Royal Dublin

Society 1899: 1 +

*Ectoparasites edited by K. Jordan and the
Hon. N. Charles Rothchild 1915: 1, nos. 1-6
(1924)

Edinburgh Medical Journal 1855: n.s. 40+

Eesti Loodusleaduse Arhiiv see Archiv fiir die
Naturkunde Estlands

Electric Journal (1-2, no. 5 as Electric Club
Journal) 1904: Westinghouse Club. [1-26];
27-34, no. 8

Electronics 1930: New York. 9, no. 11 +

Emu; official organ of the Australasian Orni-
thologists' Union 1901: 1-9; 10, pt. 2

Endocrinology ; published monthly for the As-
sociation for the Study of Internal Secre-
tions 1917: 1 +

Endokrinologie ; Zentralblatt fiir das Gebiet
der Inneren Sekretion und Konstitutions-
forschung 1928: 1 +

Entomologica Americana; a journal of ento-
mology published by the Brooklyn Entomo-
logical Society 1885: 1-6; n.s. 14, no. 3

Entomological News (1-36 as Entomological
News and Proceedings of the Entomological
Section of the Academy of Natural Sciences
of Philadelphia) 1890:"l +

Entomologische Mitteilungen ; Deutsches En-
tomologisches Museum 1912: 1-5

Entomologische Rundschau mit Societas En-
tomologica 1884: [45-54]

Entomologische Zeitschrift; Vereinigt mit
Internationale Entomologische Zeitschrift

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

1887: Internationaler Entomologischer Ver-
ein, E. V. 42-44; [45]; 46-50; [51-53]

Entomologische Zeitung; herausgegeben von
dem Entomologischen Vereine zu Stettin
(Stettiner Entomologische Zeitung) 1840:
1-21; 23-44; 56; 61-74, heft 1

Entomologisk Tidskrif t ; utgiven av Entomolo-
giska Foreningen i Stockholm (Journal En-
tomologique ; publie par la Societe Entomo-
logique a Stockholm) 1880: [2]; [16]; [18];
20-21; [23]; 24-36

Entomologist; an illustrated journal of general
entomology 1840: London. 6-18

Entomologist's Monthly Magazine 1864: En-
tomological Society of London. 8-22

Entomologist's Record and Journal of Varia-
tion; edited by J. W. Tutt, 1890: London.
17 (1905)

Enzymologia ; Acta Biocatalytica 1936: The
Hague. 1 +

Eos; Revista Espanola de Entomologia; Junta
Para Ampliation de Estudios 1925: Museo
Nacional de Ciencias Naturales, Madrid.
1-11

Ergebnisse der Anatomic und Entwicklungs-
geschichte (1-22 also as Anatomische Hefte,
Abt. 2; 23-31 also as Zeitschrift fur die
Gesamte Anatomie Abt. 3) 1891 : 1-32

Ergebnisse der Angewandten Physikalischen

Chemie 1931: 1 +

*Ergebnisse der Beobachtungsstationen an den
Deutschen Ku'sten u'ber die Physikalischen
Eigenschaften der Ostsee und Nordsee und
die Fischerei 1873: Kommission zur Wissen-
schaftlichen Untersuchung der Deutschen
Meere, in Kiel. 1873-93

Ergebnisse der Biologic 1926: 1 +
*Ergebnisse der Enzymforschung 1932: 1-8

Ergebnisse der Exakten Naturwissenschaften
1922: 1 +

Ergebnisse der Hygiene, Bakteriologie, Im-
munitatsforschung und Experimentellen
Therapie; Fortsetzung des Jahresberichts
iiber die Ergebnisse der Immunitatsfor-
schung 1914: 12 +

Ergebnisse der Kosmischen Physik see Ger-
lands Beitrage zur Geophysik; Supplement-
Band

Ergebnisse der Kreislaufforschung; Mono-
graphien aus dem Gebiete Beschreibender,
Experimenteller und Klinischer Kreislauf-
forschurig 1931: 1-5

Ergebnisse der Physiologic, Biologischen
Chemie und Experimentellen Pharmakolo-
gie; Begriindet von L. Asher und K. Spiro
(1-34 as Ergebnisse der Physiologic; 35-36
as Ergebnisse der Physiologie und Experi-
mentellen Pharmakologie) 1902: 1 +

Ergebnisse der Vitamin- und Hormonfor-

schung; herausgegeben von L. Ruzicka und
W. Stepp 1938: 1 +
*Ergebnisse und Fortschritte der Zoologie; Be-

__ griindet von J. W. Spengel 1909: 1-8, no. 3

Etudes de la Neva et de son Bassin (Issledo-
vaniia Reki Nevy i ee Basseina) 1922:
1'Institut Hydrologique de Russia. 1-6

Etudes Geophysiques see Prace Geofizyczne

Eugenical News; current record of human
genetics and race hygiene; organ of the
Galton Society, of the International Feder-
ation of Eugenic Organizations, and of the
Pan American Office of Eugenics and Homi-
culture 1916: Eugenics Research Associa-
tion, Cold Spring Harbor. 1-23
*Eugenical News; Supplement; Bibliographica

Eugenica 1927: 1-2 (1934)
*Eugenics; a journal of race betterment 1928:
American Eugenics Society, Inc. 1-4, no. 2
(1931)

Eugenics Laboratory Memoirs 1907: Univer-
sity of London; Francis Galton Laboratory
for National Eugenics. 1-7; 9-28 (1933)

Eugenics Lecture Series (1-9 as Eugenics
Laboratory Lecture Series) 1911: Univer-
sity of London; Galton Laboratory for Na-
tional Eugenics. 1-14 (1927)
*Eugenics Record Office Bulletin 1911: Car-
negie Institution of Washington, Cold Spring
Harbor. 1-27
*Eugenics Record Office Memoirs 1912: Cold

Spring Harbor. 1-2 (1912)
*Eugenics Record Office Report 1913: Cold
Spring Harbor. 1

Eugenics Review 1909: Eugenics Society, Lon-
don. 1 +

Evolution; a journal of nature 1927: New York
City. 1-4, no. 2 (1938)

Experiment Station Record 1889: United
States Department of Agriculture; Office
of Experiment Stations. 1 +
*Experimentelle Beitrage zur Morphologic;
herausg. von Hermann Braus (Heidelberg)
1906: 1-2, no. 1

Explorations des Lacs de 1'U.R.S.S. ; Institut
Hydrologique, Service Hydro-Meteorolo-
gique (Issledovaniia Ozer S.S.S.R.; Ediniia
Gidro-Meteorologicheskaia Sluzhba, Gosu-
darstvennyi Gidrologicheskii Institut) 1932:
1-7

Explorations des Mers de 1'U.R.S.S.; Institut
Hydrologique, Service Hydro-Meteorolo-
gique (Issledovanifa Morei S.S.S.R.; Edinaia
Gidro-Meteorologicheskaia Sluzhba, Gosu-
darstvennyi Gidrologicheskii Institut) 1925:
1-6; 11; 15-20

Ezhegodnik Zoologicheskogo Muzefa; Aka-
demiia Nauk SSSR see Annuaire du Muse"e
Zoologique ; Academic des Sciences de 1'URSS

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Farmers' Bulletin 1889: United States Depart-
ment of Agriculture. [2-1910] +

Fauna Brasiliense ; Museo Nacional do Rio de
Janeiro 1924: n.s. 1-2

Fauna e Flora del Golfo di Napoli; Pubblicata
dalla Stazione Zoologica di Napoli (1-34 as
Fauna und Flora des Golfes von Neapel und
der Angrenzenden Meeresabschnitte heraus-
gegeben von der Zoologischen Station zu
Neapel) 1880: 1 +

Fauna et Flora Laurentianae 1936: Station
Biologique du Saint-Laurent; Trois-Pistoles.
1 +

*Fauna Hawaiiensis; Bernice P. Bishop Mu-
seum of Polynesian Ethnology and Natural
History (also vol. 6, as Special Publication;
Bernice P. Bishop Museum) 1899: 1-3

Fermentforschung 1916: 1 +

Feuille des Jeunes Naturalistes; Revue men-
suelle d'Histoire Naturelle 1870: Paris, ser.
4, 35-44

*Field and Forest; a monthly journal devoted
to the natural sciences 1875: 1-3

Field and Laboratory; Contributions from the
Science Departments of Southern Methodist
University 1932: 1 +

Field Engineers Bulletin: United States Coast
and Geodetic Survey. 5 +

Field Museum of Natural History (Field Co-
lumbian Museum, 1894-1905) Publications;
Botanical Series 1895: 1 +

Field Museum of Natural History Publica-
tions; Geological Series 1895: 1-5; [6-7];
8+

*Field Museum of Natural History Publica-
tions; Ornithological Series 1896: 1

Field Museum of Natural History Publica-
tions; Report Series 1894: 1 +

Field Museum of Natural History Publica-
tions; Zoological Series 1895: 1-12; [13];
14+

*Finlandische Hydrographisch-Biologische Un-
tersuchungen 1907: Societas Scientiarum
Fennica. 1-10; 12-14

Fish Bulletin; State of California Department
of Natural Resources; Division of Fish and
Game; Bureau of Marine Fisheries 1917: 2-4;
8 +

Fisheries Bulletin; New Zealand, Marine De-
partment 1927: 1 +

Fisheries News Bulletin 1929: Department of
Fisheries, Ottawa. 1-2; 6+

*Fisheries Service Bulletin; United States De-
partment of the Interior; Fish and Wildlife
Service (2-289 as Fisheries Service Bulletin;
Department of Commerce; Bureau of Fish-
eries) 1915: 2-6; 8-14; 16-307

Fishery Board for Scotland; Salmon Fisheries
1910: 1920+

Fishery Board for Scotland ; Scientific Investi-
gations 1909: 1910+

Fishery Bulletin; Fisheries and Marine Bio-
logical Survey Division; Department of
Commerce and Industries 1935: Union of
South Africa. 1 +

*Fishery Circular; United States Bureau of
Fisheries 1931: United States Department
of Commerce. 1-28

Fishery Investigation (Supplementary Report)
1934: Imperial Fisheries Experimental Sta-
tion, Tokyo. 1 +

Fishery Investigations; Ministry of Agricul-
ture and Fisheries, London; series 1, Fresh-
water Fisheries and Miscellaneous 1913:
1 + ; series 2, Sea Fisheries 1914: 1 + ;
*series 3, Hydrography 1919: 1-4

Fiskeridirektoratets Skrifter; serie Havun-
ders0kelser see Report on Norwegian Fish-
ery and Marine Investigations

Fiziologicheskii Zhurnal SSSR (Journal of
Physiology of the USSR) (1-11 as Russkii
Fiziologicheskii Zhurnal, Journal Russe de
Physiologie; 12-14, Russian Journal of
Physiology) 1917: 1-15; [16-17]; 18-22;
[23]; 25 +

Flora oder Allgemeine Botanische Zeitung
1818: [86]; [117]; 118+

Flora Ottawaensis see Canadian Field-Natu-
ralist

Flora Polska; Rosliny Naczyniowe Polski i
Ziem Osciennych: Polskiej Akademji Umie-
jetnosci. 3—5

Flora y Fauna Peruanas 1940: Ministerio de
Fomento, Direccion de Agricultura, Gana-
deria y Colonization, Peru. 1 +

Folia Anatomica Japonica see Okajimas Folia
Anatomica Japonica

Folia Anatomica Universitatis Conimbrigensis
1926: Portugal. 1 +

Folia Biologica; publication del personal tec-
nico del Institute Bacteriologico del De-
partamento Nacional de Higiene 1931:
Buenos Aires. 1 +

Folia Endocrinologica Japonica 1925: 2 +

Folia Haematologica ; Internationales Maga-
zin fur Klinische und Morphologische Blut-
forschung (1-8 as Internationales Zentral-
organs fur Blut- und Serumforschung) (9-24
in 2 series: Teil 1, Archiv; Teil 2, Zentral-
Organ) 1904: 1 +

*Folia Microbiologica; Nederlandsch Tijd-
schrift voor Mikrobiologie 1912: 1-5

Folia Morphologica ; Organ Polskiego Towar-
zystwa Anatomiczno-Zoologicznego (War-
szawa) (Bulletin de la Societe Polonaise
d'Anatomie et de Zoologie) 1929: 1 +

*Folia Neuro-Biologica; Internationaal Cen-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

traalorgaan voor de Biologic van het Ze-
nuwstelsel 1907: Leipzig and Haarlem. 2-12

Folia Zoologica et Hydrobiologica ; Latvijas
Universitates Sistematiskas Zoologijas In-
stituta un Hidrobiologiskas Stacijas Raksti
(Organ des Systematisch-Zoologischen In-
stituts und der Hydrobiologischen Station
der Universitat Lettlands, Riga) 1929: 1 +

Fb'rhandlingar i Videnskapsselskapet i Kris-
tiania 1858: 1858-1924

Forschungen zur Geschichte der Optik see
Zeitschrift fur Instrumentenkunde; Beilage-
hefte

*Forschungsberichte aus der Biologischen Sta-
tion zuPlon 1893: 1-12

Fortschritte der Botanik ; Unter Zusammenar-
beit mit Mehreren Fachgenossen 1931: 1 +
*Fortschritte der Chemie, Physik und Physika-
lischen Chemie 1909: 1 +

Fortschritte der Medizin; Die Zeitschrift fiir
den Praktischen Arzt 1883: 51 +

Fortschritte der Zoologie 1935: 1 +

Fragmenta Faunistica Musei Zoologici Polo-
nici 1930: Panstwowego Muzeum Zoolo-
gicznego. 1—4, no. 13

Franklin Journal and American Mechanics'
Magazine see Journal of the Franklin Insti-
tute

Fukuoka Ikwadaigaku-Zasshi (Fukuoka Acta
Medica): Kyushu Imperial University. 25,
no. 3 — vol. 31

Fundamenta Radiologica; International Jour-
nal of Biophysics, Photochemistry, Photo-
biology and Medical Radiology (1-3 as Ra-
diologica) 1937: Berlin. 1 +

Fysiografiska Sallskapets Handlingar see
Lunds Universitets Arsskrift

Fysiografiska Sallskapets i Lund; Forhandlin-
gar (Proceedings of the Royal Physiographic
Society at Lund) 1931: 1 +

"Gann"; the Japanese Journal of Cancer Re-
search 1907: Japanese Cancer Association
and Japanese Foundation for Cancer Re-
search. 17-21; 24+

Gazzetta Chimica Italiana; Pubblicazione
mensile a cura dell'Associazione Italiana di
Chimica 1871: [51-58]; 62 +

Gegenbaurs Morphologisches Jahrbuch; eine
Zeitschrift fiir Anatomie und Entwicklungs-
geschichte (1-29 as Morphologisches Jahr-
luich) (53, no. 3+ as Jahrbuch fiir Morpho-
logic und Mikroskopische Anatomie, Abt. 1)
1875: 1 +

General Electric Review (1-8, no. 5 as Gen-
eral Electric Company Review) 1903: 1 +

Genetic Psychology Monographs 1926: [1-19]

Genetica; Nederlandsch Tijdschrift voor Er-
felijkheids- en Afstammingsleer 1919: 1 +

Genetics; a periodical record of investigations
bearing on heredity and variation 1916:
Brooklyn Botanic Garden. 1 +

Geofysiske Publikasjoner ; utgitt av det Norske
Videnskaps-Akademie i Oslo 1920: 1 +

Geografiska Annaler; utgivna av Svenska
Sallskapet for Antropologi och Geografi
1919: Stockholm. 1 +

Geographical Journal 1893: Royal Geographi-
cal Society, London. 1 +
*Geographical Magazine 1874: London. 1-5

Geographical Review; American Geographical
Society of New York 1916: 1 +

Geological Survey Bull etui 1883: United States
Department of the Interior. [1-814]

Geological Survey Water-Supply Paper:
United States Department of the Interior:
[72, 1902, -772, 1936]

Geologie der Meere und Binnengewasser;
Zeitschrift fiir Marine und Limnische Hy-
drogeologie und ihre Praktische Anwendung
1937: 1 +

Geophysical Magazine 1926: Central Meteoro-
logical Observatory, Tokyo. 1 +

Geophysical Memoirs; Meteorological Office
1912: London. [1-3]; 4; [5]; 6+

Geophysical Supplements to the Monthly No-
tices of the Royal Astronomical Society
1922: 1 +

Gerlands Beitrage zur Geophysik (1-10 as
Beitrage zur Geophysik; subtitles added as
follows: for vol. 1, only, Abhandlungen aus
dem Geographischen Seminar der Universi-
tat Strassburg; 2, Zeitschrift fiir Physika-
lische Erdkunde; 7, Zugleich Organ der Kai-
serlichen Hauptstation fiir Erdbebenfor-
schung zu Strassburg i. E; 13, 1914 -14,
1918, Beilage: Mitteilungen des Zentral-
bureaus der Internationalen Seismologischen
Assoziation) 1887: 1+ Erganzungsband
1902-04: 3; Supplement Band; Ergebnisse
der Kosmischen Physik 1931: 1 +

Gerlands Beitrage zur Geophysik; Ergan-
zungshefte fiir Angewandte Geophysik see
Beitrage zur Angewandten Geophysik
*Gidrobiologicheskii Zhurnal SSSR (Hydro-
biologische Zeitschrift d.UdSSR; heraus-
gegeben an der Biologischen Wolga-Station
unter Redaktion von A. L. Behning) (1-8 as
Russkii Gidrobiologicheskii Zhurnal) (Rus-
sische Hydrobiologische Zeitschrift) 1921:
1-9, no. 6

Giornale di Biologia applicata alia Industria
chimica see Bollettino Scientifico della Ea-
colta di Chimica Industriale. Bologna
*Giornale di Biologia e Medicina Sperimentale
1923: 1-2

Giornale di Biologia Industriale, Agraria ed

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Alimentare see Bollettino Scientifico della
Facolta di Chimica Industriale. Bologna
*Glasgow Naturalist 1909: journal of the Natu-
ral History Society of Glasgow. 1-8, no. 5

Glasnik Botanichkog Zavoda i Bashte Uni-
verziteta u Beogradu see Bulletin de 1'Ins-
titut et du Jardin Botaniques de 1'Univer-
site de Beograd

Glasnik Centralnog Higijenskog Zavoda (Zeit-
schrift fur Experimentelle und Angewandte
Medizin, hgg. v. Zentralhygienischen Insti-
tut; Revue de la Medicine Experimentelle
et Pratique, edit, par 1'Institut Central
d'Hygiene) 1926: Belgrade. 1 +

Glasnik Hrvatskoga Prirodoslovnoga Drustva
(1-19 as Glasnik Hrvatskoga Naravoslov-
noga Drustva) 1886: Societas (Historico-
naturalis) Scientiarum Naturalium Croa-
tica, Zagreb. 2-10; [12-13]; 20-21; [27]

Godisnjak Oceanografskog Instituta Kralje-
vine Jugoslav! je (Annuaire de 1'Institut
Oceanographique du Royaume de Yougo-
slavie) 1938: Split. 1 +

Goteborgs Kungl. Vetenskaps- och Vitterhets-
samhalles; Handlingar; Serien B. Matema-
tiske och Naturvetenskapliga Skrifter 1778:
ser. 5 (1928), 1 +

Growth; a journal for studies of development
and increase 1937: 1 +

Guide Leaflet Series; American Museum of
Natural History (1-15 as Supplement to
American Museum Journal; 16+ as reprint
from Natural History) 1901: 1-5; 8-10; 12-
14; [16-65]; 68 +

Guide to Nature 1908: Agassiz Association,
Sound Beach, Connecticut. 1-10; [11-22]

Halbjahrsverzeichnis der Neuerscheinungen

des Deutschen Buchhandels: 1921 +
*Half- Yearly Journal of the Mysore University

1927: 1-8

Harvard Forest Bulletin 1921: 1-3; 5 +
Harvard Meteorological Studies 1934: Blue

Hill Meteorological Observatory. 1 +
*Harvard University Tercentenary Gazette

1936: 1-8
Harvey Lectures 1905: Harvey Society of New

York. 1 +
Havsforskningsinstitutets Skrift; Merentut-

kimuslaitoksen Julkaisu 1920: Helsingfors.

1-114; 118 +
*Heart ; a journal for the study of the circulation

1909: London. 1-16
Hedwigia; Organ fur Kryptogamenkunde und

Phytopathologie nebst Repertorium fur Li-

teratur 1852: 1 +
Helgolander Wissenschaftliche Meeresunter-

suchungen ; im Au'trage des Reichsministe-

riums fur Wissenschaft, Erziehung und

Volksbildung 1937: herausgegeben von der
Biologischen Anstalt auf Helgoland. 1 +
*Helios; Abhandlungen und Mitteilungen aus
dem Gesamtgebiete der Naturwissenschaf-
ten; Organ des Naturwissenschaftlichen
Vereins des Regierungsbezirks; Frankfurt
(Oder) 1883: 21-22; 24-26

Helminthological Abstracts (incorporating Bib-
liography of Helminthology for the year
1934+) 1932: 1 +

Helvetica Biologica Acta see Verhandlungen
des Vereins der Schweizer Physiologen

Helvetica Chimica Acta; Edita a Societate
Chimica Helvetica 1918: 1 +

Helvetica Physica Acta; Societatis Physicae
Helvetica Commentaria Publica 1928: 1 + ;
Supplementum 2, 1929; 7, 1934

Hereditas; Genetiskt Arkiv; Uttgivet av Men-
delska Sallskapet i Lund 1920: 1 +

Herpetologica; a journal devoted to the study
of reptiles and amphibians 1936: Chicago
Academy of Sciences. 1 +

Highlands Museum and Biological Laboratory
Publication 1930: North Carolina. 1-3

Hilgardia; a journal of agricultural science
1925: California Agricultural Experiment
Station. 1 +

Hofmeister's Beitrage see Beitrage zur Chemi-
schen Physiologie und Pathologie

Hong Kong Naturalist; a quarterly illustrated
journal principally for Hong Kong and
South China 1930: 3, no. 2; supplement
no. 4

Hoppe-Seyler's Zeitschrift fiir Physiologische
Chemie (1-20 as Zeitschrift fiir Physiolo-
gische Chemie) 1877: 1+ see also Medici-
nisch-Chemische Untersuchungen ; Aus dem
Laboratorium fiir Angewandte Chemie zu
Tubingen herausgegeben von Dr. Felix
Hoppe-Seyler

*Horticulturist, a journal of rural life, literature,
art and rural taste 1846: New York. [2-26]

Hull Bulletins of Marine Ecology 1939: Uni-
versity College. 1 +

Human Biology; a record of research 1929: 1 +
*Humboldt Library of Science 1879: New York.
[1-150]

Huxley Memorial Lectures 1925: Imperial Col-
lege of Science and Technology. 1925 +

Hvalradets Skrifter; Scientific results of ma-
rine biological research 1931: Norske Vi-
denskaps-Akademi i Oslo. 1 +

Hydrobiologische Zeitschrift d.UdSSR see Gi-
drobiologicheskii Zhurnal SSSR

Hydrographic Review; International Hy-
drographic Bureau (La Revue Hydro-
graphique; Bureau Hydrographique In-
ternational) 1923: Monaco. 1 +

Hydrographische Mittheilungen see Annalen

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

der Hydrographie und Maritimen Meteoro
logie

Hydrological Conference of the Baltic States :
5, Finland, 1936; 6, Berlin, 1938

Hygienic Laboratory Bulletin see National In-
stitute of Health Bulletin

Ibis; a quarterly journal of ornithology 1859:
London, ser. 5, 1, no. 3; ser. 8, 1-6; ser. 9,
1-4

Idojaras: Magyar Meteorologiai Tarsasag
Folyoirata, Budapest. 29, 1925 +

Illinois Biological Monographs 1914: Uni-
versity of Illinois. 1 +

Illinois Natural History Survey Bulletin (no.
1 as Bulletin of the Illinois Museum of
Natural History; 1, no. 2 — vol. 12 as Bulle-
tin of the Illinois State Laboratory of Na-
tural History; 13-19 as Bulletin of the Illi-
nois State Natural History Survey) 1876:
1-10; [11]; 12 +

Imperial Bureau of Animal Genetics; Animal
Breeding Abstracts 1933: Edinburgh. 1, no. 3

Imperial Bureau of Plant Genetics (for crops
other than herbage); Plant Breeding Ab-
stracts 1933: Cambridge, England. Supple-
ment 1

Index-catalogue of the Library of the Surgeon
General's Office 1880: United States Army
Medical Library. 1 +

*Index Medicus; a monthly classified record of
the current medical literature of the world
1879: Carnegie Institution of Washington,
ser. 1-3 (June, 1927)

Index of Publications of the Bureau of Chem-
istry and (the Bureau of) Soils 1939: United
States Department of Agriculture. 1 (List
of titles and authors) 1862-1937

Index Universalis ; Dissertationum originalium
artis medicinae; E Libellis Periodicis Ex-
tractus; Editus a Collegii Medicinae Man-
jurici Curatorio de Compilando Indice:
Manchuria Medical College. 2, 1925 +

Indian Journal of Agricultural Science 1931:
Imperial Council of Agricultural Research,
Delhi. 1-9; [10]; 11 +

Indian Journal of Medical Research 1913: In-
dian Research Fund Association, Calcutta.
1 +

Indian Journal of Physics 1926: 1-2, no. 2;
4+ ; and Proceedings of the Indian Associa-
tion for the Cultivation of Science 1915:
10-11, no. 2; 13 +

Indian Journal of Veterinary Science and Ani-
mal Husbandry 1931: Imperial Council of
Agricultural Research, Delhi. 1 +

Indian Medical Research Memoirs 1924: In-
dian Research Fund Association, Calcutta.
1 +

Indian Meteorological Memoirs see Memoirs
of the India Meteorological Department

Indian Zoological Memoirs; on Indian animal
types 1926: Lucknow. 1-8

Indiana University Publications; Science Se-
ries 1935: 2 +

Indiana University Studies 1910: 1-23, no. 113

Industrial and Engineering Chemistry; Indus-
trial Edition (1-14 as Journal of Industrial
and Engineering Chemistry) 1909: 1 + ;
Analytical Edition 1929: 1 +

Industrial and Engineering Chemistry; News
Edition see Chemical and Engineering News
*Insect Life; United States Department of Ag-
riculture, Division of Entomology; periodi-
cal bulletin 1888: 1-7

Insektenborse ; Entomologische Zeitschrift
(Anzeigenblatt) 1884: des Internationalen
Entomologischen Yereins. 45—53

1'Institut Oceanographique de Monaco; Rap-
port pour 1'Annee: 1937 +
*Instituto de Pesca; section Laboratorio; Re-
publica Oriental del Uruguay; Montevideo
1923: 1923-25

Instrument World 1928: London. 1-5, no. 49

Instruments; the magazine of measurement
and control (1-4 as Instruments; Industrial-
Scientific; devoted to measurement and con-
trol problems) Instruments Publishing Co.
1 +

International Anatomical Congress 1905: 1-4
see in Verhandlungen der Anatomischen Ge-
sellschaft 19, 24, 39, 44

International Botanical Congress; Proceedings
(4 as Proceedings of the International Con-
gress of Plant Sciences) 1900: 3—6

International Cancer Research Foundation;
Report of Activities 1933: Philadelphia.
1933 +

International Catalogue of Scientific Litera-
ture 1901: International Council of the
Royal Society of London. L. General Biol-
ogy, 6-14; M. Botany, 14; N. Zoology, 1

International Congress for Applied Mechanics
see Proceedings of the International Con-
gress for Applied Mechanics

International Congress for Experimental Cy-
tologists see in Archiv fur Experimented
Zellforschung 6+

International Congress for Light Study; Pro-
ceedings: Wiesbaden, 1936 see in Strahlen-
therapie 61

International Congress for Theoretical and Ap-
plied Limnology see Verhandlungen der In-
ternationalen Vereinigung fur Theoretische
und Angewandte Limnologie

International Congress of Entomology; Pro-
ceedings 1910: 1-5; 7

International Congress of Fisheries ; Proceed-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

ings. 4th (1908) see in Bulletin of the United
States Bureau of Fisheries 28, pts. 1-2

International Congress of Genetics (1, 1899,
as Hybrid Conference Report, Journal of
the Royal Horticultural Society, vol. 24,
April, 1900; 2, 1902, as Int. Conference on
Plant Breeding and Hybridization, Mem-
oirs, Horticultural Soc. of New York, vol.
1, 1902; 3, 1906, as Report on the Third Int.
Conf. on Genetics, Royal Horticultural So-
ciety, London; 4, 1911, as Comptes Rendus
et Rapports, Conference int. de Genetique,
Paris, 1913; 6, 1932, Ithaca; Brooklyn Bo-
tanic Garden) 1899: 1+ (5, Berlin, 1927
see in Zeitschrift fur Induktive Abstam-
mungs und Vererbungslehre, Supplement-
band 1-2, 1928; 7, Edinburgh, 1939 see in
Journal of Genetics, Supplementary Volume
• 1941)

International Congress of Medical Sciences
(10, Berlin, 1890 as Verhandlungendes Inter-
nationalen Medicinischen Congresses vols.
1-5; 13, vol. 1, Paris, 1900 as Congres In-
ternational de Medicine, Comptes Rendus,
Section d'Anatomie descriptive et comparee,
Sec. d'Histologie et d'Embryologie, Sec. de
Physiologic, Physique et Chimie Biologique)
1867: 10; 11; 13, vol. 1 (11, Rome, 1894
see in Archives Italiennes de Biologie, vol.
22, 1895)

International Congress of Microbiology 1930:
1-3

International Congress of Oceanography, Ma-
rine Hydrography and Continental Hydrol-
ogy (1 as Congreso Internacional de Oceano-
grafia Hidrografia Marina e Hidrologia
Continental Seville) 1929: 1 (2 vols. 1930-
31)

International Congress of Phonetic Sciences;
Proceedings 1932: 1 see in Archives Neer-
landaises de Phonetique Experimentale 7,
1932

International Congress of Photography see
Proceedings of the International Congress
of Photography

International Congress of Radiology; Synop-
sized Abstracts from the Congress: 4, 1934
see in Acta Radiologica 15, 1934

International Congress of Soil Science 1927:
1 (Washington); 3, vol. 1 (London, 1935)

International Federation for Documentation;
Transactions: 14 (London) 1938

International Geodetic and Geophysical Union
see Union Geodesiqtie et Geophysique Inter-
nationale

International Hydrographic Conference see
Report of the Proceedings of the Interna-
tional Hydrographic Conference

International Medical Congress see Yerhand-
lungen des International Medicinischen
Congresses

International Neurological Congress; Pro-
ceedings 1931: 1 (Berne); 2 (London)
(1935) see in British Medical Journal, 1935,
vol. 2, pp. 223-25; 269-72

International Physiological Congress; Pro-
ceedings 1899: 1+ (1, Basel, Verhand-
lungen, Kurzer Abrisse, see in Zentralblatt
fur Physiologic, vol. 3, pp. 305-24; 2, Liege,
1892, ibid. vol. 6, pp. 395-409 and Revue
generale des Sciences pures et appliquees,
1892, pp. 734-62; 3, Berne, 1895, Zentr. f.
Physiol. vol. 9, pp. 465-80; 4, Cambridge,
England, 1898, Journal of Physiology vol.
23, Supplement and Zentr. f. Physiol. vol.
12, pp. 483-504; 5, Turin, 1901, Archives
Italiennes de Biologie, vol. 36 and Zentr.
f. Physiol. vol. 15, pp. 479-500; 6, Brussels,
1904, Arch. Int. de Physiol. vol. 2, 1904-05;
7, Heidelberg, 1907, ibid. vol. 5, 1907; 8,
Vienna, 1910, ibid. vol. 10, 1910; 9, Gro-
ningen, 1913, Arch. Int. de Physiol. vol. 14,
1913, Archivio di Fisiologia, vol. 12, 1914,
and Zentr. f. Physiol. vol. 27, Erganzungs-
heft, 1914; 10, Paris, 1920, Arch. Int. de
Physiol. vol. 15, 1914-20; 11, Edinburgh,
1923, Quart. Jour. Exp. Physiol. supple-
mentary volume; 12, Stockholm, 1926,
Skand. Arch. f. Physiol. vol. 49, 1926; 13,
Boston, 1929, American Jour. Physiol. vol.
90, 1929; 14, Rome, 1932, Archivio di Scienze
Biologiche, vol. 18, 1933; 15, Moscow-
Leningrad, 1935, Fiziologicheskii Zhurnal
SSSR vol. 21, 1936; 16, Zurich, 1938, Kon-
gressbericht I-III

International Zoological Congress; Proceed-
ings 1889: 1 +

Internationale Gesellschaft fur Biologische
Rhythmusforschung (Verhandlungen der
Zweiten Konferenz der Internationalen
Gesellschaft fur Biologische Rhythmus-
forschung): 2 (1939, Utrecht) see in Acta
Medica Scandinavica; Supplementum, no.
108, 1940

Internationale Monatsschrift fur Anatomie
und Physiologic 1884: 1-32

Internationale Revue der Gesamten Hydro-
biologie und Hydrographie ; herausgegeben
von R. Woltereck. 1+ *Biologisches Sup-
plement 1-7; Hydrographisches Supplement
1-5; *Literarisches Supplement 1-2

Internationale Seismologische Konferenz;
Verhandlungen 1901: 1-2 (Strassburg) see
in Gerlands Beitrage zur Geophysik; Er-
gangsbande 1-2, 1902-04

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Internationale Vereinigung fur Theoretische
und Angewandte Limnologie see Verhand-
lungen der Internationalen Vereinigung fiir
Theoretische und Angewandte Limnologie
*Internationale Zeitschrift fur Physikalisch-
chemische Biologie 1914: 1-3, heft 2

Internationaler Zellforscherkongress see Ver-
handlungen des Internationalen Zellforscher-
kongresses

Investigaciones de la Estacion Limnologica de
Patzcuaro 1941: Mexico. 1 +

Investigational Report; Fisheries and Marine
Biological Survey Division; Department of
Commerce and Industries: Union of South
Africa. 5 +

*Investigational Report; United States Depart-
ment of Commerce; Bureau of Fisheries
1931: 1-2

Investigations of Indiana Lakes and Streams
1928: Indiana Dept. of Conservation, Div.
of Fish and Game; Dept. of Zool., Ind.
Univ. 1 +

"Iris", Dresden see Deutsche Entomologische

Zeitschrift "Iris"

*Irish Naturalist; a monthly journal of general
Irish natural history 1892: 14-15; [16]; 17-
24; [25]; 26-28; [29-30]; 31-33

Isforholdene i de Arktiske Have samt Havets
Overfladetemperatur i det Nordlige Atlan-
terhav og Davis-Straede see Nautisk-Me-
teorologisk Aarbog

Isis; international review devoted to the his-
tory of science and civilization; quarterly
organ of the History of Science Society and
of the International Academy of the History
of Science 1913: 1 +

Issledovanifa Morei S.S.S.R. see Explorations
des Mers de 1'U.R.S.S.

Issledovanifa Ozer S.S.S.R. see Explorations
des Lacs de 1'U.R.S.S.

Issledovanifa Reki Nevy i ee Basseina see
Etudes de la Neva et de son Bassin

Iwata Institute of Plant Biochemistry Publica-
tions 1924: Tokyo. 1 +

Izvestifa Akademii Nauk Soyousa Sovetskic
Socialisticheskic Respublik see Bulletin de
1' Academic des Sciences de 1' Union des Re-
publiques Sovietiques Socialistes

Izvestifa Bakinskoi Ikhtiologicheskoi Labora-
torii (Bulletins of Ichthyological Laboratory
of Baku) 1922: 1-2, no. 2

Izvestifa Biologicheskogo Nauchno-Issledova-
tel'skogo Instituta pri Permskom Gosu-
darstvennom Universitete see Izvestifa
Permskogo Biologicheskogo Nauchno-Issle-
dovatel'skogo Instituta

*Izvestifa Botanicheskogo Sada; Akademii
Nauk SSSR (Bulletin du Jardin Botanique
de 1'Academie des Sciences de 1'URSS)

(1-29 as Izvestiia. Glavnogo Botanicheskogo
Sada SSSR) (Bulletin du Jardin Botanique
Principal de 1'URSS) 1901: 26-30

Izvestiia Gosudarstvennogo Gidrologicheskogo
Instituta; Edinaya Gidro-Meteorologiche-
skaya Sluzhba Soyuza SSR, Gosudarstven-
nyi Gidrologicheskii Institut (Bulletin de
1'Institut Hydrologique) 1921: 1-55; 60-61;
64-88

Izvestiia Leningradskogo Nauchno-Issledova-
tel'skogo Ikhtiologicheskogo Instituta see
Bulletin of the Institute of Fresh-water
Fisheries

Izvestifa na Tsarskitye Prirodonauchni Insti-
tuti v Sofifa (Bulletin des Institutions Roya-
les d'Histoire Naturelle a Sophia) (Mit-
teilungen aus den K. Naturwissenschaft-
lichen Instituten in Sofia) 1928: 3

Izvestiia Permskogo Biologicheskogo Nauch-
no-Issledovatel'skogo Instituta (Bulletin de
1'Institut des Recherches Biologiques de
Perm) (1-6 as Izvestiia Biologicheskogo
Nauchno-Issledovatel'skogo Instituta pri
Permskom Gosudarstvennom Universitete)
1922: 14-

Izvestifa po Prikladnoi Entomologii see Reports
on Applied Entomology

Izvestiia Tikhookeanskogo Nauchnogo Insti-
tuta Rybnogo Khozaistva i Okeanografii
(Vladivostok) see Bulletin (s) of the Pacific
Scientific Institute of Fisheries and Ocean-
ography

Izvestiia Tomskogo Gosudarstvennogo Uni-
versiteta see Transactions of Tomsk State
University

Izvestiia Vsesoiuznogo Nauchno-Issledova-
tel'skogo Instituta Ozernogo i Rechnogo
Rybnogo Khoziaistva see Bulletin of the
Institute of Fresh-water Fisheries

Jaarboek van de Koninklijke Belgische Aca-
demic see Annuaire de 1'Academie Royale de
Belgique

Jahrbuch der Hamburgischen Wissenschaft-
lichen Anstalten, Beiheft 2 see Mitteilungen
aus dem Hamburgischen Museum und Insti-
tut; Beihefte 3 see Mitteilungen aus den
Botanischen Staatsinstitut in Hamburg

Jahrbuch der St. Gallischen Naturwissen-
schaftlichen Gesellschaft 1858: 1904-05;
1907; 1910-18

Jahrbuch fiir Morphologic und Mikrosko-
pische Anatomic Abt. 1 see Gegenbaurs
Morphologisches Jahrbuch

Jahrbuch fiir Morphologic und Mikrosko-
pische Anatomic Abt. 2 see Zeitschrift fiir
Mikroskopisch-Anatomische Forschung

Jahrbucher der Zentralanstalt fiir Meteoro-
logie und Geodynamik 1848: N.F. 67

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Jahrbiicher des Nassauischen Vereins fur
Naturkunde (1-18 as Jahrbiicher des Ver-
eins fur Naturkunde im Herzogthum Nas-
sau) 1844: Weisbaden. 1-85

Jahrbiicher fur Wissenschaftliche Botanik;
Begriindet von Prof. Dr. N. Pringsheim
1858: 1 +

Jahresbericht; Physiologie und Experimen-
telle Pharmakologie (1-7 as Jahresbericht
iiber die Gesamte Physiologie und Experi-
mentelle Pharmakologie) 1920: 1 +

* Jahresbericht; Wissenschaftliche Biologie;

Bibliographisches Jahresregister der Be-
richte iiber die Wissenschaftliche Biologie
1926: 1-6

Jahresbericht der Gesellschaft von Freunden
der Naturwissenschaften in Gera (Reuss)
1858: 39-42; 49-52

Jahresbericht der K. Bohmischen Gesellschaft
der Wissenschaften see Vyrocni Zprava
Kralovske Ceske Spolecnosti Nauk

Jahresbericht der Kommission zur Wissen-
schaftlichen Untersuchungen der Deutschen
Meere in Kiel see Bericht der Kommission
zur Wissenschaftlichen Untersuchung der
Deutschen Meere in Kiel

Jahresbericht der Naturforschenden Gesell-
schaft in Emden (1842-52 as Jahresbericht
iiber die Verrichtungen) 1815: 1842-43;
1847-48; 1850-52; [1868-1913]

Jahresbericht der Naturhistorischen Gesell-
schaft zu Hannover 1850: 44-59

* Jahresbericht der Naturhistorischen Gesell-

schaft zu Niirnberg 1882: 1887; 1892-93;

1896; 1898-99; 1901-05
Jahresbericht der Schlesischen Gesellschaft

fur Vaterlandische Cultur 1824: Breslau.

43-44; 79; 84; 87; 89; 90
Jahresbericht des Frankfurter Vereins fur

Geographic und Statistik 1836: Frankfurt

a.M. 48-49
Jahresbericht des Naturhistorischen Vereins

von Wisconsin 1858: 1880-81
Jahresbericht (e) des Naturwissenschaftlichen

Vereins in Elberfeld 1847: 6; 9-12
Jahresbericht des Naturwissenschaftlichen

Vereins zu Bremen see in Abhandlungen

herausgegeben vom Naturwissenschaftlichen

Verein zu Bremen
Jahresbericht des Rheinischen Fischerei-

Vereins: Bonn. 1904-09
Jahresbericht des Vereins fur Naturwissen-

schaft zu Braunschweig 1879: 3; 6-19
*Jahresbericht des Vereins von Freunden der

Erdkunde zu Leipzig 1861: 1-8; 10-11
Jahresbericht iiber das K. K. Staatsgymna-

sium in Eger (Bb'hmen): 1910-11
Jahresbericht iiber die Deutsche Fischerei;

herausgegeben von Reichsministerium fiir

Ernahrung und Landwirtschaft 1924: 1925-
37

*Jahresbericht(e) iiber die Fortschritte der
Anatomic und Entwicklungsgeschichte 1 892 :
1-20

*Jahresbeiicht iiber die Fortschritte der Anato-
mic und Physiologie 1872: 1-20

* Jahresbericht iiber die Fortschritte der Ani-

malischen Physiologie (1-20 as Jahresbe-
richt iiber die Fortschritte der Physiologie)
1892: 1-22

*Jahresbericht iiber die Fortschritte der Tier-
Chemie oder der Physiologischen, Patho-
logischen und Immuno-Chemie und der
Pharmakologie 1871: 1-49

* Jahresbericht iiber die Fortschritte in der

Lehre von den Garungs-Organismen und
Enzymen 1890: 1-22

* Jahresbericht iiber die Fortschritte in der

Lehre von den Pathogenen Mikroorganis-
men, umfassend Bacterien, Pilze und Pro-
tozoen 1885: 1-27

Jahresbericht iiber die Gesamte Physiologie
und Experimentelle Pharmakologie see
Jahresbericht; Physiologie und Experi-
mentelle Pharmakologie

* Jahresbericht iiber die Leistungen und Fort-

schritte in der Anatomic und Physiologie
(1866-1907 as Jahresbericht iiber die Leis-
tungen und Fortschritte in der Gesamten
Medicin Abt. 1 ; 1908-16 as Fortsetzung von
Virchow's Jahresbericht) 1866: 1866-1916

Jahresbericht iiber die Tatigkeit der Deut-
schen Seewarte see in Annalen der Hydro-
graphic und Maritimen Meteorologie

Jahresbericht iiber die Verrichtungen (Wirk-
samkeit) und den Zustand der Naturfor-
schenden Gesellschaft in Emden see Jahres-
bericht der Naturforschenden Gesellschaft
in Emden

Jahresheft(e) des Naturwissenschaftlichen
Vereins fiir das Fiirstentum Liineburg 1865:
15-19

Jahresheft des Vereins fiir Mathematik und
Naturwissenschaften in Ulm a. D. see Mit-
teilungen des Vereins fiir Naturwissenschaft
und Mathematik in Ulm a. D.

* Japan Medical World; a monthly journal of

medicine, surgery and collateral sciences
1921: [1-2]; 3-10, no. 5

Japanese Journal of Astronomy and Geo-
physics; Transactions and Abstracts 1922:
National Research Council of Japan. 1 +

Japanese Journal of Botany; Transactions and
Abstracts 1922: National Research Council
of Japan. 1; [2]; 3 +

Japanese Journal of Experimental Medicine
(1-6 as Scientific Reports from the Govern-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

ment Institute for Infectious Diseases of the

Tokyo Imperial University) 1922: 1 +
Japanese Journal of Medical Sciences; I.

Anatomy 1927: National Research Council

of Japan. 3, no. 3 +
Japanese Journal of Medical Sciences; II.

Biochemistry 1925: ibid. 1 +
Japanese Journal of Medical Sciences; III.

Biophysics 1927: ibid. 1 +
Japanese Journal of Medical Sciences; IV.

Pharmacology 1926: ibid. [4-5]; 7 +
Japanese Journal of Medical Sciences; V.

Pathology 1926: ibid. 2 +
Japanese Journal of Medical Sciences; VI.

Bacteriology and Parasitology 1929: ibid.

1 +
Japanese Journal of Physics; Transactions

and Abstracts 1922: ibid. 1 +
Japanese Journal of Zoology; Transactions

and Abstracts 1922: ibid. 1-7; [8]; 9 +
Japanische Literatur zur Tuberkulosefor-

schung 1930: Anatomisches Institut der K.

Universitat, Kyoto. 1-5
Jenaische Zeitschrift fiir Naturwissenschaf t ;

herausgegeben von der Medizinisch-Natur-

wissenschaftlichen Gesellschaft zu Jena

(1-7 as Jenaische Zeitschrift fiir Medicin

und Naturwissenschaft) 1864: [1]; 2; [3];

4+
Johns Hopkins Hospital Reports 1896: 1-22,

no. 1

Johns Hopkins University Circular 1879: 1 +
Johnsonia 1941: published by the Department

of Mollusks; Museum of Comparative

Zoology, Harvard University, Cambridge,

Mass. 1 +
"Jornal de Sciencias Naturais; Sociedade

Portuguesa de Sciencias Naturais, Lisbon

1921: 1-3, no. 3
Journal and Proceedings of the Royal Asiatic

Society of Bengal (ser. 1 and 2 as Journal

and Proceedings of the Asiatic Society of

Bengal) (series 3 in 3 sections, Letters,

Science, and Yearbook) 1832: n.s. 1 +
Journal and Proceedings of the Royal Society

of New South Wales (1-8 as Transactions)

1867: 1-3; 6-7; 94-
Journal and Proceedings of the Royal Society

of Western Australia see Journal of the

Royal Society of Western Australia
Journal de Biologie (Moscow) see Biologiches-

kii Zhurnal
Journal de Biologie et de Medecine Experi-

mentales see Zhurnal Eksperimental'noi

Biologii i Meditsiny
Journal de Biologie Experimentale serie A see

Zhurnal Eksperimental'noi Biologii
Journal de Biologie Experimentale serie B see

Uspekhi Eksperimental'noi Biologii

* Journal de Botanique 1887: 1-22

Journal de Chimie Generale see Bulletin de
1'Academie des Sciences de 1'Union des Re-
publiques Sovietiques Socialistes; Classe des
Sciences Chimiques

Journal de Chimie Physique et de Physico-
Chimie Biologique (et Revue Generale des
Colloiides) (second title added with vol. 28,
when the two merged, dropped with 34)
1903: 1 +

*Journal de 1'Anatomie et de la Physiologie
Normales et Pathologiques de 1'Homme et
des Animaux 1864: 1-50

Journal de 1'Institut Botanique (de la Section)
de 1'Academie des Sciences d'Ukraine see
Zhurnal Institutu Botaniki Uan
*Journal de la Physiologie de 1'Homme et des
Animaux (Brown-Sequard) 1858: 1-6

Journal de la Societe Botanique de Russie see
Zhurnal Russkogo Botanicheskogo Obsh-
chestva

Journal de Pharmacie et de Chimie (1-5 as
Bulletin de Pharmacie; 6 as Bulletin de
Pharmacie et des Sciences Accessoires; ser.
2, 1-27 as Journal de Pharmacie et des
Sciences Accessoires) 1809: 1 +

Journal de Physiologie et de Pathologie Gene-
rale 1899: 1 +

Journal de Physique et le Radium (ser. 1-5 as
Journal de Physique Theorique et Appli-
quee; which with series 6 united with Le
Radium) 1872: 1 +

Journal de Psychologic Normale et Patho-
logique 1904: 23 +

Journal de Radiologie et d'Electrologie ; Revue
Medicale Mensuelle 1914: 1 +

Journal du Conseil 1926: Conseil Permanent
International pour 1'Exploration de la Mer.
1 +

Journal du Cycle Bio-Zoologique see Zhurnal
Bio-Zoologichnogo Tsiklu

Journal du Cycle Botanique de 1'Academie des
Sciences d'Ukraine see Zhurnal Institutu
Botaniki Uan

Journal Entomologique ; publie par la Societe
Entomologique a Stockholm see Entomolo-
gisk Tidskrift: utgiven av Entmologiska
Foreningen i Stockholm

Journal fiir Ornithologie 1853: 1-22; 24; 26-
86; Extraheft 1; Sonderheft 1906; 1917-18;
1920; 1924; 1926; 1928; 1930; 1932; 1934;
Jahresbericht 1-4; Erganzungsband 2

Journal fiir Psychologic und Neurologic 1902:
Leipzig. 49 +

Journal of Agricultural Research 1913: United
States Department of Agriculture with the
cooperation of the Association of Land-
grant Colleges and Universities. 1 +

Journal of Agricultural Science ; edited for the

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Plant Breeding and Animal Nutrition Re-
search Institutes at Cambridge and the
Rothamsted Research Institutes 1905: 14-

Journal of Agriculture of the University of
Puerto Rico (1-17 as Journal of the Depart-
ment of Agriculture of Porto Rico): Agri-
cultural Experiment Station, Rio Piedras.
[6-8]; 9+

Journal of Anatomy (1-52 as Journal of Anat-
omy and Physiology) 1867: London. 14-
*Journal of Animal Behavior 1911: 1-7

Journal of Animal Ecology 1932: British Eco-
logical Society. 1 +

* Journal of Applied Microscopy and Laboratory

Methods 1898: Bausch and Lomb Optical

Co. 1-6
Journal of Applied Physics (1-7 as Physics)

1931: American Institute of Physics. 1 +
Journal of Bacteriology 1916: 1 +
Journal of Biochemistry 1922: Tokyo. 14-
Journal of Biological Chemistry 1905: 1 +

* Journal of Biophysics 1923: Physiological In-

stitute of the Tokyo Imperial University.
1-2

Journal of Botany; British and Foreign 1863:
1 +

Journal of Cancer Research see American
Journal of Cancer

Journal of Cellular and Comparative Physi-
ology 1932: 14-

Journal of Chemical Education 1924: 14-

Journal of Chemical Physics 1933: American
Institute of Physics. 14-

Journal of Clinical Endocrinology 1941: 1 +

Journal of Clinical Investigation 1924: 1 +

Journal of Comparative Neurology (14-20 as
Journal of Comparative Neurology and Psy-
chology) 1891: 1 +

Journal of Comparative Psychology 1921: 1 +

Journal of Dental Research 1919: Interna-
tional Association for Dental Research. 1 +

Journal of Ecology 1913: British Ecological
Society. 1 +

Journal of Economic Entomology; official or-
gan of the Association of Economic En-
tomologists 1908: 1, no. 3; 29, no. 4

Journal of Endocrinology 1939: Oxford. 1 +

Journal of Entomology and Zoology (1 as
Pomona Journal of Entomology; 2-4 as
Pomona College Journal of Entomology)
1909: Pomona College Department of Zool-
ogy. 14-

Journal of Experimental Biology (1-6 as The
British Journal of Experimental Biology;
edited by J. Gray (1-2 by F. A. E. Crew)
1923: 1 +

Journal of Experimental Medicine 1896:
Rockefeller Institute of Medical Research.
1 +

Journal of Experimental Zoology 1904: \Yistar
Institute of Anatomy and Biology. 14-

Journal of General Physiology 1918: Rocke-
feller Institute of Medical Research. 1 +

Journal of General Psychology 1928: 14-

Journal of Genetics; edited by R. C. Punnett
(formerly by W. Bateson and Punnett)
1910: Cambridge, England. 1 +

Journal of Geophysics and Meteorology see
Zhurnal Geofiziki i Meteorologii

Journal of Helminthology 1923: London. 1 + ;
*Supplement; Protozoology 1925: 1-4

Journal of Heredity (1-4 as American Breeders'
Magazine) 1910: American Genetic Associa-
tion. 1 +

Journal of Hygiene 1901: Cambridge, England.
1 +

Journal of Immunology 1916: 14-

Journal of Industrial and Engineering Chem-
istry see Industrial and Engineering Chem-
istry

Journal of Infectious Diseases; founded by the
John Rockefeller McCormick Memorial In-
stitute for Infectious Diseases 1904: 14-;
*Supplements 2-4

Journal of Laboratory and Clinical Medicine
1915: 1 +

Journal of Mammalogy; published quarterly
by the American Society of Mammalogists
1919: 1 +

Journal of Marine Research; Sears Founda-
tion for Marine Research; Bingham Ocea-
nographic Laboratory, Yale University
1937: 14-

* Journal of Medical Research; official publica-

tions of the American Association of Pathol-
ogists and Bacteriologists (1-5 as Journal of
the Boston Society of Medical Science) (6 +
also numbered as n.s. 1 + ) 1896: 1-44

* Journal of Metabolic Research 1922: Physi-

atric Institute, Morristown, N. J. 1-8
*Journal of Microscopy and Natural Science
(7-9 also as n.s. 1-3; 10-16 also as ser. 3,
1-7) (1-2 as The Journal of the Postal
Microscopical Society) 1882: London. 1-16
Journal of Morphology (40-51 as Journal of
Morphology and Physiology) 1887: 1 +

* Journal of Mycology 1885: [1-3]; [5]; 6-8;

[9-13]; 14

Journal of Neurophysiology 1938: 14-

Journal of Nutrition 1928: 1 +

Journal of Oceanography 1929: Imperial Ma-
rine Observatory, Kobe. 1 +

Journal of Organic Chemistry 1936: 1 +

Journal of Parasitology; the official organ of
the American Society of Parasitologists
1914: 14-

Journal of Pathology and Bacteriology; the

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

official journal of the Pathological Society
of Great Britain and Ireland 1893: 1 +

Journal of Pharmacology and Experimental
Therapeutics; official publication of the
American Society, in association with the
British Pharmacological Society 1909: 1 +

Journal of Philosophical Studies see Philosophy

Journal of Philosophy; founded by Frederick
J. E. Woodbridge 1904: New York City.
7; 9-14; 16-18; 20 +

Journal of Physical Chemistry 1896: 1-9; 10,
nos. 1-3; 11, nos. 2-4; 12; 13, nos. 1-3; 14 +

Journal of Physiology; edited for the Physio-
logical Society 1878: London. 1 +

Journal of Physiology of the USSR see Fizio-
logicheskii Zhurnal SSSR

Journal of Research of the National Bureau of
Standards (1-12 as Bureau of Standards
Journal of Research) 1928: 1 +

Journal of Science and Technology, Cawn-
pore, India; a bi-annual journal published
under the management of the Scientific So-
ciety, Harcourt Butler Technological Insti-
tute 1935: 1, no. 1; 2, nos. 1-2

* Journal of Science and the Arts (American re-

print of Quarterly Journal of Science) 1817:
New York. 1-5

Journal of Science of the Hiros(h)ima Univer-
sity ; series A, Mathematics, Physics, Chem-
istry 1930: 1 +

Journal of Science of the Hiros(h)ima Univer-
sity; series B, Division 1, Zoology; Division
2, Botany 1930: 1 +

Journal of Scientific Instruments 1923: Insti-
tute of Physics, England. 1 +

Journal of Sedimentary Petrology 1931: So-
ciety of Economic Paleontologists and Min-
eralogists, a Division of the American Asso-
ciation of Petroleum Geologists. 1 +

* Journal of the Academy of Natural Sciences of

Philadelphia 1817: 1-8; series 2, 1847: 1;

6-16
Journal of the Acoustical Society of America

1929: American Institute of Physics. 1 +
Journal of the Aeronautical Sciences (8-9, no.

5 include Aeronautical Review Section)

1934: Institute of the Aeronautical Sciences,

Inc. 1 +
Journal of the American Chemical Society

1879: 1 +
Journal of the American Dental Association

1914: 23 +
Journal of the American Medical Association

1883: 1-8; [9]; [13-16]; 17; [18]; 19-21; [23];

24; [25]; [27-28]; 29; [34]; 35; [36]; 37-38;

[40]; 41 +
Journal of the American Museum of Natural

History see Natural History; magazine of

the American Museum of Natural History

Journal of the American Pharmaceutical Asso-
ciation (1940+ issued in two parts; Scien-
tific edition, 29+ and Practical Pharmacy
edition, 1 + ) 1912: [3]; [12-14]; 15 + ; Scien-
tific edition, 29+; Practical Pharmacy edi-
tion, 1 +

Journal of the Arnold Arboretum, Harvard
University 1919: [1]; 2-3; [4]; 6 +

Journal of the Association of American Medi-
cal Colleges (1-3 as Bulletin of the Associa-
tion of American Medical Colleges) 1926:
1 +

Journal of the Biological Board of Canada see
Journal of the Fisheries Research Board of
Canada

Journal of the Biological Photographic Asso-
ciation 1932: 1 +

Journal of the Bombay Natural History So-
ciety 1886: 1 +

Journal of the Boston Society of Medical

Science see Journal of Medical Research
*Journal of the Cancer Research Committee of
the University of Sydney 1929: 1-8

Journal of the Chemical Society (1878-1923
in two parts: Transactions and Abstracts)
(for continuation of Abstracts 1924+ see
British Chemical and Physiological Ab-
stracts A; 1924-25 as Abstracts of Chemical
Papers A; 1926-37 as British Chemical
Abstracts A) 1847: London. 29+ ; Proceed-
ings (1-5 as Abstracts of the Proceedings)
1885: 1885 +

Journal of the Chemical Society of Japan
(Nippon Kwagaku Kwaishi) (formerly the
Journal of the .Tokyo Chemical Society)
1880: 47 +

*Journal of the Cincinnati Society of Natural
History 1878: 1-20; [21]; 22

Journal of the College of Agriculture; Hok-
kaido Imperial University see Journal of the
Faculty of Agriculture; Hokkaido Imperial
University

Journal of the College of Agriculture ; Imperial
University of Tokyo 1909: 1 +

Journal of the College of Agriculture ; Tohoku
Imperial University see Journal of the Fac-
ulty of Agriculture; Hokkaido Imperial
University

* Journal of the College of Science; Imperial

University of Tokyo 1887: 1-45

Journal of the Council for Scientific and In-
dustrial Research 1927: Melbourne, Aus-
tralia. 1 +

Journal of the Department of Agriculture;
Kyusyu Imperial University; Hukuoka
(Fukuoka), Japan 1923: 1 +

* Journal of the Department of Agriculture;

Union of South Africa 1920: 1-12, no. 5
Journal of the Department of Agriculture of

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Porto Rico see Journal of Agriculture of the
University of Puerto Rico

Journal of the East Africa and Uganda Natural
History Society 1910: Coryndon Memorial
Museum, Nairobi. 1, no. 2-5, no. 11, 13-20;
6+; Special Supplement 4-5

Journal of the Elisha Mitchell Scientific So-
ciety 1883: Chapel Hill, N. C. 1 +

Journal of the Faculty of Agriculture; Hok-
kaido Imperial University (1-2 as Journal of
the Sapporo Agricultural College; 3-7 as
Journal of the College of Agriculture, To-
hoku Imperial University; 8-19 as Journal
of the College of Agriculture; Hokkaido
Imperial University) 1902: 2 +

Journal of the Faculty of Science; Hokkaido
Imperial University 1930: ser. 4. Geology
and Mineralogy, 1; ser. 5. Botany, 1 + ; ser.
6. Zoology, 1 +

Journal of the Faculty of Science; Imperial
University of Tokyo 1925: section 1. Mathe-
matics, Astronomy, Physics, Chemistry,
1-3, no. 6; 4, no. 2; section 3. Botany, 1-5;
section 4. Zoology, 1-4, no. 3; section 5.
Anthropology, 1, no. 1

Journal of the Fisheries Research Board of
Canada (1-3 as Journal of the Biological
Board of Canada) 1934: 1 +

Journal of the Franklin Institute devoted to
science and the mechanical arts (1-6 as
Franklin Journal and American Mechanics'
Magazine) 1826: 1 +

Journal of the Hong Kong Fisheries Research
Station 1940: 1, nos. 1-2

Journal of the Imperial Fisheries Experi-
mental Station 1930: Tokyo. 1 +

Journal of the Imperial Fisheries Institute
1893: Tokyo. 20+

Journal of the Indian Chemical Society (1-4

as Quarterly Journal) 1924: 1 +
*Journal of the Indian Institute of Science,
Bangalore 1914: 1-7; A, 8-21; B, 8-17

Journal of the Linnean Society (Botany and
Zoology) 1856: 1 +

Journal of the Marine Biological Association
of the United Kingdom 1887: Plymouth,
England. 1 +

* Journal of the Maryland Academy of Sciences
1930: 1-3

Journal of the National Cancer Institute 1940:
Public Health Service, Federal Security
Agency. 1 +

Journal of the New York Botanical Garden
1900: 1 +

Journal of the New York Entomological So-
ciety 1893: [4]; [10]; [29]; 34 +

Journal of the New York Microscopical So-
ciety 1885: 1-7; 8, nos. 1-3; 9-14, no. 1

Journal of the Optical Society of America (6-19

as Journal of the Optical Society of America
and Review of Scientific Instruments) 1917:
1 +

*Journal of the Portland Society of Natural
History 1864: Maine. 1, no. 1

Journal of the Postal Microscopical Society
see Journal of Microscopy and Natural
Science

Journal of the Royal Agricultural Society of

England 1839: 1-17
*Journal of the Royal Geographical Society of

Londoi 1830: 1-50
* Journal (s) of the Royal Institution of Great

Britain 1802: 1

*Journal of the Royal Institution of Great
Britain 1830: 1-2

Journal of the Royal Microscopical Society
1878: 1 +

Journal of the Royal Society of Western Aus-
tralia (1-10 as Journal and Proceedings of
the Royal Society of Western Australia)
1914: Perth. 1-2; 5-9; 11 +

Journal of the Sapporo Agricultural College
see Journal of the Faculty of Agriculture,
Hokkaido Imperial University

Journal of the Shanghai Science Institute
1933: 1 +

Journal of the Society for the Preservation of
the Fauna of the Empire 1904: Hertford,
England, n.s. 39

Journal of the Society of Chemical Industry
(37+ in three sections: Review, Transac-
tions and Abstracts) (for continuation of
Abstracts 1924+ see British Chemical and
Physiological Abstracts B; 43, 1924-25 as
Abstracts issued by Bureau of Chemical
Abstracts; 1926-37 as British Chemical Ab-
stracts B) 1882: London. 1 +

Journal of the Tennessee Academy of Science
1926: Nashville. 7 +

Journal of the Tokyo Chemical Society see

Journal of the Chemical Society of Japan
*Journal of the Trenton Natural History So-
ciety 1886: 1-2, no. 1

Journal of the Washington Academy of
Sciences 1911: 1 +

Journal of Tropical Medicine and Hygiene
(with which is incorporated "Climate")
1898: London. 30+

Journal of Urusvati Himalayan Research In-
stitute 1931: Roerich Museum, India. 3

Journal Russe de Physiologic see Fiziologi-

cheskii Zhurnal SSSR

*Journal Russe de Zoologie (Zoologicheskii
Vestnik) (Russian Journal of Zoology) 1916:
1-2, liv. 2; 3, liv. 1-2

Just's Botanischer Jahresbericht 1873: 1 +

Justus Liebigs Annalen der Chemie (1-32 as
Annalen der Pharmacie; 33-168 as Annalen

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

der Chemie und Pharmacie; 169-172 as
Justus Liebigs Annalen der Chemie und
Pharmacie) 1832: 1 +

*Kansas University Quarterly 1892: 1-10
Kansas University Science Bulletin 1902: 1 +
Kieler Meeresforschungen 1936: Institut fiir

Meereskunde der Universitat Kiel. 1 +
Kitasato Archives of Experimental Medicine

1917: Kitasato Institute for Infectious Dis-
eases. 1-7, no. 1
Klinische Wochenschrif t ; Organ der Gesell-

schaft Deutscher Naturforscher und Arzte

1922: 1 +
Koch's Jahresbericht see Jahresbericht iiber

die Fortschritte in der Lehre von den Ga-

rungs-Organismen und Enzymen
Kolloid-Beihefte ; Erganzungshefte zur Kol-

loid-Zeitschrift (1-32 as Kolloidchemische

Beihefte) 1909: 1 +
Kolloid-Zeitschrift; Zeitschrift fiir Wissen-

schaftliche und Technische Kolloidchemie

1906: 1 +
*K6nyvkiad6-vallalat 1873: K. Magyar Ter-

meszettudomanyi Tarsulat, Budapest. 27-28
*Korrespondenzblatt des Naturforscher-Ver-

eins zu Riga 1845: 5; 26-38; 42-45; 47-63
*Krankheitsforschung; Zwanglose Studien zur

Pathogenese 1925: Leipzig. 1-9
Kratkii Obzor Gidrologicheskikh Ekspedit-

sionno-Issledovatel'skikh i Polevykh Rabot :

Gosudarstvennyi Gidrologicheskii Institut,

Leningrad. 6, 1925; 9-10, 1928-29

Lancashire Sea-Fisheries Memoir 1899: 1-2

Lancet; a journal of British and foreign medi-
cine, surgery, etc. 1823: 1 +

Latvijas Biologijas Biedribas Raksti see Acta
Biologica Latvica

Latvijas Universitates Botaniska Darza Bardi

1926: Riga. 1

*Lavori fatti nell'Istituto di Anatomia compara
della R. Universita di Napoli 1885: 1-2; n.s.
1-3

*Leopoldina; Amtliches Organ der K. Leopol-
dinisch-Carolinischen Deutschen Akademie
der Naturforscher 1859: 2-58
*Leopoldina; Berichte der K. Deutschen Aka-
demie der Naturforscher zu Halle 1926: 1-6

Liebigs Annalen der Chemie see Justus Liebigs

Annalen der Chemie

*Lilly Scientific Bulletin 1912: Indianapolis.
1-10; ser. 2, 1

Lingnan Science Journal (1-4 as Lingnaam
Agricultural Review) 1922: Lingnan Uni-
versity, Canton. 1-20

*Linnaea Entomologica; Zeitschrift herausge-
geben von dem Entomologischen Vereine
in Stettin 1846: 1-3

Liverpool Marine Biology Committee Memoirs
on Typical British Marine Plants and Ani-
mals (1-30, 1899-1931 as Memoirs on Typi-
cal Marine Plants and Animals, Department
of Oceanography, University of Liverpool,
issued in the Proceedings and Transactions
of the Liverpool Biological Society 14-45)
1899: 1 +

*Liverpool School of Tropical Medicine; Mem-
oirs 1901: n.s. 1-3

Lloydia; a quarterly journal of biological sci-
ence 1938: Cincinnati. 1 +

Lotos; Naturwissenschaftliche Zeitschrift,
herausgegeben vom Deutschen Naturwis-
senschaftlich-Medizinischen Verein fiir
Bohmen, "Lotos" in Prag 1850: 57-58; 59,
no. 1 ; 61

*Lunds Universitets Arsskrift (Acta Universi-
tatis Lundensis) (26-40 also as Fysiogra-
fiska Sallskapets Handlingar, n.f. 1-15)
(continued in two series) 1864: 1-40

Lunds Universitets Arsskrift n.f. Afd. 2. Medi-
cin samt Matematiska och Naturvetenska-
pliga Amnen 1905: (1-30 also as Acta Uni-
versitatis Lundensis) (1+ also as n.f. 16+
of K. Fysiografiska Sallskapets Handlingar)
1 +

Luonnon Ystava; Elain-ja Kasvitieteellinen
Aikakausiehti (1900-18 as Luonnon Ystava;
Yleistajuinen Luonnontieteellinen Aikakaus-
iehti) 1897: Helsingfors. 4-28; 30+

Madras Fisheries Bulletin 1899: 1; 4 +
*Magazine of Natural History (1 — n.s. 1 as
Magazine of Natural History, and Journal
of Zoology, Botany, Mineralogy, Geology,
and Meteorology) 1828: 1-9; n.s. 1-4

Magyar Biologiai Kutatointezet Munkai (Ar-
beiten des Ungarischen Biologischen For-
schungs-Institutes) (1 as Archivum Balato-
nicum) 1926: Hungary. 1 +

Malayan Nature Journal; the journal of the
Malayan Nature Society 1940: 1, nos. 1-2

Malpighia; Rassegna Mensile di Botanica
1887: 1-28; 30 +

"Maly's Jahresbericht" see Jahresbericht iiber
die Fortschritte der Tier-Chemie oder der
Physiologischen, Pathologischen und Im-
muno-Chemie und der Pharmakologie

Marine Biological Report; Union of South
Africa; Province of the Cape of Good Hope
1912: 1-2

*Marine Investigations in South Africa (6+
included in the Annals of the South African
Museum) 1902: 1-4

Marine Observer 1924: Air Ministry; Mete-
orological Committee, London. 1-16, no. 135

Mariner's Mirror; quarterly journal of the So-
ciety for Nautical Research 1911: 20, no. 2

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Markische Tierwelt; Zeitschrift fur die Fau-
nistische Erforschung der Mark Branden-
burg (1-3 subtitle as Zeitschrift fur die
Faunistische Erforschung der Kurmarlc)
1934: 1 +

*Maryland Fisheries; published by Conserva-
tion Department of Maryland 1929: 1-11;
13-34

Massachusetts Institute of Technology; Me-
teorological Course; Professional Notes
1929: 1; 3; 5 +

Massachusetts Institute of Technology Mete-
orological Papers see Papers in Physical
Oceanography and Meteorology

Matematikai es Termeszettudomanyi Ertesito
(Mathematischer und Naturwissenschaft-
licher Anzeiger der Ungarischen Akademie
der Wissenschaften) 1882: Budapest. 49 +

Materialy k Poznaniiu Fauny i Flory SSSR
(Contributiones pro Fauna et Flora URPSS)
(Contributions a la Connaissance de la
Faune et la Flore de 1'URSS) Otdel Botani-
cheskii 1890: n.s. 1 (9) ; Otdel Zoologicheskii
1892: n.s. 1-4 (16-19)

Materialy po Izucheniiu Arktiki; Vsesomznyi
Arkticheskii Institut (Materials for the
study of the Arctic) 1931: Leningrad. 1-5

Mathematical Reviews 1940: 1 +
*Mathematische und Naturwissenschaftliche
Berichte aus Ungarn aus den Revenuen der
Graf Alexander Vigyazo Stiftung Unter-
stiitzt durch die Ungarische Akademie der
Wissenschaften (Magyar Tudomanyos Aka-
demia) 1882: Budapest. 1-38

Mathematisk-fysiske Meddelelser; Det K.
Danske Videnskabernes Selskab 1917: Co-
penhagen. 8, nos. 4-11; 9 +
*Meddelanden af Societas pro Fauna et Flora
Fennica 1876: Helsingfors. 1-20; 22-23; 25;
27; 30-50

Meddelanden fran K. Lantbruksstyrelsen;
Undersokningar Rb'rande Sveriges Fiskerie ;
Fiskar och Fiskevatten, utgivna av K. Lant-
bruksstyrelsens Fiskeribyra: Stockholm.
[195-278]

*Meddelanden fran K. Vetenskapsakademiens
Nobelinstitut 1905: Stockholm. 1-6

Meddelanden fran Statens Meteorologisk-
Hydrografiska Anstalt 1920: Stockholm. 1;
[2]; 3+

Meddelelser fra Carlsberg Laboratoriet see
Comptes Rendus des Travaux du Labora-
toire Carlsberg

Meddelelser fra den Biologiske Station ved
Dr0bak 1897: Christiania, Norway. 1-14

Meddelelser fra det Zoologiske Museum,
Oslo 1922: 1 +

Meddelelser fra Kommissionen for Danmarks
Fiskeriog Havunders0gelser, K0benhavn ;

Serie Fiskerei 1904: 1 + ; *Serie Fiskerista-
tistik 1909: 1-2; Serie Hydrografi 1904: 1 + ;
Serie Plankton 1904: 1 +

Meddelelser fra Skalling-Laboratoriet 1935:
Copenhagen. 1

Meddelelser om Gr0nland; Kommissionen for
Videnskabelige Unders0gelser i Gr0nland
1879: 1-121

Mededeelingen en Verhandelingen ; K. Neder-
landsch Meteorologisch Instituut 1905: 1 +

Mededeelingen van de Afdeeling Weten-
schappen; Koninklijke Belgische Academie
see Bulletin (s) de la Classe des Sciences;
Academie Royale de Belgique

Mededeelingen van de Landbouwhoogeschool
te Wageningen (Nederland) (12-13 as Me-
dedeelingen van de Rijks Hoogere Land-,
Tuin- en Boschbouwschool) 1908: 12 +

Mededeelingen van den Dienst der Volksge-
zondheid in Nederlandsch-Indie (1912-25
pt. 1 as Burgerlijke Geneeskundige Dienst)
1912: Batavia. 1922 +

*MededeeIingen van's Rijks Herbarium 1910:
Leiden. 1-70

Mededelingen van het Transvaal Museum see

Annals of the Transvaal Museum
*Medical Bulletin; a monthly journal of medi-
cine and surgery 1879: Philadelphia. [18-23]

Medical Parasitology and Parasitic Diseases
(Moscow) see Meditsinskaia Parazitologiiia
Parazitarnye Bolezni

*Medical Science; abstracts and reviews 1919:
Medical Research Council, London. 1-12

Medicina ;RevistaMexicana 1920: 12; [13-14];
15-19; [20]; 21 +

Medicina Sperimentale Archivio Italiano (1-5
as Archivio Italiano di Medicina Sperimen-
tale) 1937: Torino. 1 +

Medicine; analytical reviews of general medi-
cine, neurology and pediatrics 1922: 1 +
*Medicinisch-Chemische Untersuchungen ; aus
dem Laboratorium fur Angewandte Chemie
zu Tubingen herausgegeben von Dr. Felix
Hoppe-Seyler 1866: 1-4

Meditsinskaia Parazitologifa i Parazitarnye
Bolezni (Medical Parasitology and Parasitic
Diseases) 1932: Moscow. 4 +
*Medizinisch-Naturwissenschaftliches Archiv ;
Zeitschrift fiir die Gemeinsamen For-
schungsergebnisse der Klinischen Medizin
und ihrer Gesamten Nachbargebiete 1908:
1-2

Meereskunde ; Sammlung Volkstumlicher Vor-
trage 1907: Institut fur Meereskunde. [1-18]

Meereskundliche Beobachtungen auf Deut-
schen Feuerschiffen der Nord- und Ostsee;
Deutsche Seewarte: 1924-38

Memoires; Institut Royal Meteorologique de
Belgique (1 as Verhandelingen) 1925: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Memoires de 1'Academie des Sciences de
1'Institut de France (1-14 as Memoires de la
Classe des Sciences Mathematiques et Phy-
siques de 1'Institut de France) (1-16 (n.s.),
1816-38 as Memoires de 1'Academie Royale
des Sciences de 1'Institut de France) 1796:
1-n.s. 16; ser. 2, 59, no. 5
*Memoires de 1'Academie des Sciences de
1'URSS (Zapiski Akademii Nauk SSSR)
(formerly Memoires de 1'Academie Impe-
riale des Sciences de Saint-Petersbourg)
1726: ser. 7-8

Memoires de 1'Academie Polonaise des
Sciences et des Lettres; Classe des Sciences
Mathematiques et Naturelles (Cracovie);
Series A, Sciences Mathematiques 1929:
1-4; Series B, Sciences Naturelles 1928:
1-13

Memoires de 1'Academie Royale des Sciences
et des Lettres de Danemark; Section des
Sciences see Danske Videnskabernes Sel-
skabs Skrifter; Naturvidenskabelig og Ma-
thematisk Afdeling

Memoires dje 1'Institut Agronomique et Fo-
restier d'Etat de la Belarussie see Zapiski
Belorusskogo Gosudarstvennogo Instituta
Sel'skogo i Lesnogo Khozfaistva (Minsk)

Memoires de 1'Institut Hydrologique de
1'URSS (Zapiski Gosudarstvennogo Gidro-
logicheskogo Instituta) 1926: Leningrad.
1-2; 5-13

Memoires de 1'Office des Peches Maritimes;
Serie Speciale (1-8 as Memoires; Office
Scientifique et Technique des Peches Mari-
times) 1920: 1 +

Memoires de la Societe de Biogeographie
1926: 2

Memoires de la Societe de Physique et d'His-
toire Naturelle de Geneve 1821: 31, no. 2;
40+

Memoires de la Societe des Naturalistes de
Kiew see Zapiski Kievskago Obshchestva
Estestvoispytatelei

Memoires de la Societe des Naturalistes de
Moscou see Nouveaux Memoires de la So-
ciete des Naturalistes de Moscou
*Memoires de la Societe des Sciences Natu-
relles de Neuchatel 1835: 1; 3

Memoires de la Societe des Sciences Natu-
relles du Maroc; Empire Cherifien; Archives
Scientifiques des Protectorat Francois 1921 :
2 +

Memoires de la Societe Entomologique de
Belgique 1892: 23 +

Memoires de la Societe Helvetique des Scien-
ces Naturelles see Denkschriften der Schwei-
zerischen Naturforschenden Gesellschaft

Memoires de la Societe Royale des Lettres et
des Sciences de Boheme; Classe des Scien-

ces see Vestnik Kralovske Ceske Spolecnosti
Nauk

Memoires de la Societe Royale des Sciences
de Liege 1843: 8 +

Memoires de la Societe Royale Entomologique
d'Egypte (1-2, fasc. 1 as Memoires de la
Societe Entomologique d'Egypte) 1908: 1 +

Memoires de la Societe Vaudoise des Sciences

Naturelles 1922: Lausanne. 1 +
*Memoires de la Societe Zoologique de France
1888: 1-29

Memoires de la Societe Zoologique Tcheco-
slovaque de Prague see Vestnik Ceskoslo-
venske Zoologicke Spolecnosti v Praze

Memoires du Musee Royal d'Histoire Na-
turelle de Belgique (Verhandelingen van
het K. Natuurhistorisch Museum van Bel-
gie) 1900: 1-8; 32-90; 93 + ; Hors serie
1933: 1 +

Memoires et Publications de la Societe des
Sciences, des Arts et des Lettres du Hainaut
1839: [ser. 3, 6— ser. 6, 7]
*Memoires publics par la Societe Portugaise
des Sciences Naturelles 1913: 2-4; serie
Anthropologique et Archeologique 2; serie
Biologique 2-4; serie Geologique 2-3; serie
Zoologique 2-4

Memoirs; Australian Museum 1851: 2; 4
(Scientific Results of the Trawling Expedi-
tion of H. M. C. S. "Thetis" off the Coast of
New South Wales, Feb. and Mar., 1898,
parts 15-18); 5 +

Memoirs; Cornell University Agricultural Ex-
perimental Station 1913: 1-135; 153; 157-
59; 176; 180; 203; 207; 209

Memoirs; Geological Society of America 1934:

1 +
*Memoirs; New York Academy of Sciences

1895: 1-2

Memoirs and Proceedings of the Manchester
Literary and Philosophical Society (ser. 1-3
as Memoirs of the Literary and Philosophi-
cal Society of Manchester) 1785: 1 +
*Memoirs from the Biological Laboratory of the

Johns Hopkins University 1887: 1-5
*Memoirs of Natural Sciences; Museum of the
Brooklyn Institute of Arts and Sciences
1904: 1, no. 1
Memoirs of the American Academy of Arts

and Sciences 1780: 1 +
*Memoirs of the American Association for the

Advancement of Science 1875: 1
Memoirs of the American Entomological So-
ciety 1916: 1 +
Memoirs of the American Museum of Natural

History 1893: 1; 9; n.s. 1-3
Memoirs of the American Philosophical So-
ciety 1935: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Memoirs of the Asiatic Society of Bengal 1905:
1-9, no. 8

Memoirs of the Bemice P. Bishop Museum of
Polynesian Ethnology and Natural History
1899: 1 +

Memoirs of the Boston Society of Natural

History 1866: 1 +

*Memoirs of the California Academy of Sci-
ences 1868: 1-5, no. 1

Memoirs of the Carnegie Museum 1901: 1 +

'Memoirs of the Challenger Society 1909:
London. 1

Memoirs of the College of Agriculture; Kyoto
Imperial University 1926: 1 +

Memoirs of the College of Science; Kyoto Im-
perial University; Series A 1914: 9, no. 6;
18, no. 5 + ; Series B 1924: 1-10; [11]; 12 +

Memoirs of the Connecticut Academy of Arts
and Sciences 1810: 1-8, no. 2; [9-10]

Memoirs of the Faculty of Science and Agri-
culture; Taihoku Imperial University 1930:
Formosa. 1 +

Memoirs of the Imperial Marine Observa-
tory, Kobe, Japan 1922: 1 +

Memoirs of the Indian Meteorological De-
partment (2-17 as Indian Meteorological
Memoirs) 1876: 16+

Memoirs of the Indian Museum 1907: Cal-
cutta. 1 +

Memoirs of the Institute of Chemistry see
Zapiski Institutu Khemii

Memoirs of the Literary and Philosophical So-
ciety of Manchester see Memoirs and Pro-
ceedings of the Manchester Literary and
Philosophical Society

*Memoirs of the Museum of Comparative
Zoology at Harvard College 1864: 1-55

Memoirs of the National Academy of Sciences
1866: 1 +

Memoirs of the National Institute of Zoology
and Botany; Academia Sinica; Zoological
Series 1937: Nanking. 1 +

Memoirs of the National Research Institute of
Meteorology (1-10 as numbers; 11+ as
volumes) 1931: Academia Sinica, Nanking.
1 +

Memoirs of the New York Botanical Garden
1900: 1-7

Memoirs of the New York State Museum see
New York State Museum Memoirs

Memoirs of the Nuttall Ornithological Club

1886: 1-4
*Memoirs of the Peabody Academy of Science

1869: Salem, Mass. 1, nos. 1-6
*Memoirs of the Peabody Museum of Yale

University 1880: 1-2, pt. 1
*Memoirs of the Royal Meteorological Society
1926: 1-4

*Memoirs of the Royal Society of South Aus-
tralia 1899: Adelaide. 1-2

Memoirs of the San Diego Society of Natural

History 1931: 1 +

*Memoirs of the Tokio Daigaku (1, pt. 1 as
Memoirs of the Science Department; Uni-
versity of Tokio, Japan) (5, 7 as Memoirs of
the Science Department; Tokio Daigaku)
1879: 1, pt. 1; 5; 7; 11

Memoirs of the Torrey Botanical Club 1889:
1 +

Memoirs of the University of California 1908:
1-2; 5 +

Memoirs of the Wistar Institute of Anatomy
and Biology see American Anatomical
Memoirs

Memoirs on Genetics see Zbirnik Prats z
Genetiki

Memoirs on Typical Marine Plants and Ani-
mals; Department of Oceanography, Uni-
versity of Liverpool see Liverpool Marine
Biological Committee Memoirs on Typical
British Marine Plants and Animals
*Memoirs read before the Anthropological So-
ciety of London 1863: 1-3

Memoranda Societatis pro Fauna et Flora
Fennica 1924: 1 +

Memoria; Junta Para Ampliacion de Estudios
e Investigaciones Cientfficas 1916: Madrid.
1916-17; 1926-30

Memoria; R. Comitato Talassografico Italiano
1910: 1-22; 24 +

Memoria Anual; Museo Nacional de Historia
Natural "Bernardino Rivadavia," Buenos
Aires 1924: 1924

Memorial de 1'Institut National Meteorolo-
gique de Pologne see Prace Paristwowego
Instytutu Meteorologicznego

Memorias (y Re vista) de la Academia Na-
cional de Ciencias Antonio Alzate (1-52 as
Memorias (y Revista) de la Sociedad Cienti-
fica "Antonio Alzate") 1887: Mexico. [1-4];
5-8; [9]; 10-18; [19]; 20 +

Memorias de la Real Academia de Ciencias
Exactas Fisicas Naturales de Madrid 1861:
6; [13-14]; 15; [18]; 19-20

Memorias de la Real Sociedad Espanola de
Historia Natural 1903: Madrid. 1 +

Memorias de la Sociedad Cubana de Historia
Natural "Felipe Poey" 1915: 1 +

Memorias del Consejo Oceanografico Ibero-
Americano 1930: Madrid. 1-16

Memorias del Institute Espanol de Oceano-
grafia 1916: Madrid. 2-3; 7-15

Memorias del Jardin Zoologico: La Plata. 7;
9, nos. 1-2

Memorias do Institute Biologico Ezequiel Dias
1937: Brazil. 1 +

SI' RIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Memorias do Institute Butantan 1918: Sao
Paulo. 1 +

Memorias do Institute Oswaldo Cruz 1909:
Rio de Janeiro. 1-7; [8]; 9+; *Supplements
1-12

Memorie del Reale Istituto Lombardo di
Scienze e Lettere, Classe des Scienze Mate-
matiche e Natural! 1843: Milan. 10-13; [14];
15-17; [18]; 19-20; [21]

Memorie del Reale Ufficio Centrale di Met-
eorologia e Geofisica: Ministero dell'Agri-
coltura e Foreste, Rome. ser. 3, 2 +

Memorie della Accademia di Verona see Atti
e Memorie dell'Accademia d'Agricoltura
Scienze, etc.

Memorie della Classe di Scienze Fisiche,
Matematiche e Naturali; Reale Accademia
d'ltalia (1-3 as Memorie della Reale Acca-
demia d'ltalia; Classe di Scienze Fisiche,
Matematiche e Naturali) 1930: 1 +

Memorie della Reale Accademia delle Scienze
di Torino 1759: ser. 2, 42-56; [57]; 64

Memorie della Reale Accademia Nazionale
dei Lincei; Classe di Scienze Fisiche, Mate-
matiche e Naturali see Atti della R. Accade-
mia d'ltalia

Memorie della Societa Italiana di Scienze Na-
turali e del Museo Civico di Storia Naturale
diMilano 1865: 1 +

Memorie della Societa Toscana di Scienze
Naturali Residente in Pisa see Atti della
Societa Toscana di Scienze Naturali Resi-
dente in Pisa; Memorie

*Memorie Scientifiche (Supplemento al Bollet-
tino di Pesca, di Piscicoltura e di Idrobio-
logia) ; Ministero dell'Agricoltura e delle
Foreste; R. Laboratorio Centrali di Idro-
biologia Applicata alia Pesca (1-3, Direzione
Generate dell'Industria e delle Miniere)
Serie B. 1927: Rome. 1-10

*Memorie Storico-Giuridiche (Supplemento al
Bollettino di Pesca, di Piscicoltura e di
Idrobiologia) ; Ministero dell'Economia Na-
zionale ; Direzione Generale dell'Industria e
delle Miniere. Serie C. 1927: Rome. 1

*Mendel Journal 1909: 1-3

*Mera Publications; scientific and economic as-
pects of the Cornish Pilchard Fishery 1913:
St. Albans. 1-2

Merentutkimuslaitoksen Julkaisu see Havs-
forskningsinstitutets Skrift

Meteorologia i Hydrologia see Meteorologifa i
Gidrologiia

Meteorological Magazine (1-35 as Symons's
Monthly Meteorological Magazine; 36-54
as Symons's Meteorological Magazine)
1866: Air Ministry, Meteorological Office,
London. 1-75, no. 893

Meteorological Magazine; Meteorological So-
ciety of China 1926: 11-13; [14] +

Meteorological Office; Professional Notes
1918: London. 1 +

Meteorological Office Note; New Zealand
1930: 1 +

Meteorologie; Revue (Mensuelle) de Meteo-
rologie et de Physique du Globe (1-67 as
Annuaire de la Societe Meteorologique de
France) 1853: n.s. 1 +

Meteorologiia i Gidrologiia; Glavnoe Upravle-
nie Gidrometeorologicheskoi Sluzhby SSSR
pri SNK SSSR (Meteorologia i Hydrologia) :
Moscow. [1938-40] +

Meteorologische Beobachtungen ; Bulletin de
la Societe des Naturalistes de Moscou see
Bulletin de la Societe des Naturalistes de
Moscou

Meteorologische Zeitschrift: der Osterreich-
ischen Gesellschaft fur Meteorologie und
der Deutschen Meteorologischen Gesell-
schaft 1884: 1 +

*Methods and Problems of Medical Education
1924: Rockefeller Foundation. 1-21

Michigan Geological and Biological Survey
Publication; Biological Series 1910: 1-2;
4-5

Microentomology ; Contributions to Entomol-
ogy from the Natural History Museum of
Stanford University 1936: 1 +
*Microscope; an illustrated monthly designed
to popularize the subject of microscopy
1881: [1]; 2-9; [12]; n.s. [1]; 2-3; [4-5]
*Microscopic Journal, and Structural Record
1841: London. 1-2

Mikrochemie Vereinigt mit Acta Mikrochimica
(1-22 as Mikrochemie) 1923: 1 +

Mikrokosmos; Zeitschrift fur Angewandte Mi-
kroskopie, Mikrobiologie, Mikrochemie und
Mikroskopische Technik 1907: 20, no. 1;
24 +

Milbank Memorial Fund Quarterly 1923: 17,
no. 3; 18, no. 4+

Millport Marine Biological Station; Annual
Report see Annual Report; Scottish Marine
Biological Association

Minnesota Geological and Natural History
Survey Bulletin (no. 9 as vol. 2 of the Min-
nesota Geological and Natural History Sur-
vey Report; Botanical series): 1; 4; 9

'"Minnesota Geological and Natural History
Survey Report; Botanical series (2, 4, 6 also
as Minnesota Botanical Studies 1-3) 1892:
1-6; 8-9

*Minnesota Geological and Natural History
Survey Report; Zoological series 1892: 1-5

*Miscellaneous Publication; Australian Mu-
seum 1890: 10

Miscellaneous Publication; Bernice P. Bishop

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Museum see Special Publications; Bernice
P. Bishop Museum

Miscellaneous Publication of the Bureau of

Entomology of Chekiang Province 1930: 1-7

*Mitt(h)eilungen aus dem Embryologischen

Institut der K. K. Universitat in Wien 1877:4

Mitteilungen aus dem Hamburgischen Zoolo-
gischen Museum und Institut (1-32 as Mit-
teilungen aus dem Naturhistorischen, Zoolo-
gischen, Museum in Hamburg; 33-37 as
Mitteilungen aus dem Zoologischen Museum
in Hamburg; 38-46 as Mitteilungen aus dem
Zoologischen Staatsinstitut und Zoologi-
schen Museum in Hamburg) (1-10 reprinted
from Jahrbuch der Hamburgischen Wissen-
schaftlichen Anstalten; 1 1-38 published as 2.
Beihefte zum Jahrbuch der Hamburgischen
Wissenschaftlichen Anstalten) 1883: 1-47

Mitt(h)eilungen aus dem Naturwissenschaft-
lichen Verein fiir Neuvorpommern und
Riigen in Greifswald: 33-40; 46-47

Mitteilungen aus dem Zoologischen Museum
in Berlin (1 as Mitteilungen aus der Zoolo-
gischen Sammlung des Museums fiir Natur-
kunde in Berlin) 1898: 1-10; [12]; 13-23;
Sender heft 14; 22

"Mitteilungen aus den Botanischen Staatsinsti-
tuten in Hamburg; Beiheft zum Jahrbuch
der Hamburgischen Wissenschaftlichen
Anstalten 1896: 23-29

Mitteilungen aus den K. Naturwissenschaft-
lichen Instituten in Sofia see Izvestiia na
Tsarskitye Prirodonauchni Instituti v Sofiia
*Mitt(h)eilungen aus den Verhandlungen der
Gesellschaft Naturforschender Freunde zu
Berlin 1836: 1-3

*Mitt(h)eilungen aus der Zoologischen Station
zu Neapel 1879: 1-22

Mitteilungen der Aargauischen Naturfor-
schenden Gesellschaft 1863: Switzerland.
7-14; 19

Mitteilungen der Deutschen Entomologischen
Gesellschaft, E. V. 1930: 1-9, no. 1

Mitt(h)eilungen der Deutschen Gesellschaft
fiir Natur- und Volkerkunde Ostasiens
1873: Yokohama. 6; 26

Mitteilungen der Gesellschaft Deutscher Na-
turforscher und Aerzte 1924: 1+ see in
Naturwissenschaften vol. 12, 1924 +

Mitteilungen der Gesellschaft fiir Erdkunde
zu Leipzig (1872-1910 as Mittheilungen des
Vereins fiir Erdkunde zu Leipzig) 1872: 1 +

Mitteilungen der Gesellschaft fiir Vorrats-
schutzE. V. 1925: Berlin. [2]; [4]; 9-15, no. 1

Mitteilungen der Kaiser Wilhelm-Gesellschaft
zu Forderung der Wissenschaften 1932: 14-
see in Naturwissenschaften 20, 1932 +

Mitteilungen der Kommission zur Naturwis-
senschaftlichen Durchforschung Mahrens

see Zpravy Komise na Prirodovedecky Vyz-
kum Moravy a Slezska

Mitt(h)eilungen der Naturforschenden Gesell-
schaft Bern 1843: 1843 +

Mitteilungen der Naturhistorischen Gesell-
schaft in Colmar (Bulletin de la Societe
d'Histoire Naturelle de Colmar) 1860: N.F.
5; 8-10; 12

"Mitteilungen des Deutschen Seefischerei- Ve-
reins (1-10 as Mittheilungen der Section fiir
Kiisten- und Hochsee-Fischerei) 1885: 1-22;
[23]; 24-28; [29]; 30-42; [43]; 44; [45]; 46-49

Mitteilungen des Meteorologischen Instituts

der Universitat Helsingfors 1926: 1 +
* Mitteilungen des Naturwissenschaftlichen
Vereines an der Universitat Wien: 1893-94;
n.s. 1-12, no. 8

Mitteilungen des Naturwissenschaftlichen
Vereines fiir Steiermark 1863: 1-59

Mitteilungen des Vereins fiir Erdkunde zu
Leipzig see Mitteilungen der Gesellschaft fiir
Erdkunde zu Leipzig

Mitteilungen des Vereins fiir Naturwissen-

schaft und Mathematik in Ulm, a. D. (1888-

1908asjahresheft) 1888: 1 ; 4-5; 8-16; 19-21

*Monatsbericht der K. Preussischen Akademie

der Wissenschaften zu Berlin 1856: 1856-81

Monatsheft fiir Chemie und Verwandte Teile
Anderer Wissenschaften ; K. Akademie der
Wissenschaften in Wien 1880: 1+ see in
Sitzungsberichte der Akademie der Wissen-
schaften in Wien; Mathematisch-Naturwis-
senschaftliche Klasse

Monatsschrift fiir Krebsbekampfung see Miin-

chener Medizinische Wochenschrift
*Monist; devoted to the philosophy of science
1890: 1-31; 40-46

Monitore Zoologico Italiano (Pubblicazione
Italiane di Zoologia, Anatomia, Embriologia)
1890: Societa Italiana di Anatomia. 1 +

Monografii Volzhskoi Biologicheskoi Stantsii
see Monographien der Biologischen Wolga-
Station (der Naturforscher-Gesellschaft zu
Saratow)

Monograph Series; American Museum of
Natural History 1913: 1-2

Monographias do Institute Oswaldo Cruz
1937: Rio de Janeiro. 1

Monographien der Biologischen Wolga-Sta-
tion (der Naturforscher-Gesellschaft zu
Saratow) (Monografii Volzhskoi Biologi-
cheskoi Stantsii) 1924: 1; 3

*Monographien und Abhandlungen zur Inter-
nationalen Revue der Gesamten Hydrobio-
logie und Hydrographie 1910: 1-5

Monographs of the National Research Insti-
tute of Psychology; Academia Sinica 1932:
Nanking. 8-9

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Monographs of the Rockefeller Institute for
Medical Research 1910: 2-4; 7; 10-11; 13 +
Monographs of the United States Geological
Survey: Department of the Interior. 5
(1883); 8-10 (1884-85); 30 (1898)
*Monographs on the Theory of Photography
from the Kodak Research Laboratories
1921: 1-6
*Montana Agricultural College Science Studies ;

Botany 1904: 1, nos. 1-3
Monthly Abstract Bulletin 1915: Kodak Re-
search Laboratories. [2-6]; 7 +
Monthly Catalogue; United States Govern-
ment Publications (73-100 as Catalogue of
United States Public Documents): 73-125;
241-42; 249-300; 302-08; 310-18; 324; 328;
331; 333 +

*Monthly Meteorological Charts of the North
Atlantic Ocean: Meteorological Office, Lon-
don. 1923

"Monthly Microscopical Journal; Transactions
of the Royal Microscopical Society and
Record of Histological Research at Home
and Abroad 1869: London. 1-18
Monthly Notices of Papers and Proceedings
of the Royal Society of Tasmania see Papers
and Proceedings of the Royal Society of
Tasmania

Monthly Weather Review 1873: Department
of Agriculture, Weather Bureau. [15-16];
[19]; 25+; Supplement 1 +
*Morphologische Arbeiten 1891: 1-8
Morphologisches Jahrbuch see Gegenbaurs

Morphologisches Jahrbuch
Morris Arboretum Bulletin 1935: University

of Pennsylvania. 1 +

Miinchener Medizinische Wochenschrif 1 1 854 :
72 + ; Supplement ; Monatsschrift fur Krebs-
bekampfung 1933: 9 +

Mycologia; official organ of the Mycological
Society of America 1909: New York Botani-
cal Garden. 1 +

*Mycological Bulletin (1 as Ohio Mycological
Bulletin; 1-56 as Ohio University Bulletins;
1-12 also as 13-24 of University Bulletin;
Botanical Series) 1903: 1-12; 14; 30; 61-62;
65-66

*Mycological Notes; by C. C. Lloyd 1898: 1-7,
no. 10

Nachrichten von der Gesellschaft der Wissen-
schaften zu Gottingen; Mathematisch-
Physikalische Klasse ; Fachgruppe VI. Bio-
logie (1932-33 included all Fachgruppen)
1894: 1932-33; N.F. 1-2

National Geographic Magazine 1889: National
Geographic Society. [7-22]; 23-66; [67-73]

National Institute of Health Bulletin (1-7 as
Bulletin of the Hygienic Laboratory; 8-154

as Hygienic Laboratory Bulletin) 1900:
Public Health Service, Federal Security
Agency. 1 +

*National Medical Journal of China 1915:
Shanghai. 12-17

National Research Council; Annual Report of
the Chairman of the Division of Biology and
Agriculture: 1937-38

National Research Council; Division of Biol-
ogy and Agriculture; Cumulative Report of
the Committee on Effects of Radiation upon
Living Organisms: 1928-34

National Research Council; Division of Chem-
istry and Chemical Technology; Report of
the Committee on Contact Catalysis (1-9
see in Reprint and Circular Series of the
National Research Council) 1922: 1 +

National Research Council ; Division of Chem-
istry and Chemical Technology; Report of
the Committee on Photochemistry (1-3 see
in Reprint and Circular Series of the Na-
tional Research Council, nos. 81, 96, 108)
1928: 1 +

National Research Council ; Division of Geol-
ogy and Geography; Report of the Com-
mittee on Sedimentation (1927-34 see in
Reprint and Circular Series of the National
Research Council) 1927: 1 +
*National Research Council; Division of Geol-
ogy and Geography; Report of the Com-
mittee on Submarine Configuration and
Oceanic Circulation (1923-25 as Report of
Committee on Sound Sounding and Ocea-
nographic Thermographs) 1923: 1923-32

National Research Council ; Division of Geol-
ogy and Geography; Report of the Commit-
tee on the Measurement of Geologic Time
1937: 1937-40

National Research Council; Organization and
Members 1919: 1919+

Natur und Volk (1-51 as Bericht der Sencken-
bergischen Naturforschenden Gesellschaft
Frankfurt am Main; 52-56 as Aus Natur
und Museum; 57-63 as Natur und Museum)
1869: 1878-79; 1880-90; 1892-95; 1896-
1900; 1903-09; 1910-39

Naturae Novitates; Bibliographic Neuer Er-
scheinungen aller Lander auf dem Gebiete
der Naturgeschichte und der Exacten Wis-
senschaften 1879: Berlin. [1-25]; 26+

Natural History (1-18 as Journal of the Ameri-
can Museum); magazine of the American
Museum of Natural History. 1 + ; see also
Guide Leaflet Series

Natural History Magazine (British Museum)

1927: 1 +

* Natural History Review; a quarterly journal
of biological science 1853: London. 1-7;
n.s. 1-5

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

*Natural Science; a monthly review of scientific

progress 1892: New York. 1-15
*Naturaleza; periodico cientifico del Museo N.
de Historia Natural y de la Sociedad Mexi-
cana de Historia Natural 1869: ser. 1, 1-2;
ser. 2, 1; [3]; ser. 3, 1, no. 2
'Naturalist 1830: Boston. [1-2]

Naturaliste Canadien; bulletin de recherches,
observations et decouvertes se rapportant a
1'histoire naturelle et aux sciences en gene-
ral 1868: Quebec. 32-36; [37]; 38-48, no. 1;
66, no. 10 +

Nature; a weekly journal of science 1869:
London. 1 +

Nature ; Revue des sciences et de leurs appli-
cations a 1'art et a 1'industrie 1873: Paris.
1927 +

*Nature Study Review 1905: American Nature
Study Society, New York. 1-6; [7]; 8-16;
[17]; 18; [19]

Naturwissenschaften ; Wochenschrift fur die
Fortschritte der Reinen und der Angewand-
ten Naturwissenschaften 1913: 1 +
*Naturwissenschaftliche Rundschau; Wochent-
liche Berichte iiber die Fortschritte auf dem
Gesamtgebiete der Naturwissenschaften
1886: 1-27

*Naturwissenschaftliche Wochenschrift 1887:
1-37

Natuurkundige Verhandelingen van de Hol-
landsche Maatschappij der Wetenschappen
1799: ser. 3, [1-6]; 7-9

Nauka Polska jej Potrzeby, Organizacja i
Rozwoj (Science and Letters in Poland, their
needs, organization and progress) 1918: In-
stitute for the Promotion of Science and
Letters in Poland, Warsaw. 14

Naukovi Zapiski; Kiivs'kii Derzhavnii Uni-
versitet (Bulletin Scientifique; Universite
d'Etat de Kiev) 1935: Biologichnii Zbirnik
1-2 (n.s. 1, no. 3; 2, no. 2); Khemichnii
Zbirnik 3-4 (n.s. 3, no. 3; 4, no. 2); Fizichno-
Matematichnii Zbirnik 4 (n.s. 4, no. 5)

Naunyn-Schmiedebergs Archiv fur Experi-
mentelle Pathologie und Pharmakologie
(1-109 as Archiv fur Experimented Patho-
logie und Pharmakologie) (includes Ver-
handlungen der Deutschen Pharmakologi-
schen Gesellschaft: no. 1, 1921, Tagung 2 +
in vol. 92, 1922 + ) 1873: 1 +

Nautilus; a quarterly journal devoted to the
interests of conchologists 1886: [3-8]; 9 +

Nautisk-Meteorologisk Aarbog (Nautical-
Meteorological Annual) (1913-23 as Isfor-
holdene i de Arktiske Have samt Havets
Overfladetemperatur i det Nordlige Atlan-
terhav og Davis-Straede, saertryk af Nau-
tisk-Meteorologisk Aarbog; the State of the
Ice in the Arctic Seas and the Surface Tem-

perature of the Sea in the Northern Atlantic-
Ocean and in Davis-Strait, special print of
the Nautical-Meteorological Annual) 1897:
Danske Meteorologiske Institut, Copen-
hagen. 1913-23; 1934 +

Naval Medical Bulletin see United States
Naval Medical Bulletin for the Information
of the Medical Department of the Navy

Nederlandsch Kruidkundig Archief; Versla-
gen en Mededeelingen der Nederlandsche
Botanische Vereeniging 1846: ser. 1, [4-5];
ser. 2, 1-6; ser. 3, 1-2; years 1904 +
*Nederlandsch Tijdschrift voor de Dierkunde;
uitgegeven door het Koninklijk Zoologisch
Genootschap Natura Artis Magistra te Ams-
terdam 1863: 1-5, an. 1

Nekotorye Voprosy Sravnitel'noi Fiziologii;
Sbornik Rabot Laboratorii Sravnitel'noi
Fiziologii Zhivotnykh Biologicheskogo Insti-
tuta imeni K. A. Timiriazeva (Problems of
Comparative Physiology; Collected Papers
of the Laboratory of Comparative Physiol-
ogy of the Timiriasev Biological Institute)
1934: Moscow. 1

Neue Schriftenreihe ; Aus Deutschen Zuchten
1936: Deutsche Gesellschaft fur Ziichtungs-
kunde, e. B. Berlin. 1 +

*Neurologisches Zentralblatt ; Uebersicht der
Leistungen auf dem Gebiete der Anatomic,
Physiologic, Pathologie und Therapie des
Nervensystems Einschliesslich der Geistes-
krankheiten 1882: 1-40

*New England Farmer; a monthly journal de-
voted to agriculture, horticulture, and their
kindred arts and sciences 1849: 1-2; 9-11

New England Naturalist (1-79 as Bulletin of
the Boston Society of Natural History;
80-88 as Bulletin of the New England Mu-
seum of Natural History) (89+ forms new
series with the new title) 1915: 1 +
*New Hampshire Fish and Game Department;
Survey Report 1936: 1-4

New Phytologist; a British botanical journal
1902: 1 +

New York State Museum Bulletin 1892:
[1-290]; 292 +

New York State Museum Handbook 1927: 1 +

New York State Museum Memoirs (1-4 as
Memoirs of the New York State Museum)
1889: 1-16

New York Zoological Society Bulletin see Ani-
mal Kingdom

New Zealand Geological Survey; Paleonto-
logical Bulletin 1913: 1 +

New Zealand Journal of Science and Tech-
nology (20+ in two sections: A. Agriculture,
and B. General) 1918: 1 +

'News Bulletin of the Chicago Academy of
Sciences 1928: 2, nos. 1, 3-4

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

News Bulletin of the Zoological Society see
Animal Kingdom

News Edition; American Chemical Society see

Chemical and Engineering News
*Niederlandisches Archiv fur Zoologie 1871:
1-5; supplement 1

Norske Videnskabers Selskab Museet Arsbe-
retning 1874: Trondhjem. 1920+

Norske Videnskabers Selskab Museet Old-
saksamlingens Tilvekst 1927: Trondhjem.
1927 +

Norske Videnskabers Selskabs Forhandlinger
1926: Trondhjem. 1 +

Norske Videnskabers Selskabs Skrifter 1817:
Trondhjem. 1920+

North American Fauna 1889: Fish and Wild-
life Service, U. S. Department of the Interior
(formerly the Bureau of Biological Survey,
U. S. Department of Agriculture). 1 +

North American Flora 1905: New York Bo-
tanical Garden. 1 +

Notarisia ; Commentarium Phycologicum 1886:

1-5, no. 17

*Notas Preliminares ; editadas pela redaccao
da Revista do Museo Paulista 1914: Sao
Paulo. 1 , no. 3

Notas Preliminares del Museo de La Plata;
Universidad Nacional de La Plata 1931: 1-3

Notas y Resumenes, Institute Espanol de
Oceanografia: Madrid, ser. 2, 1-93; 96-106

Note; Institut Oceanographique de 1'Indo-
chine; Station Maritime de Cauda 1926:
Gouvernement General de 1'Indochine. 1 +

Note dell'Istituto Italo-Germanico di Biologia
Marina di Rovigno d'Istria (Notizen des
Deutsch-Italienischen Institutes fur Meer-
esbiologie in Rovigno d'Istria) 1932: 1 +

Notes; Station Oceanographique de Sa-
lammbo; Regence de Tunis 1925: 1 +

Notes and Memoirs; Hydrobiology and Fish-
eries; Fouad I Institute; Ministry of Com-
merce and Industry (formerly Coastguards
and Fisheries Service, Ministry of Finance)
1933: Egypt. 1 +

Notes et Rapports; Office Scientifique et Tech-
nique des Peches Maritimes (1-47 as Notes
et Memoires) 1920: 1; 3-4; 7; 10-13; 16 +
*Notice to Mariners; United States Coast
Guard: 1925-42, no. 22

Notice to Mariners; United States Hydro-
graphic Office: United States Navy Depart-
ment. 1931 +

Notizblatt des Konigl. Botanischen Gartens
und Museums zu Berlin-Dahlem (Post
Steglitz) sowie der Botanischen Zentral-
stelle fur die Deutschen Kolonieen 1895:
[3-6]

Notizen des Deutsch-Italienischen Institutes
fur Meeresbiologie in Rovigno d'Istria see

Note dell'Istituto Italo-Germanico di Bio-
logia Marina di Rovigno d'Istria

Notulae Naturae of the Academy of Natural
Sciences of Philadelphia 1939: 1 +

Notulae Systematicae ex Institute Cryptoga-
mico Horti Botanici Petropolitani see Bo-
tanicheskie Materialy Instituta Sporovykh
Rastenii Glavnogo Botanicheskogo Sada

Nouveaux Memoires de la Societe des Natu-
ralistes de Moscou (Novye Memuary Mo-
skovskogo Obshchestva Ispytatelei Prirody)
(1-6 as Memoires de la Societe des Natu-
ralistes de Moscou) 1806: 19-20

Nouvelles Archives du Museum d'Histoire
Naturelle de Paris 1865: ser. 1-5

Nouvelles Archives Italiennes de Biologic see
Revue des Archives Italiennes de Biologic

Nova Acta Leopoldina; Abhandlungen der
K. Leopoldinisch-Carolinischen Deutschen
Akademie der Naturforscher 1757: 20-110;
n.s. 1-6

Nova Acta Regiae Societatis Scientiarum
Upsaliensis 1773: K. Vetenskaps-Societeten.
ser. 3: [1-2]; 4; [6-17]; 18; ser. 4: 1 +
*Nuova Notarisia; rassegna consacrata allo
studio delle algae 1890: De Toni. 1-36

Nytt Magazin for Naturvidenskapene (29-74
as Nyt Magazin for Naturvidenskaberne)
1836: Oslo. 29-30; 31 +

Oberlin College Laboratory Bulletin 1893: 2;
5; 9; 13-14; 16-17; 20-31; 33-38; 40; 42-43;
45-54

*Occasional Memoirs of the Chicago Entomo-
logical Society 1900: 1, no. 1

Occasional Papers; Bernice Pauahi Bishop
Museum of Polynesian Ethnology and Nat-
ural History (1-8 as Occasional Papers of
the) 1898: 1 +

Occasional Papers; San Diego Society of Nat-
ural History 1936: 1 +

*Occasional Papers of the Bingham Oceanog-
raphic Collection 1927: Peabody Museum of
Natural History, Yale University. 1-3

Occasional Papers of the Boston Society of
Natural History 1869: 1 +

Occasional Papers of the California Academy
of Sciences 1890: 1 +

Occasional Papers of the Museum of Zoology;

University of Michigan 1913: 1 +
*Occasional Papers of the Natural History So-
ciety of Wisconsin 1889: 1-3
*Occasional Papers of the New York Academy
of Sciences 1913: 1-2

Occasional Publications of the American As-
sociation for the Advancement of Science
see Science; Supplement

*Ofversigt af Finska Vetenskaps-Societetens
Forhandlingar (51 + in 3 series) 1838: 1-50;

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

series A. Matematik och Naturvetenskaper
51-64

Ofversigt af K. Vetenskapsakademiens For-
handlingar 1844: Stockholm. [27-29]; 30;
38; 40-57; [58]; 59

Ohio Biological Survey Bulletin 1913: Ohio
State University. 1 +

Ohio Journal of Science (1-15 as Ohio Nat-
uralist) 1900: official organ of the Ohio State
University and the Ohio Academy of Sci-
ences. 1-37

Ohio Mycological Bulletin see Mycological
Bulletin

Okajimas Folia Anatomica Japonica (1-14,
heft 2 as Folia Anatomica Japonica) 1922:
1+

*Onderzoekingen, gedaan in het Physiologisch
Laboratorium der Universiteit te Leiden
(ser. 1, 1—3 as Onderzoekingen gedaan in
het Physiologisch Laboratorium der Leid-
sche Hoogeschool) 1869: 1-6; ser. 2, 1-10

Onderzoekingen verricht in het Zoologisch
Laboratorium der Rijksuniversiteit Gronin-
gen 1909: 1; 4

Opredeliteli po Faune SSSR see Tableaux
Analytiques de la Faune de 1'URSS

Organic Syntheses; an annual publication of
satisfactory methods for the preparations of
organic chemicals 1921: New York. 1 +

Osiris 1935: Belgium. 2

Osterreichische Botanische Zeitschrift (1-7 as
Osterreichisches Botanisches Wochenblatt)
1851: 1-3; 6; 8 +

*Ottawa Field- Naturalists' Club; Transactions
1879: 1-2

Ottawa Naturalist see Canadian Field-Nat-
uralist

Oversigt over det K. Danske Videnskabernes
Selskabs Forhandlinger see Bulletin de
1'Academie Royale des Sciences et des Let-
tres de Danemark

Palao Tropical Biological Station Studies 1937:
Japan Society for the Promotion of Scien-
tific Research, Tokyo. 1 +

Papeis Avulsos do Departamento de Zoologia
1941: Sao Paulo. 1 +

Papers and Proceedings of the Royal Society
of Tasmania (1-3, pt. 1 as Papers and Pro-
ceedings of the Royal Society of Van Die-
men's Land; 3, pt. 2 as Papers and Proceed-
ings of the Royal Society of Tasmania;
1863-74 as Monthly Notices of Papers and
Proceedings of the Royal Society of Tas-
mania; 1875-81 as Papers and Proceedings
and Report of the Royal Society of Tas-
mania) 1849: 1-3, pt. 2; 1864; 1866-78;
1880+

Papers from the Department of Marine Biol-

ogy of the Carnegie Institution of Washing-
ton see Carnegie Institution of Washington
Publications, Classified List

Papers from the Station for Experimental Evo-
lution, Carnegie Institution see Carnegie In-
stitution of Washington Publications, Classi-
fied List

Papers from the Tortugas Laboratory, De-
partment of Marine Biology of the Carnegie
Institution of Washington see Carnegie In-
stitution of Washington Publications, Classi-
fied List

Papers in Physical Oceanography and Mete-
orology (1 as Massachusetts Institute of
Technology Meteorological Papers) 1930:
Massachusetts Institute of Technology and
Woods Hole Oceanographic Institution. 1 +

Papers of the Michigan Academy of Science,
Arts and Letters 1921: 1 +

Papers on Animal Morphology see Zbirnik
Prats z Morfologii Tvarin

Parasitology 1908: Cambridge, England. 1 +
*Parergones del Institute Geologico de Mexico
1903: Secretaria de Fomento, Colonizacion
e Industria. 3-5

Park Museum Bulletin 1909: Providence, R. I.
[1-16]

Park Museum Memoirs 1921: Providence,
R. I. 1

Pathologica; Rivista M ensile 1908: 1 +

Peking Natural History Bulletin (1-4 as Pek-
ing Society of Natural History Bulletin)
1926: 1 +

Pennsylvania State College Studies 1936: 1 +
*People; a magazine for all the people 1931:
American Eugenics Society. 1, no. 1

Petermanns Geographische Mitteilungen (also
known as Petermanns Mitteilungen) 1855:
1-59; 83 +

Pfliigers Archiv fur die Gesamte Physiologic
des Menschen und der Tiere (1-131 as
Archiv fur die Gesamte Physiologic, des
Menschen und der Tiere) 1868: 1 +

Pharmaceutical Journal 1841: Pharmaceutical

Society of Great Britain. 146+
"Pharmaceutical Record 1881: 8-11

Pharmaceutisches Central-Blatt see Chemi-
sches Zentralblatt

Philippine Journal of Science 1906: 1; [2-3];
4-5; [6-7]; 8; [9]; 10-11; [12-13]; 14 +

Philosophical Magazine and Journal of Science
(London, Edinburgh and Dublin Philosophi-
cal Magazine and Journal of Science) 1798:
1 +

Philosophical Review 1892: New York. 42 +

Philosophical Transactions of the Royal So-
ciety of London 1665: 1-177; Series A.
Mathematical and Physical Sciences 178+;
Series B. Biological Sciences 178 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Philosophy (the Journal of the British Insti-
tute of Philosophy) (1-6, no. 21 as Journal
of Philosophical Studies) 1926: 1 +

Philosophy of Science 1934: Baltimore. 1 +

Photographic Journal; including Transactions
of the Royal Photographic Society of Great
Britain 1853: 66-70

Physica (series 4A of the Archives Neerlan-
daises des Sciences Exactes et Naturelles)
1933: 1, no. 1; 2, no. 1

Physical Review 1893: American Physical So-
ciety. 1 +

Physical Review Supplement see Review (s)
of Modern Physics

Physics see Journal of Applied Physics

Physics Abstracts being Science Abstracts
Section A (1-43 as Science Abstracts) 1898:
London. 1 +

Physics of the Earth ; a series of related mono-
graphs prepared under the direction of vari-
ous committees of the National Research
Council (1-6 see in Bulletin of the National
Research Council) 1931: 1 +
*Physikalisch-Chemisches Zentralblatt 1904:
1-6

Physikalische Zeitschrift; Vereinigt mit dem
Jahrbuch der Radioaktivitat und Elektronik
1899: 1 +

*Physiological Abstracts 1916: Physiological
Society of Great Britain and Ireland, with
cooperation of American Physiological So-
ciety. 1-22
*Physiological Researches 1913: 1-2

Physiological Reviews 1921 : American Physio-
logical Society. 1-f-

Physiological Zoology; a quarterly journal of
zoological research 1928: 1 +

*Phytologist; a botanical journal (o.s. 1-5 with
subtitle; a popular botanical miscellany)
1841: London. 1-5; n.s. 1-6

Phytopathology; official organ of the American
Phytopathological Society 1911: 1 +

Plant Physiology 1926: American Society of
Plant Physiology. 1 +

*Plant Science Literature; selected references
1934: compiled by the library staff of the
Bureau of Plant Industry from publications
received in the U. S. Department of Agri-
culture. 1-15

*Plant World; a journal of general botany 1897:
1-21; [22]

Planta; Archiv fiir Wissenschaftliche Botanik
(1-20 also as Zeitschrift fiir Wissenschaft-
liche Biologie Abt. E) 1925: 1 +

Planta Polonica; Materijaly do Flory Polski i
Krajow Sasiednich; wydawane przez Towar-
zystwo Naukowe Warszawskie (Contribu-
tions a la Flore de la Pologne et des Pays
Limitrophes; edition de la Societe des

Sciences et des Lettres de Varsovie) 1930:
1-7, no. 2

Poggendorff ' s Annalen der Physik see Annalen
der Physik

Pokusna Stanica za Ribnjacarstvo Crna
Mlaka; Zavod za Primijenjenu Zoologiju,
Zagreb: 1928-30

Pomona College Journal of Entomology see
Journal of Entomology and Zoology

Pontificia Academia Scientiarum ; Acta 1937:
1 ; 4, nos. 1-5

Pontificia Academia Scientiarum ; Commenta-
tiones 1937: 1; 3, no. 13; 4, nos. 1-2

Popular Science Monthly 1872: 1-87; Supple-
ment 1-18; n.s. 1

Porto Rico Review of Public Health and Tropi-
cal Medicine see Puerto Rico Journal of
Public Health and Tropical Medicine
*Prace Geofizyczne (Etudes Geophysiques)
(1-6 as Prace Meteorologiczne i Hydro-
graficzne) 1924: Towarzystwo Geofizykow
w Warszawie (Societe Geophysique de Var-
sovie). 2-4; 6-10

Prace Instytutu im. Nenckiego (Travaux de
1'Institut Nencki) 1921: Towarzystwo Nau-
kowe Warszawskie (Societe des Sciences
et des Lettres a Varsovie). 1; [2]; 3-14

Prace Moravske Prirodovedecke Spolecnosti
(Acta Societatis Scientiarum Naturalium
Moravicae) 1924: Brno, Ceskoslovensko.
1-10

Prace Naukowe Uniwersitetu Paristwowego
na Bialorusi see Trudy Belorusskogo Gosu-
darstven. Universiteta

Prace Paristwowego Instytutu Meteorologicz-
nego (Memorial de 1'Institut National Me-
teorologique de Pologne) 1930: Warsaw. 1 4-

Prace z Pracowni Neurobiologicznej 1916:
Towarzystwa Naukowego Warszawskiego,
III. Wydziat nauk Matematycznych i Przy-
rodniczych. 1

Prace Zoologiczne Polskiego Paristwowego
Muzeum Pryzrodniczego see Annales Musei
Zoologici Polonici

*Practical Entomologist 1865: The Entomologi-
cal Society of Philadelphia. 1-2

Pratsi Naukovo-Doslidnogo Institutu Biologii
(Travaux de 1'Institut de Recherches Scien-
tifiques de Biologie): Universite d'Etat de
Kiev. 2 (1939)4-^

Preslia; Vestnik Ceskoslovenske Botanicke
Spolecnosti (Bulletin de la Societe Botanique
Tchecoslovaque a Prague) (Reports of the
Czechoslovak Botanical Society of Prague)
1921: 10-12

Presse Medicale; Journal Bi-Hebdomadaire
1893: 34-36

*Preussisches Meteorologisches Institut Ab-
handlungen 1888: [4-5]; 8-10, no. 6

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Pringsheims Jahrbiicher fur Wissenschaft-
liche Botanik see Jahrbiicher fiir Wissen-
schaftliche Botanik

Problemes Biologiques; collection de mono-
graphies publiee sous le patronage du Co-
mite Technique des Sciences Naturelles
des Presses Universitaires de France 1925:
1-19; 21

Problems of Comparative Physiology; col-
lected papers of the Laboratory of Com-
parative Physiology of the Timiriasev Bio-
logical Institute see Nekotorye Yoprosy
Sravnitel'noi Fiziologii

Proceedings; Hawaiian Academy of Science
see Special Publications; Bernice P. Bishop
Museum

Proceedings; Industrial Statistics Conference
1938: 1st (Cambridge, Mass.)

Proceedings; North American Council on
Fishery Investigations 1921: Ottawa. 1-2

Federation Proceedings; published quarterly
by the Federation of American Societies for
Experimental Biology 1942: 1 +

Proceedings and Transactions of the Liver-
pool Biological Society (7+ includes Report
on the Lancashire Sea Fisheries Labora-
tory) 1886: 2 +

*Proceedings and Transactions of the Natural
History Society of Glasgow 1851 : ser. 2, 1-5 ;
ser. 3, 1-8

Proceedings and Transactions of the Nova
Scotian Institute of Science 1863: [2]; [4-7];
8 +

Proceedings and Transactions of the Royal
Society of Canada see Transactions of the
Royal Society of Canada

Proceedings (of) International Congresses see
International . . .

Proceedings of Scientific Societies 1876-97
(abstracts of) see in American Naturalist
10-31, 1876-97

Proceedings of the Academy of Natural
Sciences of Philadelphia 1841: 1 +

Proceedings of the American Academy of Arts
and Sciences 1846: 1 +

Proceedings of the American Association for
the Advancement of Science (beginning with
the 50th meeting, August 1901, addresses
and summaries of papers are given in Science
n.s. 14, 1901+) 1848: 1 +

*Proceedings of the American Chemical So-
ciety 1876: 1-2 (1879-1937) see in Journal
of the American Chemical Society; 1938 +
see in Chemical and Engineering News

Proceedings of the American Institute of Nu-
trition 1934: 1-8, 1934-41 see in Journal of
Nutrition 7-21, 1934-41; 9, 1942 + see in
Federation Proceedings 1, 1942 +

Proceedings of the American Microscopical

Society see Transactions of the American
Microscopical Society

Proceedings of the American Philosophical
Association 6, 1932+ see in Philosophical
Review 42, 1933 +

Proceedings of the American Philosophical
Society (early Proceedings 1744-1837 con-
tained in 22, 1885) 1838:^1-7]; 8+

Proceedings of the American Physiological
Society: 10-53, 1897-1941 see in American
Journal of Physiology 1-133, 1898-1941;
54, 1942+ see in Federation Proceedings 1,
1942 +

Proceedings of the American Scientific Con-
gress (5th as Proceedings of the Pan Ameri-
can Scientific Congress) 1898: 5; 8

Proceedings of the American Society for Clini-
cal Investigation: 17, 1925+ see in Journal
of Clinical Investigation 1, 1924-25 +

Proceedings of the American Society for Ex-
perimental Pathology: 14-27, 1927-40 see in
Archives of Pathology 4-29, 1927-40; 30,
1942+ see in Federation Proceedings 1,
1942 +

Proceedings of the American Society for
Pharmacology and Experimental Therapeu-
tics 1909: 1-32, 1909-41 see in Journal of
Pharmacology and Experimental Therapeu-
tics 1-72, 1909-41; 33, 1942+ see in Federa-
tion Proceedings 1, 1942 +

Proceedings of the American Society of Bio-
logical Chemists 1907: 1-35, 1907-41 see in
Journal of Biological Chemistry 3-140,
1907-41; 36, 1942+ see in Federation Pro-
ceedings 1, 1942 +

Proceedings of the American Society of Zoolo-
gists (formerly American Morphological So-
ciety) 1904: 1-13, 1 904-1 6 see in Science 19-
43, 1904-16; 14, 1916+ see in Anatomical
Record 11, 1916 +

Proceedings of the Anatomical Society of
Great Britain and Ireland 1888: 1+ see in
Journal of Anatomy 22, 1888 +

Proceedings of the Annual Congress on Med-
ical Education, Medical Licensure and Hos-
pitals 1905: American Medical Association.
1906-07; 1910; 1913-15; 1917; 1919-25;
1927; 1929-31

*Proceedings of the Annual Meeting of the As-
sociation of American Medical Colleges
1891: 32-34

Proceedings of the Association of American
Anatomists 1888: 1-14, 1888-1900; 15-20,
1901-05 see with American Journal of Anat-
omy 1-5, 1901-06; 21, 1906+ see in Ana-
tomical Record 1, 1906+

Proceedings of the Association of Economic
Biologists: 1919+ see in Annals of Applied
Biology 6, 1919 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Proceedings of the Association of Physicians
of Great Britain and Ireland 1907: 1 + see in
Quarterly Journal of Medicine 1, 1907 +

Proceedings of the Biological Society of Wash-
ington 1880: 1 +

Proceedings of the Boston Society of Natural
History 1844: 1 +

Proceedings of the Bristol Naturalists' So-
ciety 1863: ser. 3, [5-9]

Proceedings of the California Academy of
Sciences (with this Academy News Letter,
1940: 7 + ) 1854: 1 +

Proceedings of the Cambridge Philosophical
Society 1843: 3 +

Proceedings of the Cambridge Philosophical
Society; Biological Sciences see Biological
Reviews of the Cambridge Philosophical
Society

Proceedings of the Ceylon Natural History
Society 1912: 1+ see in Spolia Zeylanica 8,
1913 +

Proceedings of the Chemical Society: no. 16,
1885+ see with Journal of the Chemical
Society

*Proceedings of the Cleveland Academy of
Natural Sciences 1845: 1845-59

Proceedings of the Colorado Scientific So-
ciety see Colorado Scientific Society Pro-
ceedings

Proceedings of the Congress of the Zoologists,
Anatomists and Histologists, of the Union
of SSR see Trudy Vsesoiuznogo S'ezda Zoo-
logov, Anatomov i Gistologov

Proceedings of the Davenport Academy of

Sciences 1867: 1-7; 10; [12]
*Proceedings of the Elliott Society of Natural
History of Charleston, South Carolina 1 853 : 1
*Proceedings of the Entomological Society of

Philadelphia 1861: 1-6

*Proceedings of the Essex Institute 1848: Sa-
lem, Massachusetts. 4-6

Proceedings of the Genetics Society of Amer-
ica 1932: 1+ see in Records of the Genetics
Society of America 2, 1933 +

Proceedings of the Geological Society of
America 1888: 54 +

Proceedings of the Helminthological Society
of Washington (1911-13 see in Science 33-
37; 1914-33 see in Journal of Parasitology
1-20) 1934: 1 +

Proceedings of the Hydrological Congress of
U.S.S.R. see Trudy YsesoiTiznogo Gidrologi-
cheskogo S'ezda

Proceedings of the Imperial Academy; Japan
1912: 2 +

Proceedings of the Indian Academy of Sci-
ences; Sections A and B 1934: 1 +

Proceedings of the Indian Association for the
Cultivation of Science see Indian Journal

of Physics and Proceedings o'f the Indian
Association for the Cultivation of Science

Proceedings of the Indiana Academy of Sci-
ences 1891: 1-44

Proceedings of the International Congress for
Applied Mechanics: 1 (1924); 2 (1926); 3
(1938)

Proceedings of the International Congress of
Photography 1889: 7 (1928) (London)

Proceedings of the Iowa Academy of Sciences
1887: 1-37

Proceedings of the Japanese Pharmacological
Society: 3, 1929+ see in Japanese Journal of
Medical Sciences; IV. Pharmacology 4,
1930+

Proceedings of the Japanese Physiological So-
ciety: 1927+ see in Japanese Journal of
Medical Sciences; III. Biophysics 1, 1927 +

Proceedings of the Kossino Limnological Sta-
tion of the Hydrometeorological Service of
USSR see Trudy Limnologicheskoi Stantsii
v Kosine

Proceedings of the Linnean Society, London
1838: 1 +

Proceedings of the Linnean Society of New
South Wales (11-20 also numbered ser. 2,
1-10) 1876: 1 +

*Proceedings of the Los Angeles Zoological So-
ciety 1912: 1, no. 1

Proceedings of the National Academy of Sci-
ences (abstracts of the papers presented at
the annual meetings are published in Sci-
ence) *1, pt. 1-3, 1863-94; 1915: 1 +

Proceedings of the National Academy of Sci-
ences; India (1-3 as Bulletin of the Acad-
emy of Sciences of the United Provinces of
Agra and Oudh Allahabad; 4-5 as Proceed-
ings of the Academy etc.) 1931: Allahabad.
1 +

Proceedings of the National Institute of Sci-
ences of India 1935: 1 +

*Proceedings of the Natural History Society of
Wisconsin (1889+ see in Bulletin of the
Wisconsin Natural History Society) 1884:
1-5

Proceedings of the New England Zoological

Club 1899: Cambridge, Mass. [1-15]
*Proceedings of the Newport Natural History
Society 1883: 4-6; 8-9

Proceedings of the Northern Association for
Medical Radiology: 1932+ see in Acta Ra-
diologica 13, 1932 +

*Proceedings of the Ohio Academy of Science
1892: 4-8 (includes 12th-40th meetings)
(41st, 1931+ see in Ohio Journal of Science
32, 1932 + )

Proceedings of the Oklahoma Academy of
Science 1910: 1-12; 15; 17 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

"Proceedings of the Optical Convention 1005:

London. 1—3

Proceedings of the Pacific Science Congress
of the Pacific Science Association (3rd as
Proceedings of the Pan Pacific Science Con-
gress) 1920: 3-6

Proceedings of the Pan American Scientific
Congress see Proceedings of the American
Scientific Congress

Proceedings of the Pathological Society of
Great Britain and Ireland: 1906+ see in
Journal of Pathology and Bacteriology 12,
1907 +

Proceedings of the Perthshire Society of Nat-
ural Science see Transactions and Proceed-
ings of the Perthshire Society of Natural
Science

Proceedings of the Physical Society of Lon-
don (45+ contains Transactions of the
Optical Society 1933 + ) 1874: 1 +

Proceedings of the Physico-Chemical Sym-
posium on Photochemical Reactions 1 1 ,
1935 see in Acta Physicochimica U.R.S.S.
3, 1935

Proceedings of the Physiological Society:
1883+ see in Journal of Physiology 4,
1883 +

Proceedings of the Portland Society of Nat-
ural History 1862: Maine. 1; [2]

Proceedings of the Rochester Academy of
Sciences 1889: 1 +

Proceedings of the Royal Canadian Institute
1879: 1 +

Proceedings of the Royal Entomological So-
ciety of London (1871-85 see in Entomolo-
gist's Monthly Magazine 8-22) series A.
General Entomology 1926: 16+ series B.
Taxonomy 1926: 10 +

*Proceedings of the Royal Geographical So-
ciety of London 1855: [1]; 2-n.s. 14

Proceedings of the Royal Institution of Great
Britain 1851: 1 +

Proceedings of the Royal Irish Academy 1836:
1 +

Proceedings of the Royal Philosophical So-
ciety of Glasgow 1841: 30-34; 37-43; 45

Proceedings of the Royal Physiographic So-
ciety at Lund see (Kungl.) Fysiografiska
Sallskapets i Lund; Forhandlingar

Proceedings of the Royal Society of Edin-
burgh 1832: 8 +

Proceedings of the Royal Society of London
1800: 1-75; series A. Mathematical and
Physical Sciences 1905: 76+ ; series B. Bio-
logical Sciences 1905: 76+

Proceedings of the Royal Society of Medicine
1907: 1 +

Proceedings of the Royal Society of Victoria
1854: n.s. 1 +

Proceedings of the Royal Zoological Society of
New South Wales (191 4-32 see in Australian
Zoologist 1-7) 1933: 1933 +

Proceedings of the Section of Sciences (a
translation of the "Verslagen"); K. Neder-
landsch Akademie van Wetenschappen for-
merly K. Akademie van Wetenschappen te
Amsterdam) 1898: 1 +

Proceedings of the Society for Experimental
Biology and Medicine 1903: 1 +

Proceedings of the Society of American Bac-
teriologists: 18-26, 1916-24 see in Abstracts
of Bacteriology 1-9, 1917-25; 27, 1925 +
see in Journal of Bacteriology 11, 1926 +

Proceedings of the Sungaree River Biological
Station see Trudy Sungariiskoi Rechnoi Bio-
logicheskoi Stantsii

Proceedings of the United States National
Museum 1878: Smithsonian Institution. 1 +

Proceedings of the U.S.S.R. Congress of
Genetics, Plant- and Animal-Breeding see
Trudy Vsesofuznogo S'ezda po Genetike,
Selektsii, Semenovodstvu i Plemennomu
Zhivotnovodstvu

"Proceedings of the Washington Academy of
Sciences 1899: 1-13

Proceedings of the Zoological Society of Lon-
don (107-108, 1937-38 in three series: ser.
A. General and Experimental; ser. B. Sys-
tematic and Morphological; ser. *C. Ab-
stracts) 1830: 1 +

Proceedings of the Zoological Society of Lon-
don; Agenda and Abstracts of the Scientific
Meetings 1939:* 1-10

Proces Verbaux des Sciences; Congres de
Chimie Biologique (1-3 as Journees de
Chimie Biologique) 1927: 1+ see in Bulletin
de la Societe de Chimie Biologique 9 +

Program of Activities of the Chicago Academy

of Sciences 1930: 2-6, no. 4; 7, nos. 2-4
Progress Reports of Atlantic Biological Sta-
tion, St. Andrews, N. B. and Fisheries Ex-
perimental Station (Atlantic) Halifax, N. S.
1931: Biological Board of Canada. 1 +
Progress Reports of the Pacific Coast Stations ;
Pacific Biological Station, Nanaimo, B. C.
and Pacific Fisheries Experimental Station,
Prince Rupert, B. C. 1929: Fisheries Re-
search Board of Canada, Nanaimo. 1 +

*Progressus Rei Botanicae; Association Inter-
nationale des Botanistes 1907: 1-5
Protokoly Obshchestva Estestvoispytatelei pri
Imperatorskom lur'evskom Universitete see
Tartu Ulikooli juures oleva Loodusuurijate
Seltsi Aruanded

Protoplasma; Internationale Zeitschrift fiir
Physikalische Chemie des Protoplasten
1926: 1 +
Protoplasma-Monographien 1928: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Protozoology see Journal of Helminthology;
Supplement

Psyche; a. journal of entomology 1874: 1-10;

12; 14+

*Psychobiology ; edited by Knight Dunlap
1917: 1-2

Psychological Bulletin; Literary Section of the
Psychological Review 1904: 1-3; [4]; 5-10;
[13]; [15]; [19]

Psychological Monographs (1-8 as Mono-
graph Supplements) 1895: 8; 15-18; 29; 33-
34; 66; 69

Psychological Review 1894: [1-4]; 5-14; [15];
16-28; [29]; 30-31; [32] +

Psychometrika ; Supplement see Bulletin of

Mathematical Biophysics

*Pubblicazioni del R. Institute di Studi Super-
ior! Practici e di Perfezionamento in Fir-
enze; Sezione di Scienze Fisiche e Naturali
1877: 1-4; 6-7; 11-16; Sezione di Medicine
e Chirurgia 1876: 21-22

Pubblicazioni della Stazione Zoologica di Na-

poli 1916: 1 +

*Pubblicazioni della Stazione Zoologica di Na-
poli; Ricerche di Fisiologia e di Chimica
Biologica 1919: 1

Public Health Bulletin 1881: U. S. Public
Health Service. [19-267]

Public Health Reports issued weekly by the
United States Public Health Service 1886:
15; 16, pt. 1; 18; 20-25; [26]; 27-33; [34];
35+; Supplements 29; 52; 54-55; 61; 66;
69; 71; 74; 78; 82; 86; 89; 92-95; 138

Publicaciones de la Junta de Ciencies Natu-
rals de Barcelona, Musei Barcinonensis
Scientiarum Naturalium Opera 1917: series
Biologico-Oceanografia 1 ; series Botanica
1-2; series Zoologica 1-7; 11

*Publications ; Faculty of Medicine; Egyptian
University 1931: 12 (1937)

Publications; Faculty of Science; Fouad I
University (formerly Egyptian University)
1932: 1; 3 +

'"Publications; Puget Sound Biological Station;
University of Washington (1 as Puget Sound
Marine Station Publications) 1915: 1-7

Publications; Rockefeller Foundation; Inter-
national Health Board (2 International
Health Commission) 1914: 2; 7-11

*Publications ; Rockefeller Sanitary Commis-
sion for the Eradication of Hookworm Dis-
ease 1910: 1-3; 5-9

Publications Biologiques de 1'Ecole des Hautes
Etudes Veterinaires ; Brno see Biologicke
Spisy Vysoke" Skoly Zverole"karske

''Publications de Circonstance ; Conseil Perma-
nent International pour 1'Exploration de la
Mer 1903: 1-42; 46-91

Publications de la Faculte de Medecine de

1'Universite Masaryk see Spisy Lekarske
Fakulty, Masarykovy University

Publications de la Faculte des Sciences de
1'Universite Charles see Spisy Yydavane
Prirodovedeckou Fakultou Karlovy Uni-
versity

Publications de la Faculte des Sciences de
1'Universite Masaryk see Spisy Vydavane
Prirodovedeckou Fakultou Masarykovy
University

Publications of the American Association for

the Advancement of Science: 7-8; 10; 12-14

"Publications of the Bureau of Government

Laboratories; Philippine Islands 1902: 5;

7-8; 13-36

Publications of the Hartley Botanical Labora-
tories; University of Liverpool 1924: 5; 13

Publications of the Marine Biological Station,
Ghardaqa, Red Sea 1939: Fouad I Univer-
sity; Faculty of Science, Cairo. 1

Publications of the Nantucket Maria Mitchell
Association 1906: 1 +

Publications of the Scientific Institute of Fish-
ery and Oceanography see Trudy Azovsko-
Chernomorskogo Nauchno-Issledovatel'sko-
go Instituta Rybnogo Khoziaistva i Okeano-
grafii

Publications of the University of Oklahoma
Biological Survey 1928: 1-5, no. 4

Publications of the University of Sydney; De-
partment of Zoology; Monographs 1940: 1 +

Publications of the Wagner Free Institute of
Science 1929: 1-2

Puerto Rico Journal of Public Health and
Tropical Medicine (1-4 as Porto Rico Re-
view of Public Health and Tropical Medi-
cine; 5-7 as Porto Rico Journal of Public
Health and Tropical Medicine) 1925: School
of Tropical Medicine, University of Puerto
Rico, Department of Health. 1 +

Puget Sound Marine Station Publications see
Publications; Puget Sound Biological Sta-
tion; University of Washington

Quarterly Bulletin of Chinese Bibliography
(English Edition): National Library of
Peiping. n.s. 1, nos. 1, 3-4 (1940)

Quarterly Cumulative Index Medicus 1927:

American Medical Association. 1 +
* Quarterly Cumulative Index to Current Medi-
cal Literature 1916: American Medical As-
sociation. 1-12 (1926)

Quarterly Journal of Experimental Physiology
and Cognate Medical Sciences 1908: Lon-
don. 1-7; 9; 11 +

Quarterly Journal of Medicine 1907: Oxford.

1 +

Quarterly Journal of Microscopical Science
1852: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Quarterly Journal of Pharmacy and Pharma-
cology; incorporating the Yearbook of
Pharmacy 1928: London. 1 +
* Quarterly Journal of Science, Literature, and
Art (1-5 as Journal of Science and the Arts;
6-7, Quarterly Journal of Literature, Sci-
ence, and the Arts; 8-20, 22, Quarterly
Journal of Science, Literature, and the Arts;
21, Quarterly Journal of Science and the
Arts) 1816: London. 1-22; n.s. 1-6; 7, pt. 2

Quarterly Journal of the Indian Chemical So-
ciety see Journal of the Indian Chemical
Society

Quarterly Journal of the Indian Institute of
Science 1938: 1 +

Quarterly Journal of the Royal Meteorological
Society 1871: 1 + ; Phenological Report
1876: 1 + ; Supplement; Bibliography of
Meteorological Literature; prepared by the
Royal Meteorological Society with the col-
laboration of the Meteorological Office 1920:
1 +

Quarterly Review of Biology 1926: 1 +

Raboty Azovsko-Chernomorskoi Nauchnoi
Rybokhoziaistvennoi Stantsii see Report (s)
of the Scientific Station of Fisheries of Asov
and Black Seas

Raboty Dono-Kubanskoi Nauchnoi Rybokho-
ziaistvennoi Stantsii see Report (s) of the
Don-Kuban Station

*Raboty Murmanskoi Biologicheskoi Stantsii;
Leningradskogo Obshchestva Estestvoispy-
tatelei (Trauvaux de la Station Biologique
de Murman de la Societe des Naturalistes
de Leningrad) 1925: 1-3

Raboty Novorossiiskoi Biologicheskoi Stantsii
(Arbeiten der Biologischen Noworossijsk-
Station) 1923: 1-3

Raboty Volzhskoi Biologicheskoi Stantsii see
Arbeiten der Biologischen Wolga-Station

Radiography 1935: Society of Radiographers.
1 +

Radiologica see Fundamenta Radiologica

Radiology; a monthly journal devoted to clini-
cal radiology and allied sciences 1923: Ra-
diological Society of North America. 17 +
*Radium 1904: Paris. 1-11

Rapport; Station Biologique du St.-Laurent a
Trois-Pistoles 1931: Universite Laval, Que-
bec. 1-3

Rapport(s) et Proces-Verbaux des Reunions;
Commission Internationale pour 1'Explora-
tion Scientifique de la Mer Mediterranee
1926: 1 +

Rapport(s) et Proces-Verbaux des Reunions;
Conseil Permanent International pour 1'Ex-
ploration de la Mer 1902: 1-6; [7]; 8-10;
[11]; 12-16; [17]; 18 +

Rapport sur le Fonctionnement Technique de
1'Institut Pasteur de Brazzaville: Generate
Afrique Equatoriale Francaise. 1930-32;
1935 +

Rapport sur les Pecheries d'Egypte (Report of
the Fisheries of P3gypt); Institut Fouad I
d'Hydrobiologie et de Peche 1923: Minis-
tere du Commerce et de I'lnclustrie. 1923-
27; 1931 +

Rassegna delle Scienze Biologiche 1919:
Florence. [1-2]

Reading Public Museum and Art Gallery;
Scientific Publications 1941: Reading, Pa.
1 +

Records of Oceanographic Works in Japan;
compiled by the Committee on Pacific Ocea-
nography of the National Research Council
of Japan 1928: 1 +

Records of the American Society of Natural-
ists (1884-85 as Records of the Society of
Naturalists of the eastern United States)
1884: 1 +

Records of the Auckland Institute and Mu-
seum 1930: 1 +

Records of the Australian Museum 1890: 1 +

Records of the Genetics Society of America (1
as Genetics Society of America, Program)
1932: 1 +

Records of the Indian Museum; a journal of
Indian zoology (includes Report of the Zoo-
logical Survey of India) 1907: 1 +

Records of the South Australian Museum
1918: Public Library, Museum, and Art
Gallery, Adelaide. 1 +

Recueil de 1'Institut Zoologique Torley-Rous-
seau; Universite de Bruxelles 1927: 1 +

Recueil des Travaux Botaniques Neerlandais
1904: Societe Botanique Neerlandaise. 1 +

Recueil des Travaux Chimiques des Pays-
Bas; Societe Chimique Neerlandaise 1882:
1 +
*Recueil Zoologique Suisse 1883: 1-5

Refrigerating Engineering; Economic Applica-
tions of Refrigeration and Air Conditioning
1914: American Society of Refrigerating
Engineers, New York. '[29]; [36] f 37-42,
no. 5

Rendiconti; Real Istituto Lombardo di Scienze
e Lettere 1864: ser. 2, [1-2]; 62

Rendiconti delle sedute solenni della R. Ac-
cademia Nazionale dei Lincei see Atti della
Reale Accademia Nazionale dei Lincei; Ren-
diconto dell'adunanze solenne

Rendiconto dell'Accademia delle Scienze Fi-
siche e Matematiche; Classe della Societa
Reale di Napoli 1862: ser. 3, 9-26

Report; Council for Scientific and Industrial
Research; Division of Fisheries; Common-
wealth of Australia 1938: 3-6

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Report; Division of Fisheries; Union of South
Africa (1-14 as Report; Fisheries and Ma-
rine Biological Survey; Union of South
Africa) 1920: 1 + ; Inland Waters Fisheries
Survey (Dept. of Mines and Industry) Re-
ports 1-2

Report; Dove Marine Laboratory (preceded
by Report on the Scientific Investigations
of the Northumberland Sea Fisheries Com-
mittee) 1896: Cullercoats, Northumberland.
1909-1 l;ser. 2, 1-12; 14 +

Report; John Crerar Library 1895: 1-2; 4; 6-38

Report(s) ; Marine Biological Laboratory 1888:
Woods Hole. 1 +

Report; National Research Council of Japan
1922: 1 +

Report; Rothamsted Experimental Station;
Harpenden; Lawes Agricultural Trust 1843:
1908 +

*Report(s) and Notes of the Public Health
Laboratories; Cairo 1914: Ministry of the
Interior, Egypt. 3-6, 1920-24

Report and Proceedings of the Belfast Natural
History and Philosophical Society 1871:
[1874-1913]

Report of the Imperial Bureau of Fisheries;
Scientific Investigations 1912: Tokyo. 1-2

Report (s) of the Academy of Sciences of the
Ukrainian SSR see Dopovidi Akademii Nauk
URSR

Report of the Australian and New Zealand
Association for the Advancement of Science
(1-19 as Australasian Association) 1888:
1-5; 7-9; 11 +

Report of the British Association for the Ad-
vancement of Science 1831: 1 +

*Report of the Brush Hill Bird Club 1914: Mil-
ton, Massachusetts. 1

Report (s) of the Bureau of Applied Entomol-
ogy; State Institute of Experimental Agron-
omy, Leningrad see Report (s) on Applied
Entomology

Report (s) of the Central Scientific Institute of
Fisheries see Trudy Tsentral'nogo Nauch-
nogo Instituta Rybnogo Khoziaistva

Report of the Chief Inspector of Fisheries;
Western Australia: 1938 +

Report of the Chief of the Bureau of Biological
Survey see United States Department of the
Interior; Bureau of Biological Survey; Re-
port of the Chief

Report of the Chief of the Bureau of Plant In-
dustry see United States Department cf
Agriculture; Bureau of Plant Industry; Re-
port of the Chief

Report of the Chief of the Weather Bureau see
United States Meteorological Yearbook

Report of the Commissioner of Agriculture see

United States Department of Agriculture;
Report of the Secretary

Report of the Commissioner of Fisheries;
Province of British Columbia: 1913

Report of the Commissioner of Fisheries;
United States Commission of Fish see Re-
port of the United States Commissioner of
Fisheries

Report of the Commissioner of Health of Porto
Rico to the Governor of Porto Rico: 1926-31

Report of the Commissioners on Fisheries and
Game, Massachusetts see Annual Report of
the Commissioners on Fisheries and Game;
Massachusetts

Report of the Connecticut Agricultural Experi-
ment Station 1877: [1903-26]; 51 +

Report(s) of the Council and Auditors of the
Zoological Society of London 1829: 1935;
1937-38; 1940 +

Report of the Danish Biological Station to the
Ministry of Agriculture and Fisheries 1890:
3; 5 +

Report of the Delegates of the United States
of America to the Pan American Scientific
Congress 1908: 1; 3

Report (s) of the Don-Kuban Station; Scien-
tific Institute of the Fisheries and the Ocea-
nography of Asov and Black Seas (Raboty
Dono-Kubanskoi Nauchnoi Rybokhozfaist-
vennoi Stantsii ; Azovsko-Chernomorskii
Nauchno-Issledovatel'skii Institut Morskogo
Rybnogo Khoziaistva i Okeanografii) 1934:
1-3

Report (s) of the East-Siberian Scientific Sta-
tion of Fisheries see Trudy Vostochno-
Sibirskoi Nauchnoi Rybokhoziaistvennoi
Stantsii

Report of the Henry Phipps Institute 1903:
Philadelphia. 1-7; 9-10; 12-14; 18 +

Report (s) of the Ichthyological Laboratory see
Report (s) of the (Astrakhan) Scientific Sta-
tion of Fisheries of Volga and Caspian Sea

Report (s) of the Ichthyological Laboratory in
Kertch see Report(s) of the Scientific Station
of Fisheries of Asov and Black Seas

Report of the Ichthyological Laboratory in
Siberia see Trudy Vostochno-Sibirskoi
Nauchnoi Rybokhoziaistvennoi Stantsii

Report of the Imperial Fisheries Institute;
Scientific Investigations: Tokyo. 4; 7, no. 4

Report of the Institute of Scientific Research ;
Manchoukuo 1937: [1]; 2-3; [4] +

Report of the International Fisheries Commi-
sion; appointed under the treaty between
the United States and Great Britain for the
preservation of the Northern Pacific Halibut
Fishery 1931: 1-12

Report (s) of the Johns Hopkins Hospital see
Johns Hopkins Hospital Reports

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Report of the Librarian of Congress see Annual
Report of the Librarian of Congress

Report of the Marine Biological Station of
Port Erin, Isle of Man (1-5 as Annual Re-
port of the Liverpool Marine Biological Sta-
tion on Puffin Island; 6-46 as Annual
Report of the Marine Biological Station at
Port Erin under Liverpool Marine Biology
Committee) (1-46 see in Proceedings and
Transactions of the Liverpool Biological
Society) 1887: 1 +

Report of the Medical Research Council 1919:
London. 1933 +

Report of the National Academy of Sciences
(includes Report of the National Research
Council, 1916+) 1863: 1863-67; 1878+ ;
Report by the President on the Surveys of
the Territories 1878

Report of the National Research Council see
Report of the National Academy of Sciences
*Report of the Oceanographical Investigation
1926: Government Fishery Experiment Sta-
tion, Chosen, Japan. 1-2

Report of the Proceedings of the International
Hydrographic Conference (1, 1919, London;
also 1, 1929 as "First Supplementary",
Monaco) 1919: International Hydrographic
Bureau, Monaco. 1 +

Report of the Reelfoot Lake Biological Station
1937: Tennessee Academy of Sciences. 5

Report of the Royal Society of South Australia
see Transactions of the Royal Society of
South Australia

*Report of the Royal Society of Tasmania
(through 1855 as Report of the Royal So-
ciety of Van Diemen's Land) 1844: 1 + ;
1867-1932 see in Papers and Proceedings of
the Royal Society of Tasmania for 1867-
1932

*Report of the Science Advisory Board; Na-
tional Research Council 1933: 1-2

Report (s) of the Scientific Institute of Fish-
eries; Moscow see Trudy Nauchnogo Insti-
tuta Rybnogo Khozfaistva
*Report(s) of the Scientific Station of Fisheries
of Asov and Black Seas (Raboty, Trudy,
Azovsko-Chernomorskoi Nauchnoi Rybok-
hoziaistvennoi Stantsii) (1-3 as Reports of
the Scientific Station of Fisheries, Ichthyo-
logical Laboratory, in Kertch; Trudy Ker-
chenskoi Nauchnoi Rybokhozfaistvennoi
Stantsii, IkhtiologicheskoiLaboratorii) 1926:
1-10

Report (s) of the (Astrakhan) Scientific Station
of Fisheries of Volga and Caspian Sea (Trudy
Volgo-Kaspiiskoi Nauchnoi Rybokhoziaist-
vennoi Stantsii) (1-6, no. 1 as Reports of the
Ichthyological Laboratory) (Trudy Ikhtio-
logicheskoi Laboratorii) (Arbeiten des Ich-

thyologischen Laboratoriums der Kaspi-
Wolgaschen Fischerei-Verwaltung in Astra-
chan) (Travaux du Laboratoire Ichtyologi-
que d'Astrakhan aupres de 1'Administration
des Pecheries du Volga et de la Mer Cas-
pienne) (with various wording of titles and
no English translation for 1-4) 1909: 1 +

Report (s) of the Secretary and of the Treas-
urer; John Simon Guggenheim Memorial
Foundation 1925: 1928 +

Report of the Secretary of Agriculture see
United States Department of Agriculture;
Report of the Secretary

Report of the Secretary of the State Board of
Agriculture of the State of Michigan (1862-
79 as Annual Report; 1880+ as Biennial
Report) 1862: 1870; 1873-74; 1879; 1880-82

Report (s) of the Siberian Scientific Station of
Fisheries see Trudy Vostochno-Sibirskoi
Nauchnoi Rybokhoziaistvennoi Stantsii

Report of the South African Association for the
Advancement of Science see South African
Journal of Science

(Biennial) Report of the State Board of Fish-
eries and Game; State of Connecticut:
1922 +

Report of the State Entomologist of Minne-
sota to the Governor (1-3 as Annual Report
of the Entomologist of the State Experiment
Station of the University of Minnesota to
the Governor) 1893: 1-3; 11-12

Report (s) of the State Oceanographical Insti-
tute (Doklady Gosudarstvennogo Okeano-
graficheskogo Instituta) 1931: Moscow. 1-6

Report of the Superintendent, United States
Coast and Geodetic Survey see Annual Re-
port of the Director; United States Coast
and Geodetic Survey

*Report of the United States Commissioner of
Fisheries (since 1913 under United States
Department of Commerce; Bureau of Fish-
eries) 1871: 1871-1938

Report of the United States National Museum
(before 1885 included in the Annual Report
of the Board of Regents of the Smithsonian
Institution): 1885 +

Report of the Virginia Fisheries Laboratory of
the College of William and Mary and the
Commission of Fisheries of Virginia 1940:
1940+

Report of the Waite Agricultural Research In-
stitute, South Australia 1925: 1939 +
*Report of the (Tropical) Wellcome Research
Laboratories at the Gordon Memorial Col-
lege, Khartoum (4 in two sections; A. Medi-
cal; B. General Sciences) 1904: 1-4
*Report(s) on Applied Entomology (Izvestiia po
Prikladnoi Entomologii) (1-3 as Reports of
the Bureau of Applied Entomology; State

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Institute of Experimental Agronomy) 1921:
1-4, no. 2

*Report on Colloid Chemistry and its general
and industrial applications ; British Associa-
tion for the Advancement of Science; De-
partment of Scientific and Industrial Re-
search 1917: 1-2; 4-5

Report on Fisheries; Marine Department;
New Zealand 1928: 1 +

Report on Marine and Fresh Water Investiga-
tions; Department of Zoology; University
College of Wales; Aberystwyth 1913: n.s.
(1923) 1 +

Report on Norwegian Fishery and Marine In-
vestigations (Fiskeridirektoratets Skrifter;
serie Havunder0kelser) 1900: 1 +

Report (s) on Progress in Physics 1934: Physi-
cal Society, London. 1 +

Report on the Fisheries of Egypt see Rapport
sur les Pecheries d'Egypte

Report on the Injurious and Other Insects of
the State of New York 1881: New York
State Entomologist. 1-12; 13+ see in New
York State Museum Bulletin

Report on the Lancashire Sea Fisheries Lab-
oratory see Proceedings and Transactions of
the Liverpool Biological Society

Report on the Scientific Investigations of the
Northumberland Sea Fisheries Committee
see Report; Dove Marine Laboratory

Report to the Government of Ceylon on the
Pearl Oyster Fisheries of the Gulf of
Manaar; with supplementary reports upon
the Marine Biology of Ceylon 1903: 1-5

Report upon the Fauna of Liverpool Bay and
the Neighboring Seas; written by the mem-
bers of the Liverpool Marine Biology Com-
mittee and other Naturalists (articles in nos.
2-5 appear also in the Proceedings and
Transactions of the Liverpool Biological
Society) 1886: 1-5

Reprint and Circular Series of the National
Research Council 1919: 1-11; 13 +

Republica Argentina; Ministerio de Marina;
Servicio Hidrografico; Almanaque Nautico:
1940-42; Catalogo de Cartas y Libros Para
Navegacion: 1941; Tablas de Marea: 1940-
42; Avisos a los Navigantes: 16-17 (1942)

Research Bulletin; Agricultural Experiment
Station of the Iowa State College of Agri-
culture and Mechanic Arts 1911: 1-85; 87-
151; 153; 164-65; 202; 224; 254; 259

Research Bulletin; Agricultural Experiment
Station of the University of Wisconsin 1909:
1 +

Research Bulletin; College of Agriculture;
University of Nebraska; Agricultural Ex-
periment Station 1913: [1-93] +

Research Bulletin; Fishery Research Insti-

tute; Department of National Resources
1932: St. Johns, Newfoundland. 1 +

Research Bulletin; Saito Ho-on Kai Museum

1934: Sendai, Japan. 1 +

*Research Bulletin; State University of Okla-
homa 1909: 1-4

Research Bulletin; University of Missouri
College of Agriculture; Agricultural Experi-
ment Station: [89-335]

Research Bulletin ; University of Puerto Rico ;
Agricultural Experiment Station, Rio Pie-
dras 1941: 1 +

Research Series; American Geographical So-
ciety 1921: 6; 16

Research Studies of the State College of

Washington 1929: 1 +

*Researches of the Loomis Laboratory of the
Medical Department of the University of
the City of New York 1890: 1-2

Researches on the Ontogeny of Animals see
Zbirnik Doslidiv nad Individual'nim Roz-
vitkom Tvarin

*Reseau Mondial; monthly and annual sum-
maries of pressure, temperature, and pre-
cipitation based on a world-wide network of
observing stations (1910-21 as British Mete-
orological and Magnetic Year Book, part V)
1910: Meteorological Office. 1910-32

Resume du Compte-Rendu Annuel de la So-
ciete Royale des Lettres et des Sciences de
Boheme see Vyrocni Zprava Kralovske
Ceske Spolecnosti Nauk

Resumptio Genetica 1924: The Hague. 1 +
*Review of American Chemical Research; A. A.
Noyes 1895: 1-12

Review of Applied Entomology 1913: Imperial
Bureau of Entomology, London, series A.
Agricultural 1 + ; series B. Medical and
Veterinary 1 +

Review (s) of Modern Physics (vol. 1 as Physi-
cal Review Supplement) 1929: American
Physical Society. 1 +

Review of Scientific Instruments; new series
(old series incorporated in Journal of the
Optical Society of America and Review of
Scientific Instruments) 1930: American In-
stitute of Physics. 1 +

Review of the Society of Chemical Industry see
Journal of the Society of Chemical Industry

Revista Chilena de Historia Natural Pura y
Aplicada; Dedicada al fomento y cultivo de
las Ciencias Naturales en Chile 1897: [6];
[8]; 29; 31-32; 35

*Revista de Bacteriologia e Higiene 1912: Ins-
titute Nacional de Bacteriologia, La Paz,
Bolivia. 3-5

Revista de Biologia e Hygiene 1928: Sociedade
de Biologia de Sao Paulo. [1-5]

Revista de Ciencias ; Organo de la Facultad de

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Ciencias de la Universidad Nacional Mayor

de San Marcos 1897: Lima. 42, no. 431 +
Revista de la R. Academia de Ciencias Exac-

tes, Fisicas y Naturales de Madrid 1904:

[2-14]
Revista de la Asociacion Medica Argentina

1892: 37; 49, no. 356 +
Revista de la Sociedad Argentina de Biologia

y su Filiale la Sociedad de Rosario (1, no.

2-8, no. 1 as Revista . . . y su Filial la

Sociedad de Biologia del Literal) 1925: 1-4;

6 +
Revista de la Sociedad Mexicana de Historia

Natural 1939: 1 +
Revista de Sciencias Naturaes see Broteria;

Revista Luso Brazileira
Revista del Consejo Oceanografico Ibero-

Americana 1930: Madrid. 1-6, no. 1
Revista del Institute Bacteriologico del De-

partamento Nacional de Higiene 1917:

Buenos Aires. [1-2]; 4 +
Revista del Institute de Salubridad y Enfer-

medades Tropicales 1939: Mexico. 1 +
Revista del Museo de La Plata; Universidad

Nacional de La Plata 1890: 1-5; [7]; 11-23;

[24]; 26; 28; 30 +
Revista di Scienza see Scientia
*Revista do Museu Paulista 1895: Universidade

de Sao Paulo. 1-23
Revista Espanola de Biologia 1932: Sociedad

Espanola de Historia Natural. 1-5, no. 3
Revista Farmaceutica; Organo Oficial de la

Asociacion Farmaceutica y Bioquimica Ar-
gentina 1862: 74+

Revista Medico-Chirurgicala din lasi see Re-
vue Medico-Chirurgicale de Jassy
Revista Mexicana de Biologia; organo de la

Sociedad Mexicana de Biologia 1920: [1-17]
Revue Algologique 1924: Laboratoire de Cryp-

togamie du Museum National d'Histoire

Naturelle, Paris. 1 +
Revue Beige des Sciences Medicales 1929:

1 +
*Revue Biologique du Nord de la France 1888:

Lille. 1-7

Revue Bryologique 1874: [7-9]
Revue Canadienne de Biologic; Universite de

Montreal 1942: 1 +
Revue d'lmmunologie 1935: 1 +
Revue d'Optique Theorique et Instrumental ;

Institut d'Optique Theorique et Appliquee

1922: 1-2, no. 9
Revue de Cytologie et de Cytophysiologie Ve-

getales; publiee par A. Guilliermond 1935:

1, no. 2
Revue de la Medecine Experimentelle et

Pratique see Glasnik Centralnog Higijenskog

Zavoda
Revue de Mycologie; annales de cryptogamie

exotique, nouvelle serie 1936: Laboratoire
de Cryptogamie du Museum National d'His-
toire Naturelle. 1 +

Revue des Archives Italiennes de Biologic (1,
no. 1 as Nouvelles Archives Italiennes de
Biologie) 1938: Pisa. 1 +

Revue des Travaux de 1'Office des Peches
Maritimes; Office Scientifique et Technique
des Peches Maritimes 1928: 1 +
*Revue et Magazin de Zoologie, pure et appli-
quee (ser. 1 as Revue Zoologique par la
Societe Cuvienne) 1838: ser. 1, 8-9; ser. 2,
1-23

Revue Francaise d'Endocrinologie 1923: 4 +
*Revue Generale d'Histologie 1904: 1-4

Revue Generale de Botanique; fondee par
Gaston Bonnier 1889: [l];5-7; [12-14]; 39 +
*Revue Generale des Colloi'des; et de leurs ap-
plications industrielles 1923: 1-8

Revue Generale des Sciences Pures et Appli-
quees 1890: 1-31; 33-39, no. 9

(La) Revue Hydrographique see Hydrographic

Review

*Revue Internationale de 1'Electricite et de ses
applications 1885: 3; 5-37; 97-113; 115-120

Revue Medico-Chirurgicale de Jassy (Bulletin
de la Societe des Medecins et des Natu-
ralistes) (1-34 as Bulletin de la Societe des
Medecins et des Naturalistes de Jassy)
(Buletinul Societatei de Medeci si Natu-
ralisti din lasi) (35-42 as Revista Medico-
Chirurgicala din lasi; Buletin al Societatei
de Medeci si Naturalist!) 1887: Roumania.
1 +
*Revue Mycologique 1878: Toulouse. 1-28

Revue Scientifique 1863: ser. 5, [1-9]

Revue Suisse de Zoologie; Annales de la So-
ciete Zoologique Suisse et du Museum
d'Histoire Naturelle de Geneve ; fondee par
Maurice Bedot 1893: 1 +

Revue Zoologique Russe see Zoologicheskii
Zhurnal

Rheological Memoirs; Eugene C. Bingham,
Editor 1940: 1, no. 1

Rhodora; journal of the New England Botani-
cal Club 1899: 1 +

*Ricerche (fatte nel Laboratorio) di Anatomia
normale della R. Universita di Roma ed in
Altri Laboratori Biologici; pubblicate dal
Professore Francesco Todaro 1872: 1-19

Ricerche di Morfologia; pubblicate dal Prof.
Riccardo Versari 1920: 1-12

Rivista di Biologia; redatta da Osvaldo Poli-
manti (Perugia) 1919: 1 +

Rivista di Biologia Coloniale 1938: Rome. 1 +
*Rivista di Biologia Generale (1-2 as Rivista di
Scienze Biologiche) 1899: Turin. 1; [2]

Rivista di Parassitologia ; fondata da A. Missi-
roli 1937: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Rivista di Patologia Nervosa e Mentale; di-
retta da E. Tanzi 1896: [1]; 2-3

Rivista di Patologia Sperimentale ; f ondata da
Vittorio Scaffidi (12+ also as n.s. 1 + ) 1926:
1 +

Rockefeller Foundation Review 1917: 1917-
28; 1936+

Rocznik Towarzystwa Naukowego Warszaw-
skiego (Annuaire de la Societe des Sciences
et des Lettres de Varsovie) 1908: 26-30

Roczniki Chemii; Miesiecznik organ Polskiego
Towarzystwa Chemicznego; Jana Zawidz-
kiego (Annales Societatis Chimicae Polono-
rum) 1921: 1-19, no. 7

Roosevelt Wild Life Annals ; of the Roosevelt
Wild Life Forest Experiment Station 1926:
New York State College of Forestry. 1 +

Roosevelt Wild Life Bulletin; New York State
College of Forestry 1921: 1 +

Roux's Archiv fur Entwicklungsmechanik der
Organismus see Wilhelm Roux's Archiv fiir
Entwicklungsmechanik der Organismen

Roux' Vortrage und Aufsatze tiber Entwick-
lungsmechanik der Organismen, old series,
see Vortrage und Aufsatze iiber Entwick-
lungsmechanik der Organismen; new series,
see Abhandlungen zur Theorie der Organi-
schen Entwicklung

Rozpravy Kralovske Ceske Spolecnosti Nauk ;
Trida Matematicko-Prirodovedecka (Tra-
vaux de la Societe Royale des Sciences de
Boheme; Classe des Sciences) (ser. 1-7 as
Abhandlungen) 1775: ser. 8, 1-3

Rozpravy Vedecke Spolecnosti Badatelske pfi
Ruske Svobodne Universite v Praze see
Bulletin de 1' Association Russe pour les
Recherches Scientifiques a Prague
*Russian Journal of Eugenics (Russkii Evgeni-
cheskii Zhurnal) 1922: 1-7

Russian Journal of Physiology see Fiziologi-
cheskii Zhurnal SSSR

Russian Journal of Zoology see Journal Russe
de Zoologie

Russische Hydrobiologische Zeitschrift see
Berichte des Wissenschaftlichen Meeresins-
tituts

Russische Hydrobiologische Zeitschrift see
Gidrobiologicheskii Zhurnal SSSR

Russkii Arkhiv Anatomii, Gistologii i Embrio-
logii see Arkhiv Anatomii, Gistologii i Em-
briologii

Russkii Arkhiv Protistologii (Archives Russes
de Protistologie) 1922: 1-7

Russkii Evgenicheskii Zhurnal see Russian
Journal of Eugenics

Russkii Fiziologicheskii Zhurnal SSSR see
Fiziologicheskii Zhurnal SSSR

Russkii Gidrobiologicheskii Zhurnal see Gidro-
biologicheskii Zhurnal SSSR

Russkii Zoologicheskii Zhurnal see Zoologi-
cheskii Zhurnal

*Sbornik Nauchno-Issledovatel'skogo Instituta

Zoologii (Abstracts of the Works of the

Zoological Institute of the Moscow State

University) (1-2 as Bmlleten) 1933: Mos-

kovskogo Gosudarstvennogo Universiteta.

1-3
Sbornik Prirodovedecky ; Vydava Druha Trida

Ceske Akademie ved a Umfeni 1928: 5
Sbornik Trudov Gosudarstvennogo Zoologi-

cheskogo Muzeya (Archives du Musee Zoo-

logique de 1'Universite de Moscou) 1934: 1 +
Sbornik Vysoke Skoljr Zemedelske v Brne,

CSR (Bulletin de, 1'Ecole Superieure, 1'Ins-

titut National Agronomique, Brno, RCS)

1924: C1-C35; D1-D27
*Sbornik Zoologicky; Vydavatel, Prof. Dr.

Alois Mrazek v Praze 1917: Prague. 1, no. 1
Schmiedebergs Archiv fiir Experimentelle

Pathologic und Pharmakologie see Naunyn-

Schmiedebergs Archiv fiir Experimentelle

Pathologic und Pharmakologie
School Science and Mathematics; a journal

for all science and mathematics teachers

1901: [1]; [3-6]; [23-27]
Schriften der Gesellschaft zur Beforderung

der Gesamten Naturwissenschaften zu

Marburg 1823: 12-18
Schriften der Naturforschenden Gesellschaft,

Danzig 1820: n.f. [1-8]; 9-14; [15-16];

17-20, no. 3 (1938)
Schriften des Naturwissenschaftlichen Ver-

eins fiir Schleswig-Holstein 1873: 1 +
Schriften herausgegeben von der Natur-

forscher-Gesellschaft bei der Universitat

Tartu (through 23, 1916 as Schriften . . .

Jurjeff, Dorpat) 1884: 2 +
Science 1883: 1 + ; Supplement; Occasional

Publications of the American Association for

the Advancement of Science, no. 2, 1934; 4,

1937
*Science; a weekly record of scientific progress

1880: New York. 1, nos. 1, 20
Science Abstracts; Section A; Physics see

Physics Abstracts
Science and Letters in Poland, their needs,

organization and progress see Nauka Polska

jej Potrzeby, Organizacja i Rozwoj
Science Bulletin; Department of Agriculture

and Forestry; Union of South Africa 1911:

[3-232] +

Science Bulletin; The Museum of the Brook-
lyn Institute of Arts and Sciences 1901 : 1-3,

no. 5; 4, no. 1
*Science Gossip; Hardwick's 1865: London.

1-28

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Science News Letter; the weekly summary of
current science 1921: 19 +

*Science Progress; a quarterly review of cur-
rent scientific investigation 1894: 1—7

Science Progress; a quarterly review of scien-
tific thought, work and affairs 1906: 1-34,
no. 134, 1939

'"Science Quarterly of the National University
of Peking 1929: College of Science. 1-5
(1935)

*Science Record; a monthly magazine illus-
trated; edited by J. S. Kingsley 1882: 2

Science Reports of National Tsing Hua Uni-
versity 1931: Peiping. Series A. Mathemati-
cal and Physical Sciences 1 + ; Series B. Bio-
logical amd Psychological Sciences 1 +

Science Reports of the National University of
Peking 1936: College of Science. 1-2, no. 1

Science Reports of the Tohoku Imperial Uni-
versity; Series 1. Mathematics, Physics,
Chemistry 1911: 1; 10+; Series 2. Geology
1912: 1 + ; Series 4. Biology 1924: 1 +

Science Reports of the Tokyo Bunrika Dai-
gaku; Sections B and C 1932: Tokyo Uni-
versity of Literature and Science. 1 +

Sciences; Revue de 1'Association Francaise
pour 1'Avancement des Sciences: 64, 1936 +

Scientia; Rivista (internazionale) di Sintesi
Scientifica (1-6 as Revista di Scienza) 1907:
1 +

Scientia Genetica; Periodico di Genetica per

I Paesi Latini 1939: Torino. 1 +
*Scientific American Monthly 1920: 1-4

Scientific Investigations; Department of Agri-
culture and Technical Instruction for Ire-
land; Fisheries Branch: 1901-26, no. 1

Scientific Journal of the Royal College of

Science 1931: London. 1 +
*Scientific Man; a weekly illustrated journal of
science (Oct. 26-Nov. 23, 1878, as Man;
Scientific Supplement) 1878: New York. 1

Scientific Memoirs of the University of Perm
see Uchenye Zapiski; Permskii Gosudarst-
vennyi Universitet im. M. Gor'kogo

Scientific Monthly 1915: 1 +

Scientific Notes; India Meteorological De-
partment 1927: 1-11; 13 +
"Scientific Papers of the Bureau of Standards
(1-14 as Bulletin of the Bureau of Standards)
1904: U. S. Department of Commerce (1-8,
1912) and Labor. 1; 3-22

Scientific Papers of the Institute of Algological
Research; Faculty of Science; Hokkaido
Imperial University 1935: Sapporo. 1 +

Scientific Papers of the Institute of Physical
and Chemical Research (also contains Ab-
stracts from Rikwagaku-Kenkyii-jo Iho; the
Bulletin of the Institute of Physical and

Chemical Research) 1922: Tokyo. 1-7; [8-9];
[11]; [14-15]; 16+

Scientific Proceedings of the Royal Dublin
Society 1877: ser. 2, 1 +

Scientific Records of the Gorky State Univer-
sity see Uchenye Zapiski; Gor'kovskogo
Gosudarstvennogo Universiteta

Scientific Report (s) from the Government
Institute for Infectious Diseases of the
Tokyo Imperial University see Japanese
Journal of Experimental Medicine

Scientific Report on the Investigations of the
Imperial Cancer Research Fund 1904: Lon-
don. 1-11 (1934)

Scientific Transactions of the Royal Dublin
Society 1877: ser. 2, 4, pt. 2; 5, pt. 4, 12;
7, pt. 6, 7, 13

Scientific Worker 1919: Association of Scien-
tific Workers, London. 9+

Scottish Naturalist; a magazine devoted to

zoology 1912: 1912-39

*Scottish Naturalist (Perth); a quarterly mag-
azine of natural science 1871: 1—10

Scritti Biologici; raccolti da Luigi Castaldi

1926: Siena. 1 +

*Selected Papers from the Journal of the Insti-
tute of Electrical Engineers of Japan 1924:
1-14

Senckenbergiana; Senckenbergische Natur-
forschende Gesellschaft 1919: Frankfurt
a.M. 1-21, no. 2

Sigma Xi Quarterly see American Scientist

Silicate P's and Q's 1921 : Philadelphia Quartz
Co. [4-9]; 10-11; [12]; 13 +

Silliman's Journal see American Journal of
Science

Sinensia; contributions from the National
(Research) Institute of Zoology and Biol-
ogy; Academia Sinica 1929: Nanking. 1-6;
[7]; 8+

Sitzungsberichte der Akademie der Wissen-
schaften in Wien; Mathematisch-Naturwis-
senschaftliche Klasse (43 continued in Abts.
I, II, III) 1848: 1; 4-38 (1859); Abt. I.
Mineralogie, Biologic, Erdkunde 1863: 51
(1865) + ; *Abt II. Mathematik, Physik,
Chemie, Mechanik, Meteorologie und As-
tronomie 1860: 48 (1863)-96(1887); Abt.
IIA. Mathematik, Astronomie, Physik, Me-
teorologie und Technik 1888: 97+; Abt.
IIB. Chemie 1888: 97 + ; *Abt. 3. Anatomie
und Physiologic des Menschen und der
Thiere 1872: 65-131

Sitzungsberichte der Gesellschaft fiir Mor-
phologic und Physiologic in Miinchen 1885:
1-48

Sitzungsberichte der Gesellschaft Naturfor-
schender Freunde zu Berlin 1839: 1839-62;
1866-69; 1874-1938

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Sitzungsberichte der Gesellschaft zur Be-
fb'rderung der Gesamten Naturwissen-
schaften zu Marburg 1866: 1897, no. 2;
1898, no. 6; 1899, no. 1; 1907-37; 1939

Sitzungsberichte der Heidelberger Akademie
der Wissenschaften ; Mathematisch-Natur-
wissenschaftliche Klasse (after 1919 issued
in two series A and B) 1909: 1909-39, no. 3

Sitzungsberichte der Kgl. Bohmischen Ge-
sellschaft der Wissenschaften; Mathe-
matisch-Naturwissenschaftliche Klasse see
Vestnik Kralovske Ceske Spolecnosti Nauk

Sitzungsberichte der Mathematisch-Natur-
wissenschaftliche Abteilung derBayerischen
Akademie der Wissenschaften zu Miinchen
(1871-1924 as Sitzungsberichte der Mathe-
matisch-Physikalischen Classe) 1871: 1885,
Heft 4; 1925-38

*Sitzungsberichte der Naturforschenden Ge-
sellschaft zu Leipzig 1874: 2; 28-34; 37;
45-48

Sitzungsberichte der Naturforscher-Gesell-
schaft bei der Universitat Tartu see Tartu
Ulikooli juures oleva Loodusuurijate Seltsi
Aruanded

*Sitzungsberichte der Niederrheinischen Ge-
sellschaft fur Natur- und Heilkunde zu
Bonn 1854: [1899-1900]; [1902]
*Sitzungs-Berichte der Physikalisch-Medizini-
schen Gesellschaft zu Wiirzburg; Ofnzielle
Sitzungsprotokolle der Physikalisch-Medi-
zinischen Gesellschaft zu Wiirzburg (after
1923 see Berichte der Physikalisch-Medi-
zinischen Gesellschaft zu Wiirzburg) 1881:
1881-1923

Sitzungsbericht der Physikalisch-Medizini-
schen Sozietat in Erlangen 1865: 32; 34^3
*Sitzungsberichte der Preussischen Akademie
der Wissenschaften; Physikalisch-Mathe-
matische Klasse (1882-1921 as Sitzungs-
berichte der Preussischen Akademie der
Wissenschaften) 1882: 1883-1938

Sitzungsberichte und Abhandlungen der Na-
turforschenden Gesellschaft zu Rostock
1909: n.s. 1-7, no. 1 ; 3 Folge 1-6 (1936)
*Skandinavisches Archiv fur Physiologic ; Acta
Societatis Physiologicae Scandinavicae ; Ge-
griindet von Frithiof Holmgren 1889: 1-83

Skrifter udgivet af Universitetets Zoologiske
Museum; K0benhavn (Spolia Zoologica
Musei Hauniensis I.) 1941: 1
*Skrifter udgivne af Kommissionen for Dan-
marks Fiskeri og Havunders0gelser 1904:
Copenhagen. 3-10 (1927)

Skrifter utgitt (udgivne) av det Norske Viden-
skaps-Akademiei Oslo. I. Matematisk-
Naturvidenskapelig Klasse 1894: 1894 +

*Smithsonian Contributions to Knowledge
1848: 1; [2]; 3; [5]; 6; [7-9]; 10; [11]; 12;

[13]; 14-18; 20-21; [22-23]; 24; [25-26];
[29]; 30-33; [34]; 35

Smithsonian Institution; Bureau of American
Ethnology Bulletin 1887: 1-24; 33; 82-83

Smithsonian Miscellaneous Collections 1862:
1 +

Societas Entomologica 1886: Internationale
Entomologische Fachzeitschrift. 43-45

Socijalno-Medicinski Pregled; Izvestaji i
saopstenja o radu sanitetskih ustanova
Kraljevine Jugoslavije (CentralniHigijenski
Zavod u Beogradu) 1930: 1 +

Soil Science; founded 1916 by Jacob G. Lip-
man 1916: 1 +

South African Biological Society Pamphlet
1931: Pretoria. 1 +

South African Journal of Natural History;
South African Biological Society 1918: 1-6

South African Journal of Science; being the
report of the South African Association for
the Advancement of Science (1903-18 as
Report) 1903: Johannesburg. 1-4; 7; [8-9];
10-12; 14 +

Sovetskaia Botanika 1933: Botanicheskogo
Institut; Akademiia Nauk U.R.S.S.: 1933 +

Special Papers ; Geological Society of America

1934: 1 +

*Special Papers; Ohio State Academy of Sci-
ences 1899: 1-7

Special Publication; American Geographical
Society 1915: 4; 7-9; 11 ; 13; 16; 18-19; 21-22

Special Publication; Bernice P. Bishop Mu-
seum (1-6 as Miscellaneous Publication) (6
as Fauna Hawaiiensis) (11, 1926+ includes
Proceedings; Hawaiian Academy of Science)
1892: Honolulu, Hawaii. 2; 4-6; 9-21; 26

Special Publication ; Chekiang Provincial Fish-
eries Experiment Station 1936: China. 1-2

Special Publication; Coast and Geodetic Sur-
vey: U. S. Department of Commerce. [3-
196]; 198 +

Special Publication; International Hydro-
graphic Bureau 1923: Monaco. 1 +

Special Publication of the American Commit-
tee on International Wild Life Protection
1931: 1 +

Special Report Series; Privy Council; Medical
Research Council (1-50 as Medical Re-
search Committee, National Health Insur-
ance) 1915: London. 3; 5-10; 12-72; 74 +

Spisy Lekarske Fakulty, Masarykovy Univer-
sity (Publications de la Faculte de Medecine
de 1'Universite Masaryk) 1922: Brno. 1-17

Spisy Vydavane Pfirodovedeckou Fakultou
Karlovy University (Publications de la Fa-
culte des Sciences de 1'Universite Charles)
1923: Prague. 17; 57; 79; 95; 105; 108; 133;
143_44; 147-48; 150; 152-54; 157; 160-66

Spisy Vydavane Pfirodovedeckou Fakultou

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Masarykovy University (Publications de la
Faculte des Sciences de 1'Universite
Masaryk) 1921: Brno. 1-271

Spolia Zeylanica; Ceylon Journal of Science,
section B. Geology, Zoology, Anthropology
1903: Colombo Museum. 1 +

Spolia Zoologica Musei Hauniensis I see
Skrifter udgivet af Universitetets Zoologiske
Museum; K0benhavn

*Sprawozdania Stacji Hydrobiologiznej na
Wigrach (Comptes Rendus de la Station
Hydrobiologique du Lac de Wigry) 1922:
Instytut im M. Xenckiego; Warsaw. 1-4

Sprawozdania z posiedzeri Towarzystwa Nau-
kowego Warszawskiego (Comptes Rendus
des Seances de la Societe des Sciences et
des Lettres de Varsovie) 1908: 1-5; 11-18;
ser. 3. Matematyczno-fizycznych. 6-10;
19-30, no. 6; ser. 4. Biologicznych. 22-31,
no. 6

Sprawozdanie Paristwowego Muzeum Zoolo-
gicznego za Rok 1929: Warsaw. 1929

Sprawozdanie z Dzialalnosci w Roku Akade-
mickim; Wolna Wszechnica Polska: War-
saw. 21 (1926-27)

Stain Technology; a journal for microtechnic;
official organ of the Commission on Stand-
ardization of Biological Stains 1926: 1 +

Standards Yearbook; National Bureau of
Standards: U. S. Department of Commerce.
2-4 (1928-30)

Stanford Ichthyological Bulletin 1938: Natu-
ral History Museum of Stanford University.
1 +

*Stanford University Publications; University
Series 1908: 1-43

Stanford University Publications; University
series; Biological Sciences 1920: 1 +

Stanford University Publications; University
series; Geological Sciences 1924: 1 +

Stanford University Publications; University
series; Medical Sciences 1921: 1; nos. 1-2;
4, no. 1

Starunia 1934; Polska Akademja Umiejet-
nosci: Krakow. 1-16

State of New York; Conservations Depart-
ment; Supplemental to Annual Report:
1926-39

State of the Ice in the Arctic Seas and the
Surface Temperature of the Sea in the
Northern Atlantic-Ocean and in Davis-
Strait see Nautisk-Meteorologisk Aarbog

State University of Montana Studies 1926: 1

Statens Meteorologisk-Hydrografiska Ans-
talt; Meddelanden; Serien Uppsatser ( Com-
munications, series of papers) 1935: Stock-
holm. 1-32; 34 +
*Statistical Research Memoirs; University Col-

lege, University of London; Department of
Statistics 1936: 1-2

Stettiner Entomologische Zeitung see Entomo-
logische Zeitung

Strahlentherapie ; Mitteilungen aus dem Ge-
biete der Behandlung mit Rontgenstrahlen,
Licht und Radioaktiven Substanzen; Zeit-
schrift der Deutschen Rbntgengesellschaft
und der Gesellschaft fur Lichtforschung
1912: 1 + ; Sonderband 1912: 1 +

*Studies from the Biological Laboratory; Johns
Hopkins University (session 1878 as Scien-
tific Results; Chesapeake Zoological Labora-
tory) 1877: 1-5

'Studies from the Museum of Zoology in Uni-
versity College, Dundee 1888: 1-12

Studies from the Plant Physiological Labora-
tory of Charles University; Prague 1923:
1-6, no. 3, (1937)

Studies of the Institutum Divi Thomae 1937:
Cincinnati. 1 +

Suomalaisen elain-ja Kasvitieteellisen Seuran
Vanamon; Elaintieteellisia Julkaisuja (An-
nales Zoologici Societatis Zoologicae-Bota-
nicae Fennicae Vanamo) 1932: Helsingfors.
1-6 (1939)

*Suomalaisen elain-ja Kasvitieteelisen Seuran
Vanamon; Julkaisuja (Annales Societatis
Zoolog.-Botanicae Fennicae Vanamo) (con-
tinued in two series) 1923: Helsingfors. 1-15
(1934)

Suomalaisen elain-ja Kasvitieteellisen Seuran
Vanamon; Kasvitieteellisia Julkaisuja (An-
nales Botanici Societatis Zoologicae-Botani-
cae Fennicae Vanamo) 1931: Helsingfors.
1-14 (1940)

Suomalaisen elain-ja Kasvitieteellisen Seu-
ran Vanamon see also Luonnon Ystava

Suomen Hyonteistieteellinen Aikakauskirja
(Annales Entomologici Fennici) 1935: 1-5
(1939)

Svensk Botanisk Tidskrift; utgiven av Svenska
Botaniska Foreningen 1907: 1-33 (1939)

Svenska Hydrografisk-Biologiska Kommis-
sionens Skrifter 1903: Goteborg. 1 +

(Kungl.) Svenska Vetenskapsakademiens Ars-
bok 1903: 1903-36; 1938 +

(Kungl.) Svenska Vetenskapsakademiens
Handlingar 1739: 1 +

Symbolae Botanicae Upsalienses; Arbeten
fran Botaniska Institutionerna i Uppsala
1932: 1 +

Symons's Meteorological Magazine see Mete-
orological Magazine

Synthetic Organic Chemicals 1927: Eastman
Kodak Company. 1 +

Tableaux Analytiques de la Faune de 1'URSS
(Opredeliteli po Faune SSSR) 1927: Aka-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

demiia Nauk, Zoologicheskii Institut. 1-2;
11-14; 17-19; 21 +

Tabulae Biologicae (7-12 also as 1-6 of Ta-
bulae Biologicae Periodicae) 1925: Berlin.
1 +

*(Die) Tagliche Praxis; Monatsberichte iiber
die Gesamte Therapie und alle Klinischen
Facher; Supplement to Wiener Medizinische
Wochenschrift 1929: 3-6 (1934)

Tartu Ulikooli juures oleva Loodsuurijate
Seltsi Aruanded (Annales Societatis Rebus
Naturae Investigandis in Universitate Tar-
tuensi Constitutae) (1-11 as Sitzungsbe-
richte der Naturforscher-Gesellschaft bei
der Universitat Dorpat; 12-23 as Sitzungs-
berichte or Protokoly Obshchestva Estest-
voispytatelei pri Imperatorskom lur'evskom
Universitete) 1853: Naturalists' Society of
Tartu University. 1-2; 5-8; 12 +

Technical Bulletin; University of Minnesota;
Agricultural Experiment Station: 21 ; 33; 38;
40-75; 88-89; 95; 101; 111; 113-14; 133;
137 (1939)

Technical Publication; New York State Col-
lege of Forestry at Syracuse University
1914: 1; 3-4; 9-10; 19; 21; 26; 29-32; 34-37;
42^14 (1933)

Technologic Papers of the Bureau of Stand-
ards 1911: U. S. Department of Commerce.
[1-352]

*Technology Quarterly and Proceedings of the
Society of Arts 1887: Massachusetts Insti-
tute of Technology. 8-9 see Review of
American Chemical Research 1-2 (1895-96)
*Termeszetrajzi Fiizetek; A Magyar Tudo-
manyos Akademia Segelyevel Kiadja a
Magyar Nemzeti Muzeum 1877: Budapest.
22-25 (1902)

Terrestrial Magnetism and Atmospheric Elec-
tricity; an international quarterly journal
1896: 1 +

Thalassia 1932: Istituto Italo-Germanico di
Biologia Marina di Rovigno d'Istria, Venice
and Jena. 1 +

Tide Tables; Atlantic Coast: U. S. Coast and
Geodetic Survey. 1867; -1870; 1872-81;
1883-95; 1901-03; 1906; 1912 +

Tierziichtung und Ziichtungsbiologie ein-
schliesslich Tierernahrung; Zeitschrift fiir
Zuchtung, Reihe B (1-16 as Zeitschrift fur
Tierziichtung und Ziichtungsbiologie ein-
schliesslich Tierernahrung) 1924: 1-34

*Tijdschrift der Nederlandische Dierkundige
Vereeniging 1874: 1-6; ser. 2, 1-20; ser. 3,
1-3

Tijdschrift van het K. Nederlandsch Aardrijk-
skundig Genootschap; Amsterdam 1876:
ser. 2, 31 +

Tohoku Journal of Experimental Medicine
1920: Tohoku Imperial University. 1 +

Torreia; Museo Poey; Universidad de la Ha-
bana, Cuba; publication ocasional dedicada
al progreso de las ciencias naturales 1939:
1 +

Torreya; a bi-monthly journal of botanical
notes and news 1901: Torrey Botanical
Club. 1 +

Trabajos ; Estacion Limnologica de Patzcuaro ;
Depto. de Pesca e Indust. Maritimas 1940:
Mexico. 1 +

Trabajos del Institute Espanol de Oceano-
grafia 1929: 1-14 (1935)

Trabajos del Laboratorio de Investigaciones
Biologicas de la Universidad de Madrid see
Travaux du Laboratoire de Recherches Bio-
logiques de 1'Universite de Madrid

Trabajos del Museo Nacional de Ciencias
Naturales; Serie Zoologica 1912: Junta para
Ampliacion de Estudios e Investigaciones
Cientificas. 1-57 (1932)

Trabajos del Museo Nacional de Ciencias
Naturales y Jardin Botanico; Serie Botanica
1912: Junta para Ampliacion de Estudios e
Investigaciones Cientificas. 1—31 (1935)

Transactiones Societatis Pathologicae Japoni-
cae 1911: 23 +

Transactions and Proceedings and Report of
the Philosophical Society of Adelaide see
Transactions of the Royal Society of South
Australia

Transactions and Proceedings of the Botanical
Society of Edinburgh (1-11, 16-18 as Trans-
actions of the Botanical Society) 1841: 1-3;
4, pt. 2; 5 +

Transactions and Proceedings of the Perth-
shire Society of Natural Science (1880-86
as Proceedings; 1871-80 see in Scottish
Naturalist (Perth) 1880: [4-5]

Transactions and Proceedings of the Royal
Society of New Zealand (1-63 as Transac-
tions and Proceedings of the New Zealand
Institute) 1868: 1 + '

Transactions and Proceedings of the Royal
Society of South Australia see Transactions
of the Royal Society of South Australia

Transactions of the Academy of Science of St.
Louis 1856: 1-16; [17]; 18-19; [20]; 21-22;
[23]; 24 +

Transactions of the American Entomological
Society 1867: 1 +

Transactions of the American Fisheries So-
ciety (1-13 as American Fish-Cultural Asso-
ciation) 1870: 13; 15-16; 24+

Transactions of the American Geophysical
Union 1922: 1+ (2, 4, 6-11 see in Bulletin
of the National Research Council) (1, 3, 5
never published)

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Transactions of the American Microscopical
Society (2-13 as Proceedings of the Ameri-
can Society of Microscopists; 1892-94 as
Proceedings of the American Microscopical
Society) 1878: 1 +

Transactions of the American Philosophical
Society 1769: 1-3; 6; n.s. 1-10; 12 +

Transactions of the American Society of Tropi-
cal Medicine: 16, 1920+ see in American
Journal of Tropical Medicine 1, 1921 +

Transactions of the Arctic Institute of the
Chief Administration of the Northern Sea
Route see Trudy Arkticheskogo Nauchno-
Issledovatel'skogo Instituta Glavnogo Up-
ravleniia Severnogo Morskogo puti pri snk
SSSR

Transactions of the Botanical Society; Edin-
burgh see Transactions and Proceedings of
the Botanical Society of Edinburgh
*Transactions of the Cambridge Philosophical
Society 1820: England. 1-23

Transactions of the Central Geophysical Ob-
servatory see Trudy Glavnoi Geonzicheskoi
Observatorii

Transactions of the Congress of American
Physicians and Surgeons 1888: 2-3; 5; 7;
12; 14 (1928)

Transactions of the Connecticut Academy of
Arts and Sciences 1866: 1 +

transactions of the Entomological Society of
New South Wales 1863: 1-2 (1873)

Transactions of the Faraday Society 1905:
London. 1-11; 16+

Transactions of the Hertfordshire Natural
History Society and Field Club 1879: 1, pt.
2; 2, pt. 2, 5

Transactions of the Institute for Scientific Ex-
ploration of the North see Trudy Instituta
po Izucheniiu Severa

Transactions of the Institute of Marine Fish-
eries and Oceanography of the USSR see
Trudy Vsesoiuznogo Nauchno-Issledova-
tel'skogo Instituta Morskogo Rybnogo
Khoziaistva i Okeanografii

Transactions of the Kansas Academy of Sci-
ences 1868: 1-18; 20, pt. 2; 23-26 (1913)

Transactions of the Knipovich Polar Scientific

. Institute of Sea-Fisheries and Oceanog-
raphy see Trudy Podiarnyi Nauchno-Issle-
dovatel'skii Institut Morskogo Rybnogo
Khoziaistva i Okeanografii im. Pochetnogo
Chlena Akademii Nauk SSSR Prof. N. M.
Knipovicha

Transactions of the Laboratory of Experi-
mental Biology of the Zoopark of Moscow
see Trudy po Dinamike Razvitiia

Transactions of the Linnean Society of London
1791: 1-30; (series 2) Botany (1875) 1 + ;
Zoology (1875) 1+ (series 3) The Percy Sla-

den Trust Expedition to Lake Titicaca in
1937; under the leadership of Mr. H. Gary
Gilson (1939) 1 +

*Transactions of the Microscopical Society of
London 1844: 1-3

Transactions of the National Institute of Sci-
ences of India 1935: 1 +

*Transactions of the New York Academy of
Medicine 1847: 1896-1901

Transactions of the New York Academy of

Sciences 1881: [2-16]; n.s. 1 +
Transactions of the New York Microscopical
Society 1878: [1] see in American Quarterly
Microscopical Journal vol. 1

Transactions of the Northern Scientific and
Economic Expedition see Trudy Instituta po
Izucheniiu Severa

Transactions of the Oceanographical Institute
Moscow see Trudy Gosudarstvennogo Okea-
nograficheskogo Instituta
Transactions of the Optical Society (1933 +
continued in Proceedings of the Physical
Society 45, 1933+) 1899: London. 1-33
(1932)

Transactions of the Ottawa Field-Naturalists'
Club see Canadian Field-Naturalist

Transactions of the Pavlov Physiological Lab-
oratories see Trudy Fiziologicheskikh Labo-
ratorii

Transactions of the Philosophical Society of
New South Wales 1862: 1862-65

Transactions of the Physiological Institute
at the Leningrad State University see
Trudy Fiziologicheskogo Nauchno-Issledo-
vatel'skogo Instituta

Transactions of the Research Institute of Ex-
perimental Morphogenesis; State Univer-
sity of Moscow see Trudy Nauchno-Issledo-
vatel'skogo Instituta Eksperimental'nogo
Morfogeneza; Moskovskogo Gosudarstven-
nogo Universiteta

Transactions of the Royal Canadian Institute
1889: 1 +

Transactions of the Royal Entomological So-
ciety of London 1833: 91 +

Transactions of the Royal Irish Academy 1 787 ;
24, pt. 27-28; 29, pt. 16; 30, pt. 3; 31, pt. 1:
32, sec. A, pt. 4

Transactions of the Royal Microscopical So-
ciety 1844: 1853-68 see in Quarterly Journal
of Microscopical Science 1-n.s. 8; 1869-77
see in Monthly Microscopical Journal 1-18;
1878+ see in Journal of the Royal Micro-
scopical Society 1 +

Transactions of the Royal Photographic So-
ciety of Great Britain 1926-30 see in Photo-
graphic Journal 66-70

Transactions of the Royal Society of Canada
(1-24 as Proceedings and Transactions of

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

the Royal Society of Canada; 13-24 also
numbered ser, 2, 1-12) 1882: series 1-2, 1—
24; series 3, 1-12; 13+ as Section 3. Math-
ematical, Physical and Chemical Sciences:
13 + ; Section 4. Geological Sciences: 18-19;
Section 5. Biological Sciences: 18 +

Transactions of the Royal Society of Edin-
burgh (1-4 in 3 sections: History, appearing
in 1-5; includes Proceedings 1783-1803;
Papers of the Physical Class; Papers of the
Literary Class) 1783: 1-8; 10-15; 20-27;
29-30; 32 +

Transactions of the Royal Society of New
South Wales see Journal and Proceedings of
the Royal Society of New South Wales

Transactions of the Royal Society of South
Africa 1908: 1 +

Transactions of the Royal Society of South
Australia (2 as Transactions and Proceed-
ings and Report of the Philosophical Society
of Adelaide; 3-36 as Transactions and Pro-
ceedings and Report of the Royal Society
of South Australia; 37-61 as Transactions
and Proceedings of the Royal Society of
South Australia) 1877: 2-3; 7 +

Transactions of the San Diego Society of
Natural History 1905: [1-3]; 5 +

Transactions of the Scientific Chemical-
Pharmaceutical Institute see Trudy Nauch-
nogo Khimiko-Farmatsevticheskogo Ins-
tituta

Transactions (and Communications) of the
Society of Chemical Industry see Journal of
the Society of Chemical Industry

Transactions of the South African Philosophi-
cal Society 1877: Cape Town. [2-18]

Transactions of the State Agricultural Society
with Reports of County Agricultural Socie-
ties; Michigan 1849: 1-2; 4; 7
'Transactions of the Texas Academy of Science

1896: [1-10]
Transactions of the Wagner Free Institute of

Science of Philadelphia 1887: 1-11
Transactions of the Watford Natural History
Society and Hertfordshire Field Club 1875:
London. [2]

Transactions of the Western Surgical Associa-
tion 1896: 43 +

Transactions of the Wisconsin Academy of
Sciences, Arts and Letters 1870: 1 +

Transactions of the Yorkshire Naturalists'
Union 1877: Leeds. 23-25; 28-29

Transactions of the Zoological Society of Lon-
don 1835: 1 +

Transactions of Tomsk State University (Iz-
vestiia Tomskogo Gosudarstvennogo Uni-
versiteta) (Berichte der Tomsker Staats-
Universitat) 1889: 82; 84

Transactions on the Dynamics of Develop-

ment, Moscow see Trudy po Dinamike
Razvitiia

Transunti della R. Accademia Nazionale dei
Lincei see Atti della Reale Accademia Na-
zionale dei Lincei

Travaux Chimiques parus au Bulletin Inter-
national de 1'Academie Polonaise des Sci-
ences et des Lettres; Classe des Sciences
Mathematiques et Naturelles; Serie A.
Sciences Mathematiques 1931: 1-4

Travaux de 1'Institut Biologique de Peterhof
see Trudy Petergofskogo Biologicheskogo
Instituta

Travaux de 1'Institut de Physiologic see Trudy
Instituta Fiziologii

Travaux de 1'Institut de Recherches Scien-
tifiques de Biologic see Pratsi Naukovo-
Doslidnogo Institutu Biologii

Travaux de 1'Institut de Zoologie de 1'Uni-
versite de Montpellier et de la Station
Zoologique de Cette see Travaux de la Sta-
tion Biologique de Sete

Travaux de 1'Institut des Recherches Biolog-
iques de Molotiv see Trudy Biologicheskogo
Nauchno-Issledovatel'skogo Instituta pri
Molotovskom Gosudarstvennom Universi-
tete im. M. Gor'kogo

Travaux de 1'Institut des Recherches Scien-
tifiques a 1'Universite d'Etat, Voroneje,
URSS see Trudy Nauchno-Issledovatel'skogo
Instituta pri Voronezhskom Gosudarstven-
nom Universitete

Travaux de 1'Institut des Sciences Naturelles
de Peterhoff see Trudy Petergofskogo Bio-
logicheskogo Instituta

Travaux de 1'Institut Hydrologique d'Etat see
Trudy Gosudarstvennogo Gidrologicheskogo
Instituta

Travaux de 1'Institut Nencki see Prace Insty-
tutu im. Nenckiego

Travaux de 1'Institut Oceanographique de 1'In-
dochine; Memoire (1-3 as Travaux du
Service Oceanographique des Peches de 1'In-
dochine; Memoire) 1927: Gouvernement
General de 1'Indochine. 2 +

Travaux de 1'Institut Zoologique de 1'Academie
des Sciences de 1'URSS see Trudy Zoolo-
gicheskogo Instituta

Travaux de 1'Institut Zoologique de Lille et
de la Station Maritime de Wimereux see
Travaux de la Station Zoologique de
Wimereux

Travaux de la Commission pour 1'Etude du
Lac Bajkal see Trudy Baikal'skoi Limno-
logicheskoi Stantsii

Travaux de la Societe des Naturalistes a
1'Universite Imperiale de Kharkow see
Trudy Obshchestva Ispytatelei Prirody pri
Imperatorskom Khar'kovskom Universitete

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Travaux de la Societe des Naturalistes de
Leningrad see Trudy Leningradskogo Obsh-
chestva Estestvoispytatelei

Travaux de la Societe Royale des Sciences de
Boheme, Classe des Sciences see Rozpravy
Kralovske Ceske Spolecnosti Nauk; Trida
Matematicko-Prirodovedecka

Travaux de la Station Biologique "Borodin-
skaja" attachee a la Societe des Naturalis-
tes de Petrograd see Trudy Borodinskoi
Biologicheskoi Stantsii v Karelii

Travaux de la Station Biologique de Kara-
dagh see Trudy Karadags'koi Biologichnoi
Stantsii

Travaux de la Station Biologique de Murman
de la Societe des Naturalistes de Leningrad
see Raboty Murmanskoi Biologicheskoi
Stantsii; Leningradskogo Obshchestva Es-
testvoispytatelei

Travaux de la Station Biologique de Roscoff;
publics par Charles Perez 1923: 1 +

Travaux de la Station Biologique de Sebasto-
pol see Trudy Sevastopol'skoi Biologicheskoi
Stantsii

Travaux de la Station Biologique de Sete (1-
ser. 2, no. 61 as Travaux de 1'Institut de
Zoologie de 1'Universite de Montpellier et
de la Station Zoologique de Cette and varia-
tions of that title} 1873: ser. 1. 1-2; 5; series
2. Memoire 1-2; 5-6; 8-16; 18; 22-24; 27-
28; 30 +

Travaux de la Station Hydrobiologique (Zbir-
nik Prats ; Dniprians'koi Biologichnoi Stant-
sii) see Trudy Gidrobiologichnoi Stantsii

Travaux de la Station Ichtyologique, Sozopol,
Bulgarie see Trudove na Opitnata Ikhtio-
logichna Stantsiia V Gr. Sozopol

Travaux de la Station Limnologique du Lac
Bajkal see Trudy Baikal'skoi Limnologi-
cheskoi Stantsii

Travaux de la Station Zoologique de Ville-
franch-s-Mer (1925-30 as Travaux de la
Station Zoologique Russe de Villefranch-s-
Mer) 1925: 1925 +

Travaux de la Station Zoologique de Wime-
reux (1 has no series title) (2-4 as Travaux
de 1'Institut Zoologique de Lille et de la
Station Maritime de Wimereux) (5 as
Travaux de ITnstitut Zoologique de Lille
et du Laboratoire de Zoologie Maritime de
Wimereux) (6 as Travaux du Laboratoire de
Zoologie Maritime de Wimereux-Amble-
teuse) 1877: 1 +

Travaux de Laboratoire; Institut Solvay
(Physiologic) ; Universite Libre de Bruxelles
1897: fasc. 3; vol. 2-12, fasc. 1; 14, fasc. 2;
15, fasc. 1-2; 16, fasc. 1-2; 17, fasc. 1; 18 +

Travaux des Laboratoires ; Societe Scientif-

ique d'Arcachon; Station Biologique see
Bulletin de la Station Biologique d'Arcachon

Travaux du Laboratoire Biogeochimique de
1'Academie des Sciences de 1'URSS see
Trudy Biogeokhimicheskoi Laboratorii
*Travaux du Laboratoire de Leon Fredericq;
Universite de Liege, Institut de Physiologic
1885: 1-7

Travaux du Laboratoire de Morphologic Evo-
lutive see Trudy Laboratorii Evoliutsionnoi
Morfologii

Travaux du Laboratoire de Recherches Bio-
logiques de 1'Universite de Madrid; fonde
par S. Ramon y Cajal (1-20 as Trabajos del
Laboratorio de Investigaciones Biologicas
de la Universidad de Madrid) 1901: 1 +

Travaux du Laboratoire de Zoologie Experi-
mentale et de Morphologic des Animaux see
Trudy Laboratorii Eksperimental'noi Zoolo-
gii i Morfologii Zhivotnykh

Travaux du Laboratoire Ichtyologique d'As-
trakhan aupres de 1'Administration des
Pecheries du Volga et de la Mer Caspienne
see Report (s) of the (Astrakhan) Scientific
Station of Fisheries of Volga and Caspian
Sea)

Travaux du Laboratoire Zoologique et de la
Station Biologique de Sebastopol pres de
1'Academie Imperiale des Sciences see Trudy
Sevastopol'skoi Biologicheskoi Stantsii

Travaux du Musee Botanique; Academic des
Sciences USSR see Trudy Botanicheskogo
Muzefa; Akademiia Nauk SSSR

Travaux du Service Oceanographique des
Peches de 1'Indochine; Memoire see Tra-
vaux de 1' Institut Ocdanographique de 1'In-
dochine; Memoire

*Travaux Scientifiques du Laboratoire de Zoo-
logie et de Physiologic Maritimes de Con-
carneau 1909: 1-5, fasc. 1

Travaux sur la Morphologic des Animaux see
Zbirnik Prats z Morfologii Tvarin

Treubia; a journal of zoology, hydrobiology
and oceanography of the East Indian Archi-
pelago; 's Lands Plantentuim (Botanic Gar-
dens, Buitenzorg, Java) 1919: 1 +
Troms0 Museums Aarsberetning 1873: 1893;
1898-1908; 1910+

Troms0 Museums Aarshefter 1878: 1; 3; 6;
10-12; 20 +

Troms0 Museums Skrifter 1925: 1 +

Tropical Diseases Bulletin 1912: Bureau of
Hygiene and Tropical Diseases; London.
37 +

Tropical Plant Research Foundation Bulletin
1925: Washington, D. C. 1 +

Trudove na Chernomorskata Biologichna
Stantsiia Varna (Arbeiten aus der Biologi-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

schen Meeresstation am Schwarzen Meer
in Varna, Bulgarian) 1933: 1 +

Trudove na Opitnata Ikhtiologichna Stantsiia
V Gr. Sozopol (Travaux de la Station Ichtyo-
logique, Sozopol, Bulgaria): 5 +

Trudy Arkhangel'skogo Vodoroslevogo Nauch-
no-Issledovatel'skogo Instituta Avnii (Algal
Research Institute, Archangel) 1938: 1

Trudy Arkticheskogo Nauchno-Issledova-
tel'skogo Instituta Glavnogo Upravlenifa
Severnogo Morskogo puti pri snk SSSR
(Transactions of the Arctic Institute of the
Chief Administration of the Northern Sea
Route) 1933: [1-145]

Trudy Azovsko-Chernomorskogo Nauchno-
Issledovatel'skogo Instituta Rybnogo Khoz-
faistva i Okeanografii (Publications of the
Scientific Institute of Fishery and Ocean-
ography) : 1 1

Trudy Azovsko-Chernomorskoi, Kerchenskoi,
Nauchnoi Rybokhozfaistvennoi Stantsii,
Ikhtiologicheskoi Laboratorii see Report (s)
of the Scientific Station of Fisheries of Asov
and Black Seas

Trudy Baikal'skoi Limnologicheskoi Stantsii
(Travaux de la Station Limnologique du Lac
Bajkal) (1-3 as Trudy Komissii po Izu-
cheniiu Ozera Baikala) (Travaux de la Com-
mission pour 1'Etude du Lac Bajkal) 1918:
Akademiia Nauk. 1 +

Trudy Belorusskogo Gosudarstven. Universi-
teta (Annales de 1'Universite de Minsk)
(Prace Naukowe Uniwersitetu Paristwo-
wego na Biatorusi) 1922: 6-10

Trudy Biogeokhimicheskoi Laboratorii (Tra-
vaux du Laboratoire Biogeochimique de
1'Academie des Sciences de 1'URSS) 1930:

1 +

Trudy Biologicheskogo Fakul'teta Tomskogo
Gosudarstvennogo Universiteta (Wissen-
schaftliche Berichte der Biologischen Fakul-
tat der Tomsker Staats-Universitat) 1931: 1

Trudy Biologicheskogo Nauchno-Issledova-
tel'skogo Instituta pri Molotovskom Gosu-
darstvennom Universitete im. M. Gor'kogo
(1-3, no. 2; 6-8 as Trudy Biologicheskogo
Nauchno-Issledovatel'skogo Instituta pri
Permskom Gosudarstvennom Universitete;
3, no. 3-vol. 5 as Trudy Permskogo Bio-
logicheskogo Nauchno-Issledovatel'skogo
Instituta) (Travaux de 1'Institut des Re-
cherches Biologiques de Molotov; formerly
Perm) 1927: 1 +

Trudy Borodinskoi Biologicheskoi Stantsii v
Karelii; Leningradskoe Obshchestvo Est-
estvoispytatelei (Berichte der Biologischen
Borodin Station) (1-3, 1901-12 as Trudy
Pryesnovodnoi Biologicheskoi Stantsii Im-

peratorskago S.-Peterburgskago Obshchestva
Estestvoispytatelei; Berichte der Biologi-
schen Siisswasserstation der Kaiserlichen
Naturforscher-Gesellschaft zu St. Peters-
burg) (4, 1917 as Trudy Borodinskoi Bio-
logicheskoi Stantsii Petrogradskago Obsh-
chestva Estestvoispytatelei; Travaux de la
Station Biologique "Borodinskaja" attachee
a la Societe des Naturalistes de Petrograd)
(5, 1927 as Trudy Borodinskoi Presnovodnoi
Biologicheskoi Stantsii v Karelii; Berichte
der Akademiker Borodin Biologischen Siiss-
wasser Station) 1901: 1-8, no. 2; 9, no. 1
(1936)

Trudy Botanicheskogo Instituta Akademii
Nauk SSSR (Acta Instituti Botanici Aca-
demiae Scientiarum URSS); Serif* 1. Flora
i Sistematika Vysschikh Rastenii 1933: 1 + ;
Seriia 2. Sporovye Rasteniia 1933: 1 + ;
Serifa 3. Geobotanika 1934: 1 + ; Serifa 4.
Eksperimental'naia Botanika 1934: 1-3;
5 + ; Seriia 5. Rastitel'noe Syr'e 1938: 1 +

Trudy Botanicheskogo Muzeia; Akademiia
Nauk SSSR (Travaux du Musee Botanique;
Academic des Sciences USSR) 1902: 12-18;
21

Trudy Fiziologicheskikh Laboratorii; Akade-
mika I. P. Pavlova (Transactions of the
Pavlov Physiological Laboratories) : Aka-
demiia Nauk. [2-4]; 6 +

Trudy Fiziologicheskogo Nauchno-Issledova-
tel'skogo Instituta; Leningradskii Gosu-
darstvennyi Universitet (Transactions of the
Physiological Institute at the Leningrad
State University): 16-19; 22 +

Trudy Gidrobiologichnoi Stantsii (Travaux de
la Station Hydrobiologique) (1-6 as Zbirnik
Prats; Dniprians'koi Biologichnoi Stantsii;
also numbered separately as Academic des
Sciences d'Ukraine; Memoires de la Classe
des Sciences) 1926: Academie des Sciences
d'Ukraine; Kief. 1 +

Trudy Glavnoi Geofizicheskoi Observatorii
(Transactions of the Central Geophysical
Observatory) 1934: Glavnoe Upravlenie
Gidrometeorologicheskoi Sluzhby SSR; Le-
ningrad; Moscow. 1-15; 17-26; 28 +

Trudy Gosudarstvennogo Gidrologicheskogo
Instituta (Travaux de I'lnst'.tut Hydrolo-
gique Etat) 1936: Glavnoe Upravlenie Gid-
rometeoroiogicheskoi Sluzhby SSR; Lenin-
grad; Moscow. 1; 3; 5

Trudy Gosudarstvennogo Nauchno-Issledova-
tel'skogo Instituta Eksperimental'nogo Mor-
fogeneza see Trudy Nauchno-Issledova-
tel'skogo Instituta Eksperimental'nogo Mor-
fogeneza; Moskovskogo Gosudarstvennogo
Universiteta

Trudy Gosudarstvennogo Okeanografiches-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

kogo Instituta (Transactions of the Ocean-
ographical Institute) 1931: Moscow. 1-f

Trudy Ikhtiologicheskoi Laboratorii see Re-
port (s) of the (Astrakhan) Scientific Station
of Fisheries of Volga and Caspian Sea

Trudy Instituta Fiziologii; Nauchno-Issledo-
vatel'skii Institut Fiziologii nkp (Travaux de
1'Institut de Physiologic; Institut de Re-
cherches Physiologiques de Moscou) 1934: 3

Trudy Instituta Genetiki (Bulletins of the
Institute of Genetics) (1-9 under various
titles) 1922: Akademiia Nauk. 1 +
*Trudy Instituta po Izuchenim Severa (Trans-
actions of the Institute for Scientific Ex-
ploration of the North) (3-24 as Trudy Se-
vernoi Nauchno-Promyslovoi Ekspeditsii)
(Transactions of the Northern Scientific and
Economic Expedition) (25-30 as Trudy
Nauchno-Issledovatel'skogo Instituta po
Izuchenifu Severa): 3-49

Trudy Karadags'koi Biologichnoi Stantsii;
Akademiia Nauk URSR (Travaux de la
Station Biologique de Karadagh; Academic
des Sciences de la RSS d'Ukraine) Kief. 3-6

Trudy Komissii po Izucheni/u Ozera Baikala
see Trudy Baikal'skoi Limnologicheskoi
Stantsii

Trudy Kosinskoi Biologicheskoi Stantsii see
Trudy Limnologicheskoi Stantsii v Kosine

Trudy Laboratorii Eksperimental'noi Biologii
Moskovskogo Zooparka see Trudy po Dina-
mike Razvitiia

*Trudy Laboratorii Eksperimental'noi Zoologii
i Morfologii Zhivotnykh; Akademifi Nauk
SSSR (Travaux du Laboratoire de Zoologie
Experimentale et de Morphologic des Ani-
maux; Academic des Sciences de 1'URSS)
1930: 1-4

Trudy Laboratorii Evolmtsionnoi Morfologii;
Akademild. Nauk SSSR (Travaux du Labo-
ratoire de Morphologic Evolutive ; Academic
des Sciences de 1'URSS) 1933: [1-2]

Trudy Laboratorii Genetiki (Bulletins of the
Laboratory of Genetics) no. 9, 1932 see
Trudy Instituta Genetiki

Trudy Leningradskogo (Petrogradskogo)
Obshchestva Estestvoispytatelei (Travaux
de la Societe des Naturalistes de Lenin-
grad) (Liv. 1. Comptes Rendus des Seances;
Liv. 2. Section de Zoologie et Physiologic;
Liv. 3. Section de Botanique) (after 60,
1930, volume for each Livraison discontinued)
1870: Liv. 1. 51-60; Liv. 2. 44-60; Liv. 3.
47-60; 61-63; [64-67]

Trudy Limnologicheskoi Stantsii v Kosine
(Proceedings of the Kossino Limnological
Station of the Hydrometeorological Service
of USSR) (Arbeiten der Limnologischen
Station zu Kossino der Hydrometeorolo-

gischen Administration der USSR) (1-11 as
Trudy Kosinskoi Biologicheskoi Stantsii)
(Arbeiten der Biologischen Station zu Kos-
sino, bei Moskau) 1924: Gidrometeorologi-
cheskii Institut. 1 +

Trudy Morskogo Nauchnogo Instituta see Be-
richt(e) des Wissenschaftlichen Meeresins-
tituts

Trudy Moskovskogo Tekhnicheskogo Insti-
tuta Rybnogo Khoziaistva i Promyshlen-
nosti im A. I. Mikoiana: Moscow. 1938-39

Trudy Nauchno-Issledovatel'skogo Instituta
Eksperimental'nogo Morfogeneza; Mos-
kovskogo Gosudarstvennogo Universiteta
(Transactions of the Research Institute of
Experimental Morphogenesis; State Uni-
versity of Moscow) (1-5 as Trudy Gosu-
darstvennogo Nauchno-Issledovatel'skogo
Instituta Eksperimental'nogo Morfogeneza;
Arbeiten des Instituts fur Experimental e
Morphogenese) 1934: 1 +

Trudy Nauchno-Issledovatel'skogo Instituta
po Izuchenim Severa see Trudy Instituta po
Izuchenifu Severa

*Trudy Nauchno-Issledovatel'skogo Instituta
pri Voronezhskom Gosudarstvennom Uni-
versitete (Travaux de 1'Institut des ^Re-
cherches Scientifiques a 1'Universite d'Etat)
1927: Voroneje. 1-4

*Trudy Nauchno-Issledovatel'skogo Instituta
Zoologii; AssotsiatsiQ Nauchno-Issledova-
tel'skikh Institutov pri Fiziko-Matemati-
cheskom Fakul'tete i Moskovskogo, Gosu-
darstvennogo Universiteta (Arbeiten des
Zoologischen Forschungsinstituts ; Assozia-
tion der Forschungsinstitute d. Physico-
Mathematischen Fakultat der Moskauer
Universitat) 1925: 1-4, no. 3

Trudy Nauchnogo Instituta Rybnogo Kho-
zfaistva (Reports of the Scientific Institute
of Fisheries) (Abhandlungen des Wissen-
schaftlichen Institutes fiir Fischereiwirt-
schaft) 1924: Moscow. 1-5

Trudy Nauchnogo Khimiko-Farmatsevtiche-
skogo Instituta; Nauchno-Tekhnicheskoe
Upravlenie V. S. N. Kh. (Transactions of
the Scientific Chemical-Pharmaceutical In-
stitute; Scientific-Technical Department of
the Supreme Council of National Economy)
1921: Moscow. 9-12; 14-20

Trudy Obshchestva Ispytatelei Prirody pri
Imperatorskom Khar'kovskom Universitete
(Travaux de la Societe des Naturalistes a
1'Universite Imperial e de Kharkow) 1869:
37; 40, pt. 1-2; 41; 46

Trudy Osoboi Zoologicheskoi Laboratorii i
Sevastopol'skoi Biologicheskoi Stantsii see
Trudy Sevastopol'skoi Biologicheskoi Stant-
sii

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Trudy otdela sel'skokhoziaistvennoi mikro-
biologii see Trudy Vsesofuznogo Instituta
sel'skokhoziaistvennoi mikrobiologii

Trudy O-va Rossiiskikh Fiziologov see Be-
richt(e) der Gesellschaft Russischer Physio-
logen

Trudy Permskogo Biologicheskogo Nauchno-
Issledovatel'skogo Instituta see Trudy Biolo-
gicheskogo Nauchno-Issledovatel'skogo Ins-
tituta pri Molotovskom Gosudarstvennom
Universitete im. M. Gor'kogo

Trudy Petergofskogo Biologicheskogo Insti-
tuta (Travaux de 1'Institut Biologique de
Peterhof) (1-8 as Trudy Petergofskogo
Estestvenno-Nauchnogo Instituta) (Tra-
vaux de 1'Institut des Sciences Naturelles
de Peter hoff) 1925: 1 +

Trudy Petrogradskogo Obshchestva Estest-
voispytatelei see Trudy Leningradskogo
Obshchestva Estestvoispytatelei

Trudy Plovuchego Morskogo Nauchnogo Ins-
tituta see Bericht(e) des Wissenschaftlichen
Meeresinstituts

Trudy po Dinamike Razvitifa (Transactions on
the Dynamics of Development) (1-5 as
Trudy Laboratorii Eksperimental'noi Bio-
logii Moskovskogo Zooparka; Transactions
of the Laboratory of Experimental Biology
of the Zoopark of Moscow) 1926: 1-10

Trudy po Prikladnoi Botanike, Genetike i
Selektsii (Bulletin of Applied Botany, Gene-
tics and Plant-Breeding) (14-17, no. 2 as
Trudy po Prikladnoi Botanike i Selektsii)
(after 27, 1931, divided into series) 1908:
*14-27, no. 5; Ser. A. Plant Industry in
USSR 1932: 1-21; Ser. 1. Taxonomy, Geog-
raphy and Ecology of Plants 1933: 1 + ; Ser.
2. Genetics, Plant Breeding and Cytology
1932: 2+ ; Ser. 3. Physiology, Biochemistry
and Anatomy of Plants 1932: 1 + ; Ser. 10.
Dendrology and Ornamental Horticulture
1933: 1-2; Ser. 11. New Cultures and Ques-
tions of Introduction 1935: 1-2; Ser. 13.
Abstracts and Bibliography 1933: 1-9; Sup-
plements [28-84]

Trudy po Prikladnoi Entomologii see Works of
Applied Entomology

Trudy po Zashchite Rastenii see Bulletin of
Plant Protection

Trudy Podfarnyi Nauchno-Issledovatel'skii
Institut Morskogo Rybnogo Khozfaistva i
Okeanografii im. Pochetnogo Chlena Aka-
demii Nauk SSSR Pro!. N. M. Knipovicha
(Transactions of the Knipovich Polar Scien-
tific Institute of Sea-Fisheries and Ocea-
nography) 1938: Murmansk. 1 +

Trudy Pryesnovodnoi Biologicheskoi Stantsii
Imperatorskago S.-Peterburgskago Obsh-

chestva Estestvoispytatelei see Trudy Boro-
dinskoi Biologicheskoi Stantsii v Karelii

Trudy Sevastopol'skoi Biologicheskoi Stantsii;
Akademiia Nauk SSSR (Travaux de la Sta-
tion Biologique de Sebastopol; Academie
des Sciences USSR) (ser. 1 as Travaux du
Laboratoire Zoologique et de la Station Bio-
logique de Sebastopol pres de 1'Academie
Imperiale des Sciences de St.-Petersbourg;
ser. 2, 1915, Petrograd; ser. 2, no. 4, Russie;
ser. 3, Academie des Sciences, U.S.S.R.)
(ser. 2 as Trudy Osoboi Zoologicheskoi
Laboratorii i Sevastopol'skoi Biologicheskoi
Stantsii) 1903: ser. 1. 1-10; ser. 2. 1-13;
ser. 3. 1 +

Trudy Severnoi Nauchno-Promyslovoi Ekspe-
ditsii see Trudy Instituta po Izucheniiu
Severa

Trudy Sibirskoi Ikhtiologicheskoi Laboratorii
see Trudy Vostochno-Sibirskoi Nauchnoi
Rybokhoziaistvennoi Stantsii

Trudy Sibirskoi Nauchnoi Rybokhozfaistven-
noi Stantsii see Trudy Vostochno-Sibirskoi
Nauchnoi Rybokhoziaistvennoi Stantsii

Trudy Sredne-Aziatskogo Gosudarstvennogo
Universiteta (Acta Universitatis Asiae Me-
diae); ser. 8-a. Zoologiia 1927: Taschkent.
1-2

Trudy Sungariiskoi Rechnoi Biologicheskoi
Stantsii; Obshchestvo Izuchenifa Man'ch-
zhurskogo Kraia (Proceedings of the Sun-
garee River Biological Station; Manchuria
Research Society) (Arbeiten der Biologi-
schen Sungari-Station) 1925: Harbin. 1,
nos. 1-6

*Trudy Tsentral'nogo Nauchnogo Instituta
Rybnogo Khozfaistva (Reports of the Cen-
tral Scientific Institute of Fisheries) (Ab-
handlungen des Wissenschaftlichen Zen-
tralinstitutes fur Fischereiwirtschaft) 1931:
1-3

Trudy Volgo-Kaspiiskoi Nauchnoi Rybo-
khoziaistvennoi Stantsii see Report (s) of the
(Astrakhan) Scientific Station of Fisheries
of Volga and Caspian Sea

Trudy Voronezhskogo Gosudarstvennogo Uni-
versiteta (Acta Universitatis Voronegiensis)
1925: 1 +

Trudy Vostochno-Sibirskoi Nauchnoi Rybo-
khoziaistvennoi Stantsii (Reports of the
East-Siberian Scientific Station of Fisheries)
(2, nos. 1-5 as Trudy Sibirskoi Ikhtiolo-
gicheskoi Laboratorii; Report of the Ich-
thyological Laboratory in Siberia) (4-5 as
Trudy Sibirskoi Nauchnoi Rybokhoziaist-
vennoi Stantsii; Reports of the Siberian
Scientific Station of Fisheries) Narodnyi Ko-
missariat Snabzheniia; Krasnofarsk. [2-6]

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Trudy Vsesomznogo Gidrologicheskogo S'ezda
(Proceedings of the Hydrological Congress
of U.S.S.R.) 1924: 1-2, pt. 2

Trudy Vsesomznogo Instituta Eksperimen-
tal'noi Meditsiny 1933: 1, nos. 1-3

Trudy Vsesofuznogo Instituta sel'skokho-
ziaistvennoi mikrobiologii (Bulletin of the
USSR Institute of Agricultural Microbiol-
ogy) (1-3 as Trudy otdela Sel'skokhozfaist-
vennoi Mikrobiologii) (Bulletin of the
Bureau of Agricultural Microbiology) 1926:
1-5; [6-8]

Trudy Vsesomznogo Nauchno-Issledova-
tel'skogo Instituta Morskogo Rybnogo
Khoziaistva i Okeanografii (Transactions
of the Institute of Marine Fisheries and
Oceanography of the USSR) 1935: 1-2; 4;
8-9

*Trudy Vsesomznogo S'ezda po Genetike, Se-
lektsii, Semenovodstvu i Plemennomu Zhi-
votnovodstvu (Proceedings of the U.S.S.R.
Congress of Genetics, Plant- and Animal-
Breeding) 1929: 1-6

Trudy Vsesomznogo S'ezda Zoologov, Anato-
mov i Gistologov (1-3 as Trudy Vserossii-
skogo S'ezda Zoologov, Anatomov i Gistolo-
gov) (Proceedings of the Congress of the
Zoologists, Anatomists and Histologists, of
the Union of SSR) 1922: 1-4

Trudy Zoologicheskogo Instituta; Akademiia
Nauk SSSR (Travaux de 1'Institut Zoologi-
que de 1'Academie des Sciences de 1'URSS)
1932: 1 +

*Trudy Zvenigorodskoi Gidrofiziologicheskoi
Stantsii Instituta Eksperimental'noi Biologii
ginz'a Moskva (Arbeiten der Hydrophysio-
logischen Station des Instituts fur Experi-
mentelle Biologic, Moskau): 1928

Tschechische Chemische Forschungsarbeiten
see Collection of Czech Chemical Communi-
cations

Tufts College Studies; Scientific Series 1894:
1 +

Turtox News 1923: General Biological Supply
House, Chicago. [1-2]; 3-4; [5]; 6 +

Uchenye Zapiski; Gor'kovskogo Gosudarst-
vennogo Universiteta (Scientific Records of
the Gorky State University) 1936: 1-9

Uchenye Zapiski; Moskovskii Gosudarstven-
nogo Universiteta (Wissenschaftliche Be-
richte der Moskauer Staats-Universitat)
1933: 1-4; 8-9; 11; 13 +

Uchenye Zapiski; Permskii Gosudarstvennyi
Universitet im. M. Gor'kogo (Scientific
Memoirs of the M. Gorky State University
of Perm) 1935: 1 +

*Uchenyia Zapiski Imperatorskago Moskov-
skago Universiteta; Otde'l Estestvenno-

Istoricheskii 1880: 5-6; 10-11; 16; 22; 25,
nos. 1-3; 38

Union Geodesique et Geophysique Interna-
tionale; Conseil International de Re-
cherches (Conseil International des
Unions Scientifiques) ;
Assemblee Generale 1922: 1-6;
Association d'Oceanographie Physique ; Pro-
ces-Verbaux 1933: 1-3 ; Publication Scien-
tifique 1931: 1 + ;

Association de Seismologie ; Comptes Ren-
dus 1922: 5-6; Serie A. Travaux Scien-
tifiques: 2-5; 8-15, pt. 2; Serie B. Mono-
graphies: 4-7;

Commission pour 1'Etude des Relations en-
tre les Phenomenes Solaires et Terres-
tres; Rapport 1926: 1-4;
Section d'Hydrologie Scientifique (1-11 as
Section Internationale d'Hydrologie Scien-
tifique); Bulletin 1924: 1-4; 9-18;
"Section d'Oceanographie (1-3 as Sec-
tion d'Oceanographie Physique); Bulletin
1921: 1-17;

Section de Geodesie; Travaux: 7-8, 1930;
Section de Meteorologie; Assemblee Gene-
rale; Proces-Verbaux : 3 (1937); 4-5;
Section of Terrestrial Magnetism and Elec-
tricity; Bulletin 1919: Washington, D. C.
1-2; 4; 6 (1926);

Section of Terrestrial Magnetism and Elec-
tricity; Section of Oceanography; Stock-
holm Assembly ; Reports and Communica-
tions: Department of Terrestrial Magnet-
ism, Carnegie Institution of Washington.
1930

United States Bureau of Navigation; Navy
Department see American Nautical Al-
manac

United States Department of Agriculture; Cir-
lar 1927: [1-617] + ; *Department Bulletin
1913: [3-1500, 1913-37]; *Department Cir-
cular 1919: [35-425, 1919-27]
United States Department of Agriculture;

Leaflet 1927: [1-209] +

United States Department of Agriculture;
Miscellaneous Publications 1927: [233-450]
United States Department of Agriculture;
Numerical List of Current Publications see
United States Department of Agriculture:
Miscellaneous Publications 450, 1941
United States Department of Agriculture; Re-
port of the Secretary (1847-61 published as
a part of the Report of the Commissioner of
Patents; 1862-88 as Report of the Commis-
sioner of Agriculture; 1925+ contained in
full in Yearbook of Agriculture): 1847-94;
1896-1924

United States Department of Agriculture;
Technical Bulletin 1927: [1-791] +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

United States Department of Agriculture see
also Farmers' Bulletin; Journal of Agricul-
tural Research ; and Yearbook of Agriculture

United States Department of Agriculture;
Bureau of Animal Industry; Annual Report
1885: 3-4; 8-21; 27; Bulletin 1893: [1-163];
Circular 1897: [3-214]

*United States Department of Agriculture;
Bureau of Biological Survey; Bulletin 1889:
9-24; 26-27; 29-35; 37-45; Circular 1886:
28-29; 31; 55-58; 63-64; 67; 69-70; 72-73;
76-83; 85-90; 92-94

United States Department of Agriculture;
Bureau of Biological Survey see also North
American Fauna; United States Depart-
ment of the Interior, Bureau of Biological
Survey, and Fish and Wildlife Service

United States Department of Agriculture;
Bureau of Chemistry and Soils see Index of
Publications of the Bureau of Chemistry
and (Bureau of) Soils

*United States Department of Agriculture;
Bureau of Entomology (followed by Bureau
of Entomology and Plant Quarantine) Bul-
letin: 3-5; 7; 9; 11-32; n.s. 1-62; [63-64];
65-79; [80]; 81; [83]; 84-91; 93-105; 107;
109; 111; 113-15; [116]; 117-27; Circular:
3-14; 16-29; 31-39; 41-43; 45-53; 55-63;
66-88; 90-91; 93-117; 119-24; 127-45;
147-50; 152-59; 161-73; Report of the En-
tomologist: 1878-79; 1907-08; 1909-12;
1913-15; 1916-22; 1923-26; Technical Se-
ries 1895: 1-3; 5-17; 19-27

United States Department of Agriculture;
Bureau of Plant Industry; *Bulletin 1901:
[1-285]; ^Circular: [3-132]; Report of the
Chief: 1912-13; 1915-18; 1919-23; 1924-27

United States Department of Agriculture;
Bureau of Plant Industry see also Botany;
Current Literature; and Plant Science
Literature

*United States Department of Agriculture;
Division of Ornithology and Mammalogy;
Bulletin (1-2 as Division of Economic
Ornithology) 1888: 1-8 (1896)

United States Department of Agriculture;
Forest Service; Bulletin (formerly Bureau of
Forestry; Bulletin): 58; 61; 72; Circular:
80-81; 105; 114; 118-21; 127-28; 152; 164;
207

United States Department of Agriculture;
Library; Bibliographic Contributions: 2
(1922)-6; 8-15; 20

United States Department of Agriculture;
Library see also Botany; Current Litera-
ture; and Plant Science Literature; Bureau
of Plant Industry

United States Department of Agriculture;
Office of Experiment Stations; Annual Re-

port: 1903-06; Report of the Director: 1897-
1904; Bulletin: [24-223]

United States Department of Agriculture;
Office of Experiment Stations see also Ex-
periment Station Record
* United States Department of Agriculture;
Weather Bureau; Bulletin 1892: 1-14

United States Department of Agriculture;
Weather Bureau see also Monthly Weather
Review; and United States Meteorological
Yearbook (through June, 1935 as Report
of the Chief of the Weather Bureau)

United States Department of Commerce see
Annual Report of the Secretary of Com-
merce

United States Department of Commerce;
Bureau of Fisheries see Bulletin of the
United States Bureau of Fisheries; Fisheries
Service Bulletin; Fishery Circular; Investi-
gational Report; and Report of the United
States Commissioner of Fisheries

United States Department of Commerce;
Bureau of Standards see Circular of the
National Bureau of Standards; Journal of
Research of the National Bureau of Stand-
ards; Scientific Papers of the Bureau of
Standards; Standards Yearbook; and Tech-
nologic Papers

United States Department of Commerce;
Coast and Geodetic Survey see Annual Re-
port of the Secretary; Current Tables; Field
Engineers Bulletin; Special Publications;
and Tide Tables

United States Department of the Interior;
Bureau of Biological Survey (formerly U. S.
Department of Agriculture); Report of the
Chief 1906: 1907-14; 1916-17; 1919-20;
1938+

United States Department of the Interior;
Bureau of Biological Survey see also United
States Department of Agriculture; Bureau
of Biological Survey

United States Department of the Interior;
Fish and Wildlife Service; Annual Report
of the Director 1940: 1940+ ; Circular 1941 :
1-2; 4+; Conservation Bulletin (formerly
Bureau of Biological Survey) 1940: 1 + ;
Regulatory Announcement 1941: 1 + ; Re-
search Report 1941: 1 + ; Statistical Digest
1942: 1 + ; *Wildlife Circular (1-3 as Bureau
of Biological Survey) 1939: 1-13; Wildlife
Leaflet (154-65 as Bureau of Biological
Survey): 154; 164-92; 194-99; 201; *Wild-
life Research Bulletin (1-2 as Bureau of
Biological Survey) 1940: 1-4; Wildlife Re-
view 1935: 1 + ; see also Fisheries Service
Bulletin; and North American Fauna

United States Department of the Interior;
Geological Survey; Annual Report: 2-4; 7;

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

9; 13; [18]; [20]; 21; 29; 35; 40; 42-43; Pro-
fessional Papers: [14-186]

United States Department of the Interior;
Geological Survey see also Geological Sur-
vey Bulletin; Geological Survey Water-
Supply Papers; and Monographs of the
United States Geological Survey

United States Department of the Interior;
Geological (and Geographical) Survey of
the Territories; Annual Report (title varies
with the name of the Territory; 3-5 as Pre-
liminary, Field, Report) 1867: 1-12; Bulle-
tin 1874: 1-6; Miscellaneous Publications:
3-5; 8; 11-12; Report 1873: 1-3; 5-13

United States Meteorological Yearbook
(through June, 1935 as Report of the Chief
of the Weather Bureau): 1896-97; 1897-98;
1928-29; 1929-30; 1931-32; 1933 +

United States National Museum (Smithsonian
Institution) see Bulletin of the United States
National Museum; Contributions from the
United States National Herbarium; Pro-
ceedings of the United States National Mu-
seum; and Report of the United States
National Museum

United States Naval Medical Bulletin for the
Information of the Medical Department of
the Navy 1907: Bureau of Medicine and
Surgery. 40+

United States Navy Department; Bureau of
Navigation see z\merican Nautical Almanac

United States Navy Department; Hydro-
graphic Office ; General Catalog of Mari iers'
and Aviators' Charts and Books : 1931; 1935;
1939; 1941+ see also Notice to Mariners

United States Superintendent of Documents
see Monthly Catalogue; United States Pub-
lic Documents

United States Treasury Department; Coast
Guard see Bulletin of the United States
Coast Guard; and Notice to Mariners

United States Treasury Department; Public
Health and Marine Hospital Service; Yel-
low Fever Institute; Bulletin: 14 (1904)

United States Treasury Department; Public
Health Service see National Institute of
Health Bulletin; Public Health Bulletin;
and Public Health Reports

United States War Department; Office of
Engineers; Annual Report of the Chief,
Appendix Z; Annual Report of the Survey
of the Northern and Northwestern Lakes by
C. B. Comstock 1871-76

United States War Department; Office of the
Surgeon General; Bulletin 1913: 1-8; 10-11

United States War Department; Professional
Papers of the Signal Service 1881: 1-16; 23

University of California; College of Agricul-
ture ; Agricultural Experiment Station ; Bul-

letin: [131-616]; technical Papers 1923:
1-20

University of California Publications ; Bulletin
of the Department of Geological Sciences
1893: [7]; [10]; 25+; Anatomy 1921: 1 + ;
Biological Sciences 1933: 1 + ; Botany 1902:
1 + ; Entomology 1906: 1 + ; Geography
1913: [1-2]; 3-4; [5-6]; 7+; *Pathology
1903: 1-2; Pharmacology 1938: 1 + ; Phil-
osophy 1904: 17; Physiology 1902: 1 + ;
Public Health 1928: 1 + ; Zoology 1902: 1 +

University of Colorado Studies (26, 1938+ as
General Series A) 1902: 1 + ; Series D.
Physical and Biological Sciences 1940: 1 +

University of Florida Publications; Biological
Science Series 1930: 1 + ; Education Series
1932: [1-2]; Geography Series 1930: 1 + ;
Sociology Series 1932: 1 + ; Economic Series
1930: 1 + ; Inter- American Institute Series
1930: 1 + ; Business Administration Series
1931: 1, no. 1; Engineers Experiment Sta-
tion Series 1933: 1 +

University of Iowa Studies in Natural History
(1—7 as Bulletin from the Laboratories of
Natural History of the State University of
Iowa) 1888: 1 +

University of Michigan; School of Forestry
and Conservation; Bulletin 1932: 1 + ; Cir-
cular 1937: 1 +

University of Michigan Museum of Zoology;
Miscellaneous Publications 1916: 1 +

University of Michigan Studies; Memoirs of
the University of Michigan Museums 1928:
1 + ; Scientific Series 1914: 1-6; 9 +

University of Minnesota Agricultural Experi-
ment Station Bulletin: [9-325]

University of Minnesota Studies in the Bio-
logical Sciences 1913: 1-6; Studies in the
Physical Sciences 1912: 1-2

University of Missouri Bulletin; Education
Series 1911: 22-23; Extension Series 1913:
38-39; 44; 56; Engineering Experiment Sta-
tion Series 1910: 22; 25-26; *Science Series
1905: [1-2]; *Medical Series 1913: 10
*University of Missouri Studies 190 1 : 2, nos. 4-5

University of Missouri Studies; a quarterly of

research 1926: [8-9]
*University of Missouri Studies; Science Series

1905: [1-3]
*University of Montana; Bulletin; Biological

Series 1901: 1-12; 14-15

*University of Montana Studies in Psychology
1908: 1

University of Queensland; Papers; Depart-
ment of Biology 1939: [1]+; Department of
Geology 1939: [1-2] +

University of Texas Bulletin (formerly the
Bulletin of the University of Texas): [59-
4032]

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

University of Toronto Studies; Anatomical
Series 1900: 1-5; 7+; Biological Series
1898: 1 + ; Physiological Series 1900: 1 +

University of Washington Publications in
Biology 1932: 1 + ; *Fisheries 1925: 1-2;
Oceanography 1932: 1 +

University of Wisconsin Studies in Science
1920: 1+ see also Bulletin of the University
of Wisconsin ; Science Series

University Studies published by the University
of Cincinnati (series 1 as Bulletin) 1905:
series 2, 5, no. 3

*Uspekhi Eksperimental'noi Biologii (Zhurnal
Eksperimental'noi Biologii, seriia B) (Jour-
nal de Biologic Experimental, serie B)
1922: 1; [2]; 3-6; [7-8]

Uspekhi Sovremennoi Biologii (Advances in
Modern Biology) 1932: Moscow. 1-2; 3,
nos. 1-2; 4-6

Vereinsblatt der Deutschen Gesellschaft fur
Mechanik und Optik see Zeitschrift fur
Instrumentenkunde; Beiblatt; Zeitschrift
der Deutschen Gesellschaft fur Mechanik
und Optik

Verhandelingen ; Koninklijk Magnetisch en
Meteorologisch Observatorium te Batavia
1911: 1-2; 5-6; 8; 10-13; 15-17; 19 +

Verhandelingen der Koninklijke Nederland-
sche Akademie van Wetenschappen ; Af-
deeling Natuurkunde; Tweede Sectie (1-36
as Verhandelingen der K. Akademie van
Wetenschappen; Afdeeling Natuurkunde;
Tweede Sectie) 1892: Amsterdam. 1 +

Verhandlungen der Anatomischen Gesell-
schaft; Anatomischer Anzeiger; Ergan-
zungsheft 1887: 1 +

Verhandlungen der Deutschen Pathologischen
Gesellschaft 1898: 1+ see in Zentralblatt
fiir Allgemeine Pathologic und Pathologische
Anatomic 9, 1898 +

Verhandlungen der Deutschen Pharmakolo-
gischen Gesellschaft see in Naunyn-
Schmiedebergs Archiv fiir Experimentelle
Pathologic und Pharmakologie 92, 1922 +

Verhandlungen der Deutschen Tropenmedi-
zinischen Gesellschaft 1908: 1+ see in
Deutsche Tropenmedizinische Zeitschrift
12, 1908 +

Verhandlungen der Deutschen Zoologischen
Gesellschaft E. V. (30+ a<> Zoologischer
Anzeiger; Supplementband) 1891: 1 +

Verhandlungen der Freien Vereinigung
Schweizerischer Physiologen see Verhand-
lungen des Vereins der Schweizer Physio-
logen

*Verhandlungen der Gesellschaft fiir Erdkunde
zu Berlin (1902+ see Zeitschrift der Gesell-
schaft fur Erdkunde zu Berlin) 1873: 1-28

Verhandlungen der Internationalen Verei-
nigung fur Theoretische und Angewandte
Limnologie 1922: 1 +

Verhandlungen der Naturforschenden Gesell-
schaft in Basel 1852: 1 +

Verhandlungen der Physikalisch-Medizini-
schen Gesellschaft zu Wiirzburg see Be-
richte der Physikalisch-Medizinischen Ge-
sellschaft zu Wiirzburg

*Verhandlungen der Physiologischen Gesell-
schaft zu Berlin 1875: 1877-1907 see in
Archiv fiir Anatomic und Physiologic;
Physiologische Abteilung

Verhandlungen der Schweizerischen Natur-
forschenden Gesellschaft (Actes de la So-
ciete Helvetique des Sciences Naturelles)
1817:85-86; 88; 92-93; 100+

Verhandlungen der Zoologisch-Botanischen
Gesellschaft hi Wien 1851: 1 +

Verhandlungen des Botanischen Vereins der
Provinz Brandenburg 1859: 38-44

Verhandlungen des Internationalen Kanin-
chenziichter-Kongresses 1930: 1 (Leipzig)

Verhandlungen des Internationalen Zellfor-
scherkongresses (1 as Verhandlungen der
Abteilung fiir Experimentelle Zellforschung
des 10. Internationalen Zoologenkongresses)
1927: 1+ see in Archiv fiir Experimentelle
Zellforschung 6 +

Verhandlungen des Naturforschenden Ver-
eines in Briinn 1862: 42; 45-51

Verhandlungen des Naturhistorisch-Medizini-
schen Vereins; Heidelberg 1857: n.s. 1 +

Verhandlungen des Vereins der Schweizer
Physiologen (Comptes Rendus de la Societe
des Physiologistes Suisses) (1932-35, no. 1
as Helvetica Biologica Acta; 1935, no. 2 —
1938 as Verhandlungen der Freien Verei-
nigung Schweizerischer Physiologen (Comp-
tes Rendus de 1'Association Libre des
Physiologistes Suisses) 1932: 1 +

Verhandlungen und Mitteilungen des Sieben-
biirgischen Vereins fiir Naturwissenschaften
zu Hermannstadt 1849: 49-55; 57-60; 79-80

Veroffentlichungen des Geophysikalischen
Instituts der Universitat Leipzig; Zweite
Serie; Spezialarbeiten 1913: 2, nos. 1-2,
5—6; 3, nos. 3—6; 4, nos. 1—5; 5, nos. 1—4; 6+

Veroffentlichungen des Instituts fiir Meeres-
kunde an der Universitat Berlin; *1902:
1-15; n.s.; A. Geographisch-Naturwissen-
schaftliche Reihe 1912: 1 +

Veroffentlichungen des Meteorologischen Ins-
tituts der Universitat Berlin 1936: 1 +

Versammlung der Gesellschaft Deutscher

Naturforscher und Arzte: 88, 1924+ see in

Naturwissenschaften 12, 1924 +

*Verslag van de Gewone Vergaderingen der

Afdeeling Natuurkunde; K. Akademie van

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Wetenschappen te Amsterdam 1892: 5;
[35]; 36-42 (1933)

Vestnik Ceskoslovenske Zoologicke Spolec-
nosti v Praze (Memoires de la Societe
Zoologique Tchecoslovaque de Prage) 1934:
vl +

Vestnik Kralovske Ceske Spolecnosti Nauk;
Trida Matematicko-Prirodovedecka (Me-
moires de la Societe Royale des Lettres et
des Sciences de Boheme; Classe des
Sciences) (1884-85 as Zpravy o Zasedani
Kralovske Ceske Spolecnosti Nauk v Praze)
(1884-1917 as Sitzungsberichte der Kgl.
Bohemischen Gesellschaft der Wissenschaf-
ten ; Mathematisch-Naturwissenschaftliche
Klasse) 1884: 1884 +

Viaggi di Studio ed Esplorazioni 1933: Reale
Accademia d'ltalia 1933: 1

Victory (1-2, no. 49 as Defense) 1.940: 2, no.
44 +

Videnskabelige Meddelelser fra Dansk Na-
turhistorisk Forening i Kobenhavn (Kj0ben-
havn) (1-60 also as ser. 1-6) 1849: 1 +

Vierteljahrsschrift der Naturforschenden Ge-
sellschaft in Zurich 1856: 37, no. 2; 38, no. 2;
41; 42, nos. 2-4; 47-48; 49, nos. 3-4; 50+

Villanova College; The Mendel Bulletin:
[9-10]

Virchow's Archiv fiir Pathologische Anatomie
und Physiologic und fiir Klinische Medizin
1847: 1 +

Virchow's Jahresberichte see Jahresbericht
iiber die Leistungen und Fortschritte in der
Anatomie und Physiologie

Visnik Kiivs'kogo Botanichnogo Sadu (Bulle-
tin du Jardin Botanique de Kyiv) 1924:
Academic des Sciences de 1'Ukraine; Institut
de Botanique, Kief. 15-17

Voprosy Fiziologii i Ekologii Malfariinogo Ko-

mara 1940: Tsentral'nyi Institut, Moscow. 1

*Vortrage und Aufsatze u'ber Entwicklungs-

mechanik der Organismen herausgegeben

von Wilhelm Roux 1905: 1-22; 24-34

Vyrocni Zprava; Moravske Prirodovedecke
Spolecnosti 1925: Brno. 8-13

Vyrocni Zprava Kralovske Ceske Spolecnosti
Nauk (Compte-Rendu Annuel de la Societe
Royale des Lettres et des Sciences de
Boheme) (1899-1917 as Jahresbericht der
K. Bohmischen Gesellschaft der Wissen-
schaften) (1918-32, Resume du Compte-
Rendu) 1876: 1899+

War Medicine; a periodical containing orig-
inal contributions, news and abstracts of
articles of military, naval and similar in-
terest related to preparedness and war
service 1941: [2] +
*Washington University Studies; Scientific

Series 1913: 1-2; 3, no. 2; 4, no. 1;6;7, no. 2;
10, no. 2; 11; 12, no. 1; 13; Humanistic
Series 1913: 1, no. 1; 5, no. 1; 7, no. 1; 11,
no. 2; 12

Watson's Microscope Record 1924: W. Wat-
son and Sons, Ltd., London. 3-4; 6; 8; 10-44
(1938)

"West-American Scientist; San Diego Society
of Natural History 1884: [1-15]

West Virginia University Bulletin (includes
the Proceedings and the Bulletin of the
West Virginia University Scientific Associa-
tion; and the Proceedings of the West Vir-
ginia Academy of Science 1-5; 10) 1922:
[22-34]

(Das) Wetter; Monatsschrift fiir Witterungs-
kunde see Zeitschrift fiir Angewandte Me-
teorologie; Das Wetter

Wiadomosci Meteorologiczne i Hydrogra-
ficzne; Paristwowyo Instytut Meteorolo-
giczny; wydawane przez (Bulletin Meterolo-
gique et Hydrographique ; 1'Institut Na-
tional Meteorologique de Pologne): War-
saw. 1933 +

Wiadomosci Sluzby Hydrograficznej (Bulletin
du Service Hydrographique) 1935: Wy-
dawnictwo Instytutu Hydrograficznego
Ministerstwa Komunikacji, Warszawa. 1-3

Wiedemann's Annalen see Annalen der Physik

Wiener Entomologische Zeitung 1882: [14];
24-29; 30, nos. 2-end; 31-33; Beihefte 32

Wiener Klinische Wochenschrift 1888: [38-
40]; 41 + ; Supplement: 38-40

Wiener Medizinische Wochenschrift 1851:
81-89; 90, nos. 20-22 (1940)

Wilhelm Roux' Archiv fiir Entwicklungsme-
chanik der Organismen; Organ fiir die
Gesamte Kausale Morphologic (1-97 as
Archiv fiir Entwicklungsmechanik der Or-
ganismen; Wilhelm Roux; 98-104 as Archiv
fiir Mikroskopische Anatomie und Ent-
wicklungsmechanik) (105-30 also as Zeit-
schrift fiir Wissenschaftliche Biologie, Abt.
D) 1894: 1 +

Wilson Bulletin (old series 6+ also numbered

as new series 1 + ) 1889: 6-27 (1915)
*Wisconsin Naturalist; published by Charles
F. Carr, Madison 1890: 1, no. 5

Wissenschaftliche Berichte der Biologischen
Fakultat der Tomsker Staats-Universitat
see Trudy Biologicheskogo Fakul'teta Tom-
skogo Gosudarstvennogo Universiteta

Wissenschaftliche Berichte der Moskauer
Staats-Universitat see Uchenye Zapiski;
Moskovskii Gosudarstvennogo Universiteta
*Wissenschaftliche Meeresuntersuchungen
herausgegeben von der Kommission zur
Wissenschaf tlichen Untersuchung der Deut-
schen Meere in Kiel und von der Biologi-

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

schen Anstalt auf Helgoland; Neue Folge
(series 1, 1871-91 see Berichte der Kommis-
sion zur Wissenschaftlichen Untersuchung
der Deutschen Meere in Kiel) (N.F. 3+ in
two sections: Helgoland and Kiel) 1894: 1-2;
Helgoland N.F. 3-19; Kiel N.F. 3-22
Wissenschaftliche Veroffentlichungen der Ge-
sellschaft fur Erdkunde zu Leipzig (1 en-
titled Wissenschaftliche Yeroffentlichungen
des Vereins fur Erdkunde zu Leipzig) 1891:
1; 6; 8 +

*Wistar Institute Bibliographic Service (1-2 as
Bibliographic Service) 1917: The Wistar
Institute of Anatomy and Biology. 1-8;
Index of the Wistar Institute Advance
Abstract Card Service 1938: 1 +

*Woods Hole Index 1926: 1-2

*Works of Applied Entomology (Trudy po Prik-

ladnoi Entomologii) 1894: 13-15, no. 1
Wydawnictwa Muzeum Slaskiego W Kato-
wicach: Katowice, Poland (Silesia). 3, nos.
1-6, 8 (1930-35)

*Yale Scientific Monthly 1894: [5-9]; 10-11
Yearbook of Agriculture; United States De-
partment of Agriculture (1894-1919 as Year-
book of the United States Department of
Agriculture; 1920-22 as United States De-
partment of Agriculture, Yearbook; 1923-25
as United States Department of Agriculture;
Agriculture Yearbook) 1894: 1 +
Yearbook of the International Hydrographic
Bureau (Annuaire du Bureau Hydrographi-
que International) 1928: Monaco. 1938
Yearbook of the Public Museum of the City

of Milwaukee 1921: 1927-30
Year-book of the Royal Asiatic Society of
Bengal see Journal and Proceedings of the
Royal Asiatic Society of Bengal
Year Book of the Royal Society of Edinburgh
(formerly published in Proceedings of the
Royal Society of Edinburgh) 1940: 19404-
Year Book of the Royal Society of London
1896: 44 (1940)

Zapiski Akademii Nauk SSSR see Memoires
de 1' Academic des Sciences de 1'URSS

Zapiski Belorusskogo Gosudarstvennogo Ins-
tituta Sel'skogo i Lesnogo Khozfeistva (Me-
moires de 1'Institut Agronomique et Fores-
tier d'Etat de la Belarussie) 1923: Minsk.
3-5; 7

Zapiski Gosudarstvennogo Gidrologicheskogo
Instituta see Memoires de ITnstitut Hydro-
logique de 1'URSS

Zapiski Institutu Khemii; Institut Khemii,
Akademifa Nauk URSR (Memoirs of the
Institute of Chemistry) 1934: Kief. 3, nos.
3-4

Zapiski Kievskago Obshchestva Estestvoispy-
tatelei (Memoires de la Societe des Natura-
listes de Kiew) 1870: 8-23

Zbirnik Doslidiv nad Individual'nim Rozvit-
kom Tvarin; Trudy Institutu Zoologii (i
Biologii), Akademiia Nauk URSR (Re-
searches on the Ontogeny of Animals) Kief.
6-9; 11-13

Zbirnik Prats; Dniprians'koi Biologichnoi
Stantsii (Travaux de la Station Biologique
du Dniepre) see Trudy Gidrobiologichnoi
Stantsii

Zbirnik Prats z Genetiki; Trudy Institutu
Zoologii (i Biologii), Akademiia Nauk URSR
(Memoirs on Genetics) 1936: Kief. 2-3

Zbirnik Prats z Morfologii Tvarin; Trudy Ins-
titutu Zoologii (i Biologii), Akademiu Nauk
URSR (Travaux sur la Morphologic des
Animaux) (Papers on Animal Morphology)
1935: Kief. 1-5

Zeitschrift der Deutschen Gesellschaft fur
Mechanik und Optik see Zeitschrfit fur
Instrumentenkunde; Beiblatt

Zeitschrift der Gesellschaft fiir Erdkunde zu
Berlin (1-36 as ser. 3) (Verhandlungen
combined -with Zeitschrift 1902) 1853: 1866+ ;
Erganzungsheft 1924: 1 +
*Zeitschrif t fiir Allgemeine Physiologic ; heraus-
gegeben von Max Verworn 1902: 1-20

Zeitschrift fiir Analytische Chemie ; begriindet

von Remigius Fresenius 1 862 : 1 +
*Zeitschrift fiir Anatomic und Entwicklungs-
geschichte; herausgegeben von Dr. Wilh.
His und Dr. Wilh. Braune 1875: 1-2

Zeitschrift fiir Anatomic und Entwicklungs-
geschichte; herausgegeben von Curt Elze
(1-59 as Anatomische Hefte; Beitrage und
Referate zur Anatomic und Entwicklungsge-
schichte Abt. 1 ; Arbeiten aus Anatomischen
Instituten) (63-103 also as Abt. 1 of Zeit-
schrift fiir die Gesamte Anatomic) 1891 : 1 +

Zeitschrift fiir Angewandte Anatomic und
Konstitutionslehre see Zeitschrift fiir
Menschliche Vererbungs- und Konstitu-
tionslehre

Zeitschrift fiir Angewandte Chemie see Ange-
wandte Chemie

Zeitschrift fiir Angewandte Meteorologie ;
Das Wetter (1-44 as Das Wetter; Monats-
schrift fur \Vitterungskunde) 1884: 1 +

*Zeitschrift fiir Angewandte Mikroskopie;
herausgegeben von G. Markmann 1895: 1,
Heft 1 ; 5, Heft 1

Zeitschrift fiir Anorganische und Allgemeine
Chemie; begriindet von Gerhard Kriiss
(1-91 as Zeitschrift fiir Anorganische
Chemie) 1892: 1 +

Zeitschrift fiir Biologic; begriindet von L.

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Buhl, M. Pettenkofer, L. Radlkofer und C.
Voit 1865: 1 +

Zeitschrift fur Biologic, Moscow see Biolo-

gicheskii Zhurnal

*Zeitschrift fur Biologische Technik und
Methodik 1908: 1-3

Zeitschrift fur Botanik 1909: 1 +
*Zeitschrift fur den Ausbau der Entwicklungs-
lehre; herausgegeben von R. H. France;
Miinchen 1907: 1-3

Zeitschrift fur den Physikalischen und
Chemischen Unterricht; begriindet von
Friedrich Poske 1887: [4]; 5-9; 11; 13-19;
[20-21]; 22; 46+

Zeitschrift fur den Physikalischen und
Chemischen Unterricht; Sonderheft see
Abhandlungen zur Didaktik und Philoso-
phic der Naturwissenschaft

Zeitschrift fur Diatetische und Physikalische
Therapie see Zeitschrift fiir die Gesamte
Physikalische Therapie

Zeitschrift fiir die Gesamte Anatomic; Abt. 1
see Zeitschrift fiir Anatomie und Entwick-
lungsgeschichte; Abt. 3 see Ergebnisse der
Anatomie und Entwicklungsgeschichte

Zeitschrift fiir die Gesamte Experimentelle
Medizin; Zugleich Fortsetzung der Zeit-
schrift fiir Experimentelle Pathologic und
Therapie (23 contains index for 1-22 of
Zeitschrift fiir Experimentelle Pathologie
und Therapie) 1913: 1 +

*Zeitschrift fiir die Gesamte Physikalische
Therapie (1-8 as Zeitschrift fur Diatetische
und Physikalische Therapie; 9-26 as Zeit-
schrift fiir Physikalische und Diatetische
Therapie) 1898: 1-45

Zeitschrift fiir die Gesamte Physikalische und
Diatetische Therapie see Balneologe

Zeitschrift fiir die Gesamten Naturwissen-
schaften see Zeitschrift fiir Naturwissen-
schaften

Zeitschrift fiir Elektrochemie und Angewandte
Physikalische Chemie; Deutsche Bunsen-
Gesellschaft (1 as Zeitschrift fiir Elektro-
technik und Elektrochemie; 2-9 as Zeit-
schrift fur Elektrochemie) 1894: 1 +
*Zeitschrift fiir Experimentelle Pathologie und
Therapie (combined with Zeitschrift fiir die
Gesamte Experimentelle Medizin; 23 of
which contains index for 1-22) 1904: 1-22

Zeitschrift fiir Experimentelle und Angewandte
Medizin see Glasnik Centralnog Higijenskog
Zavoda

Zeitschrift fiir Garungsphysiologie see Chemie
der Zelle und Gewebe

Zeitschrift fiir Geophysik 1924: Deutsche Geo-
physikalische Gesellschaft. 1 +

Zeitschrift fiir Hydrologie (Hydrographie,
Hydrobiologie, Fischereiwissenschaft) ; her-

ausgegeben von der Hydrobiologischen
Kommission der Schweizerischen Natur-
forschenden Gesellschaft 1920: 1 +

Zeitschrift fiir Hygiene und Infektionskrank-
heiten (1-10 as Zeitschrift fiir Hygiene,
herausgegeben von Dr. R. Koch und Dr. C.
Fliigge) 1886: 1 +

Zeitschrift fiir Immunitatsforschung und Ex-
perimentelle Therapie (1-38 has 1. Teil;
Originale) 1908: 1 +

*Zeitschrift fiir Immunitatsforschung und Ex-
perimentelle Therapie; 2. Teil; Referate
1909: 1-10, no. 3

Zeitschrift fiir Induktive Abstammungs- und
Vererbungslehre 1908: 1-2; 3, nos. 1, 2,
4-5; 4, nos. 1-2; 5 +

Zeitschrift fiir Instrumentenkunde; Or?an fiir
Mitteilungen aus dem Gesamten Gebiete
der Wissenschaftlichen Technik 1881: 1 +
*Zeitschrift fiir Instrumentenkunde; Beiblatt;
Zeitschrift der Deutschen Gesellschaft fiir
Mechanik und Optik (1891-97 as Vereins-
blatt der Deutschen Gesellschaft fiir Me-
chanik und Optik; 1898-1916 as Deutsche
Mechaniker-Zeitung) 1891: 1891-1920

Zeitschrift fiir Instrumentenkunde; Beilage-
hefte; Forschungen zur Geschichte der
Optik 1928: 1 +

Zeitschrift fiir Krebsforschung; unter Mit-
wirkung des Reichsausschusses fiir Krebs-
bekampfung 1903: 33 +

Zeitschrift fiir Menschliche Vererbungs- und
Konstitutionslehre (1-7 as Zeitschrift fiir
Angewandte Anatomie und Konstitutions-
lehre; 8-18 as Zeitschrift fiir Konstitutions-
lehre) 1913: 1, no. 1; 20

Zeitschrift fiir Mikroskopisch-Anatomische
Forschung; Abt. 2. Jahrbuch fiir Morpho-
logic und Mikroskopische Anatomie 1924:

1 +

Zeitschrift fiir Morphologie und Anthropologie,
Erb- und Rassenbiologie ; herausgegeben
von Dr. h. c. Eugen Fischer (1-37, 1939 as
Zeitschrift fiir Anatomie und Anthropologie)
1899: 1 +

Zeitschrift fiir Morphologic und Okologie der
Tiere (1-28 also as Abt. A of Zeitschrift fur
Wissenschaftliche Biologic) 1924: 1 +

Zeitschrift fiir Naturwissenschaften ; Organ
des Naturwissenschaftlichen Vereins fiir
Sachsen und Thiiringen (1853-79 as Zeit-
schrift fiir die Gesamten Naturwissen-
schaften; herausgegeben von dem Naturw.
Vereine fiir Sachsen u. Thiiringen in Halle)
1853: [1854]; 52 (1879); 70-72; [73]; 75

Zeitschrift fiir Parasitenkunde (1-6 also as
Zeitschrift fiir Wissenschaftliche Biologie,
Abt. F) 1928: 1 +

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Zeitschrift fur Physik ; Deutsche Physikalische
Gesellschaft 1920: 1 +

Zeitschrift fur Physikalische Chemie; be-
griindet von Wilh. Ostwald und J. H. van' T
Hoff (137+ as Abt. A. Chemische Thermo-
dynamik; Kinetik; Elektrochemie; Eigen-
schaftslehre) 1887: 1 +

Zeitschrift fiir Physikalische Chemie; Abt. B.
Chemie der Elementarprozesse. Aufbau der
Materie 1928: 1 +

Zeitschrift fiir Physikalische und Diatetische
Therapie see Zeitschrift fiir die Gesamte
Physikalische Therapie

Zeitschrift fiir Physiologische Chemie see
Hoppe-Seyler's Zeitschrift fiir Physiolo-
gische Chemie

Zeitschrift fiir Psychologie und Physiologic
der Sinnesorgane (beginning with 41, 1906,
journal in two sections; Abt. 1. Zeitschrift
fiir Psychologie; Abt. 2. Zeitschrift fiir
Sinnesphysiologie) 1890: 1-40, 1906; Er-
ganzungsband 2 (1902) only

Zeitschrift fur Rassen-Physiologie ; Mitteilun-
gen der Deutschen Gesellschaft fiir Blut-
gruppenforschung 1928: 1 +

Zeitschrift fiir Sinnesphysiologie (continuation
of Zeitschrift fur Psychologie und Physio-
logie der Sinnesorgane Abt. 2) 1907: 41 +

Zeitschrift fiir Technische Biologic see Chemie
der Zelle und Gewebe

Zeitschrift fiir Tierziichtung und Ziichtungs-
biologie einschliesslich Tierernahrung see
Tierziichtung und Ziichtungsbiologie ein-
schliesslich Tierernahrung

Zeitschrift fiir Vergleichende Physiologic
(1-20 a/so as Zeitschrift fiir Wissenschaft-
liche Biologie, Abt. C) 1924: 1 +

Zeitschrift fiir Vitaminforschung zugleich
Zentralblatt fiir Vitaminologie und Ver-
wandte Ernahrungsprobleme 1932: 1, nos.
1, 3-4; 2 +

*Zeitschrift fiir Wissenschaftliche Biologie
1924-34; Abt. A see Zeitschrift fiir Mor-
phologic und Okologie der Tiere; Abt. B
see Zeitschrift fiir Zellforschung und Mikro-
skopische Anatomic; Abt. C see Zeitschrift
fur Vergleichende Physiologic; Abt. D see
Wilhelm Roux' Archiv fiir Entwicklungs-
mechanik der Organismen; Abt. E see
Planta; Archiv fiir Wissenschaftliche Bo-
tanik; Abt. F see Zeitschrift fur Parasiten-
kunde

*Zeitschrift fiir Wissenschaftliche Insekten-

biologie 1905: 1-27

Zeitschrift fiir Wissenschaftliche Mikroskopie
und fiir Mikroskopische Technik 1884: 1 +
Zeitschrift fur Wissenschaftliche Photogra-
phic, Photophysik und Photochemie 1903:
1 +

Zeitschrift fiir Wissenschaftliche Zoologie;
begriindet von Carl Theodor v. Siebold und
Albert v. Kolliker; Abt. A (Abt. A only with
141, 1932+) 1848: 1 +

Zeitschrift fiir Wissenschaftliche Zoologie;
Abt. B see Archiv fiir Naturgeschichte

Zeitschrift fiir Zellforschung und Mikrosko-
pische Anatomic; Abt. A. Allgemeine Zell-
forschung und Mikroskopische Anatomic
( 1 as Zeitschrift fiir Zellen- und Gewebelehre ;
2-28, 1938 as Zeitschrift fiir Zellforschung
und Mikroskopische Anatomic; 1-20 also as
Abt. B of Zeitschrift fiir Wissenschaftliche
Biologie) 1924: 1 +

Zeitschrift fiir Zellforschung und Mikrosko-
pische Anatomic; Abt. B. Chromosoma see
Chromosoma

Zeitschrift fiir Ziichtung, Reihe B see Tierziich-
tung und Ziichtungsbiologie einschliesslich
Tierernahrung

Zellen- und Befruchtungslehre in Einzel-
darstellungen ; herausgegeben von P. Buch-
ner 1928: 1

*Zellstimulations-Forschungen ; herausgegeben
von Prof. Dr. M. Popoff und Prof. Dr. W.
Gleisberg 1924: 1-3

Zentralblatt fiir Allgemeine Pathologic und
Pathologische Anatomic; begriindet von E.
Ziegler 1890: 1 +

*Zentralblatt fiir Allgemeine und Experimen-
telle Biologie; herausgegeben von Prof.
Dr. Heinrich Poll 1910: 1-2

*Zentralblatt fiir Bakteriologie, Parasitenkunde
und Infektionskrankheiten 1887: 1-16

Zentralblatt fiir Bakteriologie, Parasitenkunde
und Infektionskrankheiten; Abt. 1. Medi-
zinisch-Hygienische Bakteriologie und Tie-
rische Parasitenkunde (31+ in two sections;
Originale; Referata) 1895: 17 +

Zentralblatt fiir Bakteriologie, Parasitenkunde
und Infektionskrankheiten; Abt. 2. Allge-
meine, Landwirtschaftliche, Technische,
Nahrungsmittel-Bakteriologie und Myko-
logie, etc. 1895: 1 +

*Zentralblatt f iir Biochemie und Biophysik ; mit
Einschluss der Theoretischen Immunitats-
forschung (1-3 as Biochemisches Central-
blatt; Yollstandiges Sammelorgan fiir die
Grenzgebiete der Medizin und Chemie;
4-9, 1905-10 as Biochem. Centralb. etc.,
Centralblatt fiir die Gesamte Biologie Abt.
1; 10-15 as Zentralblatt fiir Biochemie und
Biophysik; mit Einschluss der Theoretischen
Immunitatsforschung, Zentralblatt fiir die
Gesamte Biologie; Neue Folge) 1902: 1-23

Zentralblatt fiir die Gesamte Biologie Abt. 1
see above; Abt. 2 see Biophysikalisches Cen-
tralblatt

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Zentralblatt fiir die Gesamte Neurologic und

Psychiatric 1910: 39, nos. 9-10 (1925)
Zentralblatt fiir die Gesamte Radiologie (Ront-
gen, Radium, Licht) ; Ref eratenorgan der
Deutschen Rontgen-Gesellschaft 1926: 12 +
Zentralblatt fur Geophysik, Meteorologie und
Geodosie 1937: 1 +

*Zentralblatt fiir Physiologic; unter Mit-
wirkung der Physiologischen Gesellschaft
zu Berlin (7-13, 1893-99, und des Physiolo-
gischen Clubs in Wien) und der Morpho-
logisch-Physiologischen Gesellschaft zu Wien
1887: 1-34; Supplement see Bibliographia
Physiologica, series 3-4

*Zentralblatt fiir Zoologie, Allgemeine und Ex-
perimentelle Biologic (continuation of Zoolo-
gisches Zentralblatt and as Zentralblatt fiir
Allgemeine und Experimented Biologic ser.
2) 1912: 1-6

Zhurnal Biobotanichnogo Tsiklu Yuan see
Zhurnal Institutu Botaniki Uan

Zhurnal Bio-Zoologichnogo Tsiklu; Vseu-
krains'ka Akademiia Nauk, Prirodnicho-
Tekhnichnii Viddil (Journal du Cycle Bio-
Zoologique; Academic des Sciences d'U-
kraine, Classe des Sciences Naturelles et
Techniques) : Kief. 3-4

*Zhurnal Eksperimental'noi Biologii Seriia A
(Journal de Biologic Experimentale. serie
A) 1925: Moscow. 1-7; Seriia B see Uspekhi
Eksperimental'noi Biologii

Zhurnal Eksperimental'noi Biologii i Medit-
siny; Gosudarstvennyi Institut Narodnogo
Zdravookhraneniia (Journal de Biologic et
de Medecine Experimentales; Institut
Scientifique de la Sante Publique, Moscow)
1925: [1-131

*Zhurnal Geofiziki i Meteorologii (Journal of
Geophysics and Meteorology) 1924: Mos-
cow. 1-6

Zhurnal Institutu Botaniki Uan (Journal de
1'Institut Botanique de 1'Academie des
Sciences d'Ukraine) (1-8 as Zhurnal Bio-
botanichnogo Tsiklu Yuan; Journal du
Cycle, de la Section, Botanique de 1'Aca-
demie des Sciences d'Ukraine) 1931: Kief
1-15

Zhurnal Russkogo Botanicheskogo Obsh-
chestva (Journal de la Societe Botanique
de Russie) 1916: 14, no. 4
*Zoe; a biological journal 1890: Zoe Publishing
Co., San Francisco. 1-4; 5, nos. 1-4; 6-11

Zoogeographica; Internationales Archiv fiir
Vergleichende und Kausale Tiergeographie
1932: 1 +

Zoologica; Original-Abhandlungen aus dem
Gesamtgebiete der Zoologie; herausgege-
ben von R. Hesse (1-8 von Rud. Leuckart

und Carl Chun; 9-20, 1897-1908, Chun; 21.
1908-21, Willy Kukenthal; 22-23, 1910,
Chun; 24, 1911-24, Kukenthal; 25-26,
1911-13, Chun; 27, 1913—22, Kukenthal)
(1-8 as Bibliotheca Zoologica) 1888: 1 +
Zoologica; scientific contribution of the New

York Zoological Society 1907: 1 +
Zoologica Poloniae; Archivum Societatis Zoo-
logorum Poloniae 1935: Lwow, Poland. 1-4,
no. 1
*Zoological Bulletin; edited by C. O. Whitman

and W. M. Wheeler 1897: 1-2
*Zoological Journal 1824: London. 1-5
Zoological Magazine; Zoological Society of

Japan see Dobutugaku Zassi
Zoological Record 1864: London. 1 +
Zoologicheskii Vestnik see Journal Russe de

Zoologie

Zoologicheskii Zhurnal (1-10 as Revue Zoolo-
gique Russe) (Russkii Zoologicheskii Zhur-
nal) 1916: Akademiia Nauk SSSR. 1-2; [3];
4-5; [6]; 7 +

*Zoologische Annalen; Zeitschrift fiir Ge-
schichte der Zoologie; herausgegeben von
Dr.'Max Braun 1904: 1-7, no. 3
*Zoologische Bausteine; Ausschnitte aus dem
Gesamtgebiet der Zoologie; herausgegeben
von Prof. Dr. Paul Schulze, Rostock 1925:
1-2, no. 1

*Zoologische Beitrage ; herausgegeben von Dr.
Anton Schneider, Breslau 1884: 1, nos. 2-3;
2, nos. 2-3; 3

Zoologische Jahrbiicher; Abteilung fiir Allge-
meine Zoologie und Physiologic der Thiere
1910: 30+; Abteilung fiir Anatomic und
Ontogenie der Thiere 1888: 3+; Abteilung
fiir Systematik, Okologie und Geographic
der Thiere (1-2 as Zoologische Jahrbiicher;
Zeitschrift fiir Systematik, Geographie und
Biologic der Thiere) (3-51 as Zoologische
Jahrbiicher; Abteilung fiir Systematik,
Geographie und Biologie der Thiere) 1886:
1 + ; *Supplement 1886: 1-16, Heft 2
Zoologischer Anzeiger; Organ der Deutschen
Zoologischen Gesellschaft; begriindet von
Victor Cams; fortgefiihrt von Eugen Kor-
schelt (vols. 26-93, 1903-31); herausge-
geben von Berthold Klatt (94, 1931+) 1878:
1 + ; Supplementband see Verhandlungen
der Deutschen Zoologischen Gesellschaft;
see also Bibliographia Zoologica
Zoologischer Bericht; Deutsche Zoologische

Gesellschaft 1922: 1 +

*Zoologischer Jahresbericht; herausgegeben
von der Zoologischen Station zu Neapel
1879: 1-35 (1913)

"Zoologisches Zentralblatt; unter mitwirkung
von Prof. Dr. O. Biitschli und Prof. Dr. B.

SERIAL PUBLICATIONS, MARINE BIOLOGICAL LABORATORY

Hatschek (ser. 2 continued as Zentralblatt
fur Zoologie, Allgemeine und Experimen-
telle Biologic) 1894: 1-18 (1911)

Zoologiska Bidrag fran Uppsala 1912: 1-18;

Suppl. Bd. 1 (1920)

*Zoologist; a monthly journal of natural his-
tory (ser. 1-2, a popular miscellany of
natural history) 1843: London, series 1-4
*Zoopathologica; scientific contributions of the
New York Zoological Society on the dis-
eases of Animals 1916: 1-2

Zprava o Cinnosti sekce pro plemenarskou
Biologii Moravskeho Zemskeho Vyzkum-
neho Ustavu Zootechnickeho v Brne (An-
nual Report about the activity of the Breed-
ing and Biological Section of the Zootech-
nical Research Institute in Brno): 1923-25

Zpravy Komise na Prirodovedecky Vyzkum
Moravy a Slezska (Mitteilungen der Kom-
mission zur Naturwissenschaftlichen Durch-

forschung Mahrens) 1905: Briinn. Oddeleni
Archaeologicko-Praehistoricke, 1-2; Bo-
tanicke, 1-10; Geologicke, 1-12; Minera-
logicke, 1-6; Zoologicke, 2-1 6j 18-21

Zpravy o Zasedani Kralovske Ceske Spolec-
nosti Nauk; Trida Matematicko-Pfirodo-
vedecka see Vestnik Kralovske Ceske Spo-
lecnosti Nauk

(Der) Ziichter; Zeitschrift fiir Theoretische
und Angewandte Genetik 1929: Kaiser
Wilhelm-Institut fiir Ziichtungsforschung,
Erwin Baur-Institut, Miincheberg i. M. 1 +

Ziichtungskunde 1926: Deutsche Gesellschaft
fiir Ziichtungskunde; unter Mitwirkung der
Tierzuchtinstitute an Deutschen Hoch-
schulen. 7 + ; see also Neue Schriftenreihe;
aus Deutschen Zuchten

Zymologica e Chimica dei Collodi see Bollettino
Scientifico della Facolta di Chimica In-
dustriale, Bologna

Vol. 84, No. 2 April, 1943

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

THE FUNCTION OF THE CORPUS ALLATUM IN

MUSCOID DIPTERA

M. F. DAY

(Department of Zoology, Washington University, St. Louis)

INTRODUCTION

Since the original work of Wigglesworth (1926) it has become increasingly
obvious that the corpora allata may exert an influence on the reproductive
processes of insects (see Scharrer, 1941, for review). Although Thomsen (1940)
concludes that in the adult muscid flics the single median corpus allatum controls
the ripening of the ovaries, it is not yet established whether this action is a direct
one, and whether there is a true sex hormone produced by the corpus allatum.
The fact that Thomsen (I.e.) was able to induce hypertrophy of the corpus
allatum by ovariectomy suggests a direct hormonal action, but cannot be taken
as proof. In an attempt to throw further light on this problem the experiments
to be described were performed. They are concerned with the role of the ring
gland in the adult fly, and while the problem is as yet unsettled, considerable
information has been gained and a tentative conclusion may be reached. The
significance of the work lies in the fact that it bears finally on a major biological
problem — the mode of action of hormones on cells and tissues.

MATERIALS AND METHODS

Two species of Muscidae have been employed. A pure strain of Lucilia
sericata Meig. maintained by mass inbreeding for over 230 generations was used
in all earlier work. Search for a more robust fly resulted in the use of a stock of
Sarcophaga securifera Villeneuve obtained from eggs deposited on meat, in the
fall of 1941 in St. Louis, Missouri. Since that time the stock has been maintained
by mass inbreeding in the laboratory.

The experiments have consisted mainly of extirpations and implantations of
adult organs. Operations were performed under a magnification of 30 diameters
and the essential instruments were No. 12 hard steel needles, appropriately
sharpened on a hard Arkansas oilstone, and fine iridectomy forceps. Flies were
etherized and held in "Permoplast" with cross pins. The corpus allatum may
be removed through the neck region if the head of the fly is bent forward; gonads
are removed by making a long transverse incision in the intersegmental membrane

127

128 M. F. DAY

between the 4th and 5th abdominal segments. With practice the mortality can
be reduced to very low figures for both operations, but I have never succeeded in
performing both extirpations on a single fly, even wrhen a day elapsed between
the experiments.

In the experiments involving the transplantation of the ring gland, portions
of the oesophagus invariably had to be included with the transplant, for the ring
gland alone was too small to be moved without injury. The site of implantation
was under the dorsal abdominal wall between the 2nd and 3rd scuta. In later
experiments, in an effort to induce innervation of the transplant, attempts were
made to place the ring gland near the brain, which was slightly injured in order to
stimulate the growth of nerves to the implant. These attempts were unsuccessful.

A total of 73 successful operations were performed on Lucilia and 102 on
Sarcophaga.

At the conclusion of all experiments the flies were fixed by injection with
alcoholic Bouin's, and 10 micron paraffin sections were stained either in Mallory's
triple stain or by Bodian's protargol technique. Results with both species of
flies were similar, unless the contrary is stated.

DESCRIPTION OF THE NORMAL HISTOLOGY

The histological effects of extirpation of the ring gland have not previously-
been described. In order that they may be more easily followed, a brief descrip-
tion of the normal histology is necessary.

A. The Ring Gland. The ring gland of Lucilia has been shown (Day, 1942)
to be composed of a single median corpus allatum fused with the corpora cardiaca
and the hypocerebral ganglion. The situation is similar in Calliphora (Thomsen,
1941) and in Sarcophaga (Figures 3, 4, and 5). Thomsen (1941) refers briefly
to the larger size of the corpus allatum in mature flies compared with newly
emerged flies (compare her Figures 3 and 4). The changes occurring during the
first seven days after emergence of adult Lucilia sericata and from emergence to
20 days of age of Sarcophaga, have been carefully followed. The most striking
changes occur in the first five days and thereafter there is not much alteration

PLATE I

Corpora allata and fat body of Lucilia sericata and Sarcophaga securifera fixed in alcoholic
Bouin, Bodian protargol method. Photomicrographs, magnification 400 diameters.

1. Corpus allatum of normal female L. sericata, transverse section. Note larger nuclei on
periphery of the gland, and below a little striated muscle from the dorsal vessel. Cell walls are
not easily seen but are present.

2. The same of a female castrated seven days. Note the hypertrophy of the cells and nuclei.
The gland approximately 50 per cent greater in diameter than the control even though there are
fewer cells in the section. The hypertrophy of the nuclei is particularly striking and is shown
by the larger peripheral ones as well as the central ones. Cell walls are clearly seen.

3. Corpus allatum of 5. securifera in which recurrent nerve was cut seven days earlier. Note
nerve fibers ramifying between cells. In comparison with Figures 4 and 5 note the hypertrophy
of cells and nuclei.

4. Corpus allatum of male castrate S. securifera.

5. Corpus allatum of female castrate S. securifera. Note that there is no hypertrophy in
either this gland or that in Figure 4 comparable with that found in Lucilia.

6. Fat body of female Sarcophaga securifera showing darkly stained oenocytes. Tissues are
essentially normal. Compare with Figure 7.

FUNCTION OF CORPUS ALLATUM

129

PLATE I

130 M. F. DAY

in the cytology of the gland. Differences between the sexes are insignificant.
In Sarcophaga all the fuchsinophilic droplets in the corpus cardiacum cells have
disappeared at the time of emergence, but persist for about three days in Lucilia
(Day, 1942). In Lucilia larger nuclei seem to occur on the periphery of the
gland, but this is not so marked in Sarcophaga. In both there is a thin sheath,
which has small flattened nuclei, surrounding the gland. The innervation of the
gland is well shown in Bodian preparations and, as in other insects, is very profuse
(see especially Figures 3 and 4). Nerves from the hypocerebral ganglion run
dorsally in the lateral walls of the aorta and ramify between almost every cell of
the corpus allatum. It is almost certain that fine nerves penetrate cells and end
near the nucleus in small terminal swellings. Cell membranes are not clearly
seen in the illustrations, but the gland is not syncytial. It is well tracheated, but
no specializations for transferring secretory products to the aorta are found.

B. The Fat Body. As will be shown later the fat body undergoes striking
changes after allatectomy. Its normal histology is therefore discussed here and
considerable attention has been paid to this tissue in all experimental animals.
Teunissen (1937) and Perez (1918) have described the changes in the fat body
of flies during the pupal period, but no adequate description is to be found of
subsequent changes. Evans (1935) has described the fat body of adult Lucilia
and Roubaud (1932) has described chemical changes in that of Cidex pipiens.
In Sarcophaga I have found that larval fat body cells persist in normal flies for
three days after emergence (Figure 7). Their fuchsinophilic contents gradually
diminish until the cytoplasm becomes clear and reduced, though the large nucleus
still makes it easy to distinguish the cells. The adult fat body cells are at first
small but during the first three days of adult life they gradually increase in size.
It should be noted that fat body cells differ in various parts of the body. This
discussion is limited to the fat body of the lateral body wall in the segments con-
taining the gonads. No substantial difference was observed between the sexes.

Young cells show most frequently four nuclei, but may possess more. A count
of the number of nuclei in fat body cells of Sarcophaga gave the following results:
Two nuclei wrere found in 15 per cent of the cells, four in 50 per cent, six in 5
per cent and eight in 30 per cent. The counts were made from acetic orcein
spreads from which counts may be made easily and accurately, while this is not
possible from sections. No significant change was found between young and
old flies, nor is there a difference between the sexes. There is, however, a marked
difference depending on where the fat body is located. Cells near the heart are
smaller and have fewer nuclei than those located on the lateral body wall. When
the fat body is abundant the cells composing it are closely appressed and their
exact connections are impossible to determine. In living spread preparations,
especially of older flies in which the fat body is less abundant, it is found that the
fat body cells are arranged in cords which branch and in which the oenocytes lie
in between almost every two cells. In young cells the nuclei are spherical and
possess a single, usually slightly eccentric nucleolus. The cytoplasm is aggregated
around the periphery of the cell. Within 24 hours after emergence the cells have
increased considerably in size, the nuclei have become more nearly centrally
located and the nuclear membrane has become slightly irregular in outline and
their nucleoli have enlarged considerably. The cytoplasm is uniform, but is
lightly aggregated around the nuclei. If the flies continue on a carbohydrate

FUNCTION OF CORPUS ALLATUM 131

diet no further change occurs. A protein meal, however, changes the appearance
of the cytoplasm considerably. It becomes more abundant and thick strands
run from the cell wall to the nuclei. The nuclei frequently regain their spherical
shape.

In Lucilia, the changes undergone by the fat body are essentially similar to
those just described (see Evans, 1935).

C. The Oenocytes. Intimately associated with the fat body are the oenocytes.
Snodgrass (1935, p. 411) made the undocumented statement that "oenocytes are
not known to occur in adult Diptera." However, oenocytes in adult Diptera
have been described by Perez (1910) in Calliphora and by Evans (1935) in Lucilia.

In the newly-emerged Lucilia they are not striking, but with the increase in
size of the fat body cells the oenocytes become more conspicuous. They are
characterized by uniform, basophilic cytoplasm. Many of the cells, which are
uniformly scattered among the abdominal fat body cells, are uninucleate, but
about 50 per cent are binucleate. More rarely three or four nuclei are found.
Nuclear size in Lucilia oenocytes with a single nucleus averages about six microns,
but is less when there are more nuclei per cell. It was early noticed that there
was a marked and constant difference between the oenocytes of the two sexes
of Lucilia. While those of the male were large and well filled with cytoplasm so
that the cell boundaries were convex, those of the females were less conspicuous
and had concave cell boundaries. This difference could be clearly seen in the
living fat body if the fly was injected with a solution of methylene blue which
stains oenocytes specifically. When a similar condition was found in such widely
separated flies as Alelophagus ovinus L. and Culex pipiens L., it was thought that
the situation might be general in the Diptera. It was so striking that it seemed
surprising that no record of this could be found in the literature. It was therefore
unexpected to find that Sarcophaga securifera did not conform in this respect, the
oenocytes being if anything more conspicuous in the female than in the male,
although there was little difference between the sexes. The explanation for this
is quite unknown but is obviously significant in the explanation of the sexual
differences, which must lie eventually in a knowledge of the function of the
oenocytes. Without diverging unnecessarily it seems that the majority of
evidence points to their functioning as organs of intermediary metabolism (see
Wigglesworth, 1939, p. 244). The validity of the interpretation of the following
results rests in part on the very plausible assumption that this is at least one of
their functions.

In normal flies the oenocytes are very constant, and no cytological evidence
of secretory changes can be seen in the adult, though there are some indications
that they show an inverse size relationship with the cells of the fat body, and vary
slightly with the nutritional state of the insect. As will be shown below, they
undergo marked changes upon extirpation of the ring gland.

D. The Ovaries. For purposes of subsequent descriptions it is necessary to
review briefly the growth of the eggs in the ovary of Lucilia. The relation
between nutrition and egg production of muscids has been discussed by Glaser
(1923) and Mackerras (1933), and the development of the ovaries of Anopheles
by Nicholson (1921). Nicholson divided the growth of the oocyte into two
stages. The first of these represents the growth up to a resting stage in which
the oocytes remain until the insect has taken a protein meal (see Trager, 1941,

132 M. F. DAY

p. 23). Histologically such oocytes are easily distinguished, for no yolk has yet
been laid down in them. In Sarcophaga the follicle attains a maximum diameter
of about 160 microns. Immediately after feeding on meat, yolk is deposited and
the oocyte increases in size to about three times the size reached in stage I. The
nurse cells undergo little change. Once yolk deposition has begun the follicles
increase rapidly in size. The follicular cells increase in number but decrease in
size. They change from cuboidal towards squamous cells. Later the chorion
is laid down.

THE EFFECTS OF EXTIRPATION OF THE RING GLAND

As mentioned above, extirpation of the ring gland of adult flies can be per-
formed with comparative ease. It is, however, not possible to determine without
histological examination whether both corpora allata and cardiaca are removed.
Thomsen records that cardiacectomy was avoided in her operations. Most
frequently in this work the corpora cardiaca were removed with the corpus
allatum. After allatectomy alone, however, the effects were indistinguishable
from complete extirpation of the ring gland. About 80 per cent of the flies
operated upon in this way live apparently normally and show no external signs
of their operation. Mortality is about 10 per cent and the remaining 10 per cent
show the water balance upset described below.

Among the 80 per cent of operated flies that survived, normal mating reac-
tions have been noted. The majority of flies (37 Lucilia, nine Sarcophaga) have
been operated upon when 24 hours old, and subsequently fed sugar and water,
and fixed after seven days. Thirteen cases in Lucilia were allowed to live to 21
days after the operation and three cases in the Sarcophaga series were fed meat
for four days. However, eggs were never developed, though in one case a little
yolk was found in an oocyte slightly enlarged beyond stage I.

In spite of their normal behavior, operated flies present an unmistakable
histological picture differing from the controls in regard to the fat body, ovary,

PLATE II

Fat body of Lucilia sericata and Sarcophaga securifera. Fixed with alcoholic Boiiin's.
Photomicrographs, magnification 400 diameters, except Figure 7 which is XI 70.

7. Fat body of normal male 5. securifera three days of age. Note the larval fatbody cell
whose fuchsinophil droplets have almost disappeared. Compare the oenocytes with those ot the
female (Figure 6). Note that the magnification is only 170 diameters.

8. Fat bod)- of female L. sericata in which recurrent nerve had been cut seven days. The re-
duced and pycnotic oenocytes, pycnotic small dark nuclei of the fat body are characteristic of flies
after extirpation of the ring gland.

9. Fat body of male 5. securifera allatectomized seven days. Note same effects as seen in
Figure 8. The separation of the cytoplasm from the cell wall is characteristic.

10. Fat body of female 5. securifera allatectomized for seven days when female was six days
old. Note less marked effect than in Figure 9, though the effects of the operation can be seen in
comparison with Figure 12.

11. Fat body of female 5. securifera seven days after the extirpation of the ring gland with
ring gland from female castrated seven days implanted for 46 hours. In comparison with Figure 9
the cytoplasm and nuclei are seen to have undergone conspicuous changes. Oenocytes, however,
are unaffected.

12. As Figure 11, except that the transplanted ring gland was from a normal seven-day old
female. Note that the fat body is almost normal, but the oenocytes still show the effects of
allatectomy.

FUNCTION OF CORPUS ALLATUM

133

a - -

PLATE II

134 M. F. DAY

and oenocytes. No effects could be seen on testes, on the nervous tissue, including
the neurosecretory cells of the brain (Day, 1940), alimentary canal, Malpighian
tubules, or on the accessory glands of the reproductive system of either the male
or the female.

A. The effects on the fat body become visible within three days after the
operation, but little change occurs from then up to 21 days. If the operation is
performed on a fly within two days after emergence, the cells of the larval fat
body never completely disappear, as they do in the normal fly. Even after the
acidophilic cytoplasmic inclusions (see Figure 7) have disappeared, the larval
cells can easily be recognized by the large size of the nuclei. Thus one of the
effects of extirpation of the ring gland is to inhibit the normal maturation of
certain tissues. The extent of the change in the adult fat body may be seen by
comparing Figures 7 and 9. Many of the nuclei of the fat body cells in the
operated flies become pycnotic, have crenulated borders, and are greatly decreased
in size. A still more striking change occurs in the cytoplasm which appears much
more sparse than in the controls. No specific stains have been used to attempt
to learn exactly what components have disappeared from the cytoplasm but the
general appearance is that of a cell whose reserves have been in large part utilized.
It would be desirable to determine in what way these changes correlate with
alterations in physiological function. The results suggest a comparison with the
findings of Pfeiffer (1941) on Melanoplus that allatectomy results in a greatly
increased fat content. While it will be seen from Figures 7 and 9 that there is
an increase in fat body cell size following extirpation of the ring gland, an increase
in fat does not appear to occur in female Lucilia or Sarcophaga after this operation
although the vacuoles observed in the fat body cells may be left by dissolved fat.

If the operation is performed on a fly six days of age, the fat body does not
regress, but the effect is still seen upon the oenocytes (compare Figure 10).

B. Even more striking changes occur in the oenocytes following extirpation
of the ring gland. Such changes have not been previously reported for any
insect. The effects are not quite comparable in Lucilia and in Sarcophaga. In
males of the former species the large oenocytes of the male are most markedly
affected, being much reduced in size. Their nuclei become pycnotic and their
cytoplasm changes from an homogeneous basophilic to a strong acidophilic
reaction. Comparable but less marked changes occur in the female Lucilia. In
Sarcophaga the cytoplasm does not become so markedly reduced, but the cell
boundaries of the oenocytes almost invariably become indistinct (Figure 9). As
in Lucilia the nuclei show varying degrees of pycnosis and in both sexes the cells
are greatly reduced in size. Feeding protein in addition to carbohydrate has
little effect on these oenocytes.

C. Extirpation of the ring gland produces an effect upon the ovaries. If the
operation is performed on young females fed only sugar, development beyond
Stage 1 is never found. The histology of the eggs usually remains normal.
However, in one case in which a female Sarcophaga was allatectomized when two
days old, considerable regression of the ovaries was found. Practically no
oocytes were present and the ovaries consisted of a mass of knotted tracheae and
a few small connective tissue and muscle cells.

If the operation is performed on a female in which the ovaries are well
developed, degeneration of certain oocytes occurs. Wigglesworth (1936) reported
a similar situation in Rhodnius. The eggs on the periphery are affected first and

FUNCTION OF CORPUS ALLATUM 135

the changes are exactly comparable to those seen in old females inhibited from
ovipositing. Apparently extirpation of the ring gland hastens this process and
causes its occurrence in a greater number of oocytes than normal. The first
indication of such degenerative changes is seen in an increase in size of the fol-
licular cells. These large cells then begin to phagocytize the acidophilic yolk
granules, and persist for a while with darkly staining masses in their cytoplasm.
Eventually they digest this material and the yolk decreases in quantity until in
the last stages that have been observed, the oocyte contains practically no yolk.

Previous authors have described changes in the secretions of the oviduct as
a result of allatectomy. These were not observed in Lucilia or Sarcophaga.
Similar changes in the fat body and oenocytes occur in males as \vell as females,
but the testes and accessory glands are unaffected.

D. As mentioned above, the effects of removing the corpus allatum just
described are sometimes modified. In about 10 per cent of the operations the
flies exhibit a marked distension of the abdomen, sometimes within five hours,
but more usually about 12 hours after the operation. Complete serial sections
of these cases shows no difference in the operation between these and the 80 per
cent of flies which do not show these unusual effects. The Malpighian tubules
are always considerably swollen and there appears to be an upset in the water
regulating mechanism. This effect is found only in those flies which imbibe water
and it is suggested that the operation may have stimulated some center, with
the result that the flies imbibe more than they normally do.

THE EFFECT OF SEVERING THE RECURRENT NERVE

The question arises of whether the normal functioning of the ring gland is
dependent upon its innervation. As shown by Day (1942) in Lucilia the recurrent
nerve which supplies the ring gland is composed of fibers of three separate nerves.
This compound nerve can easily be severed in the cervical region. This operation
results in higher mortality than after extirpation of the ring gland. In one experi-
ment, for example, 40 flies of both sexes of Sarcophaga were operated upon in
this way, and of these only 16 survived for a period of one week. The effects of
the operation are precisely similar to those following extirpation of the ring gland.
The fat body cells enlarge, their nuclei become small and pycnotic, the oenocytes
decrease in size and their cytoplasm becomes acidophilic, and the ovaries do not
develop beyond Stage 1. The effect in Lucilia is shown in Figure 8 and that in
Sarcophaga in Figure 10.

The histology of the corpus allatum after the severing of its nerve connections
shows changes from the normal (Figure 3). The nerve endings are still conspicu-
ous and little can be seen in the cytoplasm to suggest increased or decreased
hormone output. However, striking changes occur in the cell size and in the
nuclear size (Figure 3). Nuclear size is more easily measured than cell size.
The nuclei of the operated flies measure from 12 to 14 microns in diameter, an
increase of about 70 per cent over the diameter of the nuclei of the controls. The
nucleolus also increases in size.

THE EFFECTS OF TRANSPLANTING RING GLANDS

Since it appeared from the extirpation experiments that corpora allata were
concerned in maturation of the ovaries and directly or indirectly in the changes

136 M. F. DAY

undergone by the fat body, the ring glands of ten-day females were transplanted
into the abdomens of three-clay females. Striking changes were induced in the
hosts, probably attributable to the transplanted glands. The adult fat body of
the host was markedly depleted, but the larval fat body cells still present showed
a most unusual appearance as though their reserves were being mobilized more
suddenly than is normal. This effect was found in individuals fixed 48 hours
after the implantation. In flies fixed one week after the operation no larval,
and extremely little adult fat body tissue could be found in sections. These
experiments confirm the suggestion that the corpus allatum is concerned in the
maturation of certain tissues.

A more significantly experiment seemed to be the implantation of ring glands
into flies from which the ring gland had been extirpated for one week. It is
hardly to be expected that the effects of extirpation of the ring gland could be
reversed to the normal condition, for it has been shown that the normal activity
of the corpus allatum is exhibited only when the gland is normally innervated.
Two cases were fixed 46 hours after implanting the new gland. One showed an
abundance of fat body, apparently intermediate between that of the fly from
which the ring gland had been extirpated, and a normal of this age, and reduced
oenocytes, with strongly basophilic cytoplasm rather than acidophilic as in flies
after extirpation of the ring gland. The other showed a fat body and oenocytes
which were essentially normal (Figure 12).

In a later section it will be shown that castration causes cytological changes
in the corpus allatum of Lucilia, though not of Sarcophaga. However, the fol-
lowing experiment suggests that the corpora allata of castrate female Sarcophaga
are physiologically altered. Ring glands of flies castrated seven days previously
were transplanted as in the experiments just reported. Two cases were fixed
46 hours after implanting and two after seven days. Significant differences could
be observed between these and the former series, but there are still definite effects
of the implanted glands (Figure 11). These effects are sufficiently striking to
confirm the suggestion that the ring gland from the castrated female had very
different effects on the host from that from a normal female. It was noticed that
the corpora allata of these transplanted glands, when studied in serial sections
at autopsy, showed a slight indication of hypertrophy in a manner similar to
that discussed above in denervated corpora allata /;/ situ. Detailed analysis
must await further experiments, but the generalization is warranted that castra-
tion causes physiological changes in the corpus allatum of Sarcophaga.

THE EFFECTS OF CASTRATION ON CORPORA ALLATA AND OTHER TISSUES

A. The experiments of Thomsen (1940) in which she extirpated ovaries were
performed primarily on Calliphora. The operation resulted in hypertrophy of
the corpora allata. Full confirmation has been obtained in my experiments with
female Lucilia sericata, in which corpora allata showed considerable hypertrophy.
The increase in size of the cells is illustrated in Figure 2 when compared with
Figure 1. It will be noted that the increase is solely in cell size and there is no
increase in cell number. The cytoplasm exhibits no more signs of activity than
in the unoperated animals. Thomsen could offer no suggestion of the means by
which the hypertrophy was brought about. A comparison with cases of hyper-

FUNCTION OF CORPUS ALLATUM 137

trophy of these glands, for example in Ephestia moths (Schrader, 1938) or termite
royalties (Pflugfelder, 1938), does not assist, and it seems likely that a different
mechanism is involved in each of these examples. There is a change in nuclear
size comparable to that found in Sarcophaga, which results from severing the
recurrent nerve.

B. Thomsen did not report experiments with male flics. These can be
castrated even more easily than can females, for the testes are not so completely
tracheated as the ovaries. However, no hypertrophy of the cells of the corpora
allata, or any other change could be observed in male Lucilia either in behavior
or in histology. Most cases were fixed seven days after the operation, but in a
few cases even after 14 days no change could be observed.

C. Similar experiments were performed on Sarcophaga. Early observations
indicated no hypertrophy of corpora allata. More extensive and detailed
operations on both sexes were performed and the results carefully checked in
histological preparations to determine whether castration was complete. No
hypertrophy of the corpora allata comparable to that in female Lucilia was
found in either sex of Sarcophaga (Figures 4 and 5).

D. It was thought that the accessory glands might have an effect. In male
Sarcophaga the accessory glands alone and the accessory glands together with
the testes were successfully removed. No change in corpora allata cells was
found in ten operations.

In an activity apparently so fundamental in the physiology of the insect it is
surprising that two genera as closely related as Lucilia and Sarcophaga should
give such divergent results. Further discussion of their differences will be found
on p. 139.

Castration of female Sarcophaga has no visible effects on the female accessory
glands, or indeed on the majority of tissues. About 50 per cent of nuclei of fat
body cells do, however, show varying degrees of pycnosis. The cytoplasm
appears normal and the oenocytes are fully rounded and typical for this fly
(Figure 6). No histological evidence was found in Sarcophaga to compare with
the decrease in fat reported for castrated Melanoplus by Pfeifter (1941).

THE EFFECTS OF CUTTING AND REPLACING OVARIES

In Sarcophaga an attempt was made to gain some indication of the effects on
the ovaries by completely removing them from their attachments, and replacing
them in the haemocoele. The flies were fixed after a period of one week and
studied histologically. The ovaries had regained new tracheal connections, and
appeared normal in every respect, with stage I oocytes, as would be expected in
flies fed only sugar. Fat body and oenocytes were normal and no effect of the
operation was observed on the corpora allata.

GENERAL DISCUSSION

We may assume for purposes of comparison with other insects that the effects
of extirpation of the ring gland can be compared with allatectomy and cardi-
acectomy. The only report of cardiacectomy is to be found in a note by Pfeiffer
(1939, p. 452-453) stating that "delay in molting has been consistently obtained
by removing the corpora cardiaca" of Melanoplus. Allatectomy has been per-

138 M. F. DAY

formed by Wigglesworth (1936) on Rhodnius where it was shown to result in
loss of the ability to produce mature eggs. Degeneration occurred not only in
the oocytes but also in the follicular epithelium. Weed (1936) confirmed these
results with Melanoplus. However, in Dixippus (Pflugfelder, 1937) allatectomy
does not result in the loss of the ability to produce mature eggs though the fact
that the corpora allata have some effect on the ovaries is shown by subsequent
work (Pflugfelder, 1940). Subsequently Pflugfelder has reported a variety of
effects from the removal of the corpora allata of Dixippus: the pericardial glands
and ventral glands undergo considerable hypertrophy, and there are effects on
the regeneration of lost limbs (1938b). These results lead to the conclusion that
the corpora allata exert some influence on metabolism, but as Scharrer (1941)
says "the question is how far this concept may explain all the special effects
attributable to the glands."

From the present experiments there is evidence that the ring gland produces
more than a single substance. As has been pointed out they indicate strongly
that one of the primary effects of the ring gland is on the regulation of normal
maturation. This is also true in the larva, as shown by the experiments of Burtt
(1938) on Calliphora and of Hadorn and Neel (1938) on Igl Drosophila larva.
Histological examination of "permanent larvae" of Lucilia sericata inhibited from
pupating by removal of the ring gland shows a fat body which is unlike anything
seen in normal larvae. The cells contain a large number of small acidophilic
droplets, and a few larger droplets which stain with aniline blue. However, there
is no regression or deterioration of the fat body cells as found in the adult fly, and
a somewhat comparable picture is seen in a prepupa kept for one month in dry-
sand (see Mellanby, 1938). In such fat body cells many of the acidophilic drop-
lets are considerably larger, are less regular in size, and are aggregated around
the nucleus.

It is not known whether the hormone which permits normal pupation and
whose removal results in fat body cells of this type is the same as that which plays
a role in the removal of larval fat body in the adult fly. It seems unlikely that
this is the case. And there is sufficient change in the ring gland that it is not
necessary to assume that a single hormone is involved. A few experiments have
been performed of transplanting glands from larvae to adults. Though they
yielded no significant information, the conclusion that the ring gland produces a
substance concerned with normal maturation seems incontravertible.

It has been shown that the effects produced by implanting a ring gland into
a fly from which the ring gland had been extirpated are qualitatively different
from those which result from severing the innervation of a gland in situ. It is
possible that the effects may be produced by different concentrations of a single
secretion but it appears likely that the substance causing the breakdown of larval
tissues is not the same as that which affects the growth of the ovaries, the cyto-
plasm of the fat body, and the structure of the oenocytes (see also Vogt, 1940).
As has been suggested by Pflugfelder the corpus allatum seems to have some
influence on the metabolism of the insect. The obvious effects of the corpora
allata of various insects on the ovaries have led to the suggestion that they may
produce a hormone acting directly on the sex organs, at least in the female. The

FUNCTION OF CORPUS ALLATUM 139

outstanding result of the experiments reported in this paper is that many tissues
are affected by the corpus allatum. There is thus no reason to suppose that a
sex hormone is produced by the corpus allatum. In fact, it seems more plausible
to assume that the primary effect is on some general metabolic function. It is
well known that many flies are unable to mature their eggs without a protein
meal, while mature sperm are formed irrespective of the meal obtained by the
male. Spermatogenesis is in no way affected by extirpation of the ring gland,
while eggs, if formed, begin to undergo regression if the ring gland is removed
even though protein be fed to the flies.

The results of castration are difficult to interpret. In female Lucilia, the only
cells markedly affected are those of the corpora allata. In male Lucilia and in
both sexes of Sarcophaga, castration has little effect either on the behaviour of
the flies or on the cytological appearance of their tissues (see Figure 6). In the
female Lucilia the corpus allatum hypertrophies after castration, and in this fly
the oenocytes of the female are much smaller than those of the male. In Sar-
cophaga, castration does not result in any cytologically visible change in the
corpus allatum of either sex, and in this fly the oenocytes of the female are almost
indistinguishable from those of the male. This suggested that changes in the
oenocytes might be induced by castration of female Lucilia and of male Sar-
cophaga, but none were found. However, this finding does not invalidate the
general conclusion that the effect of the ring gland is probably primarily on some
general metabolic process, perhaps acting through the oenocytes rather than
directly on the ovary.

SUMMARY

1. Evidence from extirpation and transplantation experiments suggests that
the ring gland of Lucilia sericata and Sarcophaga securifera produces a hormone
concerned with normal development. Its action can be seen in the larva where
it results in puparium formation, and in the adult fly first in the changes which
occur during the breakdown of the larval fat body cells and subsequently in the
changes undergone by the adult fat body cells, the oenocytes, and the development
of the ovaries.

2. These last two activities may be under the influence of a hormone (prob-
ably different from that influencing development), whose action seems to be on
the general metabolic activity of the fly. The oenocytes undergo marked changes
after extirpation of the ring gland. If these are concerned with some general
metabolic function, as seems likely, the action may be primarily on them and the
effects on fat body cells may be altered by implanting a ring gland into the
abdomen of a fly, after extirpation of the ring gland, but this has no visible effect
on oenocytes or on ovarian development.

3. Castration of adult female Lucilia sericata results in hypertrophy of the
cells of the corpus allatum. No effect is produced in the male Lucilia sericata
or in either sex in Sarcophaga securifera.

4. Destruction of the innervation of the ring gland of Sarcophaga securifera
results in slight hypertrophy of the corpus allatum cells, and of their nuclei. The
physiological significance of this hypertrophy is not yet known.

140 M. F. DAY

LITERATURE CITED

BURTT, E. T., 1938. On the corpora allata of dipterous insects. II. Proc. Roy. Soc. London,

Ser. B, 126: 210-223.
DAY, M. F., 1940. Possible sources of internal secretions in the heads of holometabolous insects.

Anat. Rcc., 78: suppl.: 150.
DAY, M. F., 1942. Homologies of the ring gland of Diptera Brachycera. Ann. Ent. Soc. Amer.

(in press).
EVANS, A. C., 1935. Some notes on the biology and physiology of the sheep blowfly, Lucilia

sericata, Meig. Bull. Ent. Res., 26: 115-122.
GLASER, R. W., 1923. The effect of food on longevity and reproduction in flies. Jour. Exper.

Zool., 38: 383-412.
HADORN, E., AND NEEL, J., 1938. The accelerating effect of ring gland injection upon puparium

formation in normal and hybrid Drosophila larvae. Genetics, 23: 151.
MACKERRAS, M. J., 1933. Observations on the life histories, nutritional requirements and

fecundity of blowflies. Bull. Ent. Res., 24: 353-362.
MELLANBY, L., 1938. Diapause and metamorphosis in the blowfly, Lucilia sericata Meig.

Parasit., 30: 392-402.
NICHOLSON, A. J., 1921. The development of the ovary and ovarian egg of a mosquito, Anopheles

maculipennis Meig. Quart. Jour. Micros. Sci., 65: 395-450.
PEREZ, C., 1910. Recherches histologiques sur la metamorphose des Muscides. Arch. Zool.

exp. gen., 4: 1-274.

PFEIFFER, I. W., 1939. Experimental study of the function of the corpora allata in the grass-
hopper, Melanoplus differentialis. Jour. Exper. Zool., 82: 439-461.
PFEIFFER, I. W., 1941. Effect of removal of the corpora allata on the fat metabolism and water

content of the grasshopper. Anat. Rec., 81, suppl.: 57.
PFLUGFELDER, O., 1937. Ban, Entwicklung, und Funktion der Corpora allata und Cardiaca

von Dixippus morosus Br. Zeitschr. wiss. Zool., 149: 477-512.
PFLUGFELDER, O., 1938a. Untersuchungen u'ber die histologischen Veranderungen und das

Kernwachstum der Corpora allata von Termiten. Zeitschr. wiss. Zool., 150: 451-467.
PFLUGFELDER, O., 1938b. Weitere experimentelle Untersuchungen iiber die Funktion der Cor-
pora allata von Dixippus morosus Br. Zeitschr. wiss. Zool., 151: 149-191.
PFLUGFELDER, O., 1939. Beeinflussung von Regenerationsvorgangen bei Dixippus morosus Br.

durch Extirpation und Transplantation der Corpora allata. Zeitschr. wiss. Zool., 152:

159-184.
PFLUGFELDER, O., 1940. Austausch verschieden alter Corpora allata bei Dixippus morosus Br.

Zeitschr. wiss. Zool., 153: 108-135.
ROUBAUD, E., 1932. Des phenomenes d'histolyse larvaire postnymphale et d'alimentation

imaginale autotrophe chez le muostique commun, Culex pipiens. Compt. Rend. Acad.

Sci., 194: 389-391.

SCHARRER, B., 1941. Endocrines in invertebrates. Physiol. Rev., 21: 383-409.
SCHRADER, K., 1938. LTntersuchungen iiber die Normalentwicklung des Gehirns und Gehirn-

transplantationen bei der Mehlmotte Ephestia Kiihniella Zeller nebst einigen Bemerk-

ungen iiber das Corpus allatum. Biol. ZbL, 58: 52-90.

SNODGRASS, R. E., 1935. Principles in Insect Morphology. McGraw-Hill, New York.
TEUNISSEN, R. J. H., 1937. Strukturelle Veranderungen im Gewebe der Stoffwechselzellen des

"Fettkorpers" von Calliphora wahrend des Umbaues der Puppe. Cytologia, Fujii Jub.

Vol. II: 836-844.
THOMSEN, E., 1940. Relation between corpus allatum and ovaries in adult flies (Muscidae).

Nature, 145: 28.

THOMSEN, E., 1941. Ringdriise und Corpus allatum bei Musciden. Naturwiss., 29: 605-606.
TRACER, W., 1941. The nutrition of invertebrates. Physiol. Rev., 21: 1-35.
VOGT, M., 1940. Zur Ursache der unterschiedlichen gonadotropen Wirkung der Ringdriise von

Drosophila funebris und Drosophila melanogaster. Arch. Entw.-mech., 140: 525-546.
WEED, I., 1936. Removal of corpora allata on egg production in the grasshopper, Melanoplus

differentialis. Proc. Soc. Exp. Biol. Med., 34: 883-885.
\Yir.GLES\voRTH, V. B., 1936. The function of the corpus allatum in the growth and reproduction

of Rhodnius prolixtis (Hemiptera). Quart. Jour. Micros. Sci., 79: 91-121.
\YIGGLESWORTH, V. B., 1939. The Principles of Insect Physiology. Methuen, London.

CHANGES IN VOLUME AND PHYSICAL PROPERTIES OF

ALLANTOIC AND AMNIOTIC FLUIDS UNDER

NORMAL AND EXTREME TEMPERATURES

ALEXIS L. ROMANOFF AND FREDERICK W. HAYWARD

(Cornell University Agricultural Experiment Station, Ithaca, New York)

Avian embryonic membranes develop as special temporary organs. They
begin to appear early, developing quite separately from those of the embryo, and
cease to function at hatching. Their presence is indispensable because they
provide both for the protection of the embryo and for its independent existence.
They participate in nearly all metabolic activities of the embryo, such as nutri-
tion, respiration and excretion.

In spite of the biological importance of these membranes, little is known about
their development. A study of the changes in volume and physical properties of
the allantoic and amniotic fluids under both normal and extreme temperatures
would be of especial value in a better understanding of the physiology of develop-
ment of the avian embryo. Therefore, with these ideas in mind the present work
was undertaken.

EXPERIMENTAL METHODS

About 650 fertile eggs of White Leghorn hens (Callus domesticus), 200 of
Ringnecked pheasants (Phasianus torquatus), 150 of Bobwhite quail (Colinus
virginianus) , 200 of White Holland turkeys (Meleagris gallopavo) and 200 of
Pekin ducks (Anas domesticus) were used. The eggs were incubated in the
laboratory incubators previously described (Romanoff, 1932). Normal conditions
for development were a temperature of 37.5° C., relative humidity of about 60
per cent, and an air movement of about ten feet per second, only with some minor
modifications according to the specific requirements of each species. There was
a sufficient supply of fresh air and an adequate removal of carbon dioxide. All
of the eggs were turned at regular intervals three times a day.

For a more detailed study of the effect of temperature on the development of
embryonic membranes — allantois and amnion — chicken eggs were selected.
Eggs of other species were not used because of their scarcity.

The experimental temperatures for incubation of the chicken eggs were 34.5°,
36.0°, 38.5°, and 39.5° C. All of the other environmental factors were similar
to those under normal conditions.

The volumes of allantoic and amniotic fluids were measured with the aid of a
special aspirator. After removing the shell and shell membranes at the blunt
end of the egg, the allantoic membrane is pierced by the large hypodermic needle
on the aspirator. The allantoic fluid is withdrawn into the small measuring
cylinder by pressing the suction bulb until the total volume of liquid is obtained.
The amniotic fluid is withdrawn in a similar manner, except that the older
embryos may be placed in a clean Petri dish to facilitate removal of the liquid.

141

142

ROMANOFF AND HAYWARD

After the amniotic membrane is pierced, any escaping liquid can be collected
directly from the dish. On the whole, this method permits quick and accurate
measurements of the fluids and their ready use for further immediate studies.

The specific gravity of the fluids was determined by weighing them in small
standardized pycnometers of approximately 0.5 cc. capacity. These pycnometers
were made in 14 mm. lengths from glass tubing with a 3 mm. bore. In a test
determination by this method the average deviation of density was about 0.00024.

The hydrogen-ion concentration (pH) of the fluids was measured electro-
metrically, using a hydrogen electrode of special design for small quantities of
material of about 1.0 cc.

EXPERIMENTAL RESULTS

Volume of fluids under normal conditions

The actual volume of allantoic and amniotic fluids in the eggs of the various
species showed enormous variation (Figures 1 and 2), since the weight of the
eggs used varied from about 9 grams for quail to about 85 grams for turkey.

cc.

Li.

FIG. I.

SPGR.
I.OSh

1.04

1.03

1.02

1.01

1.00

FIG. 3.

CHICKEN

PHEASANT

QUAIL

- TURKEY

DUCK

GOOSE

PH
9

FIG. 5.

FIG. 4

4 -

2 -

50 70 90

pgH FIG. 6.

50 70 90

INCUBATION PERIOD CIN °/° )

FIGURES 1 TO 6. Changes in volume, specific gravity and hydrogen-ion concentration (pH)
of allantoic and amniotic fluids of avian eggs incubated under normal conditions. (Abscissae
represent the per cent of time for the full embryonic developmental period — chicken 20 days,
pheasant and quail 23 days, turkey and duck 27 days, and goose 30 days.)

ALLANTOIC AND AMNIOTIC FLUIDS 143

However, when the amounts are considered in relation to the size of the egg, it
is found that the allantoic fluid at the peak of accumulation is approximately 9
to 10 per cent of the original egg weight, while the amount of amniotic fluid at
its peak is about 8 to 9 per cent.

The peak for the allantoic fluid is reached shortly after the middle of the
incubation period, and corresponds in general with the observations of Kamei
(1927), and Ogorodniy and Penionschkevitsch (1939) on chicken eggs. The
volume of fluid shows a fairly regular rise, and, after reaching the peak, it falls
with nearly the same rate.

The duration of the period in which there is a high percentage of fluid in the
egg is apparently longer with the amniotic than with the allantoic fluid. The
peak of volume of amniotic fluid occurs somewhat later during incubation, and
there is a slight depression in the curve occurring at approximately the time of
highest allantoic accumulation. This depression was found to be greatly exag-
gerated with chicken eggs under natural incubation (Ogorodniy and Penion-
schkevitsch, 1939).

Specific gravity of fluids under normal conditions

The specific gravity of allantoic fluid rises throughout the period of incubation,
increasing most rapidly during the early and late stages of development (Figure 3).
On the other hand the specific gravity of amniotic fluid (Figure 4) reaches a peak
at about two-thirds of the incubation period and rises most sharply during the
time in which the allantoic fluid shows the least change. The data agree with a
few observations of Kamei (1927), and Ogorodniy and Penionschkevitsch (1939)
for chicken eggs.

Hydrogen-ion concentration of fluids under normal conditions

With both allantoic and amniotic fluids of chicken eggs, as well as of turkey,
duck and goose (Anser domesticus} eggs (Shklyer, 1937), there was found to be a
definite trend in hydrogen-ion concentration (pH) during the period of incubation
(Figures 5 and 6). In the allantoic fluid it decreased at a moderate rate from
high alkalinity at about the mid-period to medium acidity at the time of hatching.
On the other hand, it showed a more direct relationship with the amniotic fluid
and with the stages of incubation. In general, it dropped steadily from medium
alkalinity to slight acidity. The hydrogen-ion concentration of the amniotic
fluid decreased at a slower rate than that of the allantoic fluid and thus was
limited to a narrower range.

In some data presented by Aggazzotti (1913), Gueylard and Portier (1925)
the hydrogen-ion concentration for allantoic fluid seemed to follow a curve convex
to the abscissa but always near neutrality.

Volume of fluids under extreme temperatures

The accumulation of allantoic fluid of chicken eggs was noticeably influenced
by abnormal temperatures (Figure 7). The greatest volume of 7.3 cc. occurred
under normal temperature, while the next highest level of 6.7 cc. occurred under
slightly abnormal temperatures of 38.5° and 36° C. With increasingly abnormal
conditions at 39.5° and 34.5° C. the volume level was reduced to approximately

144

ROMANOFF AND HAYWARD

4.5 cc. Earlier work (Romanoff, Smith and Sullivan, 1938) showed a still lower
level of the fluid, less than 2 cc. at 40.5° C.

Concomitantly there occurred a shifting of peaks of accumulation, the peak
occurring early under high temperature and late under low temperature. This
coincides with the growth response of the embryo to abnormal temperatures
(Romanoff, Smith and Sullivan, 1938).

cc.

D
_l
U.

y
o

z
_i

FIG. 7.

SPGR
103

1.02
1.0 1

1.00

FIG. 9.

.39.5

FIG. II.

54.5

D
u_

10 15 2O

FIG. 10.

4 -

2 -

• 34.5

10 15 20

FIG. 12.

INCUBATION PERIOD GIN DAYS)

FIGURES 7 TO 12. Influence of incubating temperature on the volume (Figures 7 and 8),
specific gravity (Figures 9 and 10) and hydrogen-ion concentration (pH) (Figures 11 and 12)
of allantoic and amniotic fluids of chicken eggs.

Normally there were observed two successive almost equal peaks of accu-
mulation of amniotic fluid (Figure 8). The time of occurrence of the first peak
was noticeably affected by the temperature; it occurred early under high tem-
perature and late under low temperature. This also seems to follow the general
growth response of the embryo to various temperatures.

The depression occurring between the peaks of volume is wider at high and
narrower at low temperature since the second peak occurs at approximately
normal time. This second point of maximum volume is extremely exaggerated
under both high temperatures 38.5° and 39.5° C., and follows a much greater
depression than occurs under lower temperatures. Similar changes in volume of
amniotic fluid were observed by Ogorodniy and Penionschkevitsch (1939) at
incubating temperatures of about 39° C.

ALLANTOIC AND AMNIOTIC FLUIDS 145

Specific gravity of fluids under extreme temperatures

The physicochemical properties of the fluids of chicken eggs were also definitely
altered. There was a marked tendency for the specific gravity of the allantoic
fluid to increase at a faster rate with higher temperature (Figure 9). Further-
more, this increase occurred earlier during incubation with higher temperatures,
although all values increased, in general, with the corresponding morphological
age of the embryo.

The amniotic fluid at the beginning showed a slightly lower specific gravity
than the allantoic fluid under similar conditions (Figure 10). Then at the mid-
period of the development with high temperature it rose very rapidly and
remained at a somewhat higher level than under normal conditions. With low
temperature the rise in specific gravity was delayed and on a whole insignificant.
This gave great differences in value under the extreme temperatures.

Hydrogen-ion concentration of fluids under extreme temperatures

The allantoic fluid showed a delayed initial decrease in hydrogen-ion concen-
tration with a decrease in temperature (Figure 11), but the rate of decrease in
values under all conditions was at approximately the same rate.

The change in hydrogen-ion concentration of the amniotic fluid (Figure 12)
was almost lineal with development, the values decreasing more rapidly at low
and more gradually at high temperatures.

DISCUSSION

The changes in volume and composition of both allantoic and amniotic fluids
are closely associated with the water metabolism of the avian egg. From the
fact that there is a plentiful supply of water in the egg, Gray (1926) suggested
that this makes it possible for the eggs of land vertebrates to develop without
an external water supply such as that provided for their fish-like ancestors. The
formation of an amnion and an allantois in an avian egg is therefore possible,
with the aid of osmotic and other physicochemical activities within the egg, in
spite of a continuous loss of water by evaporation. According to Needham
(1931), the osmotic pressure of the embryonic body, for example, rises steadily
as development goes on, that of the amniotic liquid stands more or less stationary,
and that of the allantoic liquid greatly declines. This in turn may have an
intimate relationship to the specific gravity of allantoic and amniotic fluids.

The increase in volume of allantoic fluid is required to assist in the excretion
of uric acid (Fiske and Boyden, 1926; Romanoff, Smith and Sullivan, 1938).
The reabsorption of water from the allantois must begin very soon after the
mid-period of development, because its uric acid content is increasing, while its
volume is remaining steady or diminishing (Romanoff, Smith and Sullivan, 1938).
The uric acid maximum follows ammonia and urea maxima during ontogenesis
(Needham, 1926). In fact, the change in hydrogen-ion concentration of the
allantoic fluid during the last half of incubation from an alkaline to an acid state
has been explained in the relationship of urea and ammonia to uric acid (Aggaz-
zotti, 1913). It has been shown (Fiske and Boyden, 1926) that the allantois is
functional in the excretion of nitrogeneous waste products as early as 2.5 days.

146 ROMANOFF AND HAYWARD

The excessive secretion of allantoic fluid then apparently coincides with the
maximum for uric acid in order to assist in the excretion of the nitrogenous waste.

On the other hand, the amniotic fluid reaches what is practically its maximum
by the mid-period of incubation. It was found by Ogorodniy and Penion-
schkevitsch (1939) that penetration of large amounts of albumen into the am-
niotic cavity is responsible for this increase in volume, and also for the increase
in its protein content. These lead to a greatly heightened viscosity of amniotic
fluid. Close to the end of the developmental period, however, the embryo
swallows up a large quantity of amniotic fluid and its volume rapidly decreases.

The marked reduction in volume of allantoic fluid at both high and low tem-
peratures would indicate the underdevelopment of the allantois. This presum-
ably is associated with the developmental restrictions in extra embryonic circu-
lation. The observations of Tazelaar (1928) and Romanoff (unpublished) show
that during the first week of incubation there is a noticeable maldevelopment of
the area vasculosa. Also the presence of greater amounts of uric acid during
later stages (Romanoff, Smith and Sullivan, 1938) gives further indications that
the metabolic equilibrium of the embryo is upset by adverse thermal conditions.

The changes in physical properties of both fluids, as was anticipated, were
largely in line with the accelerated or retarded development both of the embryo
and of its membranes as induced by high or low temperatures.

SUMMARY

Under normal conditions of incubation the relative volume and physical
properties of allantoic and amniotic fluids are nearly identical in chicken, pheasant,
quail, turkey, duck and goose eggs.

Under high and low temperature the fluids are altered in respect to both
volume and physical properties. In general, the course of changes follows the
morphological age of the embryo. The volume of allantoic fluid was suppressed,
while that of amniotic fluid was excessively enlarged during the later part of
incubation under high temperature. The time of increase in density was shifted
in both fluids along with the developmental stage of the embryo; there was also a
noticeable reduction in the density of the amniotic fluid at low temperature.
The drop in hydrogen-ion concentration of allantoic fluid was nearly identical,
except for the time factor,- while the values for amniotic fluid decreased more
rapidly at low and more gradually at high temperatures.

LITERATURE CITED

AGGAZZOTTI, A., 1913. Influenza dell'aria rarefatta sull'ontogenese. Nota II. La reazione dei

liquid! dell'ovo durante lo svillupo. Arch. Entw.-mech. Org., 37: 1-28.
FISKE, C. H., AND E. A. BOYDEN, 1926. Nitrogen metabolism in the chick embryo. Jour.

Biol. Chem., 70: 535-556.
GRAY, J., 1926. The role of water in the evolution of the terrestrial vertebrates. Brit. Jour.

Exp. Biol., 6: 26-31.
GUEYLARD, F., AND P. PORTIER, 1925. Reaction ionique des differents constituants de 1'oeuf

de la Poule. Ses modifications an cours de 1 incubation. Compt. Rend. Acad. Sci.,

180: 1962-1963.
KAMEI, T., 1927. Untersuchungen iiber die physikalischen Eigenschaften und die chemische

Zusammensetzung der Amnios- und Allantoisfliissigkeit des Huhnerembryos. Zeitschr.

f. physiol. Chan., 171: 101-113.

ALLANTOIC AND AMNIOTIC FLUIDS 147

NEEDHAM, J., 1926. The energy sources in ontogenesis: II. The uric acid content and the
general protein metabolism of the developing avian egg. Brit. Jour. Exp. Biol., 4:
114-144.

NEEDHAM, J., 1931. Chemical embryology. Cambridge Univ. Press.

OGORODNIY, Y. M., AND E. E. PENIONSCHKEVITSCH, 1939. Role of protein and water metabolism
in the pathogeny of embryonic stickiness in chicks. Materials on the study of pathology
of embryonic development of birds, 2: 5-27 (in Russian).

ROMANOFF, A. L., 1932. Multiple laboratory incubator for the biological study of chick embryo.
Science, 75: 246-248.

ROMANOFF, A. L. Unpublished.

ROMANOFF, A. L., L. L. SMITH, AND R. A. SULLIVAN, 1938. Biochemistry and biophysics of the
developing hen's egg. III. Influence of temperature. Cornell Univ. Agr. Exp. Sta.,
Memoir 216: 1-42.

SHKLYER, N. M., 1937. A study of physicochemical changes in the egg during embryonic devel-
opment of birds. I. Changes in the concentration of H-ions in relation to embryonic
development in the eggs of domestic fowl (hen, turkey, duck, goose). Ukrainian Bio-
chem. Jour., 2: 379-406.

TAZELAAR, M. A., 1928. The effect of a temperature gradient on the early development of the
chick. Quart. Jour. Micr. Sci., 72 (1928-29): 419-446.

PHYSIOLOGICAL OBSERVATIONS UPON A LARVAL EUSTRON-

GYLIDES. IV. INFLUENCE OF TEMPERATURE, pH AND

INORGANIC IONS UPON THE OXYGEN CONSUMPTION

THEODOR VON BRAND1

(Department of Biology, The Catholic University of America, Washington, D. C.)

Experiments described in two previous papers of this series (von Brand, 1938,
1942) showed conclusively that a larval Eustrongylides, occurring encysted in
Fundulus, leads a predominantly oxidative life while in the fish. The evidence
was derived on the one hand from experiments comparing the glycogen con-
sumption of aerobically and anaerobically kept Eustrongylides larvae with that
of animals living under natural conditions either purely oxidatively (earthworm,
etc.) or primarily anoxidatively (Ascaris). The second approach consisted in
comparing the gaseous exchange of freshly isolated worms, with that of larvae
previously exposed to a period of anaerobiosis. The fact that this larval
nematode has an oxidative metabolism is of considerable interest, since with the
exception of Trichinella larvae (Stannard, McCoy and Latchford, 1938), most
parasitic worms that have so far been studied possess a predominantly fermen-
tative type of metabolism. It seemed of interest therefore to investigate the
influence of various factors upon the oxygen consumption of this worm.

MATERIAL AND METHODS

As in previous experiments only medium-sized to large worms were used,
and, depending upon the size, from four to five of these larvae constituted an
experimental lot. The oxygen consumption was again determined by the
Warburg method. The details of the experimental procedure have been de-
scribed adequately in the second paper of this series (von Brand, 1942). It
should be added that the vessels were shaken 100 times per minute with an
amplitude of 3 cm. Control experiments showed that this rate was sufficient to
establish a satisfactory equilibrium between air and water. The temperature
and the saline solutions used varied in the different series, the details will be given
at the appropriate places in the following sections.

INFLUENCE OF TEMPERATURE

One factor that under natural conditions will be of the greatest importance
in determining the metabolic level of Eustrongylides is the temperature. The
worms in the intermediate host are, during the varying seasons of the year,
exposed to temperatures ranging from near the freezing point to about 25° C.
Cowles (1930) found during his survey of Chesapeake Bay in his area U near
Baltimore, surface temperatures ranging from 0.3° C. to 24.8° C. and bottom

1 The author is indebted to the Elizabeth Thompson Science Fund for a grant towards the
purchase of the respiration apparatus used in this investigation.

148

RESPIRATION OF EUSTRONGYLIDES

149

temperatures varying between 0.9° C. and 24.4° C. Once the parasites enter
the definitive host, probably a heron, they are exposed to the higher, but constant
temperatures characteristic of birds. An investigation of the temperature
influence upon the oxygen consumption seemed therefore desirable and the more
so, as aquatic animals — parasites must be compared with them rather than with
terrestrial forms — encountering such a wide range of temperature fluctuations
in their normal surroundings are relatively rare. Furthermore with the exception
of a few experiments of McCoy (1930) on infective larvae of Ancylostomum
caninum no work on the temperature relationships of the respiration of helminths
seems to have been performed.

The results of the temperature experiments are summarized in Table I. For
each experiment freshly isolated worms were used. This, of course, introduces
the biological variation between various lots as a source of error. It seems,

TABLE I

Oxygen consumption of a larval Eustrongylides at different temperatures

Temperature
°C.

Determination
number

cmm. Oz consumed/gm./half hour

Mean value of
all readings

Mean value after exclusion
of first reading

4.5 ± 0.4

3.5 ± 0.3

9.1 ± 0.6

7.3 ± 0.6

17.0 ± 1.7

15. 7 ± 1.4

35 ± 4.1

32 ± 4.6

54 ± 5.7

53 ± 5.7

64 ± 10

62 ± 10

76 ± 4

75 ± 4

95 ± 8

90 ± 7

113 ± 5

107 ± 4

131 ± 13

117 ±16

however, preferable to use this method rather than to employ the same worms
for determinations at several or all temperatures. This would have required
long periods outside the host, during which time the metabolism sinks somewhat,
even under the most favorable conditions. It is believed that the number of
experiments is sufficient to eliminate gross errors due to biological variation.
Most experiments were conducted for four hours with readings at half hour
intervals. Below 17° C., the readings were taken only hourly, and at 5° in two
to two and one-half hour intervals. In these cases the experimental periods ex-
tended up to eight hours. All temperatures with the exception of that of 48° C.
were well tolerated by the nematodes. At 48°, however, the O2 consumption was
high only for two hours. It then sank rapidly, indicating an injury to the worms.
They in fact became more or less rigid and did not recover. At 45° C., on the
other hand, the worms survived well; after the experiments they were kept for
several days at room temperature and showed normal motility during this time.
At all temperatures the first reading was somewhat higher than were the
subsequent ones. This is due, as discussed previously (v. Brand, 1942), to the
repayment of a small oxygen debt contracted in the cysts. The following dis-

150

THEODOR VON BRAND

cussion is based on the mean values obtained after excluding the first reading,
but the conclusions reached would not be changed materially if the first values
were included.

An analysis of the values obtained shows that the increase in C>2 consumption
with rising temperatures does not follow Krogh's (1914) normal curve. Two
curves were necessary to express the temperature relationships adequately. The
Qio of the temperature interval 5 to 27° C. is 3.55, whereas in the range of 27 to
48° C. a Qio of 1.48 is found (Figure 1). Similarly two bisecting lines resulted if
the values were calculated according to Arrhenius' formula (Figure 2). It is of

140

120

100

o
c\I~

80
60
40
20

1.48

0 10 20 30 40 50
°C

FIGURE 1. The Qio of the oxygen consumption of a larval Eustrongylides.

32 33 34 35 36

FIGURE 2.

The oxygen consumption of a larval Eustrongylides expressed according to
Arrhenius' formula.

interest to note that 27° C. is one of the critical temperatures where according
to Crozier (1926) a break in the curves is frequently found. The n values too
are well within the range of those frequently found in biological processes
(Crozier, 1926a). These findings might be interpreted with Crozier (1925) on
the assumption that two master reactions are involved. One might even be
tempted to correlate them with the life history of the worm : One of the master
reactions would conceivably control the metabolism of the larva in the fish — the
turning point of the curves is near the highest temperature to which Fundulus

RESPIRATION OF EUSTRONGYLIDES

151

is exposed in this region, the other would be characteristic for the processes
proceeding once the definitive host is reached. I am, however, at the present
time not prepared to draw such a sweeping conclusion: it should be remembered
that several investigators (Belehradek, 1935; Ponder and Yeager, 1930) have
raised serious objections against Crozier's interpretations of the significance of
bisecting lines resulting from the application of Arrhenius' formula. Belehradek
(1935) has proposed a formula that frequently allows the temperature relation-
ships to be expressed by a straight line, where other formulae require two. The
Eustrongylides values are presented, according to Belehradek's formula, in
Figure 3. They show a fairly good fit to a straight line. It should be remem-

1.0 LI

1.7

FIGURE 3. The oxygen consumption of a larval Eustrongylides expressed according to

Belehradek's formula.

bered, however, that the constant a is purely arbitrary, and has to be chosen
differently from case to case. If it is changed even a few degrees the single line
relationship does not hold in the present case. It seems to the writer that
further progress in the question of temperature relationship might be expected
from a simultaneous investigation of various metabolic processes at different
temperatures.

INFLUENCE OF pH

Another factor that may vary considerably in the natural environments of
Eustrongylides is the hydrogen ion concentration. Although no actual data are
available it can perhaps be expected that the cyst fluid formed by the fish may,
like many other secretions of the body, have a pH fluctuating around the neutral
point. But as soon as the worm is freed from the cyst in the body of the definitive
host the situation becomes different. It is reported to live in the glands of the
fore stomach of aquatic birds or in the fat around the gizzard, but it has also been
recovered from the intestines and omentum (Jaegerskioeld, 1909; Cram, 1934).
Hunter (1937) found immature adults threaded in and out the stomach wall of
the black crowned night heron and the little green heron. Before establishing

152 THEODOR VON BRAND

itself in its final position the worm will be exposed to the digestive juices. No
data on the pH of these heron species were found in the literature, but Mennega
(1938) investigated the pH of the stomach content and stomach wall of a
European species of heron. She found after 24 hours starvation, pH values of
2.40 and 3.10 respectively for the fore stomach, while the pH of the contents
varied from 3.56 to 6.10 and those of the wall from 4.14 to 5.75 after food had
been taken in 45 minutes to 3| hours prior to the determinations. Cases in which
the respiratory rate varies in solutions of different pH are known from organisms
belonging to various phyla. In the holothurian Thyone, for example, Hiestand
(1940) found a steady increase in respiration in the pH range 5.4 to 8.8, whereas
Hiestand and Hale (1938) found an increased O2 consumption in fresh water
molluscs when the pH was lowered. Maier and Coggeshall (1941) found that
the rate of Oo consumption of a malaria parasite (Plasmodium Knowlesi) remained
constant between pH 7.0 and 8.0, but declined rapidly between pH 8.0 and 9.0.
Cook and Sharman (1930) found a marked influence of the pH on the CO2 output
of Moniezia, but their results need confirmation since the experimental periods
used exceeded by far the length of time in which these worms remain normal in
inorganic solutions according to the experience of other investigators.

The parasitic nematodes seem never to have been used for a study of this
type. Since the pH seems to be at least in some cases a limiting factor in the
distribution of these parasites in the host (Davey, 1938), a study of the influence
of pH on the Oo consumption was undertaken. In order to get and maintain the
intended hydrogen ion concentrations, Sorensen's phosphate (1/15 molar) and
citrate buffers (1/10 molar) were used. The necessary amounts of NaCl were
added to make the solutions isotonic to a one per cent NaCl solution. Three
to five worms were isolated from the fish and kept for 24 hours at 37° in about
30 cc. of the solution in order to adapt them as far as possible to these media.
After 24 hours the O2 consumption was determined over a period of four hours.
For each experiment a new batch of two to five worms was used, the temperature
was 37° C. A total of 74 experiments was performed, covering the pH range of
1 . 13 to 10.60. The worms withstood the total period of 28 hours in these solutions
without harm, their viability was controlled by transferring them after the deter-
minations to one per cent saline for two days during which time they invariably
showed normal motility. The extreme acidities and the alkalinity of pH 10.6
could, however, not be tolerated for a much longer period. Separate experiments
showed that the worms cannot be kept longer than about two to three days in
these solutions at 37° C. without permanent injuries, whereas they lived con-
siderably longer in the other solutions.

The results are given in Figure 4. It is apparent that between pH 3.4 and
8.3 the O2 consumption remains on an average quite constant, the average rate
being 76 cmm./gm./| hr. The only exception occurred at pH 4.5, where a
somewhat higher O2 consumption was found in a phosphate buffer solution. This
solution contained only KH2PO4 + NaCl. It seems possible that the increased
rate may be only an expression of the biological variability, but it is equally
possible that it is due to a stimulating effect of the K ion, which is pronounced
in pure solution as will be shown in a following paragraph. In the acid range a
distinctly increased rate of O2 consumption was found at pH 1.13 and 1.80. This,
probably, as an observation of the worms shows, is due to an increased muscular

RESPIRATION OF EUSTRONGYLIDES

153

activity. This may be of biological significance. The worms have to bury
themselves into the mucosa of the fore stomach and are then probably exposed
to a very low pH. This may then be the stimulus necessary for the initiation
of the boring movements.

An increased O2 consumption was also observed in the extreme alkaline range.
This is probably without biological significance, since it seems unlikely that the

150

IOOK
o

I 2

IO II

FIGURE 4. Oxygen consumption of a larval Eustrongylides in salines of various pH. Dots —
single determinations, crosses — mean values. P — Phosphate buffer, C — Citrate buffer.

nematodes will in their natural environments be exposed to similar conditions.
It may be assumed that this is an expression of increased activity due to
unfavorable surroundings.

INFLUENCE OF INORGANIC IONS

Experiments discussed in a previous paper (von Brand, 1942) showed that
the molecular concentration of the medium could be changed in rather wide
limits without interfering with the oxygen uptake of the parasite under con-
sideration, or without changing its life in vitro to a marked extent (von Brand
and Simpson, 1942). These findings were believed to be of biological importance,
since the complicated life cycle of this worm, although incompletely known,
includes actually a variety of natural "media." The eggs are probably passed
out from the host into water, possibly both fresh and brackish water, judging
from the regions from which the parasites have been reported. It seems likely
that two intermediate hosts are involved; the first is not yet known definitely,
but is probably a crustacean. As second intermediate host a variety of different
fish may serve, and, as already mentioned, aquatic birds are the definitive hosts.
Besides differences in concentration, differences in chemical composition, both in
regard to organic and inorganic constituents also may be expected in these various
habitats. Since inorganic ions frequently have a marked influence upon
respiration (some of the pertinent facts are reviewed in Heilbrunn, 1937, and
Canzanelli, Rogers and Rapport, 1942), the respiration of Eustrongylides was
studied in solutions of various ions.

Solutions isotonic to a 1.0 per cent NaCl solution were used throughout this
work. The following cations were used in the form of their chlorides: Na, Mg,
Ca, NH.4, K, and the following anions in the form of their sodium salts: Cl, SO4,
NO2, NO3 and PO4. The pH of all solutions was in the range not affecting the
oxygen uptake, in the case of the PCX ion this was achieved by using a mixture

154

THEODOR VON BRAND

of equal parts of NaaHPCX and Nal-hPO-j. All experiments were conducted at
37° C. and for each solution eight different lots of worms were used. Deter-
minations of the oxygen consumption were performed immediately after the
freshly isolated worms had been washed thoroughly with the respective solutions,

<— NACL

DAYS

150-

ZD
O

z
z
o

50 -

•

•z.

«r

•z.

FIGURE 5. Oxygen consumption of a larval Eustrongylides in isotonic solutions of various
anions. The upper part of the figure shows the average O^ consumption on specified days, the
lower part the average O-j consumption over the entire period of observation.

?oo

or
o

ISO

>IQO

x
z
o

150-

I 10 0 -

KCl.

C*CL2

DAYS

FIGURE 6. Oxygen consumption of a larval Eustrongylides in isotonic solutions of various
cations. The upper part of the figure shows the average Oz consumption on specified days, the
lower part the average Oa consumption over the entire period of observation.

RESPIRATION OF EUSTRONGYLIDES 155

after 24 hours and 48 hours. The parasites were kept in 30 cc. of the various
solutions at 37° C. between the determinations. It was, however, impossible to
secure the 48 hours value in the case of KC1 or the 24 and 48 hours values in the
case of NaNO2, since the worms did not survive long enough. All the other
solutions were tolerated remarkably well. It does not seem very likely that the
well known relative impermeability of the nematode cuticle is the responsible
factor since the total average survival was longest in NaCl and definitely shorter
in all other solutions. An exception is perhaps MgCl2. The worms kept in this
solution for days showed no sign of anesthesia which both in vertebrates and
many invertebrates is one of the best known effects of the Mg ion. It should be
remembered that the Mg ion is known to decrease permeability. It seems sug-
gestive that the average oxygen consumption in the MgCl2 solution was only very
slightly higher than that found in a pure NaCl solution, the difference may well
be within the limits of experimental error. The results of these experiments are
summarized in Figure 5 and Figure 6. The stimulating effect on oxygen consump-
tion is represented by the following two series:

Cations: Na = or slightly < Mg < Ca = NH4 < K.
Anions: Cl = or slightly < SO4 < NO2 = NO3 < PO4.

The increase in oxygen consumption was especially pronounced in the KC1
solution, the rate being about twice that found in an isotonic NaCl solution.
This stimulating effect of the K ion is in line with experiments reported by other
investigators on a variety of objects (literature in Heilbrunn, 1937).

SUMMARY

1. The temperature range tolerated by a larval Eustrongylides is great. The
worms were not harmed by temperatures between 5° C. and 45° C., but 48° C.
proved to be injurious.

2. The oxygen consumption was studied in this temperature range. Its
increase with rising temperature could be expressed by two lines only, if the Qio
was calculated or if Arrhenius' formula was used. A fairly good fit to a single
line resulted however if Belehradek's formula was applied.

3. The oxygen consumption remained practically unchanged in the pH range
3.4 to 8.3, but it was increased in the pH ranges 1.1 to 2 and 9 to 10.7. It is
possible that the increase in the extreme acid range is of biological significance,
while that in the extreme alkaline range is probably only a reaction to unfavorable
environmental conditions.

4. The oxygen consumption was studied in a series of isotonic solutions of
various inorganic substances. Of all the solutions tested, only NaNO2 and to a
lesser degree KC1 were definitely toxic. The oxygen consumption was stimulated
by various ions according to the following series:

Cations: Na = or slightly < Mg < Ca = NH4 < K.
Anions: Cl = or slightly < SO4 < NO2 = NO3 < PO4.

LITERATURE CITED

BELEHRADEK, J., 1935. Temperature and living matter. Protoplasma monographien 8. Berlin.
VON BRAND, T., 1938. Physiological observations upon a larval Eustrongylides (Nematoda).
Jour. Paras., 24: 445-451.

156 THEODOR VON BRAND

VON BRAND, T., 1942. II. The aerobic respiration. Biol. Bull., 82: 1-13.

VON BRAND, T., AND W. F. SIMPSON, 1942. III. Culture attempts in vitro under

sterile conditions. Proc. Soc. Exp. Biol. and Med., 49: 245-248.
CANZANELLI, A., G. ROGERS, AND D. RAPPORT, 1942. Effects of inorganic ions on the respiration

of brain cortex. Amer. Jour. Physiol., 135: 309-315.
COOK, S. F., AND F. E. SHARMAN, 1930. The effects of acids and bases on the respiration of

tapeworms. Physiol. Zool., 3: 145-163.
COWLES, R. P., 1930. A biological study of the offshore waters of Chesapeake Bay. Bull. U. S.

Bureau of Fisheries, 46: 277-381.
CRAM, E. B., 1934. Eustrongylides ignotus from the black-crowned night heron. Jour. Parasit.,

20: 71.
CROZIER, W. J., 1925. On biological oxidations as function of temperature. Jour. Gen. Physiol.,

7: 189-216.
CROZIER, W. J., 1926. Note on the distribution of critical temperatures for biological processes.

Jour. Gen. Physiol., 9: 525-529.
CROZIER, W. J., 1926a. The distribution of temperature characteristics for biological processes;

critical increments for heart rates. Jour. Gen. Physiol., 9: 531-546.
DAVEY, D. G., 1938. Studies on the physiology of the nematodes of the alimentary canal of

sheep. Parasit., 30: 278-295.

HEILBRUNN, L. V., 1937. An outline of general physiology. Philadelphia.
HIESTAND, W. A., 1940. Oxygen consumption of Thyone briareus (Holothuriodea) as a function

of oxygen tension and hydrogen-ion concentration of the surrounding medium. Trans.

Wisconsin Acad. Sci., 32: 167-175.
HIESTAND, W. A., AND D. M. HALE, 1938. Respiration studies with fresh water molluscs. II.

Oxygen consumption in relation to hydrogen-ion concentration. Proc. Indiana Acad.

Sci., 47: 293-298.
HUNTER, G. W., Ill, 1937. Parasitism of fishes in the lower Hudson area. Suppl. 26th Ann.

Rep. N. Y. St. Conser. Dept. No. XI, Biol. Survey Lower Hudson Waters, 1930: 264-273.
JAEGERSKIOELD, L. A., 1909. Zur Kenntnis der Nematoden Gattungen Eustrongylides und

Hystrichis. Nova Acta Regiae Soc. Sci. Upsaliensis, ser. IV, Vol. 2, No. 3.
KROGH, A., 1914. The quantitative relations between temperature and standard metabolism

in animals. Internal. Zschr. Phys. Chem. Biol., 1: 491-508.
MAIER, J., AND L. T. COGGESHALL, 1941. Respiration of Malaria Plasmodia. Jour. Infect.

Dis., 69: 87-96.
McCoY, O. R., 1930. The influence of temperature, hydrogen ion concentration and oxygen

tension on the development of the eggs and larvae of the dog hook worm, Ancylostoma

caninum. Amer. Jour. Hyg., 11: 413-448.
MENNEGA, A. M. W., 1938. Waterstofionenconcentratie en vertering in de maag van eenige

vertebraten. Dissertation Utrecht.
PONDER, E., AND J. F. YEAGER, 1930. The effect of temperature on certain simple haemolytic

systems. Jour. Exp. Biol., 7: 390-403.
STANNARD, J. N., O. R. McCoY, AND W. B. LATCHFORD, 1938. Studies on the metabolism of

Trichinella spiralis Larvae. Amer. Jour. Hyg., 27: 666-682.

FURTHER EXPERIMENTS ON CELLULOSE DIGESTION BY
THE PROTOZOA IN THE RUMEN OF CATTLE

R. E. HUNGATE

(University of Texas, Austin)

Studies on Diplodinium (Eudiplodinium) neglectum Dogiel have shown that
this rumen protozoan may be grown continuously in vitro and that it possesses
the capacity to digest cellulose (Hungate, 1942). Since the various species of
rumen protozoa may differ in their ability to digest cellulose it has seemed desir-
able to extend the investigation to additional forms before drawing any general
conclusions on the role played by the protozoa in the nutrition of their host.

By employing modifications of the culture technique used with D. neglectum
it has been possible to grow D. (Eudiplodinium) maggii, D. (Poly plastron} multi-
vesciculatum, D. (Anoplodinium) denticulatum, and Entodinium caudatum. These
forms have been identified with the aid of Dogiel's monograph (1927). They
correspond fairly closely to his descriptions, with one exception which is noted
below.

EXPERIMENTS WITH DIPLODINIUM MAGGII

Cultures of D. maggii were obtained by inoculating fresh rumen contents into
40 milliliters of a balanced salt solution (Hungate, 1942) containing 30 milligrams
of dried ground grass (Lolium italicum}. The cultures were incubated at 39° C.
under anaerobic conditions. D. maggii, D. neglectum, and a few Entodinium
survived and grew in this medium.

One-half of each culture was transferred every 48 hours in order to prevent
accumulation of staling products. To the transferred portion were added 20
milliliters of fresh salt solution and 15 milligrams of grass. A small sample (0.1
to 0.5 milliliter) was regularly removed and examined microscopically as a means
of following growth of the protozoa and the suitability of the culture methods
employed. The 48-hour interval between transfers was soon found to be too
long. The protozoa appeared sluggish just before transfer and occasionally dead
ones were seen. This was prevented by decreasing the time between transfers
to 24 hours.

Clone cultures were obtained by isolating single individuals in 5 milliliters
of salt solution plus grass. Special anaerobic vessels permitting frequent obser-
vations through a binocular dissecting microscope were used. In six clones the
protozoa were counted three days after isolation and counts of 5, 4, 9, 8, 10,
and 8, respectively, were obtained. The grass particles tended to hide some
individuals and so the count was probably slightly less than the number actually
present. The number seen is about that to be expected if the division rate is
of the order of magnitude of once per day. This rate is also suggested by the
fact that the concentration of individuals in the cultures remained approximately

157

158

R. E. HUNGATE

constant even though in transferring they were diluted daily with an equal
volume of fresh medium.

D. maggii has been carried in the laboratory as a mixed protozoan culture for
two months and as a clone culture for three more months. The concentration
of individuals at the time of transfer has fluctuated between 10 and 100 per mil-
liliter during most of the culture period. After the clone culture had been
carried for three months the number of individuals decreased to only one or two
per milliliter and the culture was discontinued.

During the period when the concentration of protozoa was fairly high the
clone was used for experiments on cellulose digestion by extracts prepared from
the protozoa. The number of flask cultures was increased to 32. Sixteen of
these supplied protozoa for cellulase tests whereas the others were transferred.

The flasks supplying protozoa for the extracts received at the last transfer
a small amount of a suspension of finely divided cellulose in addition to the
grass. The gas produced by fermentation of the cellulose carried both cellulose
and grass to the top of the liquid medium. The protozoa collected on the
bottom and could be pipetted off without disturbing the surface cap. They were
strained through bolting silk, washed, and allowed to settle in a large volume of
the balanced salt solution. By these manipulations the protozoa were separated
from most of the grass particles. They were either used immediately for enzyme
extracts or if it was desired to subject them first to a period of starvation (see
below) they were transferred to 50 milliliters of fresh inorganic solution in another
flask and left for a time without food.

TABLE I

Results of cellulase experiments with D. maggii

Experiment

Starvation
period

Protozoan
extract
plus
cellulose

Protozoan
extract,
no
cellulose

Boiled

protozoan
extract
plus
cellulose

Extract
of debris
plus
cellulose

Boiled
extract
of debris
plus
cellulose

4 hours

+ + +*

4 hours

+ + +

—

12 hours

+ +

! +'s indicate the approximate magnitude of the reduction; ± represents only a trace of
reducing material; — shows that no reducing material was formed.

Extracts from the protozoa were tested for their cellulolytic activity at a pH
of 5.8. Reduction of Benedict's solution served to measure the sugar formed.
In the first experiment there was almost as much reducing material formed in a
sample of the extract alone as in the extract plus cellulose. In subsequent experi-
ments the protozoa were starved for a short time before extraction. With this
precaution it was possible to decrease the amount of reduction due to the extract
itself.

The results of several experiments are shown in Table I. The number of
plus signs indicates the magnitude of the reduction observed.

These experiments provide positive evidence of a cellulase in D. maggii.
Since extracts of the debris in the culture (partially decomposed grass and eel-

CELLULASE IN RUMEN PROTOZOA

159

lulose containing numerous bacteria) show no cellulolytic action it must be con-
cluded that the demonstrated cellulase is elaborated within the bodies of the
protozoa. It cannot be ascribed to the cellulose-decomposing bacteria which
are present in the culture.

The ease with which reserve carbohydrates can be demonstrated micro-
scopically in Diplodinium (Schulze, 1924; Trier, 1926) suggested another approach
to the problem of cellulose digestion. A batch of D. maggii was washed and
then starved. After seven hours of starvation and again after 11 hours a small
sample was removed and stained with iodine. A photomicrograph of the stained
7-hour sample is shown in Figure la and of the 11-hour sample in Figure Ib.
The paraglycogen appears black in the figures.

FIGURE 1. (a) D. maggii, starved seven hours and then stained with iodine; (b) starved
11 hours; (c) starved 13 hours; (d) starved 11 hours, then fed cellulose and photographed after
two more hours.

It is evident from the figure that most of the food reserves are depleted after
11 hours, suggesting rather high metabolic requirements. The protozoa are not
entirely uniform in this respect, however. Some still contain reserve food, due
either to greater initial stores or to feeding on the few grass particles not removed
by washing. Others appear entirely devoid of paraglycogen though still living.
Some succumbed during the starvation period, as found by microscopic examina-
tion of an unstained sample.

At the same time that the sample pictured in Figure Ib was obtained two others
were also removed. One was placed in a small tube of inorganic solution and

160 R. E. HUNGATE

the other was similarly treated except that a small amount of a fine cellulose
suspension was added. The cellulose was almost instantly ingested by most
of the living protozoa. After two hours of incubation at 39° C. the protozoa were
removed from each of the tubes, stained with iodine, and photographed. The
results are shown in Figure Ic for the protozoa receiving no cellulose and in Id
for those with cellulose. The ingested cellulose itself gives a dark appearance
to the central portions of the fed animals and this should not be confused with
the paraglycogen which was deposited. The paraglycogen is present in the tips
of the cells (indicated by arrows in Figure Id) whereas the cellulose is in the large
central digestive sack. The individuals which show no cellulose or paraglycogen
are those which had succumbed to starvation. In the sample without cellulose
(Figure Ic) most of the cells are devoid of paraglycogen, in marked contrast to
the fed.

This experiment shows the rapidity with which cellulose is digested and
assimilated by D. maggii. Bacteria could hardly exert any significant digestive
action on the cellulose during the short time (two hours) between its addition and
the appearance of reserves in the protozoa, particularly when the thorough
washing of the protozoa is recalled. Thus, these observations fully support the
conclusion that D. maggii forms a cellulase.

EXPERIMENTS ON DIPLODINIUM MULTIVESCICULATUM

This interesting species shows numerous contractile vacuoles instead of the
usual two. It also has two narrow skeletal plates on the right side and sug-
gestions of skeletal structures on the left. The specimens cultured resembled
those described by Dogiel (1927) with the exception that the skeletal plates on
the left side were not as definite as in his description. They seemed to be
influenced by the state of nutrition of the protozoa and were less apparent in
poorly fed individuals.

D. multivesciculatum was found in cultures containing grass and cellulose and
also in those containing grass and starch. Later it was discovered that best
growth occurred with grass, cellulose, and ground wheat. This medium was
used to grow the protozoa during most of the period (100 days) in which the
laboratory culture was maintained.

Growth of D. multivesciculatum was slower than that of maggii or neglectum
and the concentration of the protozoa could be maintained only when 2-day trans-
fers were made. This indicates a division rate of once in 48 hours. The concen-
tration was around 100 individuals per milliliter under the most favorable
conditions.

D. multivesciculatum seemed to be more sensitive to environmental changes
than were any of the other protozoa studied. Individuals of most species showed
normal activity for some time after being removed from the culture and placed
in a depression slide for microscopic examination. In the case of multivesciculatum
only rarely was a motile individual observed even when examined immediately
after removal from the culture.

Several attempts to obtain clone cultures were unsuccessful, presumably due
to sensitivity to handling. However, multivesciculatum seemed to be fairly
resistant to high acidity and cultures containing it as only the large protozoan

CELLULASE IN RUMEN PROTOZOA 161

could be obtained from old flasks. Entodinium caudatum was the only other
species of protozoa present.

In order to test for cellulase the protozoa were raised in large numbers and
freed of Entodinium and debris by straining through suitable meshes of bolting
silk and by further washing. Extracts were prepared and gave positive tests for
cellulase. In the cultures containing grass, cellulose, and wheat it was the
cellulose that made up most of the material in the digestive sack.

EXPERIMENTS ON DIPLODINIUM DENTICULATUM

Protozoa of this species appeared in cultures containing cellulose and grass.
Growth occurred also when ground wheat was added to this medium. Main-
tenance of numbers during daily transfers indicated that the division rate was
at least once every 24 hours. The concentration of individuals varied between
100 and 300 per milliliter.

A clone culture was obtained starting with an individual showing the six
caudal spines characteristic of the species. After several weeks the descendants
of this individual were of many morphological types. Individuals with no spines,
with two poorly developed ones, and with several rudimentary spines were seen.
No individuals with the six typical spines were found in the culture at this time.
These observations fully substantiate Poljansky and Strelkow's report (1934) on
variation in this form. After two months the clone died in spite of efforts to
maintain suitable conditions.

Because of the small size no attempt was made to grow D. denticulatum in
sufficient numbers to test extracts for cellulase. However, it is probable that it
resembles D. maggii, multivesciculatum, and neglectum in being able to utilize
this material. Cellulose is required in the culture medium and large quantities
of it are ingested.

EXPERIMENTS ON ENTODINIUM CAUDATUM

Protozoa belonging to this genus were observed in most of the cultures inocu-
lated with rumen contents and transferred at daily intervals. Only a small
number were in the cultures containing grass or grass plus cellulose and they
seemed to ingest very little of these substrates. When soluble starch was added
they became more numerous and with ground wheat the concentration increased
to 2000 to 5000 per milliliter. The starch and wheat were ingested to some
extent but ingestion of large particles was not nearly as striking as in Diplodinium.
In some cases food vacuoles filled with bacteria were seen.

Individuals resembling E. simplex, E. longispinum, and E. caudatum were
observed in the cultures. An individual classified as E. simplex was inoculated
singly into a small amount of medium and a clone was obtained from it. After
two weeks the members of the clone were examined microscopically and individuals
similar to simplex, longispinum, and caudatum were observed. This is again in
entire agreement with the observations of Poljansky and Strelkow on this form
and indicates that simplex and longispinum are synonymous with caudatum.

In contrast to Diplodinium, grass and cellulose could be omitted from the
culture medium for E. caudatum when wheat was used as food. Omission of the
cellulose did not affect the concentration of the protozoa, but omission of the
grass resulted in some decrease in numbers.

162 R. E. HUNGATE

The apparent inability of E. caudatum to use cellulose in the cultures sug-
gested that cellulase was lacking. This was found to be the case when extracts
were tested. No cellulase could be demonstrated even though the extracts were
more concentrated than those giving positive tests in the case of Diplodinium.

CELLULOSE DIGESTION BY OTHER RUMEN PROTOZOA

The species of rumen protozoa reared thus far in the laboratory constitute
only a small fraction of those inhabiting the rumen. However, the demonstration
that Diplodinium species digest cellulose and that Entodinium caudatum does not,
makes it possible to draw some fairly satisfactory conclusions regarding most of
the other rumen protozoa.

Species of Diplodinium resemble each other in their habit of ingesting large
quantities of cellulosic plant materials. Out of four species of Diplodinium
studied, a cellulase has been found in three and all evidence points to its presence
in the fourth. The habit of ingesting large quantities of cellulosic materials thus
seems to be accompanied by an ability to digest cellulose. Entodinium caudatum
ingests very little cellulose and forms no cellulolytic enzyme. This correlation
between the presence of cellulase and ingestion of cellulose probably holds also
for the other rumen protozoa. Species ingesting large quantities of large plant
particles digest cellulose whereas others do not. Their capacity in this respect
can be ascertained by a direct microscopic examination of fresh rumen contents.

Among the rumen protozoa which have been examined during the present
investigation only species of Diplodinium have been observed to ingest large
quantities of plant materials. The other protozoa studied have included species
of Entodinium, Isotricha, Dasytricha, and Biitschlia and in none of them have
plant parts been observed to be ingested in an amount comparable to that in
Diplodinium. It is unlikely that any of them digest cellulose.

The question of the utility of the rumen protoa to their host has hinged largely
on whether they digest cellulose (Becker, Schulz and Emmerson, 1929). The
ruminant itself produces no cellulase and its utilization of cellulose is thus de-
pendent upon the microorganisms in the rumen. Those which digest cellulose
aid the ruminant and may be classified as symbionts, whereas others are either
slightly harmful or at best merely commensals. On this basis the various species
of Diplodinium may be regarded as symbionts but Entodinium, Isotricha,
Dasytricha, and Btitschlia are not.

SUMMARY

Diplodinium maggii, D. multivesciculatum, D. denticulatum, and Entodinium
caudatum can be grown in flask cultures using as substrates grass, cellulose, and
ground wheat, either singly or in combination, depending on the species being
cultured.

Clone cultures of D. denticulatum and E. caudatum show wide variations in
the morphology of individuals in the clone.

A rapid synthesis of food reserves from cellulose has been demonstrated in
D. maggii.

The results of cultural studies and of experiments on cellulose digestion by
extracts of the protozoa show that the three species of Diplodinium digest eel-

CELLULASE IN RUMEN PROTOZOA 163

lulose but E. caudatum does not. Microscopic observations indicate that all
species of Diplodinium digest cellulose, whereas Entodinium, Isotricha, Dasy-
tricha, and Biitschlia do not. Thus, Diplodinium is the only protozoan which
may be considered a symbiont.

LITERATURE CITED

BECKER, E. R., J. A. SCHULZ, AND M. A. EMMERSON, 1929. Experiments on the physiological
relationships between the stomach infusoria of ruminants and their hosts, with a bib-
liography. Iowa State Coll. Jour, of Sci., 4: 215-241.

DOGIEL, V. A., 1927. Monographic der Familie Ophryoscolecidae. Arch. Protistenk., 59: 1-288.

HUNGATE, R. E., 1942. The culture of Eudiplodinium neglectum with experiments on the diges-
tion of cellulose. Biol. Bull., 83: 303-319.

POLJANSKY, G., AND A. SxRELKOW, 1934. Beobactungen u'ber die Variabilitat einiger Ophryo-
scolecidae (Infusoria Entidoniniomorpha) in Klonen. Zool. Anzeiger, 107: 215-220.

SCHULZE, P., 1924. Der Nachweis und die Verbreitung des Chitins mit einem Anhang iiber das
komplizierte Verdauungssystem der Ophryoscoleciden. Zeitschr. f. Morph. u. Okol., 2:
643-666.

TRIER, H. J., 1926. Der Kohlehydratstoffwechsel der Panseninfusorien und die Bedeutung der
griinen Pflanzenteile fur diese Organismen. Zeitschr. f. vergl. Physiol., 4: 305-330.

METHYLENE BLUE, POTASSIUM CYANIDE AND CARBON
MONOXIDE AS INDICATORS FOR STUDYING THE
OXIDATION-REDUCTION POTENTIALS OF
DEVELOPING MARINE EGGS

MATILDA MOLDENHAUER BROOKS

(The Marine Biological Laboratory, Woods Hole, and the University of California, Berkeley)

The effects of inhibitors and accelerators of oxidations in marine eggs have
been studied first by Warburg (1910) using sea urchin eggs. Riinnstrom (1930)
made a detailed study of the effects of carbon monoxide, potassium cyanide, and
methylene blue. Later on this was studied by Clowes and Krahl (1940). They
used two stages of sea urchin eggs, unfertilized and fertilized. The present experi-
ments1 extend this work to further stages of Arbacia punctulata and Asterias
forbesii, and measure the effects of the mentioned reagents on their oxygen con-
sumption and development.

METHODS

Materials. Eight of these stages of Arbacia punctulata (Lam.) or Asterias
forbesii (Desor.) were used: unfertilized and fertilized eggs, early cleavages,
morula, blastula, early gastrula, late gastrula, and pluteus. The inhibitors of
respiration were CO, 99.5 per cent pure,2 and KCN, 5 X 10~4 M; and the accel-
erator, methylene blue, .002 percent. These concentrations were higher than
those used by Riinnstrom and therefore caused more pronounced effects.

Procedure. The experiments were done at Woods Hole, Massachusetts, from
June to August, 1941. The oxygen consumption of a stated volume of eggs or
larvae was measured in cubic millimeters per hour, according to the Barcroft-
Warburg method, in a standard set. The technique used was that described by
Dixon (1934). The vessels were standard conical type with wells inside to hold
the KOH (.2 co. of 20 per cent KOH) for absorption of CO2 and a side-arm or

1 In a preliminary report (Brooks, 1941) lower concentrations of KCN and methylene blue
were used, viz., 2.5 X 10~4 M and 1.2 X 10~4 per cent. These concentrations were nearer the
optimum value for antagonism experiments.

2 Carbon monoxide was generated in the usual way by the reaction between formic acid and
boiling concentrated H2SO4. This generator had been previously operated for several hours, and
the gas was led directly by way of a condenser through soda lime to the Warburg respiration
chamber. It was vented through the three-way cock at the top of the manometer, the Brody
solution in the manometer forced up to the cock and then allowed to settle back to the mark (20).
The direction of the flow was reversed several times, and the Brody solution allowed to fall to
the zero mark again under flowing CO after being forced up to the cock. The gas consumption
in the CO experiments, occurring principally in the first hour, suggests that air was trapped in
dead spaces or that oxygen diffused into the system through the rubber connections as the CO
was being led into the manometer. The amount of gas consumption in subsequent experiments
indicates that not more than 0.5 per cent Qz was present in the vessels. An analysis of the method
was made by Dr. W. B. Amberson by means of the Haldane method. This showed that there
was 0.5 per cent O2 present in the sample tested.

164

OXIDATION IN MARINE EGGS 165

onset which contained the KCN or methylene blue solution, or sea water to be
added at the expiration of the control readings. The eggs were collected by the
methods recommended by E. B. Harvey and centrifuged at about 3000X gravity
for one minute to give a concentrated suspension of loosely-packed eggs. They
were not tightly packed, to avoid injury. Subsequent fertilization tests showed
that 95 to 100 per cent fertilization membranes and normal cleavage were ob-
tained. Four cc. of this suspension so obtained was diluted with sea water to a
total volume of 30 cc. From this suspension 2 cc. was placed in each of the 12
Warburg vessels, two vessels being reserved for controls, lacking eggs. These
twelve vessels were used for the measurement of oxygen consumption of develop-
ing eggs, including the vessels being used for normal control of eggs in sea water.
The following conditions were established in one or another of the experiments:

1. Control eggs in sea water, as above, always used.

2. Eggs in sea water to which methylene blue, (.002 per cent) was added.3

3. Eggs in sea water in an atmosphere of CO (99.5 per cent).

4. Eggs in sea water to which KCN (5 X 10~4 M) was added.

5. Eggs in sea water with atmosphere of CO or in sea water plus KCN to
which methylene blue (.002 per cent) was added.

To produce these conditions two procedures were involved: a) For KCN and
methylene blue .5 cc. of a solution was placed in the side-arm of the vessel in
such a concentration as to give the desired concentration when mixed with the
eggs. This admixture was added without opening the vessels following a period
of one hour in which the oxygen consumption had been measured, b] Warburg
vessels in which CO was used were filled with CO at atmospheric pressure by
prolonged passage of freshly generated CO. Comparisons were instituted
between the oxygen consumption formed in these with and without subsequent
addition of methylene blue, and between these, CO-filled vessels and simultane-
ously air-filled vessels. The two vessels lacking eggs contained:

6. Vessels without eggs, but containing sea water.

7. Barometric control.

No attempt was made to measure the number of eggs used, as these experi-
ments were not designed for absolute measurements of single eggs, but of the
relative changes in the rates of approximately equal numbers of eggs as affected by
the listed reagents.

To make the procedure more comprehensible the details of a typical experi-
ment are here given. There were 14 manometers in all, run simultaneously.
Each set as listed in Tables I and II consisted of four different combinations in
triplicate, plus the two controls listed above (No. 6 and No. 7). For example,
there were three vessels in which eggs and sea water alone were used (controls) ;
three vessels in which eggs in sea water with an atmosphere of CO were used;
three vessels in which eggs in sea water to which methylene blue was added later;
and three vessels in which eggs in sea water with an atmosphere of CO were used

3 Fresh chemicals for each experiment were used. To dissolve methylene blue in sea water,
it was rubbed up in a few drops of distilled water and smoothed to a paste before adding the sea
water. The desired concentration was then made from this concentrated solution by adding
sea water. In this way the error caused by adding a few drops of distilled water became negligible.

166

M. M. BROOKS

TABLE I

Effect of methylene blue, CO and KCN in various combinations upon the oxygen consump-
tion of different stages of development of A sterias forbesii and Arbacia punctulata. In each set
there are four combinations in triplicate (in addition to the two controls). Columns 3 to 9
represent the average cmm. O2 consumed per hour in each set. Probable error is less than 5 per
cent of the mean. Concentration of KCN is 5 X 10~4 M; methylene blue, .002 per cent; CO,
99.5 per cent, temperature 20°; pH, 8.05; shaking by 50-60 excursions per minute of 6 cm. ampli-
tude; volume of eggs presumably between 0.10 and 0.15 cc. in 2 cc. total per vessel.

In air

In CO

In air

In CO

Without

With

Without

With

KCN

Number of

methylene

methvlene

+ KCN

+methylene

+KCN

experiments

sets

blue

cubic mm. of Qi
per hour

cubic mm. of Oi
per hour

cubic mm. of Ch per hour

Imma

>ure eggs, ;

1 sterias

—

6.1

— -

—

3.6

Unferti

lized eggs,

A sterias

II.

—

III.

• —

—

Fertii

'zed eggs, /

! sterias

IV.

4.5

—

Unfertilized eggs,

A rbacia

— -

—

Early

cleavages, .

4 rbacia

VI.

—

VII.

—

irula, Arb(

icia

VIII.

—

istnla, Arb

acia

IX.

— •

—

• —

—

Early

gastrula, ;

i rbacia

XI.

—

1.0

2.5

—

XII.

—

Late {

lastrula, A

rbacia

XIII.

—

deus, Arbc

icia

XIV.

—

XV.

—

OXIDATION IN MARINE EGGS

167

TABLE II

The relative effects of various reagents on eggs and larvae of Arbacia punctulata and Asterias
forbesii recalculated from Table I. Columns A to E represent ratios X100 of rates of O2 con-
sumption in the different experiments. Column A: the rate in the presence of methylene blue
as compared with its absence; Column B: methylene blue and CO as compared with CO alone;
Column C: KCN as compared with the control; Column D: KCN and methylene blue as com-
pared with KCN; Column E: CO as compared with the control. Roman numerals refer to
experiments in Table I, representing different stages of development from immature eggs to
pluteus. Concentration of KCN, 5 X 10~4 M; methylene blue, .002 per cent; CO, 99.5 per cent.

Ratios

M.B.

M.B.-CO

KCN

KCN-M.B.

Control

KCN

Control

Number of
experiments

Per cent

—

Immature e±

<gs, Asterias
50

—

II.
III.

200

Unfertilized <
112

',ggs, Asterias
90

50
40

IV.

138

Fertilised eg
111

gs, Asterias

—

V. '

183

Unfertilised <
111

',ggs, Arbacia
92

— •

VI.
VII.

116

117

Early cleava
120

ges, Arbacia
16

133

VIII.

122

Morula,
100

Arbacia

—

IX.
X.

122
120

Blastula
200

Arbacia
14

300

XI.
XII.

100
100

Early gastri
100

da, Arbacia
3

250

XIII.

100

Late gastru
100

la, Arbacia

—

XIV.
XV.

138
133

Pluteus,
250

A rbacia
16

300

168 M. M. BROOKS

to which methylene blue was added later. This, plus the two controls listed
above, makes 14 vessels. In this way all the combinations necessary for one
experiment were done in triplicate with samples from the same suspension, at
the same time, with the same temperature and at the same rate of shaking. The
composition of the sets varied with the reagents used, but there was always a
control for eggs or larvae in sea water, for sea water without eggs and for barometric
pressure. In fact the experiments were doubly controlled: 1) Readings were
taken for one hour before the reagents were added from the onsets, so that the
contents of each separate manometer and vessel were read with and without the
addition of the experimental solution. 2) In addition to this, manometers and
vessels were set up containing eggs and sea water only, and onsets containing
sea water only which was added at the same time the experimental solutions
were added.

The temperature was kept at 20.0 ± .1° for all experiments.

In a few of the cyanide experiments, the Krebs method was used to equalize
the HCN pressure for the concentration of KCN used. No difference could be
found between the results obtained with or without this modification.

The rate of shaking of the manometers was about 50 to 60 round trips per
minute at an amplitude of 6 cm. This was found not to cause any injury to the
eggs as shown by subsequent tests on eggs fertilized after shaking. These
produced between 95 and 100 per cent development, which was the same as the
per cent development in the unshaken eggs. This rate of shaking also allowed
constant oxygen consumption for four hours or more. The experiments in most
cases were run not more than three hours. In all the experiments, the rate of
shaking was sufficient to keep the eggs evenly distributed throughout the solu-
tion, but not enough to injure them as shown by tests of fertilizability.

In measuring oxygen consumption in air, the rate during the first hour was
taken as the basis. At this point the contents of the onsets were added and the
measurements continued for one to two hours more or even longer. Readings
were taken every 10 minutes and the change in the slope of the curves so obtained
showed whether or not an effect was produced. The number of cmm. of O?
consumed during intervals of ten minutes was calculated according to Warburg's
equation (1924). Columns 3 to 9 of each table give the average number of cmm.
of oxygen per hour for each set of experiments.

The measurement of gas consumption, or removal when CO was involved,
involves other bases which are referred to below.

The probable error of the mean was less than 5 per cent of the mean in each case.

RESULTS
Effects on the rate of oxygen consumption

The striking differences between the oxygen consumption by unfertilized eggs
and by fertilized eggs is well known (Warburg, 1910). The differences in rate
in the various stages used in the writer's experiments are also significant. These
will be discussed under the separate headings below (Tables I and II).

Table I gives the figures for the number of cubic millimeters of C>2 consumed
by the eggs. The figures are the averages of several similar experiments, their
numbers being given in column 2. Since each set was done in triplicate these

OXIDATION IN MARINE EGGS 169

figures are to be multiplied by three to indicate the number of separate experi-
ments done.

Table II is a summary and compilation of the effects of the various reagents.
For example, under "A," methylene blue raises the rate of O2 consumption of
unfertilized Asterias eggs to 200 per cent of the control. Column "C" shows
that KCN depresses the rate of O2 consumption of early gastrulas to 3 per cent
of that of the control. Column "D" shows that KCN and methylene blue
acting together allow a rate of O2 consumption in early cleavages in Arbacia
which is 133 per cent of that found when KCN is used alone. The Roman
numerals refer to experiments in Table I, and represent different stages of
development.

Effects of methylene blue. This dye accelerates oxygen consumption. Unfer-
tilized Asterias eggs showed the greatest acceleration, the rate of O2 consumption
reaching 200 per cent of the controls, while unfertilized Arbacia eggs reached 185
per cent of the rate of the controls. After fertilization, the rate of O2 consump-
tion, with methylene blue dropped to 138 per cent of that of the controls in
Asterias, and 116 per cent of that of the controls in Arbacia. During the morula
and blastula stages in Arbacia the rate of O2 consumption with methylene blue
became 122 per cent of that of the controls. There was no effect in the early
and late gastrula stages but methylene blue increases to 138 per cent in the
pluteus stage.

Effects of potassium cyanide. Three concentrations of KCN were tried: (1),
5 X 10~4 (the concentration represented in the tables), (2), IX 10~3, and
(3), 1 X 10~5. In the first two concentrations, the rate of O2 consumption in
unfertilized eggs was slightly decreased (See table for (1)) but the third concen-
tration produced either no change or an increase. Only (1) was used here in
detail. After fertilization, KCN (5 X 10~4 M) produces a considerable decrease
in the rate of O2 consumption in all the forms, but this decrease is most pro-
nounced in the gastrula stage where it falls to 3 per cent of the controls. In the
early cleavage states the rate of O2 consumption with KCN is 16 per cent, in the
blastula, 14 per cent, and in the pluteus, 16 per cent of the controls. In immature
Asterias eggs, the rate of O2 consumption was decreased by KCN to 50 per cent
of that of the controls.

Effects of carbon monoxide. Where a low concentration of oxygen is used
together with approximately one atmosphere of CO, a normal rate of respiration
might be due to an adequate supply of oxygen. Amberson (1928) found that
the critical oxygen tension for marine eggs is from 5 to 3.5 per cent. Below this
the eggs rapidly cease respiring. Riinstrom's experiments (1930) were done
above this range of O2 tension, so that in this case it may well be that there was
enough oxygen present to supply the eggs. In the writer's experiments, CO,
99.5 per cent pure2 was used; here simultaneous runs were done with air and CO-
filled Warburg vessels. The former was taken as the control. Carbon monoxide
depressed the respiration to such an extent that after a few hours respiration
ceased entirely. The effects, even so, showed that respiration did not stop
instantly. The gastrula stage seemed to be the most sensitive, shown by ces-
sation of gas consumption before the other stages. The least effect was in the
unfertilized eggs. For example in column E, Table II, No. XII, the rate with

170 M. M. BROOKS

CO falls to 4 per cent of that of the controls; where as in unfertilized Arbacia,
it is 75 per cent. In other stages the rates are higher than in the early gastrula.

It is felt that this small absorption of gas may represent oxygen consumption
supported by the traces of oxygen present in the CO atmosphere. The experi-
ments by Loeb (1895) on the effects of hydrogen and nitrogen atmospheres on
echinoderm eggs and larvae give the same picture as these experiments with CO.
It is suggested that the effects of CO may result from the exclusion of oxygen,
thereby eliminating the oxidized forms of the enzymes.

Antagonism between methylene blue and inhibitors. When methylene blue was
added to eggs or larvae in an atmosphere of CO, there was an increase in the rate
of O2 consumption. However, in the concentration used here, (0.002 per cent)
the maximum antagonistic action was not obtained. The antagonism between
methylene blue and carbon monoxide in optimum concentrations has been
discussed elsewhere (Brooks, 1935, 1941).

Additive effect of cyanide and carbon monoxide. — When cyanide and CO were
used together, the rate of uptake of gas was even less than when either was used
alone. Only immature and unfertilized Asterias eggs were used here (see Table
I, Column 9).

RESULTS

Effects on Development and Survival

After the measurements had been made on oxygen consumption, the eggs or
embryos were taken out of the manometer vessels and samples of them returned
to sea water. To do this, the eggs in the vessels were lightly swirled to give an
even suspension, after which .1 cc. of egg suspension was withdrawn and dis-
charged into 200 cc. of fresh sea water in 15 cm. finger bowls. Development
occurred at the temperature of the running sea water in the laboratory, 16 to 22°
C. depending upon the month. About one cmm. of dry sperm was suspended
in 200 cc. of sea water, well mixed, and one drop of this suspension added to
each finger bowl. After the eggs had settled, the sea water was renewed to get
rid of the sperm. The development of about 100 eggs per sample was followed.

Effects of methylene blue. Methylene blue, 0.002 per cent in sea water, had
acted upon the eggs for a time between two and three hours. Fertilization of
unfertilized eggs gave success equal to or better than that found in untreated
eggs (controls). During the main spawning season this was between 80 and 100
per cent formation of fertilization membranes, but at the beginning of the season,
this was much lower (from 10 to 50 per cent): the controls gave as low as or
lower percentages of success than the methylene blue-treated eggs.

Development was accelerated by methylene blue; when the earlier stages
were concerned, it was noted that many blastulae were produced among the
treated individuals in the time in which only a few were found in the controls.

The plutei produced from eggs or from the earlier developmental stages were
measured and the largest ten to 15 in each sample measured. It was found that
the average of the total lengths of the controls was 280 M as compared with 420 n
in the methylene blue-treated samples. Embryos experimented upon in the
gastrula stage gave plutei without arms (controls), in the time in which the
methylene blue-treated ones developed large arms. The pluteus stage was still
active at the end of nine days while the controls survived only four days. It

OXIDATION IN MARINE EGGS 171

may be noted that in toxic concentrations, methylene blue accelerates the
cytolysis of unfertilized eggs and to a less extent that of fertilized eggs.

Effects of carbon monoxide, with and without methylene blue. In these experi-
ments eggs or embryos were exposed to CO (99.5 per cent pure) alone, or together
with 0.002 per cent methylene blue, for a period of time between two and three
hours. Experimental and control lots were then returned to sea water as
described above. In contrast with cyanide which allowed substantial recovery,
CO alone stopped movement of the embryos irreversibly, and induced subsequent
cytolysis. Unfertilized eggs treated with CO alone lost the capacity to divide
when they were subsequently fertilized. The addition of methylene blue acting
simultaneously with CO, restored the capacity for subsequent fertilization.
Fertilized eggs treated with CO alone lost the power to cleave in the recovery
period and were cytolyzed. Addition of methylene blue was ineffective. Carbon
monoxide alone permanently stopped the motion of gastrulae and plutei, and led
to cytolysis within 24 hours. Addition of methylene blue failed to restore motility ,
but it did prevent cytolysis during at least the 48 hours of observation.

Effects on staining and reduction of methylene blue. Living eggs in methylene
blue alone showed stained granules in the cytoplasm but the nucleus and the
cytoplasmic matrix were not visibly stained. The only. exception to this was
in the case of cyanide and methylene blue experiments where the nucleus was
stained in addition to the granules. Since these were still swimming, they could
not be considered to be dead.

When methylene blue was used in conjunction with carbon monoxide, the dye
was presumably reduced since no color remained in either the cells or in the sur-
rounding solution while the manometer vessels were closed. Methylene blue is
not reduced in an atmosphere of carbon monoxide or in the absence of oxygen,
in the absence of living cells. Living cells must therefore furnish the hydrogen
for the reduction of the dye.

DISCUSSION

In the past it has been customary to account for the fact that cyanide fails
to block oxygen consumption of many types of cells beyond a minimum by
inferring that there exists a "cyanide-resistant" respiration, in addition to the
"cyanide-sensitive" respiration. Many cases of "cyanide-resistant" animals or
plants have been reported. For example, Lund (1918) found that cyanide does
not affect the respiration of Paramoecium; Shoup and Boykin (1931), confirmed
this in detail; Pett (1936), showed that a certain yeast is not affected by cyanide.
Dixon and Elliott (1929), found that only a portion of the respiration of at least
one strain of yeast is affected by cyanide. When cyanide does have an in-
hibitory effect as shown for rat liver by Dixon and Elliott (1929), this could be
completely reversed by quickly rinsing the tissue with buffer solution, even when
the concentration of cyanide was M/30. A resume of some cyanide-resistant
tissues is given by Dixon and Elliott. In some cases, activation is produced by
cyanide. This has been shown by Reynolds (1924) in the case of Fusarium; by
Hanes and Barker (1931) for potato tubers; by Tomkins (1932) for moulds; by
Watanabe (1932) for certain algae; and by Kisch (1933) in the case of certain
mammalian tissues. An interesting point is that the formation of flavin in yeast
is activated by cyanide as shown by Pett (1936).

172 M. M. BROOKS

The extent of this cyanide respiration suggested to some an explanation on the
basis of a reversible combination between CN~ and one enzyme according to
Warburg's postulation, a reaction whose extent can be predicted with the use
of a reaction constant. Ross (1938) has offered such a calculation on Nitella
oxygen consumption. Fisher and Ohnell (1940) have developed this idea in
greater detail.

However, since cyanide gives different effects not only in a wide variety of
tissues but also in the different stages of the developing echinoderm eggs and
embryos, it is felt that these results can be explained on a basis quite independent
from the postulation of several different enzyme systems, each with its com-
bination constant.

The interpretation offered by the writer is based upon the relation between
the rate of oxygen consumption and the redox potential surrounding the living
cells (Heymans and Heymans, 1922; Genevois, 1928; Barron, 1930). In develop-
ing this theory, the terminology of Clark (1927) is used. According to this, the
redox potentials (Eh values at pH 7.0) for observed aerobic cells were found to
be not far from 0.0 volts (Needham and Needham, 1925; Brooks, 1926). The
workers mentioned above found that as the redox potential becomes more
positive, within certain, limits of Eh and pH, the oxygen consumption increases.

When methylene blue acts on living tissues whose Eh is less than that of the
methylene blue, the poising influence of methylene blue tends to raise the Eh
toward that of methylene blue. The couple, methylene blue: leuco methylene
blue, has an Eo of around 0.0 volts at pH 7.0. The oxidized form, methylene
blue, has been added in these experiments, and should poise the solution and eggs
toward positive levels. It is logical to conclude that a rise in redox potential
induced by this dye is due to this poising effect of the dye. At a constant con-
centration of the dye, this rise in Eh would increase with increasing disparity
between the Eh level of the dye and that of the living cell.

Conversely, if there is no effect of methylene blue, when added to suspensions
of living cells, one may well conclude that the Eh of the cells is at the same level
as that of the dye solution itself. Therefore, we may essay an explanation on
the basis that the Eh levels of living cells are shown by the magnitude of the
effect of methylene blue on their oxygen consumption.

The following deductions are accordingly made on this basis. Unfertilized
Asterias and Arbacia eggs have a lower redox potential than fertilized ones,
since methylene blue produces a pronounced increase in the rate of O2 consump-
tion. The redox potential of the larvae in the early stages after fertilization is
below optimum but rises in the gastrula stage to optimum. Here, there is no
effect of methylene blue in these experiments. In the pluteus stage, there is
again a decrease in the redox potential. It should be remembered that there
must be an optimum Eh, above or below which a decrease in O2 consumption
occurs. A small change in Eh around the optimum must have practically no
effect, while an equal change at more or less positive potentials would produce
increasingly marked effects in oxygen consumption.

The lowered redox potential 4 of KCN, produced by its poising action in

4 The work of Pett (1936) who found that cyanide increases the flavin content of yeast can
be interpreted from the point of view of redox potentials; namely that the cyanide produced a
more negative potential which was more favorable to the production of flavin.

OXIDATION IN MARINE EGGS 173

connection with an unknown oxidant (Barnard, 1933), rather than the reactions
of a stable chemical combination of CN~ and the iron of the heme radical of the
respiratory enzyme, can be used as a basis in interpreting the reduction of
oxygen consumption caused by cyanide solutions. When there is no effect of
KCN on the rate of oxygen consumption, it appears that the redox potential is
already negative and is not lowered further by the addition of the reagent.
Here the normal redox potential within the cells appears to be essentially the
same as that which would be produced by KCN in the concentration used.
This seems to be the case in unfertilized eggs. This conclusion agrees with the
results with methylene blue, which show a large increase in the rate, indicating
that methylene blue raises the potential to a considerable extent.

It is also interesting that in the gastrula stage KCN reduces the rate of oxygen
consumption to its lowest level, whereas methylene blue causes no change in the
rate. It seems as though this stage has the highest redox potential of all those
investigated. These experiments with KCN and methylene blue therefore
appear significant in that KCN produces a lower potential, and the dye" a higher
one. This explanation has been offered previously by the writer (Brooks, 1935,
1941) to explain the action of methylene blue when cyanide is added to cells.
It is merely a balancing between the two reagents to keep the redox potentials
normal. Since different cells have different redox potentials, it will be necessary
to use appropriate concentrations of KCN and methylene blue as determined by
experimentation.

Here it is further suggested that these two reagents can be used as indicators
of the redox potential of a cell. Since the cell's redox potential becomes more
negative with increasing concentrations of KCN (within limits), one can ascertain
the original redox potential by noting the effect of KCN on the rate of oxygen
consumption. Conversely, methylene blue can be used to change the EH in the
opposite direction.

In these experiments no attempt was made to obtain the maximum antago-
nistic effects of methylene blue with KCN and CO. This has been done in other
papers (Riinnstrom, 1930; Brooks, 1935). This paper shows merely the effects
of adding the reagents in the concentration used in this paper. There is an
antagonistic effect in each case, but optimum rate (Qo2) has not been sought.

In the system of oxidative and reductive enzymes in the egg and embryo
of Arbacia and Asterias, the concept of the optimum must be applied. However
it is necessary to assume that all of these enzymes must be in equally favorable
states. For a given enzyme the highest rate of reaction, here thought of in terms
of optimum oxygen consumption, occurs at the Eh where the reduced and
oxidized states of this enzyne are equal in (active) concentration. One of the
enzymes of such a system may be nearly oxidized and another at the same Eh
may be nearly reduced. But at some optimum Eh, the best integration of their
action must occur. This optimum is more nearly attained in fertilized eggs of
these forms rather than in unfertilized eggs. It does not seem reasonable to
assume that there are little or no respiratory enzyme systems in unfertilized eggs
because they have a low rate of oxygen consumption. On the contrary, since
methylene blue produces such a profound increase in the oxygen consumption,
it would appear that this reagent poises the potential at an optimum where the
activities of oxidants and reductants of the respiratory enzyme systems more

174 M. M. BROOKS

nearly approximate each other. It would further appear that the process of
fertilization in these eggs raises the redox potential to a more positive level which
is closer to the optimum. Unless the unfertilized egg does not contain any
respiratory enzyme (cytochrome oxidase), then any concentration of KCN should
produce a decrease in the rate, if it is true that CN~ combines with the cytochrome
oxidase as is postulated by Warburg (I.e.) and repeated by many others for
example, Barren (1937), Krahl, Keltch, Neubeck and Clowes (1941), Henderson
(1938), Wendel (1933, 1934). It seems preferable to think of these living cells
of this nature as containing such an enzyme system, and the hypothesis of com-
bination seems to be unessential.

The present experiments may be further tested in the light of additional
evidence against the assumption that the effect of KCN on living cells is due to
a combination of CN~ with the Fe of the heme of the cytochrome oxidase
(respiratory enzyme), thereby inactivating it. This enzyme has not been isolated
and there are no direct experiments to prove its existence. The evidence is
derived from measurements of oxygen consumption. Those who favor the
hypothesis that CN~ unites with the heme of the cytochrome oxidase, base their
experimental evidence on chemically isolated compounds such as the hemo-
xhromogens and combinations of CN~ with these at high pH values, i.e., 9.0 to
13. (Hogness, Zscheile, Sidwell and Barron (1937), Barren (1937), Clark
(1939), Clark, Taylor, Davies, and Vestling (1940). It appears quite evident,
however, that such results at these unphysiological pH values can not be used to
explain the conditions found in living cells. Theorell (1940) using cytochrome c
(an oxidation link in most cells, which is also a heme compound and has been
isolated), showed that CN~ forms combinations with this compound only in
acid or alkaline solutions but not at pH values around 7.0 such as occur in living
cells. This can be considered as further evidence against the hypothesis that
CN~ combines with a heme such as the cytochrome oxidase which is supposed to
exist in living cells.

A further observation made here can be understood on the basis of this
concept: fertilized eggs to not cleave in CN~-containing media, but do form
multiple asters without cell division. This suggests that division requires a
higher redox potential, whereas aster formation goes on at lower redox potentials.

The effect of CO on the oxygen consumption of eggs and embryos of Asterias
and Arbacia points to an interpretation which also obviates the assumption of
combination of CO with an enzyme. Mere absence of oxygen suffices to explain
these observed results. The fact that Theorell (1940) found that cytochrome c
does not combine with CO at physiological pH ranges supports this.

It is of interest to recall in this connection the case of animals having hemo-
globin. It is generally conceded that the principal effect of CO in stopping
respiration is due to its affinity for hemoglobin, forming carbohemoglobin which
is a stable compound. In proportion to this conversion the blood fails to carry
oxygen to the tissues (Henderson, 1938). The effect of CO upon the oxidative
enzyme is therefore secondary through the inactivation of the hemoglobin. If
there is no hemoglobin present as in the case of marine eggs, one is led to conclude
that in this case also the effect is due to lack of oxygen rather than to a specific
effect such as combination of CO with the cytochrome oxydase. At the present
writing it is not known whether there exists in Arbacia eggs a substance similar

OXIDATION IN MARINE EGGS 175

in action to the hemoglobin of mammals to combine with CO. The probability
is, however, that the principal effect of CO on these cells is due to its replacement
of oxygen.

CONCLUSION

The experiments here described appear to find their simplest explanation on
the basis of the relation between the redox potentials and the rate of oxygen
consumption of the cells. Development of the larvae falls into this picture.
The effects of carbon monoxide seem to result only from the practical exclusion
of oxygen. The converse action of cyanide and methylene blue supports this
concept: cyanide stops oxygen use most effectively when acting on the stage of
eggs or larvae least accelerated by methylene blue (the gastrula) and least
effectively when acting on the stage (unfertilized eggs) most strongly accelerated
by methylene blue. This relation is borne out in detail in all stages; and is sup-
ported by minor observation.

Changes in redox potentials within the eggs are considered to be established
by the presence of reduced and oxidized metabolites, and reduced and oxidized
links in the enzyme chain of oxidation. All of these redox participants must be
affected by the introduction of redox agents like methylene blue and cyanide
which act through their poising action. Their effects must lead toward or away
from an optimum EK in which the oxidizing enzyme chain acts most rapidly.

The interrelationship between the oxidation chain and the prevalent Eh values
is a master key in understanding the changes in rate of oxygen consumption
during development of echinoderm eggs, normally and under the influence of
reagents. This concept is of general applicability.

SUMMARY

The oxygen consumption of eggs and larvae of Asterias forbesii and Arbacia
punctulata was measured by the Warburg-Barcroft technique, in sea water, in
sea water solutions of KCN (5 X 10~4 M) and methylene blue (0.002 per cent)
and with atmospheres of carbon monoxide (99.5 per cent pure). Eight stages
were studied: unfertilized and fertilized eggs, first cleavages, morula, blastula,
early and late gastrula, and pluteus. Subsequent development after a period of
two to three hours exposure to these reagents was followed in sea water.

Methylene blue increased oxygen consumption most when acting on unfer-
tilized eggs, did not increase it for gastrula, and increased it slightly in the other
stages. When transferred to sea water, the effects of methylene blue persisted
in increasing the rate of development of larvae, prolonged their life and produced
larger plutei.

Cyanide decreased oxygen consumption most strongly when acting on
gastrulae, less so for other stages, and had little or no effect on unfertilized eggs.

These two agents have a converse action. This was shown by the antagonism
between these two, as shown in this paper by the promotion of motility in cyanide
paralyzed forms.

Carbon monoxide prevented gas consumption, subsequent fertilization and
produced cytolysis. Methylene blue promoted the subsequent fertilization of
CO-treated eggs.

176 M. M. BROOKS

These results are most simply accounted for on the assumption that the redox
potential changes to a more positive level during progress from the egg to the
gastrula stage and thereafter drops slightly. Methylene blue raises and cyanide
depresses the positive redox potential. Carbon monoxide, as used in these
experiments, indirectly depresses the redox potential by preventing the oxidized
forms of the enzymes to exist. These effects take place in an oxidative enzyme
chain whose members undergo reduction or oxidation. The whole system suffers
changes under the named reagents, leading towards or away from the optimum
EH levels and maximum oxygen consumption.

Acknowledgment is made for the assistance granted by the Ella Sachs Plotz
Fund and the Board of Research of the University of California. The technical
assistance was done by Miss Patricia Perkins and Mr. R. R. Ronkin. My
thanks are herwith expressed to Dr. W. B. Amberson for his kindness in making
the Haldane analysis for oxygen.

LITERATURE CITED

AMBERSON, W. R., 1928. The influence of oxygen tension upon the respiration of unicellular

organisms. Biol. Bull., 55: 79-91.

BARNARD, R. D., 1933. The effect of cyanide and a variation in alkalinity on the oxidation-
reduction potential of the hemoglobin-methemoglobin system. Jour. Gen. Physiol., 16:

657-675.
BARRON, E. S. G., 1930. The catalytic effect of dyes on the oxygen consumption of living cells.

Jour. Gen. Physiol., 13: 483-494.
BARRON, E. S. G., 1937. Oxidation-reduction potentials of blood hemin and its hemochromogens.

Jour. Biol. Chem., 121: 285-312.
BROOKS, M. M., 1926. Studies on the permeability of living cells. Amer. Jour. Physiol., 76:

360-379.
BROOKS, M. M., 1935. Methylene blue and hemoglobin derivatives in asphyxial poisoning.

Amer. Jour. Physiol., 114: 160-178.
BROOKS, M. M., 1941. Infra-red spectrophotometric studies on hemoglobin as affected by

cyanide, methylene blue and carbon monoxide. Amer. Jour. Physiol., 132: 311-320.
CLARK, W. M., 1927. Studies in oxidation-reductions. Hygienic Lab. Bull. 151, U. S. Pub.

Health Service, Washington, D. C.
CLARK, W. M., 1939. Potentiometric and spectrophotometric studies of metalloporphyrins in

coordination with nitrogenous bases. Cold Spring Harbor Monograph, 7: 18-32.
CLARK, W. M., J. F. TAYLOR, T. H. DAVIES, AND C. S. VESTLING, 1940. Metalloporphyrins.

Jour. Biol. Chem., 135: 543-68.
CLOWES, G. H. A. AND M. E. KRAHL, 1940. Oxygen consumption and cell division of fertilized

sea urchin eggs in the presence of respiratory inhibitors. Jour. Gen. Physiol., 23: 401-

411.

DIXON, M., 1934. Manometric methods. Cambridge University Press.
DIXON, M., AND ELLIOTT, K. A. C., 1929. The effect of cyanide on the respiration of animal

tissues. Biochem. Jour., 23: 812-830.
FISHER, K. C., AND OHNELL, R., 1940. The steady state frequency of the embryonic fish heart

at different concentrations of cyanide. Jour. Cell. Comp. Physiol., 16: 1-13.
GENEVOIS, L., 1928. Coloration vitale et respiration. Protoplasma, 4: 67-87.
HANES, C. S., AND BARKER, J., 1931. The physiological action of cyanide. Proc. Roy. Soc.,

B, 108: 95-118.

HENDERSON, Y., 1938. Adventures in respiration. Williams and Wilkins, Baltimore, Md.
HEYMANS, J. F., AND HEYMANS, C., 1922. Hyperthermie et augmentations du volume respira-

toire.et de relimination de 1'anhydride carbonique par le bleu de methylene. Archiv.

Inter, pharmacodyn. Therap., 26: 443.
HOGNESS, T. R., F. P. ZSCHEILE, A. E. SIDWELL, AND E. S. G. BARRON, 1937. Cyanide hemo-

chromogen. Jour. Biol. Chem., 118: 1-14.

OXIDATION IN MARINE EGGS 177

KISCH, B., 1933. Steigerung der Gewebsatmung durch kleine cyanmengen. Biochem. Zeit.,

263: 187-197.
KRAHL, M. E., A. K. KELTCH, C. E. NEUBECK, AND G. H. A. CLOWES, 1941. Cytochrome

oxidase activity in eggs of Arbacia punctulata. Jour. Gen. Physio!., 24: 597-617.
LOEB, J., 1895. Untersuchungen iiber die physiologischen Wirkungen des Sauerstoffmangels.

Archill, ges. Physiol., 62: 249-294.
LUND, E. J., 1918. The rate of oxidations in Paramoecium caudatum and its independence of

the toxic action of KCN. Amer. Jour. Physiol., 45: 365-373.
NEEDHAM, J., AND NEEDHAM, D. M., 1925. H-ion concentration and the oxidation-reduction

potential of the cell. Proc. Roy. Soc. London, B, 98: 259-286.

PETT, L. B., 1936. Studies on yeast grown in cyanide. Biochem. Jour., 30: 1438-1445.
REYNOLDS, E. S., 1924. Some relations of Fusarium lini and KCN. Amer. Jour. Bot., 11:

215-218.
Ross, E., 1938. The effects of sodium cyanide and methylene blue on oxygen consumption of

Nitella. Amer. Jour. Bot., 25: 458-463.
SHOUP, C. S., AND BOYKIN, F. T., 1931. The insensitivity of Paramoecium to cyanide and the

effects of iron on respiration. Jour. Gen. Physiol., 15: 107-118.
RUNNSTROM, J., 1930. Atmungsmechanismus und Entwicklungserregung bei dem Seeigilei.

Protoplasma, 10: 106-173.

THEORELL, H., 1940. Structure and function of some enzymes. Amer. Assoc. Adv. Sci., Pub-
lication No. 14: 136-146.
TOMKINS, R. G., 1932. The action of certain volatile substances and gases on the growth of

mould fungi. Proc. Roy. Soc. London, B, 111: 210-226.
WARBURG, O., 1910. Uber die Oxydationen in lebenden Zellen nach Versuchungen am Seeigelei.

Zeitschr.f. physiol. Chem., 66: 305-340.
WARBURG, O., 1924. Verbesserte Methode zur Messung der Atmung und Glykolyse. Biochem.

Zeitschr. 152: 51-63.
WATANABE, A., 1932. Uber die Beeinflussung der Atmung von einigen griinen Algen durch

Kaliumcyanid und Methylenblau. Ada phytochim, Tokyo, 6: 315-335.
WENDEL, W. B., 1933. Methylene blue and cyanide poisoning. Jour. Amer. Med. Assoc., 100:

1054.
WENDEL, W. B., 1934. The mechanism of the antidotal action of methylene blue in cyanide

poisoning. Science, 80: 381 .

DEVELOPMENT OF THE PRIMARY GONADS AND DIFFEREN-
TIATION OF SEXUALITY IN TEREDO NAVALIS AND
OTHER PELECYPOD MOLLUSKS

WESLEY R. COE

(Osborn Zoological Laboratory, Yale University, and Scripps Institution of Oceanography,

University of California, La Jolla x)

In connection with studies on the sexual differentiation and changes of sex
in several genera of bivalve mollusks the writer has observed certain morpho-
logical characteristics of the gonads which seem not to have been reported
previously. These concern particularly the types of cells which compose the
gonads in the early stages of development and the provisions for the nourishment
of the forming gametes. They pertain also to the earliest sexual differentiation
of the primary gonia and their transformation into the functional gametes of the
primary sexual phase. Some confusion exists in the literature because of failure
to interpret correctly the significance of the two types of sexual cells which in
young individuals of Teredo navalis and other ambisexual mollusks characterize
this initial phase of functional sexuality.

ORIGIN AND EARLY DEVELOPMENT OF GONAD

In all the pelecypods examined the gonads originate from a group of cells
situated in the posterior portion of the body, near the visceral ganglion and on
the ventral side of the pericardium. As these primordial germ cells multiply
they become more or less completely separated into two groups, situated sym-
metrically on the two sides of the body. By the continued multiplication of their
constituent cells each group grows anteriorly in the surrounding mesenchyme
or vesicular connective tissue to form the branching system of tubular follicles
which characterizes the gonads of the pelecypods (Figure 1).

Two rather distinct types of gonads are found in these mollusks, each type
being characteristic of certain genera. In Teredo, Bankia, Mya, Petricola,
Barnea and some other members of the order Teleodesmacea the cells of the
gonad soon become differentiated into large, vacuolated follicle cells and primary
gonia. The follicle cells function as accessory nutritive cells. They occupy most
of the space within the tubular follicle, the primary gonia being scattered along
the periphery or near the central axis (Figures 1 to 4). In Ostrea, Pecten,
Mytilus, Yolsella and some other Prionodesmacea, on the contrary, the gonad is
composed almost entirely of gonia, with only minute follicle cells (Figures 5,6).
In these forms the gametogenic cells obtain their nourishment directly from the
surrounding vesicular connective tissue. An intermediate condition is found in
Anomia and some other genera in which the gonadal follicles are associated with

1 Contribution from Scripps Institution of Oceanography, New Series No. 193.

178

DIFFERENTIATION OF SEX IN MOLLUSKS

179

vesicular nutritive cells, often arranged in follicles (Figure 7), or they may be
composed of follicles with relatively few accessory nutritive cells.

The genital ducts of the adult open into the mantle cavity near the site of the
original gonadal primordium or, in some species, into the kidneys and thence to
the mantle cavity. When the individual has become sexuallv mature the branch-

FIGURE 1. Portion of section through posterior region of body of young Mya arenaria,
showing early stage in development of the tubular, branching gonads (g) composed of large,
vacuolated follicle cells with primary gonia (pg) scattered along the periphery; bl, vesicular con-
nective tissue; cvn, cerebro-visceral nerve cords; go, genital openings; k, kidney; /, intestine;
me, epibranchial chamber; pc, pericardial cavity. (Modified from Coe and Turner, 1938.)

ing gonadal follicles may extend throughout the mesosoma and, in the mussels
and a few other pelecypods, even into the mantle beneath one or both valves of
the shell.

SEXUAL DIFFERENTIATION

In Teredo navalis, which normally experiences alternating male and female
phases of functional sexuality, the gonadal primordia consist of groups of cells,
all of which are identical in appearance. But as soon as the primordia begin to
branch out to form the tubular follicles, as described in a preceding paragraph,
two distinct types of cells become distinguishable. At the end of each growing

180

WESLEY R. COE

follicle there is a cap of undifferentiated nuclei in a continuous mass of cytoplasm
(Figures 2, 4). As growth proceeds some of these nuclei become ecnlosed in large,
vacuolated cells which function thereafter as accessory nutritive cells. A smaller
number retain a larger portion of the terminal cytoplasm and become the primary
gonia. As the follicle increases in length these primary gonia are left behind on
the periphery of the follicle cells which otherwise fill the entire follicle (Figures
2, A, 4). The primary gonia multiply slowly as the follicles increase in length.

FIGURE 2. Development of primary gonad in Teredo navalis. A, sexually undifferentiated
branching follicle, composed of large, vacuolated follicle cells (fc) with primary gonia (pg) scat-
tered on the periphery; n, terminal cap of undifferentiated proliferating nuclei; vet, vesicular
connective tissue. B, later stage with differentiated ovocytes (oc) and spermatogenic cells (sp)
derived from the primary gonia; fc, remaining follicle cells with disintegrating nuclei and products
of abnormal spermatogenic cells. C, follicle of primary male phase, with spermatogenic cells
(sp) and spermatozoa filling the lumen and large ovocytes (oc) on the periphery.

No sexual differentiation can usually be detected in the primary gonia until
the young teredo has attained a length of about 10 mm. In the warm season
of the year, when the water is above 18 degrees C., this length may be reached
within three to four weeks after entering the wood. Dwarfed individuals become
sexually differentiated at a smaller size but at approximately the same age.

The spermatogonia are recognized as soon as the primary gonia begin pro-
liferation into groups of nuclei within a cytoplasmic syncytium. These soon
form primary spermatocytes with characteristic phases of synapsis (Figure 2, B).

DIFFERENTIATION OF SEX IN MOLLUSKS 181

Approximately half the total number of primary gonia become activated in this
manner. An equal number differentiate into ovogonia and their resulting
ovocytes.

Each of the rapidly growing ovocytes remains attached to the wall of the
follicle until fully mature. The spermatogenic cells, on the contrary, multiply
rapidly and soon fill the axis of the follicle (Figure 2, B). The follicle cells have
meanwhile begun to undergo cytolysis and disintegration. Their nuclei first
become pycnotic and are later cytolyzed. The collapse of the follicle cells forms
a central lumen in the follicle and in this cavity spermatogenesis is completed
(Figure 2, C).

With the continued growth of the ovocytes and a vast increase in the number
of spermatogenic cells the follicles become greatly distended and the former lumen
packed with spermatozoa (Figure 2, C). This is a typical hermaphroditic gonad
but since the spermatozoa will usually become ripe and will be discharged before
the ova are fully mature the individual is properly recognized as being in the
primary, or protandric, or ambisexual male phase.

• If all individuals experienced the same aspects of sexual differentiation there
could be no difference of opinion as to their status, but an examination of the
gonads of more than 3000 individuals shows wide variations in the proportion of
spermatogenic and ovogenic cells present. This is true not only for different in-
dividuals but for different follicles of the same individual (Coe, 1933, 1934a).
Photographs of some of these variations are shown by Coe (1933).

At one extreme are the so-called true males, in which the ovogonia, if present,
fail to produce ovocytes. At the other extreme there are proterogynic individuals
which form ovocytes exclusively in their first functional sexual phase, the usual
primary male phase being aborted or inhibited (Coe, 1935). In exceptional
individuals both spermatogonia and ovogonia are activated simultaneously,
producing functional hermaphrodites. Some of these are capable of self-
fertilization (Coe, 1941).

Both the true males and the proterogynic individuals must be considered as
exceptions to the great majority of the population in which each individual
normally functions first as male and later as female. The female phase will be
followed by a second series of male and female phases if the life of the individual
be sufficiently prolonged. This alternation or rhythm of sexual phases may be
considered as characteristic of the species, although the mortality is usually very
high before even two of the phases have been completed (Coe, 1934a).

Furthermore some of the typically protandric individuals approach more or
less closely the true males in having relatively few ovocytes in the gonads. They
retain the male phase longer than others which bear a closer resemblance to the
proterogynic individuals in the appearance of the primary gonads (Coe, 1934a,
1936).

Because of this variability in the expression of ambisexuality, as well as associ-
ated differences in the time necessary to complete any one of the sexual phases,
the proportion of individuals in each phase found in any sample of the population
would seem to have less significance than has sometimes been assumed (Grave
and Smith, 1936; Coe, 1936). It is obvious that if in such a sample twice as
many individuals are found in one sexual phase as in another it may be merely
indicative that the one phase lasts twice as long as the other. It might also

182

WESLEY R. COE

indicate that some individuals are older than others, since the sexual phases
appear in sequence during the life of the individual, with the exceptions already
mentioned.

During the summer months there will ordinarily be fully three to five times
as many individuals in the primary male phase as in the female phase at the first
spawning period. This may occur as early as six weeks after metamorphosis.
Three or four weeks later, after the change of sexual phase, the same individuals
may be expected to show an approximation to a reversed proportion of male and

FIGURE 3. Portions of primary gonads of Petricola pholadiformis. A, sexually undifferen-
tiated stage; follicle composed mainly of vacuolated, nutritive follicle cells (Jc) with primary
gonia (pg) along the central axis or lumen (lu). B, later stage in young male; most of the follicle
cells have been cytolyzed to supply nourishment for the proliferating spermatogenic cells (sp).

FIGURE 4. Development of primary gonad in Mya arenaria; central follicle with terminal
cap of undifferentiated nuclei (n), from which both follicle cells (fc) and primary gonia (pg)
originate; lower portion of follicle shows later stage in which the follicle cells are being replaced
by spermatogenic cells (sp), with spermatozoa adjacent to the lumen (In). Note blood vessels
(bl) and small amount of connective tissue between follicles.

female phases. There will be many deviations, however, since, as already men-
tioned, the primary sexual phase will be more prolonged in some individuals than
in others and some will change quickly to the third (ordinarily male) phase. In
the meantime younger members of the population will be functioning in the
primary male phase.

The proportion of individuals in which the first sexual phase is female is
thought by Grave (1942) to be larger than formerly reported (Coe, 1936) but the
data presented do not seem to justify such a conclusion.

DIFFERENTIATION OF SEX IN MOLLUSKS

183

The following scheme, modified from Coe (1935), indicates such variations in
the sequence of sexual phases as are inferred from a study of more than 3000
individuals at various ages. It is impossible to follow these phases in any single
individual because the teredo dies soon after it is removed from the wood for
examination. The presence of larvae in the gill chambers, however, is sufficient
proof that the preceding sexual phase of the gonad was female.

A ^

FIGURE 5. Ostrea lurida. A, portion of primary gonad showing typical ambisexual male-
phase condition, with peripheral layer of ovocytes (oc) and developing sperm balls (sp) in the
lumen; gd, ciliated genital duct. B, terminal portions of two follicles of secondary gonad in early
female phase surrounded by much vesicular connective tissue (vet).

FIGURE 6. Ostrea virginica. A, portion of primary gonad in ambisexual condition. B,
terminal portion of follicle surrounded by much vesicular connective tissue in early male phase
of adult; letters as in Figure 5.

1. True male phase (exceptional) . . . Second male phase . . .

2. Ambisexual male phase . . . First female phase . . . Second male phase
. . . Second female phase (if life be sufficiently prolonged).

3. Functional hermaphroditic phase (exceptional) . . . Male or female
phase . . .

4. Female phase (exceptional) . . . First male phase . . . Second female
phase . . .

5. Female phase (exceptional) . . . Second female phase (?)...

Many other pelecypods resemble the teredos in having the primary gonads
composed largely of nutritive follicle cells. In some of these, of which Petricola
pholadiformis may be taken as an example, the primary gonia are scattered along
the central axis of the follicle (Figure 3). In others, as Mya arenaria (Coe and
Turner, 1938), they are distributed along the periphery (Figure 4). In both
cases the follicle cells are cytolyzed during gametogenesis. With the exception

184

WESLEY R. COE

of an occasional hermaphrodite, all individuals of both species are strictly uni-
sexual and the two sexes are approximately equal in number.

A different type of gonad is found in both the larviparous and oviparous
oysters (Ostrea), in scallops (Pecten), mussels (Mytilus, Volsella), and numerous
other bivalves. In these the gonads are composed almost entirely of gametogenic
cells which receive their nourishment directly from the sourrounding vesicular
connective tissue. The follicle cells are few and small (Figures 5, 6). As the
gonadal follicles increase in size the surrounding nutritive connective tissue is
utilized.

The larviparous oysters, of which Ostrea lurida may serve as an example,
exhibit changes in sexuality during life, the sequence of sexual phases being similar

FIGURE 7. Anomia simplex. A, portion of primary gonad surrounded by nutritive tissues
(nf). B, follicles of mature ovary in which the nutritive tissues ' (nf) have been almost assimi-
lated; bv, blood vessels; m, mantle; gd, genital duct.

to those mentioned for Teredo navalis (Coe, 1934). In the oviparous oysters, as
0. mrginica, there is likewise a strong tendency toward protandry, since about
70 per cent of the young individuals first function as males (Coe, 1938). The
primary gonad usually contains antecedent cells of both sexual types (Figure 6, A).
Functional hermaphroditism occurs occasionally and a few individuals change
from the male to the female phase during their first spawning season. Thereafter
the individual functions in one sexual phase or the other during all of each spawn-
ing season, but not infrequently the sexual phase changes between two spawning
seasons (Burkenroad, 1937; Galtsoff, 1938; Coe, 1938; Loosanoff, 1942). In

DIFFERENTIATION OF SEX IN MOLLUSKS 185

Venus the sexes are separate, following a juvenile, usually protandric, sexual
phase (Loosanoff, 1937).

Gonads in some respects intermediate between the other two are found in
Anomia and in some other genera. Here the gonadal follicles, which are com-
posed almost entirely of gametogenic cells, are surrounded by nutritive tissues
of similar configuration (Figure 7). As in the other types of gonads the nutritive
tissues are utilized during the course of gametogenesis (Figure 7, B).

SUMMARY

Two more or less distinct types of primary gonads are found in bivalve
mollusks. The simplest type occurs in several families of the order Prionodes-
macea, where the profusely branching follicles on each side of the body are
composed mainly of gametogenic cells and each follicle is nourished by the sur-
rounding mesenchyme or vesicular connective tissue.

In the second type, found mainly in the order Teleodesmacea, the branching
follicles of the primary gonads are composed principally of large, vacuolated
follicle cells of a nutritive nature, the primary gonia being scattered along the
central axis or on the periphery. These nutritive cells are cytolyzed during
gametogenesis.

In some bivalves intermediate conditions are found, with associated gameto-
genetic follicles and nutritive tissues.

In the ambisexual or hermaphroditic species, as Teredo navalis, the primary
gonia are early differentiated into the two sexual types. Of these the spermato-
gonia usually, but not invariably, proliferate and complete gametogenesis in
advance of the ovogonia, giving each follicle the appearance of a spermary with
a layer of ovocytes on the periphery. As a general rule the ovocytes do not
become fully mature until after the discharge of the spermatozoa. There are
many variations in the relative proportion and time of spawning of the two types
of gametes, however, and in exceptional cases the gonad is almost exclusively of
the male or of the female type. Most individuals function first in the male
phase, then change to the female phase, while some individuals experience in ad-
dition a second sequence of male and female phases. Less frequently a primary
female phase precedes the first male phase. Individual differences in the com-
binations of the modifying hereditary sex factors are presumably responsible for
most of these variations.

Each species studied, except those that are strictly unisexual, shows some
variations in the sequence of male and female or functionally hermaphroditic
phases.

LITERATURE CITED

BURKENROAD, M. D., 1937. The sex-ratio in alternational hermaphrodites, with especial refer-
ence to the determination of sexual phase in oviparous oysters. Jour. Marine Research,
1: 75-84.

COE, W. R., 1933. Sexual phases in Teredo. Biol. Bull., 65: 283-303.

COE, W. R., 1934. Alternation of sexuality in oysters. Am. Nat., 68: 236-252.

COE, W. R., 1934a. Sexual phases in the pelecypod mollusk Teredo. Science, 80: 192-193.

COE, W. R., 1935. Sequence of sexual phases in Teredo, Ostrea and Crepidula. Anal. Rec.,
Suppl., 64: 81.

COE, W. R., 1936. Sequence of functional sexual phases in Teredo. Biol. Bull., 71: 122-132.

186 WESLEY R. COE

COE, W. R., 1938. Primary sexual phases in the oviparous oyster (Ostrea virginica). Biol.

Bull., 74: 64-75.

COE, W. R., 1941. Sexual phases in wood-boring mollusks. Biol. Bull., 81: 168-176.
COE, W. R., AND HARRY J. TURNER, JR., 1938. Development of the gonads and gametes in the

soft-shell clam (Mya arenaria). Jour. Morph., 62: 91-111.
GALTSOFF, PAUL S., 1938. Sex change and physiological intersexuality in Ostrea virginica.

(Abstr.) Anal. Rec., Suppl., 72: 42.
GRAVE, BENJAMIN H., 1942. The sexual cycle of the shipworm, Teredo navalis. Biol. Bull.,

82:438-445.
GRAVE, BENJAMIN H., AND JAY SMITH, 1936. Sex inversion in Teredo navalis and its relation to

sex ratios. Biol. Bull., 70: 332-343.
LOOSANOFF, VICTOR L., 1937. Development of the primary gonad and sexual phases in Venus

mercenaria Linnaeus. Biol. Bull., 72: 389-405.
LOOSANOFF, VICTOR L., 1942. Seasonal gonadal changes in the adult oysters, Ostrea virginica,

of Long Island Sound. Biol. Bull., 82: 195-206.

AN IMPROVED METHOD OF ASSAYING MELANIN IN FISHES J

F. B. SUMNER AND PETER DOUDOROFF

(Scripps Institution of Oceanography of the University of California, La Jolla)

Some five years ago, the authors described a method of evaluating the melanin
content of fishes (Gillichthys) kept under various experimental conditions, along
with some preliminary results obtained by the use of this method (Sumner and
Doudoroff, 1937). In the following year we published a brief account of similar
experiments, conducted upon another teleost, Gambusia affinis (Sumner and
Doudoroff, 1938). In these last it was found advisable to revise our earlier
procedure in important ways.

Despite certain obvious shortcomings in the technique employed in those
earlier studies, interesting quantitative relations were shown to obtain between
the melanin extracted from our fishes and the optic stimuli to which they had
been subjected. Whatever the precise nature of these relations, there could be
no reasonable doubt that fishes (of at least two species) which had lived for some
weeks upon black or dark gray backgrounds had a much higher melanin content
than fishes kept upon various paler backgrounds. This was in full agreement
with experiments involving the counting of melanophores, through which it had
been shown that both the number of these cells per unit area of skin and the
amount of melanin per cell may be greatly influenced by the background.2 Much
remained to be learned, of course, regarding the details of these relations.

In quantitative studies of melanin, gravimetric determinations seem to be
ruled out by the impossibility of isolating this substance without sacrificing a
large proportion of it during the process of isolation. Various methods of obtain-
ing pure melanin have been developed by chemists whose object has been to
purify it for chemical analysis, rather than to measure accurately the amount
present in a given sample of tissue (e.g., Gortner, 1910, 1911; Salkowski, 1920;
Heinlein, 1924).

Colorimetric methods were consequently resorted to early in our endeavors
to assay melanin. We know of only two previous workers (Kudo, 1922; Vilter,
1931) who had attempted the measurement of changes of melanin resulting from
experimental optical conditions, at least in fishes and amphibia. Recently, how-
ever, Dawes (1941) has published the results of some interesting experiments
upon Rana temporaria.5 His findings, so far as the effects of black and white
backgrounds are concerned, fully confirm our own. Considering the facts just
stated, however, it is obvious that Dawes is over-enthusiastic in his claim that
his work "affords conclusive evidence for the first time that prolonged exposure
of one of the lower vertebrates to ... black background results in a marked
increase in the melanin content of the skin."

1 Contributions from the Scripps Institution of Oceanography, New Series, No. 194.

2 The work of ourselves and others in this field has been summarized by Sumner (1940b).

3 Dawes's technique involved peptic digestion of the frogs' skins.

187

188 SUMNER AND DOUDOROFF

The employment of colorimetric methods in "melaninometry" is rendered
possible by the solubility of this substance in dilute alkali.4 Admittedly these
methods involve some rather serious difficulties. Some of the primary require-
ments in the preparation of such a melanin solution are: (1) to retain without
loss and unaltered the melanin contained in each sample of material; (2) to
eliminate all cloudiness and obtain a perfectly clear solution; (3) to eliminate any
colored materials other than melanin; (4) to avoid the production of pseudo-
melanins ("melanoidins") which sometimes result from the action of strong acids
upon proteins.

The procedure outlined below is the outcome of experimentation by the
authors, which was commenced in 1935. While it is empirical in many details,
we believe that the various steps are theoretically justifiable. Many of these
steps are based upon the methods of previous workers, but the combination is ours.

Comparison with our earlier report (1937) shows that somewhat extensive
changes have been made in this procedure. Since little melanin is contained in
the more massive tissues of the body (muscles and viscera), the presence of which
increases the difficulty of preparing clear solutions, it was decided to exclude
these tissues; hence paragraphs 4, 5, and 6 of our revised procedure (cf. Sumner
and Fox, 1935). 5 Again, peptic digestion, followed by centrifuging, was replaced
in our revised technique by boiling in 6 per cent HC1, followed by dialyzing
(paragraphs 22 to 25).

Our most recent procedure follows:

(1) The fishes (unfed for at least two days) were killed in chloroform vapor;
(2) dried on paper towels; (3) each individual measured, and each lot (those
combined to form a single sample) weighed; (4) dipped into boiling distilled
water f minute, then cooled in cold water; (5) the skin was removed from the
entire body and head and placed (along with fins) in distilled water; (6) water
warmed to 60° C. and beakers placed in oven at that temperature for one hour
(to remove gelatin); (7) 95 per cent alcohol substituted for water, changed once,
material bottled and kept in the dark, sometimes as long as five or six weeks;
(8) alcohol poured off and material subjected to Soxhlet fat-extration (in 150 cc.
thimbles) for three hours, using 250 cc. of alcohol-ether mixture (2:1); (9) ma-
terial left in thimbles and dried in oven at 60° for several hours; (10) decalcified
in one per cent HC1 for one hour at 60° (5 cc. per gm. of original weight of fish,
not of skin) ; (11) acid filtered off through no. 2 sintered-glass filter, residue washed
with 500 cc. distilled water; (12) residue from filter hydrolyzed by boiling one
hour in 0.2 per cent NaOH (5 cc. per gm. of original weight of fish) in three-liter
flask, under reflux condenser; (13) resulting mixture filtered through no. 2 glass
filter, to remove undissolved impurities; (14) filtrate, when of sufficient volume,
divided into two parts ("a" and "b") to be run separately as checks; (15) HC1
added, drop by drop, to each lot, until neutral point is just past and a precipitate
forms which includes the melanin (important that approximately the same pH

4 Strictly speaking, these are not molecular solutions, but very fine colloidal suspensions.
They may be very clear, however, and there is little or no Tyndall-effect unless the suspensions
are highly concentrated.

6 As we were primarily interested in changes in the external pigmentation (that of the skin),
we had no object in retaining the considerable amount of melanin contained in the peritoneum
or in the eyes.

ASSAYING MELANIN IN FISHES 189

is reached for all samples); (16) material (liquid and precipitate) transferred to
centrifuge tubes (approximately 100 cc. capacity), centrifuged 15 minutes at
approximately 1760 r.p.m., liquid6 poured off (precipitate adheres closely to
bottom of tube) ; if all material from one sample is not, at first, contained in a
single tube, it is combined into one tube and re-centrifuged; (17) 95 per cent
alcohol carefully poured onto precipitate in tubes (4 cc. per gm. of original weight
of fish), left at least \ hour; (18) this alcohol removed by suction (using a glass
tube, finely drawn out), and replaced by same amount of absolute alcohol, left
at least one hour; (19) alcohol again removed and ether (the same quantity) sub-
stituted, precipitate thoroughly stirred, left at least one hour; (20) materials
centrifuged \ hour, and ether removed by suction; (21) dried in oven at 60°;
(22) dissolved (while still in centrifuge tubes) in 0.2 per cent NaOH (1 cc. per
gram of original weight of fish); (23) equal volume of 12 per cent HC1 added,
making a concentration of (nearly) 6 per cent acid; (24) transferred to flasks
and boiled one hour under reflux condensers; (25) liquid, with precipitate, trans-
ferred to celloidin ("Parloidin") dialyzers, suspended in one-liter beakers of dis-
tilled water. Left at least three hours, during which water is changed five times;

(26) material poured (and rinsed) from each dialyzer into graduated cylinder,
enough 5 per cent NaOH added to make a 0.2 per cent solution, when liquid has
been brought to final required volume (3 cc. per gm. of original weight of fish) ;

(27) solutions returned to flasks and boiled for one hour under condensers (if
necessary the condensers are left off for a time and liquid evaporated sufficiently
to bring the volume down to somewhat less than the required final volume, to
allow for rinsing) ; (28) solutions returned to graduates and enough distilled
water added to bring them as nearly as possible to required volume (3 cc. per
gram of fish); (29) passed through no. 2 sintered-glass filters, previously dried
in oven (no material loss of melanin by adsorption is found to take place in a
filter of this grade of porosity).

The melanin "solutions" are now satisfactorily clear and ready for direct
colorimetric reading by transmitted light. This is obviously more satisfactory
than our earlier, more involved procedure (1937).

The colorimeter employed in these studies (the Ives Tint-Photometer) has
been employed by the senior author and collaborators for a number of years in
several lines of investigation. Since it has already been described rather fully
(Sumner and Fox, 1933, and earlier), no detailed account is called for. One beam
of light, reflected from a block of white magnesium carbonate, is passed through
an absorption-cell containing the solution under examination, another beam
through a neighboring cell containing distilled water. An adjustable diaphragm
controls the amount of light emerging from the latter cell so that the intensity of the
beams emerging from the two cells can be brought to equality. Before reaching
the eyes of the observer however, both beams are passed through one of a series
of color filters. Using each of these filters in turn, the two halves of the field
are balanced and the readings recorded. For the present studies, three filters

6 This liquid is ordinarily clear and of a pale straw-color, quite different from that of a diluted
melanin solution. Solutions from "black" fishes (i.e., ones from black background) were found,
however, to be about 80 per cent more deeply tinted than ones from "white" fishes when several
pairs of samples were compared. At most, the coloration is feeble.

190

SUMNER AND DOUDOROFF

have been employed: red, green, and blue.7 It is evident that in the use of this
instrument high readings represent low melanin values and vice-versa. The
highest (100 per cent) readings are obtained from pure water.

These successive readings constitute the primary data upon which our assays
of melanin are based. Each observer repeats his readings several times. When
two observers work in collaboration, their readings are averaged.8

For the interpretation of these figures for color-values obtained with the
tint-photometer it is necessary to prepare a set of curves based upon melanin
solutions of known concentration. Through the kindness of the late Professor R.

100

4,0

6,0

9 0

FIGURE 1. Tint-photometer readings of melanin solutions of various concentrations. The
highest concentration ("100%") was prepared by boiling dried melanin for three hours in 0.2
per cent NaOH solution in the proportion of 0.667 gms. to 100 cc. This was variously diluted
with NaOH solution. Abscissas = tint-photometer readings; ordinates = dilutions (expressed
as percentages of "100%" solution).

A. Gortner, of the University of Minnesota, we obtained a sufficient quantity
of purified melanin, prepared by him from the skin and other tissues of the "silky"
fowl. Curves based upon tint-photometer readings of a series of dilutions of a
solution (.0667 gm. per 100 cc. 0.2 per cent NaOH) are shown in Figure 1. These
are the curves which have been employed by the senior author in deriving the
values reported for Girella and Fundulus in this issue (pp. 195-205). The

7 Specifications for these filters, taken by means of the Bausch & Lomb Visual Spectropho-
tometer are as follows:

Red

Green

Blue

Maximum transmission (70% ±) ......................... 760-650

Lower limit (1% transmission) ........................... 602

Maximum (18% ±) .................................... 530-520

Upper limit (1%) ....................................... 610

Lower limit (1%) ............................. 475

Points of half transmission ............................... 500, 565

Maximum (31% ±) .................................... 460-450

Upper limit (1%) .............................. 515

(The blue has been referred to as "blue-violet" in our earlier publications.)
8 There was, not unexpectedly, a personal equation in these readings. In the case of the
last ten samples studied by us, melanin values based upon the readings of the senior author were
1.7 per cent higher than those obtained by the junior author. The mean differences, regardless
of sign, averaged 2.0 per cent.

ASSAYING MELANIN IN FISHES 191

melanin values therein presented are expressed in milligrams per gram of the
original weight of the fish.

Allowance must be made for the probability that melanins derived from dif-
ferent organisms are not all chemically identical. Hence, these figures for fish
melanin, based upon comparisons with the melanin of a bird, may be no more
than rough approximations. In our present studies, however, we are chiefly
concerned with relative rather than absolute values, our main object being the
discovery of relations among our various experimental groups.

We feel justified in basing our melanin titer upon the weight of the fishes,
rather than upon the surface area of the skins, for the reason that the skin of an
animal has a third dimension, thickness, which probably varies roughly in pro-
portion to its other dimensions. (It is obvious that the wet weight of the
scraped-off skins would be of no significance for this purpose.) It is of interest,
however, that when the melanin content, per square of body length, is computed
(in the absence of any actual measurements of surface areas), the essential rela-
tions among the various experimental lots remain unchanged.

Previous writers have called attention to the production of dark "melanoid"
substances through the action of strong acids upon proteins. These would
obviously interfere with colorimetric measurements of melanin. That boiling
with 6 per cent HC1 can have had no such effect upon our material seems to be
proved by a special test which was made for this purpose. Twelve goldfishes,
very nearly devoid of melanin in their skins, were subjected to our standard
procedure in three lots of four fishes each. These agreed in yielding solutions
hardly tinted enough to permit of readings with the tint-photometer. The
slight coloration was about 2 per cent of that of the "100 per cent" standard
melanin solution (Figure 1). Moreover, in six cases, one to two additional hours
of boiling in HC1 (step 24, p. 189) resulted in a slight mean loss, rather than
increase in the melanin titer, though this difference was of doubtful significance.

The same cannot be said, however, of extending the time of boiling in sodium
hydroxide. Six samples which were boiled for three hours gave melanin values
averaging 7.4 per cent lower than portions of the same solutions which were boiled
the usual one hour.

Furthermore, as we have reported earlier (Sumner and Doudoroff, 1938)
alkaline solutions of melanin invariably deteriorate (become paler) at ordinary
room temperatures, even when kept in the dark. Thus a set of three samples
from black-adapted fishes underwent a mean apparent loss in melanin content of
9 per cent in 24 hours, 18.6 per cent in five days, and 24.3 per cent in 12 days.
These figures are based upon the means for the three color filters. Actually,
there were wide differences between the figures for loss, when based upon readings
with the different filters. Thus, at the end of 12 days, the figures seem to show
a decrease of 33 per cent, if readings with the red filter alone are considered; only
16 per cent if the blue ones alone are considered. This obviously implies a change
in the color as well as in the density of our solutions.

Readings taken of our "standard" melanin solutions at the end of 12 days
presented much the same picture. In the "70 per cent" solution9 (corresponding
approximately to that derived from black-adapted Girella) the seeming loss in

9 Meaning 70 per cent of the concentration of the strongest solution (taken as 100 per cent)
upon which Figure 1 is based.

192

SUMNER AND DOUDOROFF

melanin concentration, when based upon the mean of readings with the three
filters, was 20.5, a figure not far from that shown by the solutions of fish melanin.
Here, too, the separate figures, based upon the three filters, ranged from 27 per
cent (red) to 15.7 per cent (blue).

Some further optical properties of a melanin solution, both when freshly
prepared and after standing in the dark for 12 days, are portrayed in Figure 2.
This is based upon readings with a Bausch and Lomb Visual Spectrophotometer
of a solution having the same concentration as the "100 per cent" solution of

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

63 64 65 66 67 68 69 70 71 72 73 74

FIGURE 2. Curves of transmission of various wave-lengths by "100%" solution of purified
melanin (see legend for Figure 1). Lower curve = freshly prepared solution. Upper curve =
same solution after 12 days. Abscissas = wave-lengths at lO-m/z intervals; ordinates = percent-
ages of transmission in comparison with distilled water.

Figure 1. The "curve" of transmission, at least for the freshly prepared solution,
turns out to be a nearly straight line, with a practically uniform gradient from
blue through red.

When a fresh "70 per cent" solution is compared with a 12-day-old solution
of the same original strength, we find an increase of 22 per cent in the reading
with the red filter, an increase of 41 per cent in the reading with the blue. This
might be construed as an increase in transmission toward the blue end of the
spectrum. It must be remembered, however, that with increasing dilution, the
transmission of the various wave-lengths through a colored solution tends to
equalize, and that the least transmitted wave-lengths (in this case blue) will
increase much more rapidly than the more transmitted ones (in this case red).
In passing from a "70 per cent" to a "51 per cent" melanin solution, for example,
we find an increase of 22 per cent in the "red" reading, and an increase of 89 per
cent in the "blue" reading. Thus the much smaller increase in the transmission
of blue rays through the 12-day-old sample, in comparison with the diluted fresh
sample, denotes a shift toward the red on the part of the former. The figures
upon which Figure 2 is based likewise reveal a greater relative transmission of the
red rays than would result from simple dilution.

This lability of melanin solutions makes it necessary to employ boiling periods
of uniform length in preparing them, and to make the photometric readings as
soon as possible after the solutions are prepared — three or four hours at most.
The unavoidable losses are thus kept constant, and the figures for the various
lots may be regarded as strictly comparable.

A chief reason for our dissatisfaction with our earlier procedures (1937, 1938)
was the frequent appearance of considerable differences in the yield of samples
from which identical results had been expected. So far as such unexpected dif-
ferences related to different lots of fishes having the same experimental history,

ASSAYING MELANIN IN FISHES 193

there was, however, no real ground for concern. Later studies of melanophore
density (Sumner, 1940, a and b} have emphasized the enormous variability in this
respect within each of the experimental lots. There was considerable overlapping,
for example, between the frequency distributions of melanophore numbers in even
the "black" and "white" series of Lebistes.

Of more serious import are differences in results obtained from portions of
the same sample of material which, after division, have been subjected to identical
treatment. Such discrepancies, which were earlier encountered to a discouraging
degree, have been largely, though not wholly, eliminated in our later studies.
Comparisons of figures for "a" and "b" portions of the same sample (step 14,
above) give us something of an index of the degree of constancy of our results.
In 34 cases in a recent study, in which readings from two such portions were
obtained, the mean difference between these portions was 3.2 per cent. This
figure, it is true, indicates only a moderate degree of precision, but it is small
compared with most of the experimentally produced differences upon which our
conclusions are based. In 28 of these 34 cases, the differences between our "a"
and "b" sub-samples were under 5 per cent. In the remaining six, they were 5
per cent or more, the largest figure being 12.3 per cent. No explanation can be
given for these exceptional cases. Fortunately, they seem to become less frequent
with increasing experience.

The results of a rather extensive application of the technique here described
are presented in another paper in this issue.

We make grateful acknowledgment of the kindness of the late Professor R.
A. Gortner of the University of Minnesota in supplying us with samples of purified
melanin, and for valuable information; to our colleague, Dr. D. L. Fox for the
use of the spectrophotometer under his charge as well as for advice on various
matters, and to Mr. Sheldon Crane for assistance in the photometric work.

SUMMARY

A method is described of preparing a solution (transparent colloidal suspen-
sion) of the melanin of fishes, and of assying this colorimetrically. The method
includes hydrolysis of the tissues in a boiling alkaline solution, precipitation, cen-
trifuging, further hydrolysis in acid and removal of acid and digestion products
by dialysis; finally dissolving the melanin in dilute NaOH. Readings of the
various samples were made with a tint-photometer, but a spectrophotometer
could have been employed, perhaps, to advantage.

For the interpretation of these readings, curves were drawn, based upon
solutions of purified melanin of known concentration.

The transmission "curve" of the various wave-lengths through such a melanin
solution was likewise obtained by the use of a spectrophotometer. This proved
to be a nearly straight line from a low point in the blue to a high point in the red.

With lapse of time, alkaline "solutions" of melanin rather rapidly deteriorate,
even in the dark, becoming increasingly transparent to all wave-lengths, but pro-
portionately more to the red than would result from mere dilution.

LITERATURE CITED

DAWES, B., 1941. The melanin content of the skin of Rana temporaria [etc.]. Jour. Exper.
Biol., 18: 26-49.

194 SUMNER AND DOUDOROFF

GORTNER, R. A., 1910. Studies of melanin. Jour. Biol. Chem., 8: 341-363.

GORTNER, R. A., 1911. On melanin. Biochem. Bull., 1: 207-215.

HEINLEIN, H., 1924. Zur Kenntnis melanotischer Pigmente. Biochem. Zeitschr., 154: 24-34.

KUDO, T., 1922. Veranderung der Melaninmenge beim Farbwechsel der Fische [etc.]. Arch.
f. Entwick. d. Org., 50: 309-325.

SALKOWSKI, E., 1920. Ueber die Darstellung und einige Eigenschaften des pathologischen Mela-
nins. Virchow's Arch., 227: 121-137.

SUMNER, F. B., 1940a. Further experiments on the relations between optic stimuli and the
increase or decrease of pigment in fishes. Jour. Exper. Zool., 83: 327-343.

SUMNER, F. B., 1940b. Quantitative changes in pigmentation, resulting from visual stimuli in
fishes and amphibia. Biol. Rev., 15: 351-375.

SUMNER, F. B., AND P. DOUDOROFF, 1937. Some quantitative relations between visual stimuli
and the production or destruction of melanin in fishes. Proc. Nat. Acad. Sci., 23:
211-219.

SUMNER, F. B., AND P. DOUDOROFF, 1938. Some effects of light intensity and shade of back-
ground upon the melanin content of Gambusia. Proc. Nat. Acad. Sci., 24: 459-463.

SUMNER, F. B., AND D. L. Fox, 1933. A study of variations in the amount of yellow pigment
(xanthophyll) in certain fishes [etc.]. Jour. Exper. Zool., 66: 263-301.

SUMNER, F. B., AND D. L. Fox, 1935. Studies of carotenoid pigments in fishes. II. Investiga-
tions of the effects of colored backgrounds [etc.]. Jour. Exper. Zool., 71: 101-123.

VILTER, V., 1931. Modifications du systeme melanique chez les axolotls [etc.]. C. r. Soc. de
biol, 108: 774-777.

A FURTHER REPORT UPON THE EFFECTS OF THE VISUAL
ENVIRONMENT ON THE MELANIN CONTENT OF FISHES1

F. B. SUMNER
(Scripps Institution of Oceanography of the University of California, La Jolla)

INTRODUCTION

That a sufficiently long sojourn upon a black or white background may result
in a marked increase or decrease in the pigmentation of fishes and amphibia has
been shown by several investigators (Cf. recent review by the present author,
19406). Sumner and Wells (1933) found pronounced changes of this sort in
Lebistes reticulatus, in which not only the number of melanophores per unit area
of skin was found to be markedly affected, but also the amount of melanin per
cell. These changes were portrayed unmistakably in our published photographs,
but no attempt was made at that time to measure their extent.

For two other fishes, Gillichthys mirabilis and Gambusia affinis, Sumner and
Doudoroff (1937, 1938) made quantitative determinations of the amount of
melanin present in animals of different experimental history. These deter-
minations were beset with considerable obstacles \vhich seriously affected their
precision. Certain conclusions were, however, established with reasonable
certainty in both of these series of experiments. (1) Fishes of "black" history
yielded considerably more melanin than fishes of "white" history, while those
from intermediate backgrounds (grays) were intermediate in this respect.
(2) Within broad limits, the intensity of the incident light was a minor factor in
the production of such differences.

A more definite quantitative relation was indicated as possible between the
albedo2 of the background and the amount of melanin produced (or retained).
In Figure 3 (Sumner and Doudoroff, 1937) and Figure 1 (Sumner and Doudoroff,
1938), it is evident that the relation between these two variables is not a linear
one. If the figures for albedo stated on page 213 (1937) be converted into per-
centages of "white" (the latter being regarded as 100) and if 0.74 be taken as the
value for "black," the Gillichthys values of Table I, "A" (means), form a nearly
perfect logarithmic series.3 This relation does not hold at all well for the "B"
fishes, however, though the arrangement here is likewise of the "hollow curve"
type.

The relations shown by the "A" set of these fishes suggested as possible the
rule that the amount of pigment produced (or retained) varied inversely as the

1 Contributions from the Scripps Institution of Oceanography, New Series, No. 195.

2 By albedo is meant the specific reflectivity of a given surface, i.e., the proportion of incident
light which that surface reflects.

3 This mode of treating the data is not in the least arbitrary. Our original attempt to state
the albedo of our "white" in absolute terms was not successful and has been abandoned. But
the figures for "albedo," referred to "white" as a standard, are fairly exact. The figure of 0.74
for "black" is taken from Sumner (1940a). While only approximate, it is certainly a fairer figure
than 0.

195

196 F. B. SUMMER

logarithm of the albedo of the background. The analogy between such a formu-
lation and the so-called "Weber-Fechner Law" in human sense-physiology was
at once recognized.

Because of the difficulties encountered in our earlier attempts to assay melanin,
the present author next resorted to counting melanophores in definite areas of the
skin of Lebistes reticulatus (Sumner, 1939, 1940a). Accurate counting was
rendered possible by causing the pigment-masses of the melanophores ("melano-
somes") to concentrate through the action of adrenalin.

In these experiments, the previously observed gradient in the effects of the
various backgrounds, from black to white, was clearly manifest. And again, the
logarithmic relation between stimulus and pigmentation was plainly indicated,
though the values for the "black" fishes were somewhat too low (1940a, Figures
3 and 4). Once more, any differences due to the intensity of general illumination,
if significant at all, were slight in comparison with those resulting from the shade
of the background.

While there could be no doubt that the melanin content of black-adapted
fishes was greater than that of white-adapted ones, it was plain that more exact,
quantitative expressions for these differences in melanin content were desirable.

Further endeavors to perfect a method of assaying melanin were resumed by
Sumner and Doudoroff, who have discussed the outcome of their completed task
in this issue of the Biological Bulletin (pp. 187-194). The present paper reports
the results of two series of experiments in which this method has been applied
successively to two species of fish, Fundulus parvipinnis and Girella nigricans.
Since, of these two, the results from the latter species were of far greater sig-
nificance, they wrill receive the principal attention.

I must here acknowledge the valuable assistance of Dr. Peter Doudoroff and
Mr. Carl I. Johnson in the preparation of some of the equipment here employed,
and of Mr. Urless Lanham and Mrs. J. F. Wohnus in connection with the care
of the fishes and some of the subsequent laboratory procedure. Acknowledg-
ment is also due Messrs. P. S. Barnhart and C. W. Palmer for obtaining the
supply of fishes.

METHODS

In the case of Girella, eight to ten of the fishes, averaging (two months later)
about 60 mm. in length, were placed in each of the 24 experimental bowls.
Running sea-water was supplied to these. The fishes were fed three times a
week, chiefly with beach-worms (Thoracophelia) and canned shrimps. Debris
(feces and food remains) was removed from the bottoms of the bowls by siphon-
ing, nearly every day, and the bowls were scoured once a week to remove bacterial
and algal growths.

The bowls were kept in six cabinets, painted white within and lighted by
electric lamps overhead. Four bowls of the same albedo were kept in each -of
five cabinets, lighted by a 100-watt lamp. A second set of four black bowls was
kept in the sixth cabinet, lighted by a 10-watt lamp. The cabinets were all kept
lighted night and day.

In the case of Fundulus, only five cabinets were employed, all with 100-watt
lights, and the extra set of black bowls was omitted.

The intensity of illumination in the more strongly lighted cabinets, as deter-

VISUAL ENVIRONMENT AND MELANIN CONTENT

197

mined with' a Weston Photronic Cell placed at the customary water-level in the
bowls, the latter being covered by a ^-inch screen, was about 33 foot-candles.
In the less brightly lighted cabinet, it was about 2 foot-candles.

The bowls were of clear, uncolored glass, with nearly straight sides, 24 cm.
in diameter and 15 to 17 cm. deep. As has been our practice, these bowls were
painted on the outside with several coats of auto enamel. Black and white
enamel were used, singly and in mixtures which provided the three shades of gray.
In order to eliminate bright gleams, from surface reflection, the bowls were given
a frosted surface by sand-blasting on the inside. This surface becomes trans-
parent when covered with water.

Albedos were determined by a method described more fully earlier (Sumner,
1940a). Light reflected from the bottoms was measured by the use of a photronic
cell and galvanometer. The readings thus obtained did not reveal the absolute
albedos of the surfaces in question (i.e., ratios of reflected to incident light), since
it was impracticable with available equipment to measure the incident light
reaching the bottom of a bowl nearly filled with water. This was not necessary,
however, because the important thing to know in these experiments was the
relative reflectivity of these various surfaces, as compared with one another.
As already stated, these figures were reduced for present purposes to a common
standard, the reflectivity of the white bowls being taken as 100.

Since, as already indicated, the bottoms of the bowls were unavoidably
befouled part of the time, another set of albedo readings was taken with the
bowls in this state. For this purpose, an attempt was made to reproduce their
average condition preceding the — usually daily — siphoning off of the debris.

TABLE I
Albedos of the various backgrounds, relative to white as a standard

Without debris

With debris

Approximate
average
condition

White . .

10000

100.00

Pale gray

38.17

37.31

37.74

Medium gray

17.84

17.77

17.80

Dark gray

6.56

7.22

6.90

Black

0.94

1.94

1.42

The result was necessarily only a rough approximation. Table I gives the figures
for each type of bowl, with and without the debris. The figures in the third
column, which represent an average of the clean and the dirty conditions of the
bowls, are the ones here employed in considering the relations between albedo
and pigmentation.4

The present table gives the albedos of the bowls used for Girella. The set
used for Fundulus received the same shades of paint (many of the bowls were
identical), with the exception of the "dark gray" bowls, which were uninten-
tionally made considerably paler, having an albedo of 11.79, instead of 6.90.

4 In Table I of Sumner, 1940a, the figures in the column headed "With Debris" are the ones
employed in the text discussions of that paper, but those figures are based upon a less extreme
condition of fouling than that dealt with in the present paper.

198

F. B. SUMNER

In the^Girella experiments, about half of the fishes were removed for the
melanin assay two months (59 to 61 days) after the commencement of the experi-
ment, the remainder being removed two months later (a total of 122 to 123 days).
Four samples were taken from each type of background on each of these occasions.
Each sample consisted of from three to five fishes. Two series of Fundulus were
likewise assayed, after periods of 24 to 26 days and 57 to 59 days, respectively.
Here the four samples from each background consisted of five to seven fishes.

The treatment to which this material was subjected has been described by
Sumner and Doudoroff in the present number of the Biological Bulletin (pp.
187-194).

RESULTS

For Girella, notes were made on the appearance of the living fishes near the
close of each period of the experiment.

(1) Differences of shade, resulting from the influence of the backgrounds,
were very marked. Most of the fishes in the black bowls were so black as to be
nearly invisible against these backgrounds. Those in the white bowls were very
pale, appearing almost white (for a few seconds only) when transferred to a black
bowl. Fishes in the intermediate bowls were correspondingly graded.

(2) Except in the black bowls, and to a less degree in the dark gray ones,
however, the adaptation was far from complete, even after four months. The

TABLE II

Melanin content (mg.lgm.) of Girella killed after two months

Black (10W)

Black (100W)

Dark gray

Medium gray

Pale gray

White

1.35
1.40
1.60
1.61

[0.91]

1.37
1.52
1.55

0.86
0.95
1.03
1.21

0.78
0.82
0.93

0.63
0.68
0.80

0.81

0.82
0.89
0.93

1.49

1.48

1.01

0.84

0.70

0.86

TABLE III
Girella killed after jour months

Black (10W)

Black (100W)

Dark gray

Medium gray

Pale gray

White

1.33

1.22

0.88

0.63

0.50

0.61

1.51

1.40

1.02

0.71

0.57

0.62

1.51

1.55

1.12

0.73

0.61

0.65

1.58

1.57

1.22

0.91

0.64

0.79

1.48

1.43

1.06

0.74

0.58

0.67

white-adapted fishes, while very pale, appeared far from white, on their own
backgrounds, and could not even be regarded as well concealed. The fishes on
pale and medium gray were also considerably darker than their respective back-
grounds.

VISUAL ENVIRONMENT AND MELANIN CONTENT 199

(3) Great individual differences of shade were sometimes manifested among
fishes in the same bowl, particularly in the medium and dark gray bowls. Among
the latter it was noted that the two extremes could even be characterized as
"pale" and "very dark," relative to their background. Whether or not such
visible differences resulted from actual differences in the amount of melanin was
not determined. Apparently, they were more or less permanent for the indi-
viduals concerned.

(4) The greater part of the induced differences of shade, even after four
months, was of the transitory ("physiological") type. Strikingly rapid changes
were still visible, following the transfer of a fish from one background to another.
Again, the effect of the chloroform vapor with which the fishes were killed was
to level down these differences, the pale fishes becoming much darker and vice-
versa. While no figures are available for the color differences of the living fishes,
it is certain that the difference between those of "black" and "white" history
was many times the maximum difference (2^ : 1) shown by the melanin extracted
from these same lots.

Figures for the melanin content of the skins of the various lots of Girella are
shown in Tables II and III,5 and some of the relations among these values are
depicted graphically in Figures 1 and 2. Each of the values comprised in the
tables is derived from one of the "samples" of three to five fishes referred to above,
and in most cases it represents the mean of two sub-samples, into which the
dissolved material was early divided. The figures in each column are arranged
in order of magnitude, without reference to the chronological order of the analyses.

The range of magnitude displayed in each of the columns of these tables is
plainly considerable. This despite the fact that each of these figures represents
the mean condition of several individuals. Considering the very great individual
variability of some fishes in respect to pigmentation, however, these differences
among the melanin values for small samples are not surprising (Sumner and
Doudoroff, this issue, p. 192).

It seems probable that the figure in brackets in Table II (0.91) is due to some
accident or error of procedure, though no such error was perceived at the time.
This low value is not approached by any other of the 16 figures comprised in the
four columns for "black" fishes in the two tables. It will also be noted that the
mean for the column containing it (black, 100W.), when the aberrant figure is
omitted, agrees rather closely with the other three means for "black." If this
(possibly correct) figure is included, the mean for its column is 1.34.

The two vacant spaces in Table II result from the accidental loss of the cor-
responding samples of material.

Aside from one feature, the curves (Figure 1) show much the same form as
those presented in previous papers (Sumner and Doudoroff, 1937, 1938; Sumner,
1940, a and 6). From black to pale gray, the arrangement of the values ap-
proaches very closely a logarithmic one, at least for the 4-month series (Figure 2).

In one respect, however, the curves based upon these experiments differ
markedly from any of those shown in our earlier papers. In the present case,
there is an actual rise from pale gray to white, the reality of this relation being

6 These melanin values were obtained by reference to curves based upon "standard" solu-
tions of melanin derived from silky fowls (see preceding paper, p. 190). It can hardly be assumed
that they represent with any exactness the absolute quantities of melanin in our fishes.

200

F. B. SUMNER

emphasized by the fact that it is shown in both the 2-month and the 4-month
Here we have an abrupt departure from the logarithmic relation. The

series.

possible significance of this will be discussed later.

1.40-
1.30 —
1.20 —
I. IG-
LOO—
.90 —
80—
.70—
.60-
.50

Dj.

I
W.

FIGURE 1. Melanin content (milligrams per gram of original weight of the fishes) of Girella,
after sojourn on various backgrounds; broken line after two months, continuous line after four
months. Abscissas = albedos; ordinates = melanin.

140
130
120
1.10
1.00
90
80
.70
.60

II I

100 50
W P.S.

BK.

FIGURE 2. Melanin content of the 4-month series plotted against the logarithms of the albedos

of the backgrounds.

Comparison of the 2-month and the 4-month series brings out certain points
of interest. In respect to the values for black and dark gray, the two series differ
but slightly from one another. Beyond the latter point, however, the two curves
diverge steadily, that for the 4-month fishes falling well below that for the 2-month
ones. This is not surprising when we consider that freshly caught fishes, when
subjected to these artificial conditions, commonly lose pigment freely upon the

VISUAL ENVIRONMENT AND MELANIN CONTENT 201

paler backgrounds, but undergo little increase upon the darker ones.6 This loss,
as might have been expected, has progressed considerably further in four months
than in two. It is not impossible, indeed, that longer subjection to the experi-
mental conditions would have resulted in depressing the value for "white" below
that for "pale gray."

To consider these differences quantitatively, we find that after two months
the melanin yield of the "black" fishes was about If times that of the "white"
ones, and more than twice as great as that of the "pale gray" ones. For the
4-month series, the figures are 2 + and 2|, respectively. As already stated, these
differences are slight in comparison with the very great differences in the ap-
pearance of the living fishes.

Fundulus, as already stated, yielded far less instructive results than Girella.
Earlier experiments had shown that visible responses to backgrounds were much
less pronounced in the former than in the latter. Indeed, the living "black"
fishes were far from black and did not seem to darken further after the first few
days.

Mean melanin values (mg./gm.) for the 2-month period are as follows:

Black 0.37

Dark gray 0.40

Medium gray 0.36

Pale gray 0.34

White . 0.26

These figures are based upon four samples (five to seven fishes each) in
the case of the three grays, only three samples in the case of white and black.
The significance of any of these differences may well be doubted, with the ex-
ception of that between "white" and the other four members of the series. In
general, the variability among the figures for each background is high.

Comparing the figures for Fundulus with those for Girella, it is plain that
the former, in all cases, is a much less highly pigmented fish than the latter.
The ratio, indeed, is nearly 1 : 4. Again, the influence of the background is
much less pronounced in Fundulus than in Girella. In the former it has been
seen that there is little or no difference in melanin content between the fishes
kept on black and those kept on the various shades of gray, while the "white"
fishes seem to have lost only 30 per cent of their melanin.

DISCUSSION

Despite the marked differences shown by the different fishes in our present
and similar previous experiments, certain common features are obvious in their
reactions to the background. In every case, among these various fishes (Gil-
lichthys, Gambusia, Lebistes, Girella, Fundulus) quantitative changes have been
brought about in the amount of melanin contained in the skin. In all of these,
except Fundulus, the greatest melanin content was shown by the black-adapted
fishes, and in all except Girella the lowest melanin content was shown by the
white-adapted fishes. For the most part, too, the intermediate values for this

6 However, fishes which have become depigmented as a result of subjection to pale back-
grounds will regain this pigment when transferred to black (Sumner and Wells, 1933).

202 F. B. SUMMER

pigment were graded in the order of decreasing albedo of the background. Ex-
ceptions to this were the position of "white" in the "B" series (but not the "A")
in Gillichthys, the transposed relation between "dark gray" and "medium gray"
in Gambusia, the position of "white" in Girella and the position of "black" in
Fundulus. It should be added, however, that in no case did one of these values
deviate more than one place from its expected position in the series.

In none of these fishes were the melanin values, when plotted against albedo,
arranged in linear fashion. In every case except Fundulus, they were so arranged
as to form a "hollow" curve. (Since Fundulus displayed such limited pigmental
reactions, it may be omitted from further discussion of this subject.)

In our most extensive experiments — those upon Gillichthys, Lebistes and
Girella — the whole or greater part of this curve was of the logarithmic type. In
series "A" of the Gillichthys experiments this arrangement was nearly perfect.
In the Lebistes experiments it held fairly well except for "black," the value for
which was somewhat too low. In the Girella experiments — the most extensive
of all — it held very closely except for "white," the value for which was too high,
so much so as to throw it out of proper alignment in the series (Figures 1 and 2).

We have more than once pointed out the possible analogy between the rela-
tions shown in these pigmental responses of fishes to albedo and the phenomena
of human sense physiology which have been generalized as the "Weber-Fechner
Law." Throughout a considerable range of stimuli, it has been found that
equal increments of sensation (just perceptible differences) result from propor-
tional rather than equal increments of the stimulus. In Fechner's well-known
formulation, Sensation = C log Stimulus.

It is commonly recognized that this is a generalization of limited application,
and that at best it holds for a limited range of stimuli. Particularly is it known
that the relation in question breaks down at low intensities of the stimulus, and
sometimes also at high intensities (Woodworth, 1938, pp. 430 et seq.). Whether
or not the low melanin value for "black" in the Lebistes experiments and the
high value for "white" (i.e., lessened depigmentation) in the Girella experiments
could be regarded as examples of the "breakdown" of any such general rule at
low and high intensities of stimulation is, of course, quite questionable. For
the present, we can say only that the analogy between these phenomena of pig-
mentary response and the phenomena comprised under the "Weber-Fechner
Law," however suggestive this may be, is perhaps an entirely superficial one.

That the intensity of incident light, within wide limits, is much less effective
than albedo in causing differences in pigmentation, has already been insisted upon.
It is possible, however, that different species differ from one another in the degree
to which light intensity is effective in this matter. In the Gillichthys experiments,
it was found that 7 per cent more pigment was formed in the cabinet which was
lighted by two 200-watt lamps than in the cabinet which was lighted by two 10-watt
lamps. In the Lebistes experiments, likewise, a small but inconstant difference
was shown, fishes from the more highly lighted cabinet (64(±) foot-candles)
averaging slightly higher in melanin content than those from the less highly
lighted one (0.24 f.c.). On the other hand, the rather unsatisfactory experiments
with Gambusia and the recent far more thorough experiments with Girella furnish
no evidence that in these fishes the wide differences of illumination resulted in any

VISUAL ENVIRONMENT AND MELANIN CONTENT 203

pigmental differences.7 In the former the illuminations ranged, approximately,
from 90 to 0.25 foot-candles; in the latter they were 33 and 2 f.c. respectively.
It cannot be stated positively, however, that all these differences in results are
due to differences of species. Several other factors (time and temperature, as
well as light) varied from one experiment to another.

In respect to the amount of pigmental change brought about by these differ-
ences of albedo, we find once more that the various species employed differed
rather widely from one another. Here again, we must bear in mind the reserva-
tion expressed in the preceding paragraph, although specific differences have
certainly played an important role.

In counts of the melanophores in Lebistes (Sumner, 1940a), the mean number
of melanophores in a definite area of skin was 1\ times as great in the black-
adapted fishes as in the white-adapted ones. In some lots of the same species
reared by Sumner and Wells (1933), however, the differences, while not deter-
mined quantitatively, appear from photographs to have been far greater than
this. In the present Girella series, we may repeat, the differences in melanin
content were 2 and 1\ : 1. In the Gillichthys experiments of Sumner and
Doudoroff (1937), on the other hand, the melanin content of the "black" fishes,
after 87 days, exceeded that of the "white" ones by only about 30 per cent.
Finally, in the recent Fundulus experiments, there were no certain differences
among any of the first four albedos (black to pale gray), while the average of
these exceeded the "white" value by about 42 per cent (of the latter).8

Before concluding, we may dispose of one possible serious criticism of our
experimental procedure. It is needless to say that, when a number of fishes are
kept together in bowls of the size here used, a not inconsiderable part of the visual
field of every fish is occupied by its neighbors. It may be asked, accordingly,
whether this circumstance may not be sufficient to invalidate any conclusions
based upon the albedos of the bowls themselves.

Against any such possible objection there are rather strong arguments. It
was long ago shown for flatfishes by the present writer (Sumner, 1911) that the
chromatic changes of fishes are far more influenced by stimuli received from the
bottom of a container than from its lateral surfaces. That this type of response
is not restricted to bottom-dwelling fishes was amply shown by N. A. Wells and
myself for Fundulus parvipinnis in 1930. In these experiments (results unpub-
lished), two sets (four each) of 5 X 7-inch battery-jars were painted, one with
the bottoms black and the walls white, the other in the reverse condition. Fishes
(four in each jar) were kept for 22 days under these conditions, and were com-
pared several times during this period in all-white jars, the two contrasted lots
being poured into the latter simultaneously. With probably not a single ex-
ception, all of the fishes from the white-bottomed jars were paler than any of

7 That is, if the bracketed figure in Table II is left out of consideration. If included, it would
change the mean figure in the "wrong" direction, i.e., give a lower value for the more highly lighted
fishes.

8 Dawes (1941) reports a difference of 60 per cent in skin melanin between black-adapted
and white-adapted frogs, after about five weeks. He believes that a greater change from their
original condition occurred in his black-adapted than in his white-adapted specimens, since the
former depart more from "the mean animal which was not kept on any particular background" ( !).
Dawes's claim for complete priority in the demonstration of such changes can hardly be taken
seriously in view of the facts reported in the preceding pages.

204 F. B. SUMNER

those from the black-bottomed ones. This despite the fact that the area of the
walls of the jars, below the water line, exceeded that of the bottoms in a ratio
of more than 3:1.

In our various experiments with painted aquaria, one fish's neighbors com-
monly formed part of its lateral field of vision; much less commonly did they
obscure its view of the bottom or any considerable part of this. Moreover, as
adaptation to the background proceeded, the biological portion of this background
came to contrast less and less with the remainder.

SUMMARY

The results are presented of recent experiments upon Girella nigricans and
Fundulus parvipinnis, together with a comparative discussion of previous similar
experiments by the author and collaborators.

In this latest series, Girella was exposed for two and for four months to back-
grounds of five albedos (black, three grays and white), lighted by 100-watt
electric lamps; while another set was exposed to black backgrounds only, lighted
by a 10-watt lamp. In the Fundulus series, only the brighter lights were
employed.

The effects of this treatment upon melanin production (or loss) were much
more pronounced in Girella than in Fundulus. In Girella, the amount of melanin
in the skin, after four months, was 1\ times as great in fishes from the black con-
tainers as in those from the pale gray containers. This difference in melanin
content was trivial, however, in comparison with the difference in appearance of
the living fishes. The latter was due, for the most part, to the transitory dis-
position of the pigment within the chromatophores.

The minimum melanin content in Girella was not obtained from the occupants
of the white containers, but from those of the pale gray containers. The four
values from "black" to "pale gray" formed, however, a descending series having
a distinctly logarithmic arrangement.

In Fundulus no such arrangement was found, there being only one significant
difference, that between fishes of "white" history and all of the others. The
mean value of the latter is less than \\ times that of the former.

The data from this and similar previous experiments show that in four of the
five species studied, the melanin values, when plotted against albedo, form
"hollow" curves, and that this arrangement, in some of the cases, is definitely
logarithmic. The possible analogy between this tendency and the "Weber-
Fechner Law" is discussed.

Fishes kept in black and dark gray bowls showed little change after two months.
Those from the other bowls, most of all from the white, showed considerable
further decreases between the 2-month and the 4-month periods. It is probable
that the differences between the dark-adapted and pale-adapted fishes resulted
less from increase of pigment in the former than from decrease in the latter.

No probable difference existed between black-adapted fishes kept under
100-watt lights and the same when kept under 10-watt lights, the lighting here
being in a ratio of about 16 : 1.

Reasons are given for believing that the chromatic response of a fish to its
background is not much interfered with by the presence of other fishes in the same
container.

VISUAL ENVIRONMENT AND MELANIN CONTENT 205

LITERATURE CITED

DAWES, B., 1941. The melanin content of the skin of Rana temporaria [etc.]. Jour. Exper.

Biol., 18: 26-49.
SUMNER, F. B., 1939. Quantitative effects of visual stimuli upon pigmentation. Amer. Nat.,

73: 219-234.
SUMNER, F. B., 1940a. Further experiments on the relations between optic stimuli and the

increase or decrease of pigment in fishes. Jour. Exper. Zool., 83: 327-343.
SUMNER, F. B., 1940b. Quantitative changes in pigmentation, resulting from visual stimuli in

fishes and amphibia. Biol. Rev., 15: 351-375.
SUMNER AND DOUDOROFF, 1937. Some quantitative relations between visual stimuli and the

production or destruction of melanin in fishes. Proc. Nat. Acad. Sci., 23: 211-219.
SUMNER AND DOUDOROFF, 1938. Some effects of light intensity and shade of background upon

the melanin content of Gambusia. Proc. Nat. Acad. Sci., 24: 459-463.
SUMNER AND WELLS, 1933. The effects of optic stimuli upon the formation and destruction of

melanin pigment in fishes. Jour. Exp. Zool., 64: 377-403.
WOODWORTH, R. S., 1938. Experimental Psychology. Henry Holt, New York, 889 pp.

Vol. 84, No. 3 June, 1943

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

THE EFFECT OF CARBON DIOXIDE AND LACTIC ACID ON THE

OXYGEN-COMBINING POWER OF WHOLE AND HEMOLYZED

BLOOD OF THE MARINE FISH TAUTOGA ONITIS (LINN.)

R. W. ROOT AND LAURENCE IRVING

(The Department of Biology, College of the City of New York;
The Edward Martin Biological Laboratory, Swarthmore College;

and
The Marine Biological Laboratory, Woods Hole)

Hemoglobin combines with oxygen less readily in the presence of carbon
dioxide. This influence of carbon dioxide on mammalian hemoglobin is frequently
called the Bohr effect after its first observer (Bohr, Hasselbalch and Krogh,
1904). In the blood of some fish the effect of carbon dioxide upon hemoglobin is
more pronounced than in mammals (Krogh and Leitch, 1919), and it is particu-
larly conspicuous in the blood of several marine fishes (Root, 1931), in which a
change in carbon dioxide tension of only a few millimeters greatly reduces the
affinity of the hemoglobin for oxygen. Because the consequences have an im-
portant significance for respiratory transport, Green and Root (1933) made a
theoretical study of the carbon dioxide effect in fish blood. They concluded that
the effect of carbon dioxide could be described in terms of the acidification which
is produced if it were assumed that acidity suppressed the acid dissociation of the
fish hemoglobin and only the ionized hemoglobin combined with oxygen. Ac-
cording to this view, the effect of carbon dioxide could be reproduced by any
other acid.

Several subsequent observations add new information pertinent to the carbon
dioxide effect. The blood of a number of fresh water and marine fish, but not
equally of all, is sensitive to the effect of carbon dioxide upon oxygen combination
(Willmer, 1934; Black, 1940; Root, Irving and Black, 1939; Benditt, Morrison
and Irving, 1941). Hemolysis of the blood of several fish was found to render
their hemoglobin quite insensitive to carbon dioxide (Black and Irving, 1938), so
that it appeared that the condition of the hemoglobin in the erythrocyte was
essential for the special sensitivity of fish hemoglobin. Elimination of the carbon
dioxide effect by hemolysis is not always complete, however, for Benditt, Morrison
and Irving (1941) found that after hemolysis of the blood of Atlantic salmon the
carbon dioxide effect was reduced, but still evident. The oxygen-combining
power of the hemoglobin of hemolyzed blood of some marine fishes is not strikingly

207

208 ROOT AND IRVING

affected by acidification with phosphate buffers (Hall and McCutcheon, 1938).
Other influences than acidity, such as the formation of carbamino compounds
(Roughton, 1935) and the influence of certain ions beside those of hydrogen
(Barron, Munch and Sidwell, 1937; Sidwell, Munch, Barron and Hogness, 1938)
on mammalian hemoglobin can strongly affect oxygen combination.

These recent studies have emphasized the influence of factors other than
hydrogen ion concentration upon the combination of oxygen with hemoglobin.
It seemed desirable, therefore, to examine further the part of hydrogen ion con-
centration in the effect of carbon dioxide upon fish blood. The studies have been
carried out on the blood of the marine fish Tautoga onitis (Linn.). Earlier studies
had shown that the whole blood of this fish was much affected by carbon dioxide,
but that there was little effect of carbon dioxide up to 75 mm. Hg pressure when
the blood had been hemolyzed (Root, Irving and Black, 1939). We have sub-
jected the hemolyzed blood of the tautog to pressures of carbon dioxide up to 500
millimeters and determined its ability to combine with oxygen. These effects are
compared with those obtained when lactic acid is substituted for carbon dioxide,
in both whole and hemolyzed blood, in order to determine how the effect of carbon
dioxide on the hemoglobin is related to acidity. The results show that hemolyzed
blood, although little affected by low tensions of carbon dioxide, reacts in a man-
ner similar to whole blood when the carbon dioxide pressure is raised sufficiently.
Furthermore, similar effects can also be produced with lactic acid.

METHODS

The methods used in obtaining blood, equilibrating it, and analyzing the gas
phases were the same as those described in a previous paper (Root and Irving,
1940). Hemolysis was secured by the addition of a few drops of highly con-
centrated saponin solution. When lactic acid was used, a measured quantity of
blood was placed in a tonometer and rapidly whirled as the acid was added
drop by drop. Correction was made for dilution of the blood when determining
the oxygen content of the samples.

As a criterion of the ability of any sample of blood to combine with oxygen,
the blood was equilibrated with 155 millimeters oxygen (approximately air
tension) at 15° Centigrade, and the percentage HbO2 determined. The value
obtained by such a procedure will be used to indicate the "oxygen-combining
power" of the blood sample.

The pH of the blood samples equilibrated with carbon dioxide was determined
either by use of the Henderson-Hasselbalch equation, assuming a pK value of 6.27
at 15° Centigrade, or by measurement with the glass electrode. The pH of the
lactic acid-treated blood was in all cases necessarily measured with the glass
electrode. A remark is necessary concerning the validity of the calculated pH
values. We have plotted the logarithms of the carbon dioxide tensions against
both the calculated and the measured pH values for a number of the carbon
dioxide-treated blood samples. In either case, a straight line relationship ob-
tained for almost the entire range of pH values. Although the curves for the
calculated and the measured pH values were not identically placed, the calculated
values being usually 0.1-0.2 pH unit higher than those measured, they paralleled
each other nicely, indicating that disparity between the two could be removed
by the use of a slightly different constant in the calculations.

EFFECT OF CO, ON FISH BLOOD

209

The stability of the more acid blood samples was checked by redetermining
their oxygen capacity following treatment with carbon dioxide or lactic acid.
For lactic acid samples, it was necessary to neutralize the acid by an equivalent
amount of NaHCO3. Our experience has shown that some methemoglobin was
likely to be formed in blood more acid than approximately pH 6.5, and that it
was necessary to make allowance for this in calculating the percentage HbOo
present in these samples. Methemoglobin formation proved to be more trouble-
some with hemolvzed blood than with the whole blood.

RESULTS

It can be seen from Figure 1 that whole blood begins to lose oxygen-combining
power rapidly as the pH falls below 7.7 (carbon dioxide tension about 2 mm. Hg).

CD
I

WHOLE BLOOD

I 1 1 1
400 200 100 50

1
10

PCQHEMOLrZED

BLOOD

150 100

50 10 4 2

Prn WHOLE BLOOO
C02
i i i i 1 1

1.0. 62 64 8.6

6.8 7.0 7 2 7.4 7.6 7,8

8.0

FIGURE 1. The relation between oxygen-combining power of hemoglobin at 155 mm. O2-
pressure and pH in whole and hemolyzed blood of the tautog at 15° Centigrade. The pH was
modified by the addition of carbon dioxide, the approximate tensions being indicated in the graph,
and was calculated by means of the Henderson-Hasselbalch equation.

Hemolyzed blood, on the contrary, shows little loss in oxygen-combining power
until the pH goes below 6.5 (carbon dioxide tension approximately 100 mm. Hg).
This is in agreement with Hall and McCutcheon's (1938) observation that

f\J

O 60

WHOLE BLOOD

7.0

FIGURE 2. The relation between the oxygen-combining power of hemoglobin at 155 mm. O:-
pressure and pH in whole and hemolyzed blood of the tautog at 15° Centigrade. The pH was
modified by carbon dioxide and was measured by means of the glass electrode.

hemolyzed tautog blood in phosphate buffers showed little loss in oxygen-affinity
through a pH range of 6.8-7.4. Below pH 6.5 it begins to lose oxygen-combining

210

ROOT AND IRVING

power rather rapidly. By the time 50 per cent of the oxygen-combining power
has been lost the pH of whole blood is about 7.1, whereas the pH of hemolyzed
blood is as low as 6. The curves remain similar in shape, and the similarity sug-
gests correspondence in the behavior of the hemoglobin inside and outside of the
cell.

The data of Figure 2, obtained by measurement of pH with the glass electrode,
agree with the results shown in Figure 1. These curves cannot be exactly super-
imposed on the corresponding curves of Figure 1, since, as pointed out previously,
the measured and calculated pH values disagreed by a constant.

The data of Figure 3 show nearly the same effect on hemoglobin when the pH
is modified bv lactic acid instead of carbon dioxide. This holds for both whole

FIGURE 3. Comparison of the relationship between oxygen-combining power of hemoglobin
at 155 mm. Ch-pressure and pH in whole and hemolyzed blood of the tautog at 15° Centigrade
when the pH (measured) is modified either by carbon dioxide or by lactic acid.

and hemolyzed blood. There is no great difference in the effect of the acidity
produced by the two acids on the oxygen-combining power of hemoglobin in
whole blood. Hemolyzed blood is also affected according to pH, although at a
lower pH than in whole blood.

DISCUSSION

The difference in the tension at which carbon dioxide begins seriously to affect
the oxygen-combining power of hemolyzed tautog blood, as compared with the
whole blood, led to the earlier conclusion (Root, Irving and Black, 1939) that
hemolysis renders this blood insensitive to carbon dioxide. At that time, hemo-
lyzed blood was treated with less than 100 millimeters carbon dioxide (maximum
about 75 millimeters), and, as the present work indicates, there is no considerable
loss in oxygen-combining power under these conditions. It can still be stated
that hemolyzed blood is insensitive to moderately low carbon dioxide tensions;
but with tensions greater than 100 mm. Hg hemolyzed blood shows the same
general phenomenon as is exhibited by whole blood at a much lower partial
pressure of carbon dioxide, namely, a rapid falling off in oxygen-combining power.
This finding indicates that hemolysis does not so modify the properties of hemo-
globin that it is incapable of responding to acidity. It suggests that, in whole
blood, the pH of the cells may be considerably less than that measured in the
plasma. If that view is correct, when hemoglobin is released into the plasma, the

EFFECT OF CO2 ON FISH BLOOD 211

carbon dioxide tension causing a given loss in oxygen-combining power will exceed
the tension required for an equivalent loss from whole blood.

To examine this view, it may be assumed that the introduction of carbon
dioxide varies only pH, and that the difference in the response of whole and
hemolvzed blood is only a matter of difference between plasma and red cell pH.
The pH which produces 50 per cent loss of oxygen-combining power in hemolvzed
blood would be that prevailing in the red cell when the whole blood also suffers
50 per cent loss. Figure 1 shows that for hemolvzed blood the pH for 50 per cent
loss is approximately 6. For a corresponding loss in oxygen-combining power,
the whole blood has a pH slightly above 7, and the pH of the red cell should be 6
when the pH of whole blood or plasma is 7. We have calculated the pH of the
red cell from data on its carbon dioxide content given by Root and Irving (1940).
The calculation was based upon determinations made at a carbon dioxide pressure
sufficient to cause a 50 per cent loss in oxygen-combining power of whole blood.
The calculated pH in the cell was 6.9, or 0.9 higher than in hemolyzed blood with
the same oxygen-combining power.1

The results obtained with lactic acid, since they parallel those obtained with
carbon dioxide, make it appear that the response of whole blood to carbon dioxide
is essentially an acid response. The results give no indication that the anions of
lactate and bicarbonate differ in their effect on the hemoglobin of fish blood.

One of the most urgent requirements yet remaining to further knowledge on
the effect of carbon dioxide on fish blood is the determination of the pH inside the
red cell for any given carbon dioxide pressure and pH of the plasma. The fact
that hemolysis causes considerable drop in the carbon dioxide-combining power
of the blood indicated that the cell was more acid than the plasma (Root and
Irving, 1940), but calculations have failed to reveal that it is more acid than
0.1-0.2 of a pH unit.

We are indebted to the following individuals for assistance during this in-
vestigation: Dr. S. W. Grinnell, for measurements of pH with the glass electrode;
Virginia Safford Black and Henry Brown, for technical aid in the routine analyses
of the blood. We also wish to thank Dr. P. S. Galtsoff, Director, and Mr. Robert
Goffin, Superintendent, of the U. S. Bureau of Fisheries at Woods Hole, for their
generous co-operation in the matter of laboratory space and facilities.

SUMMARY

A study has been made of the effect of carbon dioxide and lactic acid on the
oxygen-combining power of whole and hemolyzed tautog blood. The data pre-
sented show the change in oxygen-combining power of the blood as a function of
pH, when the pH is modified either by the addition of carbon dioxide, or lactic
acid. Both whole and hemolyzed blood lose much of their ability to combine
with oxygen as the pH is lowered. The effect of carbon dioxide and lactic acid is

1 Prof. A. C. Redfield, who kindly read the manuscript of this paper, has suggested that there
may be a change in the acid dissociation of the hemoglobin upon hemolysis. This could account
for the marked difference in the behavior of the whole and hemolyzed blood toward acidity, without
the necessity of assuming a large difference in pH between the plasma and the red cell. At the
same time it would be quite in line with the theory advanced by Green and Root (1933) to account
for the marked effect of CO2 on the oxygen-combining power of the whole blood. We are grateful
for this suggestion.

212 ROOT AND IRVING

quite similar. The similarity between the effects of carbon dioxide and lactic acid
suggests that carbon dioxide and anions lactate and bicarbonate have no special
effect beyond the result of acidity.

The contrast in oxygen affinity of whole and hemolyzed blood is shown by the
fact that hemolyzed blood must be made one pH unit lower than the calculated
pH of the cells to produce the same reduction of oxygen affinity.

LITERATURE CITED

BARRON, E. S. G., R. MUNCH AND A. E. SIDWELL, 1937. The influence of electrolytes on the

oxygen dissociation of hemoglobin. Science, 86: 39-40.

BENDITT, EARL, PETER MORRISON AND LAURENCE IRVING, 1941. The blood of the Atlantic sal-
mon during migration. Biol. Bull., 80: 429-440.
BLACK, EDGAR C., 1940. The transport of oxygen by the blood of freshwater fish. Biol. Bull.,

79: 215-229.
BLACK, E. C., AND LAURENCE IRVING, 1938. The effect of hemolysis upon the affinity of fish

blood for oxygen. Jour. Cell, and Comp. Physiol., 12: 255-262.
BOHR, CHR., K. HASSELBALCH, AND A. KROGH, 1904. Ueber einen in biologischer Beziehung

wichtigen Einfluss, den die Kohlensaurespannung des Blutes auf dessen Sauerstoffbindung

iibt. Skand. Arch. Physiol., 16: 402-412.
GREEN, A. A., AND R. W. ROOT, 1933. The equilibrium between hemoglobin and oxygen in the

blood of certain fishes. Biol. Bull., 64: 383-404.
HALL, F. G., AND F. H. McCuTCHEON, 1938. The affinity of hemoglobin for oxygen in marine

fishes. Jour. Cell, and Comp. Physiol., 11: 205-212.
KROGH, A., AND I. LEITCH, 1919. The respiratory function of the blood in fishes. Jour. Physiol.,

52: 288-300.
ROOT, R. W., 1931. The respiratory function of the blood of marine fishes. Biol. Bull., 61:

427-456.
ROOT, R. W., AND LAURENCE IRVING, 1940. The influence of oxygenation upon the transport of

CO2 by the blood of the marine fish Tautoga onitis. Jour. Cell, and Comp. Physiol., 16:

85-96.

ROOT, R. W., LAURENCE IRVING AND E. C. BLACK, 1939. The effect of hemolysis upon the com-
bination of oxygen with the blood of some marine fishes. Jour. Cell, and Comp. Physiol.,

13: 303-313.
ROUGHTON, F. J. W., 1935. Recent work on carbon dioxide transport by the blood. Physiol.

Rev., 15: 241-296.
SIDWELL, A. E., JR., R. H., MUNCH, E. S. G. BARRON AND T. R. HOGNESS, 1938. The salt effect

on the hemoglobin-oxygen equilibrium. Jour. Chen;., 123: 335-350.
WILLMER, E. N., 1934. Some observations on the respiration of certain tropical fresh-water

fishes. Jour. Exp. Biol., 11: 283-306.

INTAKE AND LOSS OF IONS BY LIVING CELLS. I. EGGS AND

LARVAE OF ARBACIA PUNCTULATA AND ASTERIAS FORBESI

EXPOSED TO PHOSPHATE AND SODIUM IONS1

S. C. BROOKS

(From the Marine Biological Laboratory, Woods Hole, and the University of California, Berkeley)

In the summers of 1940 and 1941 the writer attempted to apply the tracer
technique to measure the permeability of marine eggs and larvae to inorganic ions.
The 7-emission of most suitable ions prevented transmission of these ions through
the mails or by express, and in consequence it was decided to try the phosphate ion.
Activated phosphorus, i5P32, emits /3-particles, but no detectible 7-rays; the /3-par-
ticles are effectively screened by ordinary packing.

The use of the phosphate ion involves on the other hand its low solubility in
sea water. The ion used was predominantly HPO4=, since the sodium phosphate
solution prepared to a pH of 7.35 was brought to a pH of about 8.0 on solution in
sea water. The solubility of CaHPO4, the first salt to appear on adding Na2HPO4
to sea water, is about 0.2 gm. L"1, equivalent to 1.41 mM. No figures have been
found relating to the effects of the other ions on the solubility of CaHPO,j. The
imposed limits of solubility of phosphates made it necessary to use concentrations
materially less than 1.4 mM. Here we have used 0.195 to 0.81 mM. When eggs
are immersed in such dilute solutions it appears that such protoplasmic constitu-
ents as the proteins would usually be capable of combining with ions greatly in
excess of the amount of the phosphate ion likely to be found in the eggs. This
will be referred to in the discussion.

METHODS

Living materials. Eggs of Arbacia punctulata (Lam.) were obtained by re-
moval of ripe ovaries to fresh sea water. The shed eggs were passed through
gauze, and concentrated by gentle centrifugation ; (2400 X gravity for 15 seconds).
These eggs had a mean diameter of 72 n, and were surrounded by a tenuous jelly
12 to 20 /z thick. This was almost completely removed by the process of con-
centrating eggs.

When eggs were to be fertilized or larvae reared dry sperm was collected, sus-
pended in sea water approximately 0.5 per cent. About 0.2 ml of this suspension
was added to 100 ml of sea water containing 1 ml of eggs. Fertilization tests were
run in all experiments, and usually a success of 98 per cent or more was obtained.
No experiments yielding less than 94 per cent success are considered here. Forma-
tion of the fertilization membrane was counted as a success. The eggs of Asterias
forbesi (Desor.) were obtained in much the same way as those of Arbacia. But

1 This work has been supported by grants from the Research Committee of the University of
California and greatly helped by facilities provided by'the Marine Biological Laboratory of
Woods Hole. Both of these are gratefully acknowledged. In this work the writer was assisted
by Dr. L. J. Mullins, Mr. A. H. Whiteley, and Mr. Aser Rothstein.

213

214 S. C. BROOKS

these eggs are obtained in an unripe state, in which their diameter is about 130 n\
on standing in sea water the eggs ripen, and shrink to about 120 /u- The present
experiments were done with a mixture of ripe and unripe eggs. The eggs are
enveloped in a jelly whose thickness is about 12 ^. This jelly is practically all
removed from the eggs during their collection and concentration. In all other
ways these experiments are like those with Arbacia eggs.

Reagents. Woods Hole sea water and water distilled at the Marine Biological
Laboratory were used. Sodium phosphate was obtained through the kindness of
the Radiation Laboratory of the University of California, and was thus provided
as a solution either 0.210 or 0.105 M and adjusted to the pH of human blood
plasma, viz., 7.35. Neutron bombardment of i5P31 transformed about one ten
millionth of the atoms to i5P32. The phosphorus thus activated was oxidized to
phosphoric acid and partially neutralized with NaOH. The phosphate solutions
received at Woods Hole had originally activities of 130-470 mC L"1. This
isotope emits /3-particles of a maximum energy of 1.72 M.E.V. The activity was
measured against the 7-emission of radium in equilibrium with its products.
The activity at the time of each experiment was calculated using a decay con-
stant of 0.0479 per day; the half life of i5P32 is 14.2 days. Phosphorus containing
i5P32 will be designated as P*. A sample of radioactive NaCl was generously
furnished by Prof. K. T. Bainbridge of the Department of Physics, Harvard
University, and was used in two experiments. The characteristics of this isotope
(nNa24) have been described previously (Brooks, 1939).

Solutions. Eggs or larvae were immersed in solutions of this phosphate in
sea water. Concentrations used lay between 0.195 and 0.81 mM, the concen-
trations being dictated by the radioactivity of the dilution. They are well below
the solubility of CaHPO4 which is 0.2 gm. L"1 or 1.4 mM L"1. No precipitate
was observed in the experimental solutions, at least during the duration of the
experiments. Similarly no significant decrease in activity of these solutions was
noted in the same times, the decay being negligible. The activities of these solu-
tions at the beginnings of the experiments lay between 0.29 and 0.045 mC L"1.
These values lie well below the levels indicated for toxicity of Na* by Mullins
(1939). This ion had been calibrated by comparison of the 7-radiations of this
and radium in equilibrium with its products, and since its /3-activity is about 20
times its y-activity we may say that the toxic limit for Na* is of the order of 20
mC L-1. The maximum energies for Na* (1.40 M.E.V.) and P* (1.72 M.E.V.)
are comparable. It is safe to assume that so far as we know the radiation of P
in our solutions was not a factor, unless the present experiments should furnish
valid evidence of such an effect.

Procedure. Three methods of exposing the material to the phosphate-sea
water solutions were used: a) "common dish method." All the eggs or larvae
were put into a 600 ml beaker in roughly 100 ml of solution, kept suspended by
occasional swirling, and samples of 5 ml each were withdrawn at intervals. In all
except the last (Exp. 13) these samples were centrifuged 20 seconds in Hopkins
tubes, the solutions replaced by isotonic erythritol, centrifuged 30 seconds and
the erythritol decanted. From the sediment 0.02 ml was transferred to a de-
pression slide for measurement. The finding that much of the phosphate was
removed in the erythritol solution led to a method used for one experiment (Exp.
13), in which the eggs were centrifuged once as above; centrifuged again and the

INTAKE OF IONS BY MARINE EGGS 215

last of the supernatant fluid above the eggs removed, and the eggs themselves
removed until 0.02 ml was left. These eggs were cytolyzed in distilled water
and transferred to a depression slide, b) "Continuous method." Not satisfied
with the above procedure, we attempted a procedure in which equal portions
of an egg or larvae suspension were put into coarse Buchner funnels (Pyrex 3G3)
and the phosphate-sea water was slowly passed through this material, removed
quickly by suction and followed by isotonic sucrose sucked through in a few
seconds. The whole sample in situ was compared with a filter alone treated
identically. This was not quite satisfying and a new procedure was devised, c)
"Separate dish method." Identical samples (5 ml) of a suspension of eggs were
mixed in a Syracuse watch glass with 5 ml of a phosphate-sea water solution, thus
avoiding the disturbances set up in the common dish method. The separate
samples were collected at intervals, and to do this they were centrifuged and
cleared of excess solution and otherwise treated as in Exp. 13 cited above.

In all cases the first decantate was saved, samples of 0.02 ml of this taken, and
pH observed in the remainder, and attention was paid to a cloudiness which in
the earlier experiments appeared to consist of fragmented eggs and possibly some
egg jelly. No significance was found for the appearance or non-appearance of this
cloudiness, nor of the pH which varied from 7.6 to 8.0. The -activities of samples
of 0.02 ml each were measured.

The sediments which in the first and third methods consisted of 0.03 ml and
0.07 to 0.10 ml, respectively, were collected with uniform centrifugation and
found to contain 65 per cent eggs and the remainder of a fluid identical with the
decantate. The activity of this fluid must be deducted from the observed ac-
tivity, leaving an activity due to phosphate in the eggs or larvae themselves. The
volumes of eggs in the samples were obtained by adjustment, or only noted and
appropriate corrections were made in the calculations.

Measurements. Measurements of the phosphate content were made by a
Geiger-Miiller counter with a scale of eight. A definite number of impacts,
usually 200, was counted, and the elapsed time noted. Comparison between the
samples of eggs, supernatant, and the original phosphate-sea water solution,
whose concentration was known, made it possible to translate the values from
activities to concentrations. Otherwise this procedure is identical with that
previously described (Brooks, 1939).

Errors in measurement. Variations in background radiation or in variations
within the counter operate to change the reading. An idea of the possible magni-
tude of the error due to these factors can be got from seven counts of a single
sample of phosphate-sea water over a period of one hour. The range of variation
was 10.9 per cent of the mean. In one experiment a series of eight readings on
separate samples of different fluids were repeated four hours later. The devia-
tions here from the mean of each pair were between 0.5 and 5.5 per cent, or 0.0004
and 0.0085 mM. This confirms the above.

In addition to the counter error discussed above, there is an error in the taking
of small samples. The collection of the 0.02 ml samples, used for solutions, was
done in a hemocytometer pipette. Operating on seven samples of a single solution
of phosphate-sea water an error of 19 per cent was found.

The egg samples were adjusted to graduations in Hopkins tubes, whose bottom
cylindrical portion was graduated to 0.01 ml, an error not less than the first cited,

216

S. C. BROOKS

but probably of the same order of magnitude. It is felt that observed readings
may well vary up to 20 per cent, and even that single readings, not supported by
the adjoining samples in the succession of samples, should be not seriously re-
garded. The levels indicated by a series of successive samples has significance.

EXPERIMENTAL DATA: ARBACIA

Eggs and young larvae. Figure 1 shows the total concentration of phosphate in
successive samples of a lot of Arbacia eggs inseminated 25 minutes before the
start of exposure to the phosphate. This experiment was done by the common-
dish method. The early low values seem to show that no phosphate has been
taken in until the time of the first cleavage, which at the temperature of 20.2° C.
took place at about 50 minutes on the figure. The fact that much of the phos-
phate had been washed out into the erythritol solution used in washing more or

LEAVAGE

LJL

o /

O o °

o
o

0 0

o
°o IN'

/ 0°

FERCELLU

LAR

0 4(

D 6

0 8

3 100

FIGURE 1. Concentrations of radioactive phosphate in samples of Arbacia eggs (ordinates)
during the first 100 minutes of immersion in sea water plus '0.81 mM phosphate (abscissas). The
eggs were freshly inseminated at the beginning and showed cleavage at about 50 minutes. The
sloping line is the basis for the calculation of the intake constant, K. Common-dish method;
washed with an isotonic erythritol solution. Temperature 20.2° C.

less invalidates this conclusion. Cleavage can be thought of as causing an in-
crease in permeability to the ion, or may be thought of only as interference with
the washing out of the ion. In the first case we may calculate the intake after
cleavage to be 0.71 X 10~10 moles cm~2 hr^1. All values of the intake constant K
are assembled in Table I. It was noted that there was no injurious effect exerted
by the phosphate insofar as is shown by the comparative development of treated
and untreated control eggs.

Figure 2 represents a similar experiment except that no washing was done,
and that the eggs were inseminated only one hour before the end of the experi-
ment. At the temperature of 19.1° the first cleavage should not have occurred
during the experiment. No observation was made on this point. At the time
of insemination, after one hour exposure to 0.81 mM phosphate, tests showed
94 per cent success. No great change in the general slope of the curve has been
noted at the time of fertilization or elsewhere. Calculated as above we find the
permeability of the egg prior to cleavage to have various possible values, depend-

INTAKE OF IONS BY MARINE EGGS

217

ing on the degree of confidence in individual points, or the stage of the eggs.
Various possibilities are shown in Figure 2 as K\, K2, etc., the intake constants.
The value of Ki (calculated as above) is 70 X 10~10 moles cm~~ hr~l, a value much
greater than that found in the previous figure and experiment. K2 is about the
same. Both intake rates must be attributed to the early intake as distinguished
from late intake. There is usually an early maximum, sometimes two, sepa-
rated by a phase of loss of this ion, from a late ion intake always slower than the
first. A similar phenomenon has been noted for Nitella and the alkali metal
cations (Brooks, 1939), and Spirogyra, Urechis eggs, and amebas (Brooks, 1940).

0.8

"06

0,4

INTER

CELLULAR

100

FIGURE 2. Concentrations of radioactive phosphate in samples of unfertilized Arbacia eggs
(ordinates) during the first two hours of immersion in sea water plus 0.81 mM phosphate (abscissas).
Four possible bases for intake constants are given. Common-dish method; no washing. Tem-
perature 19.1°.

Apparently the washing with erythritol obscures the normal ion intake, and conse-
quently the intake constant taken from Figure 1 is fallacious. The true late
intake constants for this experiment must be about K3 = 3.3 X 10~n and K4
= 1.8 X 10~10 for unfertilized and fertilized eggs.

Experiments done by other methods show the course of intake much better.
Thus the results of the two experiments done by the separate-dish method are
shown in Figure 3. Both experiments show the existence of an early maximum,
a loss phase and a late maximum. Experiment SI gives KI = 100 X 10~10 and
K2 = 10 X 10-10 moles cm-2 hr^1, while Experiment S2 gives Kr = 33 X lO"10
and K22 = 14.0. The caption of Figure 3 explains the sub- and superscripts.
It is apparent that these constants are not far from those found from Figure 2, in
which an experiment was done with a different method (the common-dish method)
but not utilizing erythritol solution washing. Washing disturbs the ion intake.

218

S. C. BROOKS

The slightly greater K values in Figure 3 may be connected with the lower con-
centration and radioactivity of the immersion fluid. (See Table I.)

5
£

*2 X

INTERCELL

LAR

120

FIGURE 3. Concentrations of radioactive phosphate in samples of unfertilized Arbacia eggs
(ordinates) during the first 90 or 125 minutes immersion in sea water plus 0.262 mM phosphate
(abscissas) in two experiments. Tangents at four points indicated give the bases for the cor-
responding intake constants, namely, the early and late (subscripts 1 and 2) for Experiment SI
(superscript 1), and similarly for Exp. S2. Separate-dish method; no washing. Temperature 22°.

E
60

NTERCELLULAR-

FIGURE 4. Concentrations of radioactive phosphate in samples of developing Arbacia larvae
(ordinates) during the first 48 hours immersion in sea water plus 0.70 mM phosphate (abscissas).
The stages attained are noted in this figure. A tangent at the indicated point gives the basis for
the intake constant, K. Common-dish method; washed with an isotonic erythritol solution.
Temperature 20°.

Egg to early pluteus. One experiment extended over a long period, 24 hours, in
which the fertilized eggs reached the early pluteus stage. The eggs were in-

INTAKE OF IONS BY MARINE EGGS

219

seminated at the beginning of the experiment, and the larvae survived in the
solution more than 24 hours, but were dead within 48 hours. The stages attained
are noted in the figure (Figure 4). This experiment was done by the common-
dish method with washing, and apparently the early intake is obscured. But the
late intake appears and can be measured.

The phosphate content of these larvae enormously exceeded that in the sur-
rounding fluid. Soon after cleavage had started the intake accelerated, and

TABLE I

Intake and permeability constants of Arbacia and Asterias eggs and larvae for the HPO4= ion. The
subscripts of K refer to the order in which these values occur during exposure. The superscripts

refer to experiment number

Concentra-

Radio-

Experiment
number

Figure number
+ constant

Stage

tion of ex-
perimental
fluid

activity of
experimen-
tal fluid

Intake
constant

Permeability
constant

mC/L

KX1010

PX106

Arbacia 1 2

(1) K,

2-cell

0.81

0.160

0.71

0.88

Arbacia 13

(2) KO

70.0

96.0

K3>

1-cell

0.81

0.160

3.3

4.1

K4J

1.8

2.2

Arbacia SI

(3) K,M

100.0

36.0

K2M

unfertilized
egg

0.262

0.096

10.0
33.0

3.7
13.0

Arbacia S2

K,2f

K32J

14.0

5.5

Arbacia 1 1

(4) K!

blastulae

0.70

0.240

3.7

5.3

Arbacia C2

(5) K22

2-cell

0.195

0.045

83.0

426.0

Arbacia Cl

(5) K2i

gastrulae

0.195

0.094

15.0

77.0

Arbacia C5

(5) K,«

plutei

0.350

0.055

13.3

38.0

Asterias 3

(6) K23

unfert. eggs

0.175

0.246

6.5

28.0

Asterias 2

(6) K,*

unfert. eggs

0.175

0.260

400.0

2280.0

phosphate apparently passed into the larvae at a uniform rate up until the forma-
tion of plutei. In the next 12 hours the larvae died in this experiment. The ac-
celerated intake may be referred to the increase in surface area of the protoplasm
of the larva, and modified by the deep position of certain masses of cells. Differ-
ent tissues of a larva may have different permeabilities.

The intake constant can be stated for comparative purposes, by using the
approximate surface area of the egg, and calculation gives us a value of 10.8
X 10~10 moles cm~2 hr-1. This is not greatly unlike the values obtained in the
previously cited experiments. As a matter of fact, the superficial area of develop-
ing larva up to the beginning of the pluteus stage should not exceed 2 or 3 times

220

S. C. BROOKS

as great as that of the egg. If we modify the figure given above by using the
factor of 3, we obtain a K about 3.7 X ICT10 moles cm~2 hr"1, a figure which
comports well with K3 of Figure 2, which was 3.3 X 10~10.

But the experiments by the continuous method give higher permeabilities:
Figure 5 shows the intake of phosphate on a reduced scale of the ordinates. All
three of the valid experiments show the initial peak and loss spoken of above
(p. 217), but the slope of initial rise does not show intake constants higher than
the later values given here. The experiment with fertilized eggs in the 2-cell
stage gives a very high permeability taken from the rising phases of the curves:
assuming that two blastomeres have 2 X the surface of the egg we deduce intake
constant K!2 = 83 X 1Q-10.

_i
\

30
25
20
1.5
1.0
05

i
l
i

(2 CELLED)

SOLUTION

FIGURE 5. Concentrations of radioactive phosphate in samples of fertilized eggs, (1), gas-
trulae (2) and plutei (3) of Arbacia (ordinates) during the first 35 or 60 minutes of immersion in
sea water plus 0.195 (1 and 2) or 0.350 (3) mM phosphate. Continuous method; washed with an
isotonic sucrose solution. Temperatures about 22°.

Gastrulae and plutei. The curve for gastrulae (Figure 5), taken about the
middle of this phase, gives an intake of about 15 X 10~10 using a factor of 3 for
the probable superficial area of the gastrulae. This experiment is not very
satisfactory. The plutei, allowing them a superficial area of 4X that of the egg,
gives an intake of 13.3 X lO"10 given by the slope of the curve at K25 (Table I).

Comparative permeability to the sodium cation and the phosphate anion. Two
experiments were done to compare the permeability to sodium and phosphate ions.

INTAKE OF IONS BY MARINE EGGS

221

Both were done before the realization that erythritol solution washing was remov-
ing the ions to a great extent. The closest approximation made on the basis of
the rate of intake of these ions in the first 2 or 4 minutes, corresponding to the
first peak, has been made on Experiment 5, not here otherwise shown. The rates
of intake were sodium: 24.5 X 10~8; phosphate: 0.29 X 10~8 moles cm~2 hr"1.
Compensating for the different concentrations used we obtain permeability con-
stants for sodium: 9.8 X 1Q-6; phosphate: 0.51 X 1Q-6 moles cm~2 hr"1" (GM
L"1)"1- It appears possible that the sodium cation penetrates about 17 times as
easily as the phosphate dianion.

Too many considerations enter into this picture to allow drawing conclusive
values. For example, the mobility of phosphate calculated from conductivity,
diffusion, etc. is between those of K+ and Na+, e.g. the equivalent conductivity of
3/2 HPO4= is 57 while K+ and Na+ show 65.0 and 43.4. This would be expected
from the dimensions (Figure 6) of hydrated K+ (1H2), Na+ (7 H2O) and HPO4=

HPO

FIGURE 6. The van der Waals volumes of sodium (Na+), potassium (K+), and phosphate
(HPO4=) ions, drawn to scale, with the mean number of water molecules attached to them in
moderately concentrated aqueous solution. An ion approaching a plasma membrane would carry
this hydration. Removal of this water (which may be necessary for passage) requires expenditure
of work.

(unhydrated) ; whereas the deduction from Figure 5 would require hydration of
HPO4= presumably about 3 H2O to account for the lower penetrability of this ion.
This envisages a plasma membrane structure which will act as an ultrafilter whose
available (existent or potential) free spaces are about 7-9 A in diameter. This
corresponds generally with the results with chiefly water soluble substances, but
different cells or tissues seem to vary considerably in permeability with pore size
and the importance of the ultra-filter action of their plasma membranes. Possi-
bly in passing through the plasma membrane the unhydrated ion is slowed by its
divalent character, leaving only the univalent H2PO4~, present in a relatively
small proportion, to pass through the plasma membrane. Obviously much work
is still needed here.

EXPERIMENTAL DATA: ASTERIAS EGGS

The intake of phosphate by the eggs of Asterias forbesi in two experiments is
shown in Figure 7. In this figure as in Figures 1 to 4 a deduction was made during

222

S. C. BROOKS

calculation to account for intercellular phosphate in the samples. Since these
tares differ in the two experiments they are shown separately in Figure 7.

The intake curves in both experiments, like those shown in Figures 2, 3, and 5,
show an initial peak ion content, followed by a loss. After this the eggs absorb
phosphate at a steady, but relatively slow rate. Two intake constants have been

SOLUTION

INTERCELLULAR
INTERCELLULAR

•"'V

Exp 3
EXP. 2

FIGURE 7. Contents of radioactive phosphate in samples of unfertilized eggs of Asterias
(ordinates) during 5 (Exp. 2) or 18 (Exp. 3) hours of immersion in sea water plus 0.175 mM
phosphate (abscissas). Tangents at points shown by Ki2 and K23 give the bases for intake con-
stants for early intake in Experiment 2 and late intake in Experiment 3. Common-dish method;
washed with an isotonic erythritol solution. Temperatures about 15°.

calculated to represent the typical condition; they have been calculated from the
slopes shown by K23 = 0.26 X 1Q-10, and Kr = 400 X 10"10 moles cm"2 hr"1.
KI is the initial, K2 the later permeability, and the superscripts 2 and 3 refer to
the two experiments. The remaining intake rates are not clearly indicated but
inspection shows that they will have about the same magnitudes, respectively.

DISCUSSION

Injury. In all the experiments dealing with unfertilized eggs, tests were made
of the fertility of these eggs. In all cases a success of 94 per cent or better in all
stages of Arbacia; imperfect ripening of Asterias eggs led to lower degrees of suc-
cess, but in no case was there any evidence of lowering by phosphate of the fer-
tility. About 50 per cent of the Asterias eggs formed fertilization membranes
after insemination.

Effect of the method used. Among other' factors, some of which will be men-
tioned below, it appears that washing with erythritol as done in the common-dish
method, was largely responsible for the apparent low permeability of the cleaving
eggs of Figure 1. We forbear from comparing this with the remaining experiments
cited here.

Effect of ^-radiation. The assemblage of constants in Table I, columns 6 and
7, suggests the possibility that these eggs are so sensitive to /3-radiation that the
solutions with the higher activities yield the lowest permeabilities. Column 7
gives the permeability constants assuming that the only driving force is the con-

INTAKE OF IONS BY MARINE EGGS 223

centration gradient across the plasma membrane. They are presumably more
representative of the properties of the plasma membrane than are the intake con-
stants (column 6).

Effect of phosphate concentration. Columns 4 and 7 of Table I illustrate the
role of the concentration. Comparison of K3 or K2 of Figure 2 whose concentra-
tion is 0.81 mM L"1 with K22 of Figure 5 whose concentration is 0.195 might be
thought to indicate an effect of phosphate concentration on permeability. The
continuous method deals with masses lying in streaming solutions, and the dif-
ference in permeability may be due only to diffusion factors. But here we are
dealing with quite different methods, and are unsure of the cause for the difference.
A similar effect is shown, however, between K2 or K3 of Figure 2 and K3 or K4 of
Figure 3. But here again we find ourselves with rather different methods.

The results are so unclear that we may only suspect such a relation. Repeti-
tion of this work on an extensive scale ought to establish or negate the reality of
this effect.

Permeability in different stages. Some of the evidence which can be found in
Table I favors the idea that the gastrulae and plutei are less permeable than
blastulae and less developed stages including the unfertilized egg (Figure 5).
Blastulae appear to have about the same permeability as uncleaved eggs (Figures
2 and 4). These conclusions are far from being final.

Initial peak and losses. In Figures 5 and 6 and suggested in Figure 2 there
appear an initial rapid uptake of phosphate, followed within a few minutes by a
loss of this ion. This loss may proceed to levels suggesting nearly complete re-
moval of this ion. This is so like the curve shown in previous publications for
Nitella (Brooks, 1939, 1940) and Spirogyra and Urechis eggs and ameba (Brooks,
1940) to Na+, K+, and Rb+ as to lead us to accept this as a reality. In these
publications I have suggested that the initial rise is due to an inorganic ion
exchange (Steward's "induced absorption"); no explanation has been offered for
the loss of such an ion.

The rate of such an inorganic ion exchange will be affected by (a) penetrability
of the radioactive ion, and its activity gradient across the plasma membrane;

(b) similar properties of all available free intracellular ions available for exchange;

(c) similar properties of competing ions and (d) the properties, number, and dis-
tribution of ion binding groups. If we could ascertain all of these it should be
possible to account for the different rates of entrance of ions. In the case of the
phosphate, both H2P*O4~ and HP*O4= being present, the presence of competing
ions and the possibility of prompt combination of phosphate in metabolism make
this problem still insoluble.

An explanation of the loss of ions during the so-called "loss phases" will be
offered in a following paper.

Later absorption of phosphate. A frequently raised question is: how much of
this ion can be combined with the surface of the egg? Toward answering this
question, it is possible to calculate the number of moles of HPO4= combined with
the proteins and fats of the plasma membranes. Let us allow that one Svedberg
unit of a protein, whose diameter is 30 A, can combine with 30 equivalents of an
anion. A value of 10 to 15 equivalents would probably be more applicable at the
pH of the protoplasm or of sea water, and for all anions with all of which the
phosphate must compete. The plasma membrane, according to Parpart and

224

S. C. BROOKS

Dziemian (1940), should also contain lipids to about % the weight of proteins,
while Schmitt and Palmer (1940) show that the plasma membrane contains
enough protein to form a layer 60 A thick. These data were found for the ery-
throcyte. These figures are rough approximations, and the actual amounts in
Arbacia eggs may differ somewhat. We use the surface 1.63 X 10~4 cm2, and
volume, 1.174 X 10~6 cm3, as calculated from the observed diameters of these
eggs. The plasma membrane is assumed to be 200 A thick. We neglect the
combining power of the lipids, which is about % that of proteins, so as to offset
the large allowance made above for the anion combining power of proteins. A
liter of eggs could combine in their plasma membranes with about 0. 1 mM of HPO4=.
Table II shows how much phosphate got into the eggs. Only 6 out of 9 of
these values exceed the calculated amounts (0.1 mM L"1) which could be con-

TABLE II

The highest recorded contents of HPO4= in eggs of Arbacia and Asterias during immersion in
solutions of radioactive sodium phosphate in sea water (pH 7.8-8.0)

HPO4-

Maximum

Time for

Species

Experiment
number

Figure
number

content of
external

recorded
HPO4-

attaining
this

fluid

content

maximum

hours

Arbacia punctulata

0.81

0.039

0.7

Arbacia punctulata

0.81

0.050

0.8

Arbacia punctulata

0.262

0.047

1.0

Arbacia punctulata

0.7

8.3

24.0

Arbacia punctulata

0.195

4.35

0.58

Arbacia punctulata

0.195

0.92

0.58

Arbacia punctulata

0.35

1.90

1.00

Asterias forbesi

0.175

0.35

0.17

Asterias forbesi

0.175

0.48

18.0

ceived of as combined with the plasma membrane. Nevertheless it seems im-
probable that this phosphate is strictly confined to the plasma membrane. So
many considerations enter into the picture, all greatly reducing the theoretical
figure, that it seems to be more reasonable to think of the phosphate as combining
less with the plasma membrane but passing through into the cortex or interior
cytoplasm. The whole egg has an ample calculated combining power to take
care of much more phosphate than has entered in any of these experiments.

It is interesting that the experiments which show the highest phosphate tend
to show most clearly the separation between the initial peak in phosphate content
followed by loss of this ion. It is as though the ion had passed into the egg, and
was released by a change within the egg.

But it seems probable that the phosphate is combined by metabolic processes,
notably the formation of substances like hexose phosphates, base phosphates, the
formation of phospholipins, and the formation of skeletal elements. This process
reduces the free phosphate content of the egg, and hence favors entrance of the ion.

It will be noticed that in six of the experiments of Table II the content of
phosphate notably exceeds that in the bathing solution. This condition can be
attained by the operation of metabolic processes, as mentioned above, or by an

INTAKE OF IONS BY MARINE EGGS 225

organic-inorganic ion exchange, as postulated in other cases (Brooks, 1939, 1940).
We feel that all three aspects of accumulation are probably operative, i.e. inor-
ganic- and organic-inorganic ion exchange, and combination of the entering and
reacting ion.

SUMMARY

(1) Eggs and larvae of Arbacia punctidata and A sterias forbesi were immersed
in sea water containing low concentrations of radioactive sodium phosphate
(0.175-0.81 mM) and the phosphate content in subsequently collected samples
after intervals up to 48 hours was determined by measuring the /3-radiation from
the samples.

It was found that:

a) Phosphate was taken in often in at least two distinct periods, the first
within the first half hour, followed by a loss of the ion, and secondly in the later
stages.

b) The permeabilities during early absorption are generally greater than those
during late absorption.

c) If it be assumed that the only driving force is the concentration gradient
across the plasma membrane (a very imperfect assumption), the early permeabili-
ties vary from 5,3 to 96 X 10~6 moles cm~2 hr"1 (GM L^1)"1 for Arbacia and
2280 X 10~6 for Asterias. The late permeabilities of both range from 2.2 to
426 X 10-6.

d) The maximum concentrations found in eggs or larvae of both vary from
0.050 to 8.3 mM for Arbacia, and 0.35 to 0.48 mM for Asterias.

(2) Inverse correlations are intimated between external concentration or radio-
activity and permeability. The effects of radiation are discussed.

(3) The part played by the stage of the egg or larva, the effect of the methods
used, the dimensions of ions, and theories of absorption are discussed.

LITERATURE CITED

BROOKS, S. C., 1939. Ion exchanges in accumulation and loss of certain ions by the living proto-
plasm of Nitella. Jour. Cell. Comp. Physiol., 14: 383-401.

BROOKS, S. C., 1940. The intake of radioactive isotopes by living cells. Cold Spring Harbor
Sympos. Quant. Biol., 8: 171-177.

MULLINS, L. J., 1939. The effect of radiation from radioactive indicators on the penetration of
ions into Nitella. Jour. Cell. Comp. Physiol., 14: 403-405.

PARPART, A. K., AND A. J. DZIEMIAN, 1940. The chemical composition of the red cell membrane.
Cold Spring Harbor Sympos. Quant. Biol., 8: 17-22.

SCHMITT, F. O., AND K. J. PALMER, 1940. X-ray diffraction studies of lipide and lipide-protein
systems. Cold Spring Harbor Sympos. Quant. Biol., 8: 94-99.

INTAKE AND LOSS OF IONS BY LIVING CELLS. II. EARLY
CHANGES OF PHOSPHATE CONTENT OF FUNDULUS EGGS1

S. C. BROOKS

(From the Marine Biological Laboratory, Woods Hole, and Department of Zoology,

University of California, Berkeley)

Immersion of eggs of Fundulus spp. and other marine eggs in sea water con-
taining a radioactive ion such as the phosphate or alkali metal ions reveals rapid
intake, followed after a few minutes by outward migration of the marked ion and
subsequent increases and decreases in content of this ion. In the summers of
1940 and 1941, during the course of work on the intake of the radioactive phos-
phate ion,2 the experimental procedure was varied to obviate as far as possible
the effects of handling, and special tests were made to evaluate sources of error.
This paper concerns primarily the eggs of Fundulus heteroclitus (L.) ; experiments
were also done on eggs of F. majalis (Walbaum). An earlier paper treats of
similar investigations on the eggs of Arbacia and Asterias (Brooks, 1943).

METHOD

Eggs were obtained by stripping the fish. The ripe eggs of F. heteroclitus
consist of a membranous coat, the chorion, whose mean outside diameter was 1.80
mm. ; outside it is a layer of fibers which serve to moor the eggs to each other and
other objects; the thickness of this membrane was of the order of 0.05 mm. In-
side this lies the perivitelline fluid and the egg itself, whose mean diameter was
1.69 mm.; the egg nearly fills the chorion. Unless specially mentioned, these
experiments deal only with unfertilized eggs.

Some of the unfertilized eggs were obtained in the unripe condition, and
ripened gradually in sea water. The unripe eggs contain closely packed spheres
of a material of refractive index higher than the rest of the egg. On ripening this
material disappears either by changes in refractive index or by solution from the
surfaces of these spheres or in both ways. Unripe eggs placed in distilled water
become white and opaque in 5 to 10 seconds, while ripe eggs remain clear. This
may be due to changes in permeability of the chorion to ions, which rapidly leave
the unripe eggs, allowing precipitation of globulins or similar material inside the
eggs, as suggested by Gray for trout eggs (Gray, 1932). Only in the last few of
our experiments were the unripe eggs eliminated. However, there is no good
evidence that the results were perceptibly affected by the presence of unripe eggs.

The eggs of F. majalis were slightly larger than those of F. heteroclitus, but

1 This work has been supported by grants from the Research Committee of the University of
California and greatly helped by facilities provided by the Marine Biological Laboratory of Woods
Hole. Both of these are gratefully acknowledged. In this work the writer was assisted by Dr.
L. J. Mullins and Mr. Aser Rothstein.

- In this paper the term "phosphate ion" denotes both HoPO-T and HPO4=, both of which
were present at the pH values used.

226

INTAKE OF IONS BY MARINE EGGS 227

apparently otherwise did not differ. Experiments done with F. majalis are not
different from those of F. heteroditus. Only the latter are cited in this paper.

The freshly obtained eggs were at first used within about 15 minutes, but a few
of the later experiments were started one to 1^2 hours later to allow for ripening
and the elimination of unripe eggs. Their viability was established by fertiliza-
tion and subsequent observation. It is felt that lots of eggs giving 80 per cent
or more of development are satisfactory in this work. All experiments cited gave
at least this degree of fertility.

Reagents. Woods Hole sea water and water distilled at the Marine Biological
Station were used. A sodium phosphate solution, 0.105 M and isotonic with
human blood, was generously furnished by the Radiation Laboratory of the
University of California. To make this, phosphorus element had been bombarded
with neutrons, oxidized to phosphoric acid and partially neutralized with NaOH
to a pH of 7.35. This isotope is described in the previous paper (Brooks, 1943).

Solutions. Eggs were immersed in solutions of sodium phosphate (containing
both H2P*O4~ and HP*O4=) in sea water. The concentrations used were made
up to give approximately the same radioactivities. This was dictated by the
sensitivity of the measuring device and the amount of egg material practicable for
a single test. The limits of concentrations were 0.143 and 0.42 mM. These are
well below the saturation concentration of this salt in sea water. The activities
used varied between 0.17 and 0.26 mC L"1. The radioactive phosphate was
measured against the y radiation of radium in equilibrium with its products.

Experimental procedures. Three general plans were used: a) The "common-
dish" method in which about 50 to 100 eggs were drained and covered with 40
to 100 ml. of phosphate sea water. For each sample three eggs were withdrawn,
washed in distilled water, blotted with filter paper and set aside to dry on depres-
sion slides. Such samples were collected at different intervals from one or 2
minutes up to 60 minutes and in some cases up to 24 hours, b) Hoping to ob-
viate the mechanical disturbance involved in removing, often after tearing loose
the eggs removed for successive samples, each sample was put into Syracuse watch
glasses. This is called here the "separate dish method." Three eggs were put
into each dish in 5 ml. of sea water; to start the experimental exposure 5 ml. of
sea water containing radioactive phosphate ions were added . Each of two workers
handled one "series" of samples, and collected them according to an accurately
timed schedule. The three eggs were transferred by a medicine dropper with
minimum fluid into 100 ml. of distilled water which was then swirled. Then the
eggs were removed to depression slides, and the excess fluid was removed by a
capillary pipette. This whole process took 12 to 18 seconds. Two "series" of
samples were thus obtained. This method yielded the most satisfying results.
However, it was suspected that it still involved enough mechanical disturbance to
create changes in permeability, and a method (c) was devised in which lots of
three eggs each were placed in short capillaries placed in siphon tubes. This will
be referred to as the "continuous method," but will not be described in detail
because with this no significant changes in the results were detected.

Determinations of the phosphate content were made by a Geiger-M tiller
counter with a scale of eight, as described in the previous paper (Brooks, 1943).

S. C. BROOKS

ERRORS

Errors in counts have been shown to be usually within limits of ±20 per cent
(Brooks, 1943). Differences in size of the eggs were found to lie within 14 per cent
of the mean size. About 50 eggs were measured. Since three eggs were used
for each sample, the error should not exceed about 5 per cent, but errors up to
14 per cent are possible. The combined error might reach 25 to 30 per cent.

Errors due to the effects of pH changes: It has been found in experiments on
Funclulus embryos 3 that increases in pH to levels exceeding that of normal sea
water may greatly affect the phosphate intake by the embryos. These changes
in pH were produced by the addition of reagents not used in the experiments
under discussion here. But it was found that the sodium phosphate, which
as furnished has a pH of about 7.4, acidifies the solution to about 7.75, and by the
action of decreased pH might directly or indirectly affect the result. In the case
of enhanced pH, the results may be due to the precipitation of calcium or mag-
nesium phosphates, which in turn produced solutions unbalanced toward univalent
cations and hence toward the observed increased permeation of ions. This may
also be due to disturbance in equilibria in solution, or to diminished H-ion. When
the pH is diminished we may suspect that permeation is decreased somewhat
below normal. We have no evidence that changes in pH during the progress of
an experiment are responsible for significant changes in permeability, at least in
Fundulus eggs. Such changes in pH seem to have been very small, of the order
of 0.1 pH units, and within the error of determination as done.

DATA

Experiments on eggs. Figure 1 shows the progressive changes in one experi-
ment in the phosphate content of whole eggs, calculated as though uniform
through the whole egg. Two curves are shown each corresponding to one of two
lots of eggs, and handled by one of two operators using the separate dish method.
All lots were taken from a single pooled batch of eggs. The phosphate content
of these samples rises during 15 minutes to a crest at about 0.20 mM L-1, then drops
to lower levels around 0.05 to 0.12 mM L"1 after 20 to 26 minutes, and thereafter
tends to rise to higher levels. The curves connecting samples of single series rise
generally but also show swings up and down, and the two series seem to reach
these later crests and dips at different times.

Mean values calculated for these duplicate samples still show the first peak,
the first dip, and to a lesser extent the subsequent increase in phosphate content,
but it seems as though the individual history of a single sample is obscured by
combining them to obtain the means. Until it is possible to measure the radio-
active isotope concentration repeatedly in individual living eggs, not affected by
mechanical handling nor desiccated (as is necessary to allow the full radiation to
reach the counter cell), will it be possible to follow the uptake and loss of an ion
by an individual egg. This method has been found possible in the case of Nitella
(Brooks, 1939) but not so far for the larger fish eggs.

Figure 2 shows the results of two quite independent experiments, each with
two series as before. They also were done by the separate dish method. They

3 These experiments related to the effects of inhibitors of oxygen consumption, and have not
been published so far.

INTAKE OF IONS BY MARINE EGGS

229

show the same phenomena as Figure 1 : an initial peak followed by a dip in phos-
phate concentration, followed irregularly by higher concentrations. The two
series of each experiment tend to follow each other at first, and then to diverge.

0.20

INI ERCE ..ULAR

0.05

TIME,MINUTES

FIGURE 1. Phosphate concentrations in duplicate samples of unfertilized Fundulus eggs
(ordinates) during the first 60 minutes of immersion in sea water plus 0.42 mM phosphate
(abscissas). Tangents to the curves were drawn at the points indicated, and formed the bases for
Ki and Ko. Separate dish method. Temperature 17.2°.

Even the time of the first peak may vary somewhat as to the time of its appear-
ance, viz., in one of these experiments, at 7 to 10 minutes, and in the other at
15 minutes.

230

S. C. BROOKS

Figure 3 shows an experiment done by the common dish method. Here two
peaks occur at 2 and at 15 minutes, respectively. The swings are wider than in

3O 4O 5O 6O

TIME, MINUTES

FIGURE 2. .Phosphate concentrations in duplicate samples of each of two experiments on
unfertilized Fundulus eggs (ordinates) during the first 60 minutes of immersion in sea water plus
0.42 mM phosphate (abscissas). The slopes of the curves at the indicated points are the bases
for Ki and K2. Separate dish method. Temperature 17.3°.

2.0

INTERCELLULAR

TIME,MIN.

FIGURE 3. Phosphate concentrations in samples of unfertilized Fundulus eggs (ordinates)
during the first 60 minutes of immersion in sea water plus 0.262 mM phosphate (abscissas).
Three slopes indicated in this figure form the bases of Ki and K2 and K3. Common dish method.
Temperature 18.5°.

the previously cited experiments, and the peak phosphate concentration exceeds
that of phosphate in the immersion fluid. The phosphate concentration in the

INTAKE OF IONS BY MARINE EGGS 231

immersion fluid was lower in this experiment, 0.262 vs. 0.42 mM for the already
cited experiments. The observed results in this experiment may be due to this
lower concentration.

Three experiments were done with the continuous flow method. The first
such experiment showed a lower phosphate content but essentially the same as
the previously cited ones (maximum 0.153 mM L"1) ; the second shows a little less
intake (maximum 0.094 mM L"1); and the last gave a maximum phosphate con-
tent of 0.140 mM L"1. The period for recovery from mechanical disturbance
(allowed the eggs in position in flowing sea water before applying phosphate sea
water) was not important: the first twTo continuous experiments allowed about
one half-hour; the last allowed over 3 hours. The first differs from the last two,
so that there is no correlation between these differences and the length of the
recovery period. No cause for this rather low ion intake is apparent. It may
depend upon "physiological" conditions associated with the lateness in spawning
period, or possibly like those occurring in the advanced embryos.

Experiments on 8 to 10-day embryos. These experiments were done exactly
like those with unfertilized eggs using the separate dish method. The immersion
fluid had a concentration of 0.40 mM, and activities of 0.215 and 0.195 mC L~'.
The rinsed and dried eggs gave low intakes: the first had a phosphate content of
0.03 mM L"1 after 4 minutes of immersion and rising gradually to 0.06 mM L"1
after one hour (the end of the experiment) ; the second gave 0.035 and 0.06 mM L^1
after 4 and 15 minutes but gradually lost phosphate thereafter reaching 0.04 mM L"1
after one hour. Both experiments were done in duplicate. The two samples at a
given time gave agreement within 20 per cent (= ±10%) in all but 5 of the total
20 readings of the two experiments. The observed changes were greatly in excess
of the differences usually found for a single point.

The intake of phosphate in these two experiments failed to show any marked
separation between early and later periods separated by loss of phosphate.
The maximum contents are like those of the experiments by the continuous
method cited above.

DISCUSSION

In all of the figures it can be seen that in the later stages the phosphate content
is or sometimes decreases to less than the amount needed to fill the space outside
the egg itself at a concentration equal to that of the immersion fluid. Similar
phosphate contents at the beginning of the experiments can be thought of as
being due to incomplete diffusion of phosphate into this space, but the later oc-
currences seem to need special explanation.

The most obvious explanation is that marked differences in permeability of
the chorion itself might lead to exclusion of phosphate in some cases, and not in
other cases. But the work of Sumwalt (1929, 1933) on the potential differences
across this chorion shows that potential differences are set up across this mem-
brane when the egg is placed in salt solutions. This potential difference is not
affected by the change from a chloride to a sulfate, but is halved by the change
from K to Ca; equimolecular solutions of halides and sulfates give the same po-
tential difference, but alkali and alkaline earth chlorides yield potential differ-
ences varying with the physico-chemically determined mobilities of the cation,
and dilution of the bathing solution increases the observed plus potential. This

232 S. C. BROOKS

has been interpreted as due to selective permeability to cations, the chorion being
impermeable to anions.

It may be suggested that anions are fixed by chemosorption within the
chorion, and relatively few are allowed to pass through. The chorion with a
volume of about 0.50 mm3, conceived as consisting of about 10 per cent protein
of whose amino acids 10 per cent combine with anions, could account for about
2 X 10~7 equivalents of phosphate. Now, for example, the experiment given in
Figure 1 shows that one egg contains newly acquired phosphate in 15 minutes to
the extent of about 10~10 moles, or twice as many equivalents if we calculate on
the phosphate diion. Figure 3 shows a larger content, but still less than 2 X 10~7.
This means that all of the phosphate found in the egg can easily be kept within
the chorion itself by chemosorption or combination. This comports well with
the (Sumwalt's) concept of the chorion being anion impermeable. If the protein
of the chorion can combine to the estimated extent with anions the whole absorp-
tion can be thought of as combination of phosphate with the chorion. The losses
following the initial and later peaks in this case might be due to changes in the
combining power of the chorion, a point which will be reserved for later discussion.

It has been suggested that the radioactivity of the isotope may be responsible
for this phenomenon. The /3-activity of the external phosphate solution, deter-
mined by comparison with the /3-activity of uranium X, was between 0.18 (exp.
14) and 6.29 mC L~l (exp. 1). Muir (1942) has found that radioactive phosphate
increases the viscosity of Spirogyra protoplasm (defining viscosity as resistance of
chromatophores against centrifugal displacement), and alters the phosphate con-
tent when the activity of the immersion fluid was 4.0-17.0 mC L~', but has no
apparent effect when its activity was 2.0 mC L~l. This activity was calibrated
with the same uranium X standard of about 0.25 IJLC, which was used in my work.
Since this exceeds materially the activity of the most active solutions used in the
present experiments, it seems that injury, due to /3-radiation, of Fundulus eggs
at this strength must be considered to be improbable until more definite experi-
mental proof is available for the Fundulus egg.

This question may also be investigated from the basis given for the toxicity
of radioactive sodium for Nitella (Brooks, 1939). Here the sodium ion, of ac-
tivity equal to or less than 1 mC L-1, 7-ray measured, is non-toxic. Since the
ratio of /3- to 7-radiation of sodium is of the order of 20 we may calculate that the
/3-radiation of sodium of 20 mC L"1 is harmless. This is only a first approximation,
questions of absorption, energy of the /3-rays and so on complicate the picture.
The maximum energies of the /3-radiations of P32 and Na24 are substantially alike,
1.72 and 1.40 MEV., so that no great difference is to be expected on this count.
From this point of view also, no toxic effect of /3-radiation in the present experi-
ments it to be expected.

EXOSMOSIS INTO SEA WATER OF ABSORBED PHOSPHATE

Procedure. About 100 eggs, obtained in the usual way, were placed in a solu-
tion of phosphate, 0.32 mM L"1 in sea water, and lots of 10 eggs each were taken
out at intervals up to 40 or 60 minutes. They were washed in 200 ml. of dis-
tilled water for 5 to 10 seconds, transferred to depression slides, and freed from
distilled water. Then 0.5 ml of sea water was added, and after three minutes the
eggs were removed to another depression slide and freed of sea water as perfectly

INTAKE OF IONS BY MARINE EGGS

233

as possible with a capillary pipette, this fluid returned to the first slide. It was
found that some of the phosphate came out into the washing sea water. Washing
preliminary to this at this stage was avoided, so that the sum of the readings for
eggs and sea water would represent the total content of the eggs when they were
put into the sea water.

Readings were made on the lots of 10 eggs and on the corresponding sea water,
0.5 ml. A sample of 0.02 ml of the original phosphate-sea water had an activity
of 0.24 mC L-1.

Results. Figures 4 and 5 represent the results of the two experiments done.
In each there are plotted the initial phosphate concentration, just before im-

1.5

1.0
05

1
o
c
o
o
0.3O

0.20
010

0
X

0.5
OA
03
0.2
0.1

\ \

\ ^

s i

\\ /

\\* /

A-

./ \

(

{ Y'

IO 2O 3O 4O 50

LENGTH OF PREVIOUS IMMERSION.M1N.

FIGURE 4. Rates of exosmosis from unfertilized Fundulus eggs during immersion for 3
minutes in sea water when these eggs contained phosphate in concentrations obtained as the sum
of eggs plus sea water, (•) the K values calculated from measurements on eggs (o) or on the sea
water (X) (ordinates) after various times up to 40 minutes of immersion in sea water plus 0.32 mM
phosphate (abscissas), together with values of K X mean egg phosphate concentration (4-) all
plotted as ordinates.

mersion in the 0.5 ml of sea water (•) ; a diffusion constant, K, per minute, assum-
ing exponential exosmosis (o and X); and the product of the two ( + ). The
constant was calculated in two ways: from the decrease of phosphate in the eggs,
and the increase of phosphate in the sea water. These agree within the errors of
calculation.

We note that the diffusion constant is low when the phosphate concentration
initially in the eggs is high, and vice versa. Comparing the two experiments it is
apparent that the K values differ greatly and inversely to the concentrations of
phosphate in the eggs. Actual figures for means of all readings were K: 0.64 and
0.21; concentrations: 0.09 and 0.26 mM; ratios 3.0 and 1/2.9. The products of
concentrations by the constants do not give uniform figures, but these are more
so than either constants or concentrations in virtue of their inverse relation.
Their mean values for all determinations were 0.037 and 0.027, with a ratio of
1.37 in contrast to ratios of about 3 for concentrations and constants.

234

S. C. BROOKS

Discussion. These results may be interpreted as showing a) a high phosphate
within a cell lowers the permeability of the cell, or b) exosmosis of phosphate is
dominated principally by a constant rate of passage of this ion independent of
the concentration gradient across the surface of the egg. This latter might mean
that exosmosis depends on a metabolic process, or on saturation utilization of
relatively few available attachment points within the chorion. The latter hypo-
thesis is consonant with the conclusion that phosphate penetrates into and is held
in the chorion only (p. 231).

The process of exosmosis can also be thought of as an ionic exchange, a concept
closely related to the latter of these suggestions. For this the presence of counter

0.30

0.20

0.10

'0.03

0.02
0.01

10 2O 3O 5O 6O

LENGTH OF PREVIOUS IMMERSION,MIN.

FIGURE 5. Rates of exosmosis from unfertilized Fundulus eggs during immersion for 3 minutes
in sea water when these eggs contained phosphate in concentrations obtained as the sum of eggs
plus sea water, (•) the K values calculated from measurements on eggs (o) or on the sea water
(X) (ordinates) after various times up to 60 minutes of immersion in sea water plus 0.32 mM
phosphate (abscissas), together with values of K X mean egg phosphate concentration (+) all
plotted as ordinates.

ions to replace phosphate, i.e. CT~ and SO4=, both within the chorion or the plasma
membrane, and in the washing solution, here sea water, would be necessary.
Data are lacking for definitive discussion ; suggestions can be gotten from the
work of Jenny (1936), Jenny and Overstreet (1939), Graf (1937), Hendricks
(1941), and others.

Among such suggestions we may mention the possibility that replacement of
univalent chloride ion by the phosphate diion may lead to dehydration of either
the chorion or the plasma membrane, by simultaneous attraction of two groups
or molecules (lipins or proteins possibly) by the divalent ion. Schmitt and
Palmer (1940) cite the effect of Ca++ in contrast with K+ in dehydrating cephalin
suspensions. An effect of this type would seem to reduce the permeability of the
plasma membrane. The surface layer or plasma membrane is here thought of as
made up of hydrated proteins, lipins, etc. The chorion may resemble this ade-

INTAKE OF IONS BY MARINE EGGS

235

quately for this argument. This argues in favor of the first of the interpretations
here given just above.

GENERAL DISCUSSION

In this discussion it must be kept in mind that the phosphate may not pene-
trate through the chorion of the Fundulus egg during the first minutes or hours.
Longer immersion surely allows this ion to reach the embryo, as had been shown
by photographic tests. These demonstrate the presence of the radioactive phos-
phate in the skeleton of the Fundulus embryo.4 We are not in a position to deny
that the same ion passes through the chorion even during the first few minutes of
immersion. For this reason we shall speak of the permeability of the egg, without
attempting to trace the immediate destination of the phosphate ion.

Like the echinoderm egg (Brooks, 1940, 1943) and other materials (Brooks,
1940) which absorb cations, the Fundulus egg absorbs radioactive phosphate ions
in two or more distinct periods. The intake and permeability constants are given
in Table I, calculated from the slopes shown in Figures 1 to 3 at the indicated

TABLE I

The intake and permeability constants for phosphate into unfertilized eggs of Fundulus heteroclitus,
together with the maximum concentration, assumed uniform, within the eggs

Experiment
number

Figure
number

Constant

Concentration
of
experimental
fluid

Radio-
activity of
experimen-
tal fluid

Intake
constant

Permeability
constant

Maximum
concen-
tration
reached

mC/L

KX105

PX102

0.420

0.25

2.6

6.2

0.126

0.420

0.25

1.16

2.8

0.134

0.420

0.25

5.13

12.2

0.087

0.420

0.25

4.74

11.3

0.080 •

0.262

0.193

18.8

71.8

0.41

0.262

0.193

6.97

27.6

—

0.262

0.193

1.11

4.4

1.69

positions. The volume and surface of the ripe egg itself is used. Only minor
changes in these constants would result from the use of those of unripe eggs or
those of the chorion.

Table I shows that the rate of intake of phosphate was of the order of 1-7
X 10~5, a figure which can be compared with the values for Arbacia eggs of 2-100
X 10~10 moles cm~2 hr"1; and the permeability constants for Fundulus eggs were
0.03-1.69 X 10-2 compared with 2.2-42.6 X 1Q-6 moles cm-2 hr"1 (GM L-1)'1.
Asterias eggs show a permeability (Brooks, 1943) as high as 0.228 X 10~2. The
permeability constants here, as previously, are arbitrarily based on the assumption
that the only driving force is the concentration gradient into the egg. It should
be noted that dimensions of this constant reduce to cm hr"1, which differs from
those of the diffusion constants only by cm (the diffusion constants are expressible

4 Personal communication from Dr. L. J. Mullins.

236 S. C. BROOKS

as cm2 hr^1. It has been noted (Brooks, 1943) that if the thickness of the plasma
membrane is known the permeability constant can be multiplied by this thickness,
becoming expressible as cm2 hr"1. Here however it is doubtful that a plasma
membrane is the structure involved.

The maximum concentrations found in the Fundulus eggs vary from about
1/5 to about 6 times that of the immersion fluid (Table I). In the case of the
echinoderms (Brooks, 1943) we find maxima varying from about 1/26 to 22 times
that of the immersion fluid. In other words, the product of these extremes is
about one in each case, that is, they vary about unity. The nature of the causes
for such variations is still in doubt.

The fact that the permeabilities for Fundulus eggs are so much greater than
those for Arbacia and Asterias eggs suggests strongly that quite different mem-
branes are responsible for these magnitudes. It seems probable that the values for
Fundulus eggs are dominated by the chorion, while those for the echinoderm eggs
are properties of their plasma membrane, or possibly of the surface layer or the
whole cortex of the egg (Chambers, 1938). Just has emphasized the importance
of the egg cortex (Just, 1939) but it is by no means certain that the egg's per-
meability is a property of the whole egg cortex.

The fact that the chorion of a Fundulus egg has so ample a possible capacity to
combine, or fix by chemosorption of anions that all the content could easily be
bound, makes it seem possible that relatively little phosphate passes through this
membrane during the first few hours of experimentation. If this conclusion is
applicable to such univalent anions as chloride, this confirms the conclusion of
Sumwalt (1929, 1933).

The experiments then suggest strongly that they deal primarily with the
capacity of the chorion to absorb and lose the phosphate ion and to delay its
penetration into the egg by virtue of its capacity to bind this ion.

The occurrence of an early peak in ion intake, closely followed by a loss, is
more or less general, and, although it has not been found by many workers, still
it seems to seek a rational explanation. Pantanelli (1918) has noted such pheno-
mena when Valonia utriculosa, Saccharomyces sp., or Vicia Faba and other vascular
plants (whole plants with their roots in the bathing solution) are allowed to take
up ions from the bathing solutions. Analysis of these fluids showed early intake
within an hour or less, followed by loss and later by further intake of ions. Na+
K+, Mg++, Ca++, Ba++, Zn++, NO3-, SO4=, and HPO4= show this in one or more
cases. No radioactive ions were used, so that radioactivity cannot be blamed
for this type of intake which has been widely observed. Recently Leibowitz
and Kupermintz (1942) noted that Escherichia coli in a buffered glucose solution
containing KC1 would absorb K+ strongly within the first 5 minutes, and then
release it within an hour. The authors felt that this behavior was associated with
the formation of a polysaccharide, but the brief communication did not allow
convincing proof of this point.

In this connection it is of interest to note that amides and urea decrease the
viscosity of the ectoplasm (cortex) of Spirogyra sp. followed by return to about
the initial value. This was followed by repeated wide variations in viscosity
(Northen, 1940). Failure to maintain the normal position of the chloroplasts
during centrifugation was considered to be evidence of decreased viscosity. This
whole picture resembles that of the ion content of living cells during ion absorption.

INTAKE OF IONS BY MARINE EGGS 237

This parallelism suggests that the ion content is intimately connected with the
state of the proteins. Northen (1940) speaks of "protein dissociation." It may
be that the release of fatty materials from the Arbacia egg by the action of NH3
(Heilbrunn, 1937), the bringing out of the double diffraction by lipins in the
Golgi apparatus of snail spermatocytes by the use of chrysoidin, an amino con-
taining dye (Monne, 1939), the similarity of the action of the K and NH4 ions
and acetyl choline in breaking down the cell membranes at nerve synapses (see in
this connection Mann, Tennenbaum, and Quastel, 1939) and the general toxicity
of NH4 salts all reflect physico-chemical changes in proteins, presumably globulins
or possibly nucleoproteins. Such a change of state by a protein is shown when
the rodlets of tobacco mosaic virus, revealed by the electron microscope technique,
lose their ordered configuration when they are suspended in NaOH or NH4OH
containing solutions (Stanley, 1935; Stanley and Anderson, 1941). The behavior
of this nucleoprotein is due to the presence of NH4+ or NH3, and Na+ or possibly
the OH~ ion. Inactivation, which is related to this type of conversion of tobacco
mosaic virus, occurs also in urea solutions (Stanley and Lauffer, 1939), and in
alkaline suspensions produced by addition of NaOH (Stanley, 1935).

At least one of the alkali metal ions appears to be effective, and if all such ions
have similar properties in this case, as experiment shows in similar cases, then the
exposure of living cells to unbalanced solutions rich in such cations as Na+, K+,
Rb+, Cs+, NH4+, and probably the organic cations such as choline, guanidine,
amines, urea, the basic amino acids, etc.. would entrain a change in the protein
molecules ("conversion "). This, by analogy with the observed effects on viruses,
would probably decrease the capacity of these proteins to participate in the
molecular structure of the plasma membranes. The loss of elongated molecules
should deprive the plasma of essential, mechanically necessary components.
This effect might be produced directly, or by releasing NH3 or NH4+ which are
continuously present in any synthesizing cell, and allowing them to affect the
plasma membrane secondarily. Organic cations might well be an equally im-
portant factor (Hendricks, 1941). The mutual replacement of ions has been
studied carefully by soil students. Gieseking and Jenny (1936) found that the
NH4+ ion is very easily replaced by such ions as Na+ and K+. The alkaline earth
ions are more resistant to replacement, and their function can well be pictured
on this basis. For example, the presence of enhanced proportions of these ions
should stabilize the plasma membrane, and delay or prevent the conversion of
proteins such as we picture.

This breakdown in the plasma membrane is envisioned as marking a change
from a membrane permeable to ions by ionic exchange, that is, a membrane having
pores of about 7-9 A diameter (Brooks, 1943), presumably left between protein
molecules and preponderantly plus or minus charged, to a membrane which
allows ion pairs to diffuse through it, i.e. with larger and little charged pores
("mass movement" of Fenn, 1940). When a cell is placed in a solution contain-
ing suitable cations or anions we find that the ions of this solution are soon seen
in the protoplasm itself (e.g. Brooks, 1940), and tend to accumulate in excess of
the concentrations in the bathing solution. Soon, however, another process
supervenes and results in the movement of same ions (and perhaps others) out of
the protoplasm into the bathing solution. The first can be accounted for by ionic
exchange (by segregated cation exchange and anion exchange) utilizing as counter

238 S. C. BROOKS

ions such things as NH4+ or organic cations, and any of the organic anions or Cl
and phosphates, all of which are known to occur or are produced within living
cells generally. The second process is the loss of these accumulated ions by
allowing the free diffusion of salts to occur, involving the simultaneous movement
of the oppositely charged ions. The observed repetition of these gain and loss
phases strongly suggests that the fundamental changes in the proteins of the
protoplasma membrane and of the cortex can be reversible, or that converted
proteins are replaced by similar unconverted proteins from the deeper lying regions
in the cell. Newly made proteins may also serve in this replacement. This
hypothesis accounts qualitatively for the observed course of ion absorption where
accumulation and loss of ions occur. The loss phases might well be absent in
the cases where they were not observed either because of the absence of active
intracellular cations, or because of a more stabile plasma membrane.

This idea is also of importance in a case like the Fundulus egg. We have
noted that unripe eggs have a chorion which may be easily permeable to ions.
But on ripening the chorion should attain sufficient closeness of their fabric to
allow some degree of ion accumulation. Like the plasma membrane it may during
an experiment suffer a change in its proteins and allow loss of salts by mass move-
ment. Such a similarity between the chorion and its parent substances is ade-
quate to account for the similarity of the gain and losses of ions when Fundulus
eggs are placed in suitable salt solutions.

•

SUMMARY

Unfertilized eggs of Fundulus heteroclitus were immersed in sea water con-
taining low concentrations (0.193 to 0.25 mM) of radioactive sodium phosphate
and the phosphate content in subsequently collected samples after the intervals
from one-half minute to one hour was determined by measuring the 0-radiation
from the samples.

It was found that:

(a) The phosphate ion was taken in during two or more periods, separated
by periods during which the ion was lost.

(b) During the first rise the permeability was high (6.2-71.8 X 10~2 moles cm~:
hr"1 (Gm L"1)"1) while the later rises have lower permeabilities (2.0-4.4 X 10~2).

(c) The maximum concentrations found in these eggs varied from 0.080 to
1.69 mM or about 1/5 to about 6 times that of the immersion fluid for the re-
spective experiments.

The great rates of penetration (permeabilities) found, considered with the
relatively high combining power of the chorion has led to the tentative conclusion
that during the first hour or so of such experiments very little phosphate pene-
trates through the chorion to the egg cell itself.

A tentative theory as to the nature of the processes leading to intake and often
accumulation of an ion, its subsequent loss, and repetition of this cycle is proposed.
It depends on the assumption that proteins suffer reversibly or irreversibly a
"conversion" during ion intake, and that this is due to the action of the ions
concerned.

LITERATURE CITED

BROOKS, S. C., 1939. Ion exchanges in accumulation and loss of certain ions by the living proto-
plasm of Nitella. Jour. Cell. Comp. Physiol, 14: 303-401.

INTAKE OF IONS BY MARINE EGGS 239

BROOKS, S. C., 1940. The intake of radioactive isotopes by living cells. Cold Spring Harbor

Sympos. Quant. Biol., 8: 171-177.
BROOKS, S. C., 1943. Intake and loss of ions by living cells. I. Eggs and larvae of Arbacia

punctulata and Asterias forbesi exposed to phosphate and sodium ions. Biol. Bull 84-

213-225.
CHAMBERS, R., 1938. The physical state of protoplasm with special reference to its surface.

Amer. Naturalist, 72: 141-159.
FENN, W. O., 1940. The distribution of radioactive potassium, sodium and chlorine in rats and

rabbits. Bull. Conference Appl. Nuclear. Phys., p. 30.
GIESEKING, J. E., AND H. JENNY, 1936. Behavior of multivalent cations in base exchange. Soil

Science, 42: 273-280.

GRAF, E., 1937. Uber den Basenumtausch an Kasein. Kolloid Beih., 46: 229-310.
GRAY, J., 1932. The osmotic properties of the egg of the trout (Salmo fario). Jour. Exp. Biol ,

9: 277-299.

HEILBRUNN, L. V., 1937. Protein lipid binding in protoplasm. Biol. Bull., 71: 299-305,
HENDRICKS, S. B., 1941. Base exchange of the clay mineral montmorillonite for organic cations

and its dependence upon adsorption due to van der Waals forces. Jour. Phys. Chem.,

45: 65-81.
JENNY, H., 1936. Simple kinetic theory of ionic exchange. I. Ions of equal valence. Jour.

Phys. Chem., 40: 501-517.
JENNY, H., AND R. OVERSTREET, 1939. Surface migration of ions and contact exchange. Jour.

Phys. Chem., 43: 1185-1196.

JUST, E. E., 1939. The biology of the cell surface. Philadelphia. Blakiston's.
LEIBOWITZ, J., AND N. KUPERMINTZ, 1942. Potassium in bacterial fermentation. Nature, 150:

233.
MANN, P. J. G., M. TENNENBAUM AND J. H. QUASTEL, 1939. Acetylcholine metabolism in the

central nervous system. The effects of potassium and other cations on acetylcholine

liberation. Biochem. Jour., 33: 822-835.
MONNE, L., 1939. Polarisationsoptische Untersuchungen iiber die Golgiapparat und die Mito-

chondrien mannlicher Geschlechtszellen einiger Pulmonatenarten. Protoplasma, 32:

184-199.
MUIR, R. M., 1942. Effect of radiation from radioactive isotopes in the protoplasm of Spirogyra.

Jour. Cell. Comp. Physiol., 19: 244-247.
MULLINS, L. J., 1939. The effect of radiation from radioactive indicators on the penetration of

ions into Nitella. Jour. Cell. Comp. Physiol., 14: 403-405.

NORTHEN, H. T., 1940. Effects of protein-dissociating agents on the structural viscosity of proto-
plasm. Biodynamica, 3: 10-27.
PANTANELLI, E., 1918. Decorso dell'assorbimento di ioni nelle piante. Bull. Orto. Bot., R. Univ.

Napoli, 6: 1-37.
SCHMITT, F. O., AND K. J. PALMER, 1940. X-ray diffraction studies of lipide and lipide-protein

systems. Cold Spring Harbor Sympos. Quant. Biol., 8: 94-99.
STANLEY, W. M., 1935. Chemical studies on the virus of tobacco mosaic. III. Rates of inacti-

vation at different hydrogen-ion concentrations. Phytopathol., 25: 475-492.
STANLEY, W. M., AND T. F. ANDERSON, 1941. A study of purified viruses with the electron micro-
scope. Jour. Biol. Chem., 139: 325-338.
STANLEY, W. M., AND M. A. LAUFFER, 1939. Disintegration of tobacco mosaic virus in urea

solutions. Science, 89: 345-347.
SUMWALT, M., 1929. Potential differences across the chorion of the Fundulus egg. Biol. Bull.,

56: 193-213.
SUMWALT, M., 1933. Ion effects upon ion permeability of the Fundulus chorion. Biol. Bull., 64:

114-123.

BILIARY AMYLASE IN THE DOMESTIC FOWL

DONALD S. EARNER

(Department of Zoology, University of Wisconsin, Madison)

Although the question of the secretion of a biliary amylase by higher verte-
brate animals is discussed extensively in earlier physiological literature it has
attracted little attention in recent years. The general assumption among physi-
ologists is that bile has no enzymatic function in the processes of digestion.
Loehner (1929) presented a critical discussion of the earlier literature and recorded
the results of his own investigations in which he was able to prove the presence of
an amylase in the bile of cattle and sheep. Fossel (1931) demonstrated that this
amylase was hepatic in origin and not due to contamination with amylase from
the intestine. There is little information on biliary amylase in birds. Jacobson
(1856) reported its presence in the domestic fowl, domestic duck, and domestic
goose. Because of the methods used by him the probability of bacterial action
as the cause of amylolysis is not precluded. Stresemann (1934) stated that amy-
lase is found in the bile of the domestic goose. When preliminary investigations
by the author gave positive results he decided to conduct further experiments
which would give information concerning the relative amylolytic activity of the
bile and the general occurrence of biliary amylase in the domestic fowl.

MATERIALS AND METHODS

White Leghorns, Rhode Island Reds, and Barred Plymouth Rocks from the
stocks of the Department of Poultry Husbandry were used as experimental
animals.

Bile from the gall bladder was obtained by removal of the intact organ. The
bile was then drained directly into sterile vials (toluol added) by means of a small
incision in the wall of the bladder.

Bile was also obtained from the hepatic duct which leads directly from the
liver to the intestine. To secure this the bird was anesthetized with nembutal or
ether and placed on its left side. An incision was made on the right side posterior
to the last rib and parallel to the sternum at such a level that the duodenal loop
could be reached. This loop was then drawn to the opening made by the incision,
thus exposing the cystic, hepatic, and pancreatic ducts. A glass cannula with an in-
side diameter of about 0.7 mm. and a slight enlargement about 5 mm. from the end
was inserted through a small incision in the duct and tied in place. The purpose of
the slight enlargement near the end of the cannula was to prevent its slipping out
of the duct after it had been tied in place. A gum rubber collecting chamber, 15
cm. long and 1.5 cm. in diameter, containing a few drops of toluol was then at-
tached to the free end of the cannula. Usually the collecting chamber was
attached to the cannula before it was inserted into the duct. The chamber was
carefully placed among the loops of the intestine and the incision sutured. After
24 hours the bird was killed and the chamber removed. Seven successful opera-

240

BILIARY AMYLASE IN THE DOMESTIC FOWL

241

tions yielded 4 to 10 ml. of bile each. The principal difficulty encountered and
the reason for most of the failures was the clogging of the cannulae due to their
necessarily small diameters.

Pancreatic juice was obtained from the largest pancreatic duct by means of a
similar operation. The recovery of the birds was surprising. In more than 30
operations there were only three deaths, two during anesthesia and one from
hemorrhage.

Bile and pancreatic juice were used immediately for tests or were stored at
1° C. with toluol until used. No differences could be detected between the fresh
and stored samples.

Amylolytic activity was estimated quantitatively from the reducing sugar
produced by the action of the bile on one per cent boiled starch substrate (toluol
added) with 0.01 M phosphate buffer and 0.02 M NaCl at 40° C. Quantitative
estimations of reducing sugar were made according to the method of Shaffer and
Somogyi (1933). The only modification of this method necessary was the ex-
tension of the heating time of the digest sample with the copper reagent. An
arbitrary "amylase unit" was established for convenience in expression of results.
This unit is defined as that amount of amylolytic activity which will in one hour
produce 25 per cent of the maximum amount of reducing sugar from one ml. of one
per cent boiled starch at pH 7.10 (0.01 M phosphate buffer) with 0.02 M NaCl
at 40° C. Trial digests were made to establish the concentration of bile which
would give a linear relation with time for a period of about one hour after the
addition of the bile to the substrate.

Similar digests were made for qualitative tests. Reducing sugar was detected
by Benedict's qualitative test. All tests whether quantitative or qualitative
were made within one hour after the addition of bile to the substrate.

Experiment 1

In order to ascertain whether or not the biliary amylase was hepatic in origin,
bile was collected from the hepatic duct by means of a cannula thus excluding the

TABLE I

Relative amylolytic activity of bile and pancreatic juice

Activity in amylase units*

Group

Juice

Number
of birds

Optimum
pHt

/ml. juice

/mg. dr. weight

Gall bladder bile

15§

15-90

70-500

7.1 -7.2

Hepatic duct bile

10-30

200 700

7.1 -7.2

Pancreatic juice

800-6000

4000-600,000

7.05-7.15

* Arbitrary unit for comparative purposes. See text.

t With 0.01 M phosphate buffer and 0.02 M NaCl.

| Groups A and B have 4 birds in common.

§ Samples from 10 of these pooled and treated as a single sample.

possibility of contamination by amylase from the intestine. Samples of hepatic
duct bile from six birds were compared quantitatively with the gall bladder bile
from four of these birds as well as with gall bladder bile from 11 others. The

242

DONALD S. FARNER

samples from ten of the latter were pooled and treated as a single sample. The
data recorded in Table I show that the hepatic duct bile contained amylase in
amounts comparable to that in the gall bladder bile thus proving its origin in the
liver. Comparisons were made on a volumetric as well as dry weight basis. For
the purposes of studying relative activity, samples of pancreatic juice from three
different birds were analyzed quantitatively. The relative amylolytic activities
of bile and pancreatic juice as recorded in Table I reveal that the action of bile on
starch is small when compared with that of pancreatic juice.

The optimum hydrogen ion concentrations for the amylolytic actions of gall
bladder bile, hepatic duct bile, and pancreatic bile were determined by buffering
the substrate to various hydrogen ion concentrations with 0.01 M phosphate
buffer in the presence of 0.02 M NaCl. The optimum hydrogen ion concentration
as recorded in Table I agrees well with the accepted value for pancreatic amylase
(pH 7.1) in the presence of the same concentration of NaCl (Tauber, 1937).

Experiment 2

The general occurrence of amylase in the bile of the domestic fowl was studied
with qualitative tests on 50 birds in three age groups as shown in Table II.

TABLE II

General occurrence of biliary amylase in the domestic fowl

Source of bile

Age of birds

Number
of birds

Occurrence of amylase in bile*

present

not present

Hepatic duct
Gall bladder

adult
adult

6
20

6
19

0
1

Gall bladder
Gall bladder

8 weeks
4 weeks

12
12

2t
5

* Digest with 0.1-0.3 ml. bile and 1.2 ml. starch substrate with conditions as described in text.
t Both contained traces of amylase.

Samples which showed only traces of amylolytic activity were regarded as nega-
tive. Indications from these tests are that its occurrence is irregular among
younger birds.

DISCUSSION

Since bile obtained by the cannulation of the hepatic duct contained amylase
the possibility of its presence being due to contamination by amylase from the
intestine is eliminated. This is in agreement with the results obtained by Fossel
(1931) in cattle and sheep. His observation that due to reabsorption of water
the concentration of amylase in gall bladder bile is higher than that of hepatic
duct bile appears to be supported by the results of these experiments. Loehner's
observation that the amylolytic activity of the bile is not great when compared
to that of the pancreatic juice is certainly supported by the data obtained in this
investigation. However, the function of biliary amylase in the processes of
starch digestion should not necessarily be designated as unimportant until the

BILIARY AMYLASE IN THE DOMESTIC FOWL

243

relative rates of secretion of bile and pancreatic juice are known. No information
concerning this is available for the domestic fowl at the present time.

Since the liver is generally supposed to contain a starch-splitting enzyme,
"glycogenase," the question arises as to the possibility that biliary amylase and
"glycogenase" are identical. The author has no evidence concerning this. Any
consideration of this possible identity must take into account the conclusion of
Cori and Cori (1938) that glycolysis in the liver is due to the action of an enzyme
system rather than to the action of a single enzyme, "glycogenase," as formerly
supposed.

SUMMARY

The bile of the domestic fowl contains an amylase which is secreted by the
liver. Its optimum hydrogen ion concentration is similar to that of pancreatic
amylase. The amylolytic activity of pancreatic juice was found to be 10-800
times that of bile.

A cknowledgments

The author wishes to thank the Department of Poultry Husbandry for the
experimental birds, and Professor C. A. Herrick, Departments of Zoology and
Veterinary Science, for laboratory facilities and for advice and assistance in
performing operations.

LITERATURE CITED

CORI, G. T., AND C. F. CORI, 1938. Enzymatic breakdown of glycogen in liver extracts. Proc.

Soc. Exp. Biol. Med., 39: 337-338.
FOSSEL, M., 1931. Gallen- und Gallenwegstudien. V. Ueber die Herkunst der Gallenamylasen.

Pflueg. Arch. ges. Physiol, 228: 764-771.
JACOBSON, J., 1865. De sacchari formatione fermentoque in jecore et de fermento in bile. Inaug.

Dissert. Koenigsberg, 1865.
LOEHNER, L., 1929. Gallen- und Gallenwegstudien. IV. Zur Entscheidung der Gallenamylasen-

frage. Pflueg. Arch. ges. Physiol., 223: 436-449.
SHAFFER, P. A., AND M. SOMOGYI, 1933. Copper-iodometric reagents for sugar determination.

Jour. Biol. Chem., 100: 695-713.
STRESEMANN, E., 1934. In Kuekenthal und Krumbach, Handbuch der Zoologie, 7, II, Aves,

p. 487.
TAUBER, H., 1937. Enzyme Chemistry. John Wiley and Sons, New York, 139-141.

^LIBRARY

ACCELERATION OF CLEAVAGE OF ARBACIA EGGS BY

HYPOTONIC SEA WATER

IVOR CORNMAN

(Marine Biological Laboratory, Woods Hole, and Department of Zoology,
University of Michigan, Ann Arbor)

During the summer of 1938, a series of experiments was undertaken at Dr.
Robert Chambers' suggestion, with a view to determining something of the
mechanism by which decreased tonicity slows mitosis. In the dosage-rate curves
there was noted an aberration which seemed to indicate an acceleration of cleav-
age in slightly diluted sea water. Detailed investigation showed the acceleration
to be real,1 and the results are presented here.

I am indebted to Dr. Robert Chambers for continued helpful advice, and to
Dr. Alvalyn Woodward for sharing laboratory space supplied by the Faculty Re-
search Fund of the University of Michigan during the summer of 1941.

MATERIALS AND METHODS

The eggs of Arbacia punctulata were used throughout. For each experiment,
the washed, concentrated eggs were measured into control and hypotonic solutions
in quick succession. All eggs were examined after 24 to 48 hours for normal
development. Hypotonicity is represented in percentage of normal sea water:
96 per cent being 4 parts of distilled water in 96 of sea water. Both concentration
and time of exposure were varied experimentally. .

The solutions and eggs were brought to a uniform temperature before mixing,
and the temperature maintained with a water bath. There was no difference
among the solutions measurable within a tenth of a degree Centigrade, and the
positions of the flasks were rotated to prevent favoring of any one by an imper-
ceptible temperature difference. In some of the earliest experiments, in which
a small water bath was used, the temperature rose as much as 3° C. during the
course of the experiment, but frequent measurements of the solutions were made
to insure an identical temperature in the different solutions. The temperatures
employed ranged from 18.5° to 23° C., being kept close to that of the running
water in the tanks to avoid temperature shock in transferring the eggs from the
sea urchins to the solutions.

It should be emphasized that alteration in rate of cleavage always refers to a
comparison of experimental and control lots of eggs from a single female, run at
the same time and subject to the same variations in temperature or other environ-
mental factors. To check on variability in the method, experiments were run

1 A search of the literature revealed that this accelerating effect of hypotonicity had received
considerable attention, but no experiments upon the cleavage rate of marine eggs had been re-
ported. Not until the work upon Arbacia eggs had already been reported (Cornman, 1940) and
the full account was ready for press was it discovered that the acceleration of cleavage in Arbacia
eggs had been extensively studied ten years earlier in the physiology class at Woods Hole under
Dr. M. H. Jacobs' direction. The effect had been noted before that by Dr. Walter E. Carrey.

244

HYPOTONIC ACCELERATION OF CLEAVAGE 245

with sea water in all flasks. The curves thus obtained from these six identical
controls were exactly superimposable, and treated statistically showed no signifi-
cant differences. Besides sea water controls, isotonic controls were also used in
some experiments to check against the effect of electrolyte concentration. Iso-
tonic sodium chloride was used as a control for total electrolyte content. These
solutions were introduced in the same quantities as distilled water in the experi-
ments with tonicities of 96 per cent and 95 per cent.

In the crucial experiments the pH of the media was checked with a glass elec-
trode at the beginning and at the end of the experiments. Between the concen-
trations 100 per cent and 88 per cent sea water, the range in which acceleration of
cleavage occurs, there was found to be a drift of only 0.1 pH unit toward alkalinity
upon dilution, and no further change during the course of the experiment. The
natural buffers of sea water were adequate to prevent any greater change in pH.

Comparison of the rates of cleavage was made by statistical analysis of sam-
ples fixed at successive intervals of two, three, or five minutes. This was done by
pipetting samples from each of the control and experimental solutions, into one
per cent formaldehyde, all within 10 to 15 seconds of each other. In each of
these samples, 200 eggs were counted. In order to obtain samples that were as
representative as possible, the experimental solutions were stirred after each
sampling, and the killed eggs were thoroughly mixed before they were transferred
to the counting slide. In counting the eggs, a regular pattern was followed which
covered the entire slide. From these samples, the percentage of eggs cleaved
within each period was determined. Arranged in a frequency table, these per-
centages supplied the distribution, mean, and median of the time between fer-
tilization and cleavage. In these statistical calculations, the percentage of eggs
cleaved during the period between the moments of sampling constituted the fre-
quencies of the classes, and the times at which the samples were taken constituted
the class limits. Thus, the data could be handled by conventional statistical
methods. Although not entirely satisfactory, it gives a preliminary estimate of
the validity of results. Complete analysis involves special statistical problems
which cannot be dealt with here. In all, over 500,000 eggs were counted.

OBSERVATIONS

The results of typical experiments are represented graphically in Figure 1.
Connecting the cleavage percentages are steep ogives, tapering somewhat more
near the 100 per cent cleavage mark than near zero. Experiment 1-12
(Figure 1) show a series of curves revealing a clear-cut acceleration in 96 per cent
and 92 per cent sea water, no acceleration in 88 per cent, and retardation in 84
per cent.

In general, acceleration of the first cleavage was found to occur in concentra-
tions between 100 per cent and 88 per cent sea water. Acceleration was obtained
with treatments beginning anywhere from four minutes after insemination, up to
the diaster of the first cleavage. There was no sharply defined peak of accelera-
tion at any one tonicity, but acceleration in 96 per cent was most frequently the
highest. Moreover, the maximum acceleration for any single experiment oc-
curred at 96 per cent: a statistically calculated acceleration of 3.36 minutes, which,
referred to the control mean cleavage time of 65.36 minutes, represents a shorten-
ing of the cleavage time by 5.1 per cent. Accelerations obtained varied from this

246

IVOR CORNMAN

5.1 per cent to zero. Particularly during the summer of 1941 the eggs showed
little response to hypotonicity, although the experimental procedure was the same
as previously used. It should be emphasized that the variation was between zero
and statistically significant accelerations. In the several hundred experiments
there were few decelerations obtained in 98 per cent to 94 per cent sea water, and
none of these was significant insofar as the statistical methods employed could

100

MINUTES rROM INSEMINATION

FIGURE 1. Typical curves for first cleavage, showing a moderate hypotonicity effect, the
amount of scattering about the ogive, and the slight variations in slope. The curves are smoothed
by eye in accordance with the general trend of a large number of curves.

1-12: Cleavage curves for 84 per cent to 100 per cent sea water. They show acceleration in 96
per cent and 92 per cent, no effect in 88 per cent, and retardation in 84 per cent. The acceleration
in 96 per cent, determined statistically, was 2.85 ± 0.43 minutes. Treatment begun 14 minutes
after insemination. Temperature 19° C.

V-4: Cleavage curves for 96 per cent and 100 per cent sea water, and for the isotonic sodium chloride
control for 96 per cent. There is a decided acceleration in the hypotonic sea water, but none in
the isotonic control. The statistically determined acceleration in 96 per cent was 2.31 ± 0.36
minutes; in the isotonic control, —0.04 ± 0.41 minutes. Treatment begun 13 minutes after
insemination; temperature 18.5° C.

show. In the range 92 per cent to 88 per cent, on the contrary, some experimental
sets were retarded, while others were accelerated. This possibly represents a dif-
ferent threshold of response among the different sets. Dilutions to 84 per cent
and more always gave decelerated cleavage, as did all hypertonic solutions.

At 19° to 20° C., cleavage between 5 per cent and 95 per cent is completed in
from four to 20 minutes in normally developing eggs. (The spread for the steep
section of the ogive in experiment 1-12 is about 8.5 minutes.) There are much

HYPOTONIC ACCELERATION OF CLEAVAGE 247

smaller differences of spread within a single experiment (Figure 1). These dif-
ferences in slope have not been analyzed for significance.

Changes in electrolyte content and electrolyte balance caused no acceleration.
Cleavage curves for 96 per cent sea water, and sea water diluted to 96 per cent
by isotonic NaCl, are compared with cleavage in 100 per cent sea water in Figure
1, experiment V-4. Both maltose and sucrose isotonic controls retarded cleavage
slightly, but probably not significantly. Lillie and Cattell (1929) found no con-
siderable alteration of cleavage rate, even with electrolytes reduced to 60 per cent
with isotonic sucrose.

When eggs remain in the hypotonic solutions, the acceleration is not duplicated
in the second and third cleavages. Fewer figures are available for these cleav-
ages, but they indicate definitely that after acceleration of the first cleavage, sub-
sequent cleavages are not accelerated to the same extent. Also, if the first cleav-
age does not respond to hypotonicity, the second cleavage does not. However,
the second cleavage will sometimes respond to hypotonicity if the eggs are im-
mersed after completion of the first cleavage. The acceleration is not as marked
as that obtained in the first cleavage, reflecting, perhaps, the changed condition
of the egg, and the shorter time available for the hypotonicity to act.

DISCUSSION

The amount and consistency of acceleration obtained show clearly that cleav-
age of Arbacia eggs proceeds faster in hypotonic sea water than in normal sea
water. However, dilution of sea water has effects other than reduction of the
tonicity, so it remains to be shown that tonicity, and not some other dilution
effect is responsible for the acceleration. Electrolyte concentration is one of
these factors which must be distinguished from simple osmotic activity, since the
electrolytes in sea water affect cells in many ways other than by their osmotic
pressure. To test this possibility, it is a simple matter to reduce the electrolyte
content without reducing the tonicity, by introducing isotonic nonelectrolytes
into control solutions. Maltose and sucrose were used in these controls. The
results with both were the same: a slight retardation of doubtful statistical
significance. Electrolyte balance may also be involved to a slight degree, since
some salts occur in sea water in much lower concentrations than do others. So-
dium chloride is the most plentiful, and so diluting sea water might have the effect
of changing the salt balance in favor of NaCl, particularly if one of the less abun-
dant salts were brought near a physiological threshold of the Arbacia egg. If
this is the case, then diluting sea water with isotonic NaCl should effect an even
greater NaCl preponderance without any tonicity change. In controls diluted,
no significant difference from the sea water control was in evidence. Unless we
assume that sugars or sodium chloride in some way counteract a stimulation
caused by changes in electrolyte content or salt balance, we must conclude thaJ
electrolyte alterations play no part in the acceleration of cleavage by dilution.

There remains the question of pH effect, since a very slight trend toward
alkalinity occurred in the range of dilutions which produced acceleration. This
shift amounted to 0.1 pH unit upon dilution to 90 per cent, from which interpola-
tion gives a 0.05 unit shift for the 96 per cent and 95 per cent dilutions. While it
is extremely small, this pH shift must be taken into consideration, because a

248 IVOR CORNMAN

number of investigators have found an acceleration of cleavage in sea water made
more alkaline. There is by no means unanimous agreement upon this accelera-
tion by alkalinity. Jacques Loeb (1913) concluded that the natural hydroxyl ion
concentration is optimum for the development of sea urchin eggs, although he had
earlier reported (1898) that addition of sodium hydroxide accelerated develop-
ment. Of the several papers reporting acceleration of cleavage with increase in
pH, the work of Smith and Clowes (1924) with Arbacia eggs is the most applicable,
since they determined the precise pH values involved. In their Figure I, a pH
increase of 0.05 corresponds to an acceleration of less than one per cent. Even
part of this small increase must be a hypotonicity effect because 0.02 N NaOH was
used to increase the pH, whereas an isotonic concentration would be around 0.5 N.
So while tonicity cannot account for the marked acceleration obtained in basified
sea water, nor pH for the acceleration obtained with slight dilutions, there remains
the possibility that the results of Smith and Clowes, and those presented here,
represent to a slight degree combined effects of pH and tonicity, in vastly different
proportions.

Since the accelerations in the various hypotonicities do not form a smoothly
graded series when different experiments are compared, it should be emphasized
that these results were obtained under a variety of experimental conditions.
Temperatures, chosen to conform with that of the incoming sea water to avoid
temperature shock, ranged from 18.5° to 23° C. Eggs from different sea urchins
vary in response, and there possibly is a seasonal difference. There is even a
yearly difference; eggs in the summer of 1941 showed much less response than in
1940 and 1939. Particularly important is the natural variation in sea water con-
centration along the coast (Garrey, 1915). These variables do not affect the
validity of the results, since results are stated in terms of controls run at the
same time with eggs from the same material.

From the work of other authors we can conclude that hypotonic stimulation of
cell division is not an isolated phenomenon. It speeds up regenerative as well as
embryonic processes. Jaques Loeb, in 1892, reported accelerated growth and
regeneration of Tubularia hydranths kept in hypotonic sea water (one-third dis-
tilled water), and suggested that growth in general decreases with decrease in
water content and increases with limited increase in water content. Morgulis
(1911) demonstrated that there is actually a high water content in the regenerat-
ing tails of the polychaete, Podarke, and the salamander, Triturus. Goldfarb
(1907) obtained maximum regeneration with Eudendrium and Pennaria hydranths
in sea water diluted to 95 per cent to 80 per cent. Sayles (1928) found that new
tissue formation during regeneration in Lumbriculus increased as the medium was
diluted below the tonicity of the body fluid. Injecting distilled water into the
body cavity (Sayles, 1931) produced the same cellular picture that was found in
regenerating worms. Increase in mitoses in the digestive tract was one of the
responses to hypotonicity. Aisenberg (1935) obtained increase in mitoses in frog
epithelium after immersion in distilled water, or injection of distilled water.

Following the work of Carrel and Burrows (1911), a number of workers verified
these authors' finding of an increase in growth of tissue cultured in diluted media.
In most cases, however, growth was measured by the area of the outgrowth, and
the role of mitosis in this increase was not studied. Lambert found no increase
in cell division and attributed the increase in area of the cultures entirely to migra-

HYPOTONIC ACCELERATION OF CLEAVACK 249

tion of the cells. The experiments of von Mollendorff with fibrocytes of adult
rabbits also revealed no acceleration in hypotonic culture media. On the con-

trary, Zivago, Morosov, and Ivanickaja (1934) found that cells of human em-
bryonic heart do proliferate more rapidly in diluted culture medium. Olivio and
Gomerato (1932) also found an increase in mitoses in hypotonic tissue culture
media. In the first transplant into plasma diluted to half, they found the mitotic
index of chick heart was nearly twice that of similar tissue grown in undiluted
plasma. In subsequent transplants, the hypotonic cultures from seven-day
hearts maintained a higher mitotic index throughout the 11 days of culture (four
transplants). However, the mitotic index of the three-day heart dropped below
that of the control after the first transplant. Knake (1933) found a selective
effect of hypotonicity upon cultures of chick pancreas, resulting in increased
growth of epithelium and decreased growth of fibroblasts.

The effects of hypotonicity upon echinoderm development have been reported
only for stages later than the initial cleavages. Vernon (1895), working with
Strongylocentrotus, and Medes (1917), with Arbacia, obtained larger larvae in
slightly diluted sea water. Bialaszewicz (1921), however, reports only retarda-
tion of Strongylocentrotus and Echinus between the 4 to 12 blastomere stage
and the blastula, in all dilutions.

There are some clues as to the mode of action of the hypotonicity. One point
at which it takes effect is within the mitotic' cycle, inasmuch as cleavage can be
accelerated by treatment begun as late as the diaster stage. The greater accelera-
tion obtained when treatment is begun earlier may merely reflect the longer time
of action, or may result from a second point of action during the fertilization
process. Churney's studies (1940) suggest that the elongation of the egg prepara-
tory to cleavage may be the process sensitive to hypotonic acceleration. He
found that elongation of the eggs is proportional to the dilution. If hypotonicity
acts at this point, it must do so by allowing the elongation to occur sooner, since
merely speeding the elongation once it has begun could not produce accelerations
as large as those obtained.

The type of action by which hypotonicity takes effect would seem to be an
improvement of the intracellular conditions, bringing them nearer optimum for
cleavage, at least. If such is the case, one would expect that each successive
division would be equally accelerated. Some of the results reviewed above,
particularly those of Olivio and Gomerato, must represent such a sustained effect.
Possibly in Arbacia the available energy is quickly exhausted, so that only one
cleavage can be hastened. Varying supplies of energy could also explain the
large variations between different sets of eggs. However, these experiments are
not designed to eliminate the possibility that the acceleration results from a single
stimulus rather than a sustained optimum. It is hoped that interrupted dosage,
after which the eggs are returned to normal sea water, will reveal whether stimulus
or optimum is the accelerating mechanism.

SUMMARY

1. In sea water diluted to a concentration 98 per cent to 94 per cent that of
normal sea water, the eggs that responded showed acceleration of the first cleavage

250 IVOR CORNMAN

as high as 5.1 per cent of the normal time between insemination and cleavage.
Eggs from some sea urchins did not respond, but none showed deceleration in
this range of concentrations.

2. In concentrations 92 per cent to 88 per cent, some sets of eggs were accel-
erated, while others were retarded. This reveals a threshold of antagonism be-
tween accelerating and decelerating effects.

3. Concentrations 84 per cent and less always retarded, as did concentrations
hypertonic to sea water.

4. Sea water diluted with isotonic electrolyte or nonelectrolyte did not produce
an acceleration.

5. The response could be obtained with treatment begun shortly after entrance
of the sperm, or as late as the diaster of the first cleavage. The latter indicates
that the acceleration results in part, at least, from action upon some phase of
mitosis.

6. The second cleavage could be accelerated separately, but eggs left in hypo-
tonic sea water did not show cumulative accelerations for each cleavage. This
shows that limited energy is available, or the effect is of the nature of a stimulus.

LITERATURE CITED

AISENBERG, E. J., 1935. De 1'effet de 1'hypo et de 1'hypertonie sur les mitoses. Bull. Histol.

Appl., 12: 100-122.
BIALASZEWICZ, K., 1921. L'influence de la pression osmotique sur la vitesse du developpement

des embryons. Travaux du lab. physiol. Inst. M. Nencki (Soc. Sci. Varsovie), 1: 1-14.
CARREL, A., AND M. T. BURROWS, 1911. On the physico-chemical regulation of growth of tissues.

The effects of dilution of the medium on the growth of the spleen. Jour. Exper. Med., 13:

562-570.
CHURNEY, L., 1940. Mitotic elongation. II. Osmotic and salt effects. Physiol. ZooL, 13: 95-

101.
CORNMAN, I., 1940. Acceleration of cleavage by hypotonic sea water (abstract). Anal. Rec.,

78: suppl. p. 76.

GARREY, W. E., 1915. Some cryoscopic and osmotic data. Biol. Bull., 28: 77-86.
GOLDFARB, A. J., 1907. Factors in the regeneration of a compound hydroid Eudendrium ramosum.

Jour. Exper. ZooL, 4: 317-356.
KNAKE, E., 1933. Uber die Wirkung einer Veranderung des osmotischen Druckes, untersucht an

Gewebekulturen. Archiv f. exper. ZeUforsch., 14: 611-615.

LAMBERT, R. A., 1914. The effect of dilution of plasma medium on the growth and fat accumula-
tion of cells in tissue cultures. Jour. Exper. Med., 19: 398-405.
LILLIE, R. S., AND W. CATTELL, 1929. The relation between the electrical conductivity of the

external medium and the rate of cell division in sea urchin eggs. Jour. Gen. Physiol., 5:

807-814.
LOEB, J., 1892. Untersuchungen zur physiologischen Morphologic II, Chapter VIII. Hertz,

Wiirzburg.
LOEB, J., 1898. Uber den Einfluss von Alkalien und Sauren auf die embryonale Entwickelung und

das Wachsthum. Archiv f. Entw-mech., 7: 631-641.

LOEB, J., 1913. Artificial parthenogenesis and fertilization, p. 35. LTniv. Chicago Press.
MEDES, G., 1917. A study of the causes and the extent of variations in the larvae of Arbacia

punctulata. Jour. Morph., 30: 317-432.
VON MOLLENDORFF, W., 1938. Zur Kenntnis der Mitose. Der Einfluss von hypo- und hypertonie

auf den ablauf der Mitose sowie auf den Wachstumsrhythmus von Gewebekulturen.

Zeitschr. ZeUforsch. mikr. Anal., 28: 512-546.
MORGULIS, S., 1911. Contributions to the physiology of regeneration. IV. Regulation of the

water content in regeneration. Jour. Exper. ZooL, 10: 321-348.
MORSE, H. N., 1914. The osmotic pressure of aqueous solutions. Carnegie Inst. Wash. Publ. 198.

HYPOTONIC ACCELERATION OF CLEAVAGE 251

OLIVIO, O. M., AND G. GOMERATO, 1932. Coefficients mitotico dell'accrescimento delle colture

in vitro in plasma ipotonico. Boll. Soc. Ital. Biol. Sperim., 7: 482-484.
SAYLES, L. P., 1928. Regeneration of Lumbriculus in various Ringer fluids. Biol. Bull., 55:

202-208.
SAYLES, L. P., 1931. Double nucleoli and mitoses in cells of the alimentary tract of Lumbriculus

following dilution of the body fluids. Jour. Exper. Zoo/., 58: 487-494.
SMITH, H. W., AND G. H. A. CLOWES, 1924. The influence of hydrogen ion concentration on the

development of normally fertilized Arbacia and Asterias eggs. Biol. Bull., 47: 323-332.
VERNON, H. M., 1895. The effect of environment on the development of echinoderm larvae.

Proc. Roy. Soc. London., 57: 382-385.
ZIVAGO, P. L, B. D. MOROSOV, AND A. F. IVANICKAJA, 1934. Uber dieEinwirkungder Hypotonie

auf die Zellteilung in den Gewebekulturen des embryonalen Herzens. Comptes rendus

Acad. Sci. U.R.S.S., 3: 385-386.

THE INDEPENDENT DIFFERENTIATION OF THE SENSORY
AREAS OF THE AVIAN INNER EAR

HIRAM J. EVANS

(Biological Laboratories, Harvard University, Cambridge)

INTRODUCTION

The differentiation of the otocyst of the chick embryo when isolated and trans-
planted to the chorioallantoic membrane has been studied by Hoadley (1924).
Grafts of primordia taken from 48-hour embryos showed membranous labyrinths
which, while they were irregular in shape, displayed considerable histogenetic
differentiation. This was particularly true of the sensory areas, which presented
an histological picture directly comparable to that of the control. As neither
nerves nor ganglion cells were found in the grafts Hoadley concluded that in that
respect the sensory areas were independent differentiations of the otic epithelium.
Fell (1928) studied the development of the 72-hour chick otocyst in tissue culture
and obtained labyrinths which showed good histological differentiation but only
slight advance in morphogenesis. She reported that the sensory areas of the
explanted inner ears were nearly comparable to the sensory areas of the control.
No observations on isolated labyrinths which were explanted and treated with a
specific nerve stain are recorded by either of these investigators.

The results obtained by Hoadley and by Fell indicate that differentiation of
the sensory areas of the chick labyrinth may take place independent of innerva-
tion. This cannot be proved, however, until it is demonstrated beyond doubt
that there are no nerve fibers present in the immediate vicinity of or associated
with well differentiated sensory areas of explanted labyrinths. It is difficult
to demonstrate this without resorting to techniques which selectively stain the
nerves, many of which are not satisfactory for embryonic preparations. The
method of Bodian (1937), however, has yielded excellent results. The following
experiments were made in an effort to test the validity of the conclusions of
Hoadley and of Fell. The results of the experiments confirm those conclusions.

MATERIAL AND METHODS

In the following experiments grafts to the chorioallantoic membrane were
made essentially as described by Hoadley. Chick embryds of ages ranging from
12-somites to 43-somites were placed in warm mammalian Ringer's solution on a
warm stage on the stage of a binocular dissecting microscope. Fine knives made
from dissecting needles were used in the operations which were performed by aid
of a low magnification of the microscope. The otic region was removed and
treated in either of two fashions. Some of the transplants were composed of the
otocyst together with the surrounding mesenchyme and a portion of the adjacent
myelencephalon ; in other cases the otocyst was freed and then cleaned of as much
of the adhering mesenchyme tissue as was possible in view of the haste required
in the operation and the desire to avoid mechanical injury to the primordium. In

252

DIFFERENTIATION OF THE AVIAN EAR 253

most of the later transplantations, the otocyst alone was wrapped in a mantle of
somatopleure taken from the lateral plate region of the area pellucida. Such
treatment materially increased the percentage of successful grafts, apparently by
protecting the donor tissues in the interval before complete incorporation by the
host membrane. An envelope of splanchnopleure was also tested in the trans-
plants but the somatopleure proved to be superior for the purpose. Control
transplants of both somatopleure and splanchnopleure alone as well as of mes-
enchyme from the otic region were made and examined.

The grafts were fixed in Allen's P.F.A.3 fixative, sectioned at 10 n and stained
with activated protargol according to the method of Bodian (1937).

EXPERIMENTAL RESULTS

Transplants of inner ear rudiments isolated from embryos of several different
ages have been made and numerous grafts have been recovered. Identification
of parts of these graft ears has been made largely on the basis of histological dif-
ferentiation, but in some instances, the morphology of the labyrinth has been used
to supplement the histological picture.

Since many of the younger otocysts were wrapped in either somatopleure or
splanchnopleure from the lateral plate region before transplantation and all ex-
plants had some mesenchyme cells clinging to them, it was thought well to deter-
mine what embryonic structures might arise from these tissues when transplanted
to a foreign environment. To do this, some transplants were made of somato-
pleure alone, some of splanchnopleure, and some of the mesenchyme adjacent to
the otocyst. Graft A 184-41 is of somatopleure taken from the lateral plate
region of a 31 -somite donor and has a control age of 9^ days. There is no dif-
ferentiation of embryonic structures in the graft. The same result was obtained
when splanchnopleure of the lateral plate region of a 30-somite (ca.) donor was
grown for 7 days.

Considerable differentiation is found in some grafts of mesenchyme taken from
the region of the otocyst. Two cases are worthy of mention, because ganglion
cells and cartilage are present in both. Unfortunately, the exact region from
which the mesenchyme was taken is not recorded in the protocols. Case A192-41
is of mesenchyme taken from a 31-somite donor and has a control age of 9| days.
It contains an elongated nodule of well -differentiated cartilage and beyond one end
of this cartilage is a ganglion. Case A182-41 is of mesenchyme from a 28-somite
donor. It has a control age of 9 days. A cartilage nodule is present with
ganglion cells localized at one side of it. There are several epithelial pearls in
this graft.

The presence of cartilage in the grafts of mesenchyme indicates that by 28
somites the mesenchyme in the region of the otocyst possesses the capacity to form
cartilage even though it is isolated from the influence of the membranous laby-
rinth. The ganglion cells which are present in both of these grafts probably mean
that some cranial neural crest material or some cells from the field of the acoustico-.
facialis ganglion were transplanted with the mesenchyme.

Since the grafts recovered are too numerous for complete description, they
have been divided into convenient groups based on the stage implanted and typical

254

HIRAM J. EVANS

examples from each group will be fully described. Information secured from
some of the other grafts which will not be described is presented in Table I.

It is extremely difficult to determine the specific identity of a sensory area in
a graft. Because of this, thickenings of the otic epithelium which tend to re-
semble cristae are referred to as cristae and the macula-like sensory areas are
designated as maculae. It should be borne in mind that these terms are used in a
descriptive sense and do not always imply a positive identification of a specific
sensory area.

TABLE I

Data on some cf the grafts of the otic region which are not described in the text

Case number

Donor age

Control
age in
days

Remarks

A2-40

17S

Several sensory thickenings and most of them are innervated.

A247^1

18S

Two otic pits and brain implanted. Both inner ears have dif-

ferentiated sensory areas. Some of these are innervated.

A327-41

25S

Good morphological differentiation. No ganglion cells in graft

and no differentiation of sensory areas.

A 140-41

26S

Two sensory areas with no nerves to their epithelia.

A244-41

28S

Nerves and ganglion cells present but no differentiation of sen-

sory areas.

A 169-41

31S

8£

Shows utriculus, sacculus, canal rudiment and endolymphatic

evagination. Half-moon of cartilage around labyrinth. No

nerves in graft. Contains a well differentiated crista and a

macula.

12-20 Somites. Case A299-41 is a 7-day graft of an otic placode isolated from
a 12-somite donor. Before transplantation the placode was wrapped in somato-
pleure. The membranous labyrinth consists of two vesicular structures of un-
equal size connected by a small lumen and has an endolymphatic duct and sac.
There is no development of sensory areas in the epithelium of the smaller vesicle,
but the larger vesicle has several sensory areas. The high columnar epithelium
of the endolymphatic sac is more convoluted than in the control. No canal
rudiments are present. A rod-shaped cartilage mass is found along one side of
the ear. There are a few ganglion cells and short nerve processes between lobular
outpocketings of the endolymphatic sac but these neurones are localized and no
nerves approach the sensory epithelium. Three cristae which appear to be dif-
ferentiations of a large patch of thickened epithelium are present in the larger
vesicle. There is no definite development of hair and supporting cells, but some
short dark-staining cells which reach the surface of the epithelium suggest the
initial differentiation of hair cells. No nerves enter the sensory areas. These
sensory areas do not project as far into the lumen as the cristae of the control.

Near one end of the larger vesicle there are two small, flattened epithelial
thickenings with neither otolithic membranes nor differentiated hair cells. Their
form is similar to that of a macula but the small size and lack of differentiation
make their identity somewhat problematical.

Another graft (A302-41) in this group is of an otic pit isolated from an 18-
somite embryo. This was not wrapped in either somatopleure or splanchnopleure

DIFFERENTIATION OF THE AVIAN EAR 255

before implantation. The control age is 8| days. This inner ear is vesicular and
has an endolymphatic duct and sac. Well differentiated cartilage forms a half-
moon around the side of the labyrinth which is away from the endolymphatic duct
(Figure 1). Ganglion cells and nerves are present in the graft but they are not
found in the immediate vicinity of the sensory epithelium. A crista (Figures 1
and 2) has a slight indication of a covering membrane but hair and supporting
cells are not clearly differentiated. No nerve elements can be distinguished among
the connective tissue cells which underlie the sensory epithelium.

The nerve fibers in this graft originate from ganglion cells located in the
mesenchyme to one side of the otic epithelium. From these cell bodies the fibers
pass through a break in the cartilage to a ganglion located beside the endolym-
phatic sac. Some nerves from this latter ganglion pass close to the epithelium
of the endolymphatic sac and are lost in the surrounding connective tissue. An-
other branch continues through the break in the cartilage and passes in the oppo-
site direction along the outer edge of the cartilage to a brain-like mass of nervous
tissue. The presence of nerve elements indicates an incomplete isolation of the
otic pit. At this stage it is difficult to clean the ear rudiment before transplanta-
tion. A significant feature of both these labyrinths is the complete separation
of definite sensory areas from nerve fibers and ganglion cells.

21-25 Somites. The grafts in this age group show more complete morpho-
logical differentiation than do those described in the preceding section (cf. Water-
man and Evans, 1940). Correlated with this is an increase in the completeness of
histogenesis.

The most extensively developed graft, A397-41, is of an otic pit isolated from
a 21-somite donor. Its control age is 8f days. A utriculus, a sacculus and two
canal rudiments are present. A portion of the saccular region has been inter-
preted as a lagena, because of the similarity of its sensory epithelium to a papilla
basilaris. There is no indication of development of the recessus labyrinthi.
This is unusual, for this part is one which persists in practically all grafts of the
inner ear. The otic capsule is represented by three nodules of cartilage.

There are four foci of thickened epithelium which represent the sensory areas
but none of these sensory areas is innervated. A crista is found near the utricular
end of a canal rudiment and the other canal rudiment has a macula near its base.
This latter sensory area is in a small pocket between the utricular ends of the two
canal rudiments. The sensory area of the lagena has some clear cells to one side
which are similar to those of a papilla basilaris. There is differentiation of hair
and supporting cells, but the membrane which covers this sensory area resembles
an otolithic rather than a typical tectorial membrane. The rest of the thickened
epithelium of this lagenar sensory area resembles a macula but has no differentia-
tion of hair or supporting cells.

Beyond and to one side of the blind ends of the canal rudiments are three
groups of nerve cell bodies. There are 2-4 cells per group and capsule cells are
evident at the periphery of these cell bodies. A few nerve fibers run toward these
groups of cells from the region of the otic epithelium but the fibers are not stained
heavily enough in the vicinity of the cell body to trace them into the cell itself.
In the other direction, these fibers disappear in the mesenchyme. A second
region of ganglion cells lies between the epithelium of the lagena and the edge of
the graft. The most prominent of these cells is a bipolar neurone lying close to the

256

HIRAM J. EVANS

• $&

^vSw

^*^^S£^

PLATE I

1. Section through graft A302-41. The endolymphatic sac is represented by convoluted
epithelium near the top of the photograph. Two oval areas in the cartilage at the right mark the
nerves which pass through the cartilaginous capsule. A well formed crista is seen on the lower
side of the lumen. X 45.

2. Higher power view of the crista of Figure 1. X 195.

3. Section through graft A120-41. The macula is at the extreme left of the photograph.
A crista is present slightly to the right and below the macula. X 60.

4. Higher power view of the macula and crista seen in figure 3. X 112.

5. Section through the innervated sensory area of graft A163-41. Nuclei of hair cells may
be seen near the surface of the macula which is on the left of the lumen. Nerve fibers in the
area underlying the macula appear as faint lines in the figure. X 178.

6. Section through graft A163-41 at another level. A crista is present at the lower margin
of the lumen. The mass of cells at the left which projects into the lumen represents the termina-
tion of the innervated sensory area seen in Figure 5. X 110.

DIFFERENTIATION OF THE AVIAN EAR 257

lagenar epithelium. One process of this neurone bends back on itself and joins
other nerve fibers which run through the thickened sensory epithelium of the
sacculus just inside and parallel to the basement membrane. They soon leave
the sensory epithelium and pass along the closed end of the lagena into the
mesenchyme.

Case A151-41 is of an inner ear rudiment which was isolated from a 24-somite
donor and wrapped in somatopleure before transplantation. Its control age is
9 days. The labyrinth is somewhat vesicular but histologically a utriculus, sac-
culus and an endolymphatic duct and sac are distinguishable. There are no
canal rudiments. The cartilage capsule is confined to the utricular end of the
ear. It surrounds the utriculus at its distal end but forms only a nodule at one
side of the proximal region. Poor development of the cartilage capsule is taken
as evidence that little adjacent mesenchyme was transplanted with the otic
rudiment.

A crista is found in the utriculus and a macula is present in the sacculus. The
crista shows no evidence of subordinate differentiation or of innervation. There
are some ganglion cells outside of the cartilage capsule. From these cells, nerves
pass through a break in the capsule, run beneath the macula and end in the adja-
cent mesenchyme. Just beneath the free surface of this macula and parallel to
it are some fibers which stain black with Bodian's technique. Although their
cell bodies are not evident, these are interpreted as nerves and the sensory area is
probably innervated by these fibers. The innervation is not typical. There is
some suggestion of hair and supporting cells in this macular area and the epithe-
lium is thrown into shallow humps at two places. A poorly developed otolithic
membrane is present.

27-30 Somites. Operations performed on embryos of these ages are easily
accomplished by shelling out the otocyst, but it is difficult to free the inner ear
rudiment from all of the ganglion cells. The exact relation of otocyst and gang-
lion cells is indefinite in control embryos and the number of grafts in this group
which showed nerve cell bodies and nerves connecting with the otic epithelium
make it reasonably certain that the nervous elements were not successfully ex-
cluded in the majority of cases.

Case A336-41 is a 6^ day graft of an otocyst isolated from a 27-somite embryo.
Utriculus, sacculus, endolymphatic sac and duct, lagena and three canal rudiments
can be distinguished. The cartilage capsule is almost complete, especially
around the lagena and sacculus. The endolymphatic sac and duct are inside the
cartilage capsule next to the otic epithelium instead of being external to the cap-
sule as in the control. In all, five sensory areas are represented.

Two cristae and one macula are found in the utriculus. The cristae are on
opposite sides of the utriculus with the macula beside one of the cristae. Both
of the cristae are innervated and show slight differentiation of hair and supporting
cells. The presence of cupular material is questionable. The macula is poorly
differentiated with no clearly defined hair or supporting cells and no innervation.

The sacculus is a bilobed sac-like structure which joins the utriculus opposite
the point of entry of the endolymphatic duct. It has a macula which is in-
nervated and shows the initial differentiation of both hair and supporting cells.
The lagena appears as a direct continuation of the utriculus and contains a fairly
well differentiated sensory area which is similar to a papilla basilaris. Beside

HIRAM J. EVANS

the lagena there is a large ganglion from which nerve fibers run to the cristae and
to the sensory areas in the sacculus and in the lagena. A few nerve cell bodies
are found beside the sacculus and some more are adjacent to the lagena.

A second graft of an otocyst from a 27-somite donor (Al 20-41) has only one
of its three sensory areas innervated. This otocyst was wrapped in splanchno-
pleure before implantation and has a control age of 8J days. That this was a
more successful isolation than case A336-41 is suggested by the fact that the
cartilage capsule is less complete than in the previous case. The cartilage forms
a half-moon around the labyrinth except at one extremity where it assumes an
elongate rod-shaped form (Figure 3). A utriculus, sacculus, endolymphatic ap-
pendage and two canal rudiments may be distinguished.

Two cristae and a macula are found in the utriculus. The cristae are on op-
posite sides of the utriculus and are not innervated. They show no differentiation
of hair and supporting cells. The macula is located near one of the cristae and
has both hair and supporting cells (Figures 3 and 4). On opposite sides of the
macula are two sets of nerve cell bodies which are connected by nerves. These
nerves pass through the sensory epithelium and it is probable that they give off
some branches which innervate this sensory area although the innervation is not
typical.

The third graft to be described (A238-41) is of an otocyst isolated from a 30-
somite donor. Its control age is 9| days. Utriculus, sacculus, lagena, endo-
lymphatic duct and sac and one canal rudiment are represented. The cartilage
capsule forms a half-moon around the labyrinth. A non-innervated crista is
present in the utriculus and the sacculus contains extensive sensory epithelium
of the macular type. Adjacent to the sacculus is a ganglion containing the cell
bodies of several nerves which run beside the macula but enter its epithelium at
only a few points. The macula is covered by an otolithic membrane. The
lagena is identified by some clear columnar cells which are typical of the papilla
basilaris but this papilla basilaris is poorly differentiated and is not innervated.

31-43 Somites. In transplants of older otocysts, there tend to be more well-
differentiated sensory areas than were present in grafts of younger stages. A
striking thing about this present group is the relatively few sensory areas which
show signs of innervation. In general, morphogenesis has proceeded further in
these transplants than in transplants of younger otocysts, although vesicular
inner ears are found in the grafts.

Case A163-41 is an otocyst isolated from a 32-somite donor and wrapped in
somatopleure before implantation. Its control age is 8^ days. This inner ear is
an ovoid vesicle with an endolymphatic duct and sac entering from one side.
There are no canal rudiments nor is there any epithelial differentiation of the pars
inferior labyrinthi. At one end of the labyrinth the cartilage capsule completely
surrounds the otic epithelium but the rest of the labyrinth has only a half-moon
shaped capsule around the side opposite to the endolymphatic duct and sac.

There are three general regions of thickened epithelium which represent four
sensory areas. Two of them are well defined, but the third is spreading and is
composed of a crista and a macula. At least a part of all of these sensory areas is
apparent in the portion of the otic epithelium which is surrounded by cartilage,
and two of them extend into the region of the labyrinth which has only a half-

DIFFERENTIATION OF THE AVIAN EAR 259

moon of cartilage around it. The macular portion of the large spreading area of
sensory epithelium begins in the portion of the labyrinth where the capsule is
complete. Here the sensory epithelium is stratified and has a suggestion of a
covering membrane. The sensory area has a flattened macula-like appearance
with both hair and supporting cells present. The macula (Figure 5) is innervated
by nerve fibers which pass through a break in the cartilage capsule; the cell bodies
of these nerves were not identified. The sensory epithelium soon increases in
area and changes from a flattened to a hillock-like form which is interpreted as a
crista. It has both hair and supporting cells as well as a cupular remnant. This
sensory epithelium terminates just before the endolymphatic duct enters the
vesicular portion of the labyrinth. In the region where the complete cartilage
capsule becomes half-moon shaped, there is a thickening of the otic epithelium
into a crista-like sensory area (Figure 6). There is evidence of both hair and
supporting cells, although the presence of sensory hairs is questionable. The
smallest sensory area is a thickening of the epithelium in which stratified cells
form a small crista. This is not well differentiated, for neither hair nor supporting
cells can be distinguished and cupular material is lacking. This labyrinth is
noteworthy because of the large areas of well differentiated sensory epithelium
which are not innervated.

A graft of an otocyst from a 43-somite donor (A382-41) shows more mor-
phological differentiation than does the preceding case. This otocyst had the
endolymphatic rudiment cut off prior to implantation. The control age is 12
days. A utriculus, sacculus, lagena, two canal rudiments and a complete canal
are present. The sensory areas are well differentiated. The cartilage capsule
surrounds the portion of the utriculus which communicates with the complete
semicircular canal but in other places it is made up of several cartilage nodules
which differ in size.

Seven thickenings of the epithelium probably represent sensory areas. Four
of them are widely separated from each other, but the other three are on the same
side of the labyrinth and are so close together that they may well have arisen by
a splitting of a single area of sensory epithelium. In the pars inferior portion
of the inner ear are found a macula as well as some neuroepithelium which re-
sembles a papilla basilaris. The macula has stratified epithelium, is flattened
and probably represents the macula sacculi. Supporting cells and hair cells with
sensory hairs which project above the surface of the macula are evident. The
macula is covered by an otolithic membrane. The sensory area of the lagena is
identified by its flattened shape and stratified epithelium surmounted by a tec-
torial membrane. This membrane has been separated from its attachment point
but its position is similar to the tectorial membrane of the control. Well defined
hair and supporting cells are present. The hair cells are close to the surface as in
the papilla basilaris of the control and sensory hairs project above the epithelium.

The utriculus contains five sensory areas. The one which is close to the sac-
cular region is flattened and shows both hair and supporting cells. Sensory hairs
and an otolithic membrane are present. This sensory area is interpreted as a
macula utriculi. Almost in continuity with this sensory area is another flattened
region of well differentiated neuroepithelium which resembles the macula de-
scribed above. The macula is continuous with a well differentiated crista which

260 HIRAM J. EVANS

occurs at the entrance of the complete canal. The histological picture suggests
an ampulla with its crista. Above the crista is a covering which may represent
cupular material but it does not resemble a typical cupula. A sixth area of
neuroepithelium is found at the base of a canal rudiment. It has a typical crista
shape and is well differentiated. There are both hair and supporting cells as well
as sensory hairs. As in the case of the other crista in this graft, there is some
dark-staining material above the epithelium which is interpreted as a cupular
remnant. The seventh sensory area is a crista at the opposite side of the utriculus
from the two well differentiated cristae which are described above. Hair and
supporting cells are not well differentiated and the presence of sensory hairs is
questionable. There are no nerves in this graft.

An interesting feature of this group of transplants is the absence of nerves in a
large proportion of the grafts. In two cases (A377-41, A163-41) there is only one
innervated sensory area in each labyrinth and case A187-41 has three innervated
sensory areas which are near each other. Three other grafts examined in this
group had no nerve elements present.

The differentiation of the components of the ear was also tested by transplant-
ing portions of the otocyst from donors of this age group. Two grafts (A375-41,
A380-41) of the endolymphatic rudiments of otocysts from 43-somite donors show
that this portion may survive and grow in a graft. The epithelium is convoluted
and the columnar cells are much taller than in the endolymphatic sac of the 12-day
control. Case A382-41, previously described in this group, had the endolym-
phatic rudiment removed before implantation. This labyrinth showed no evi-
dence of an endolymphatic duct or sac. Apparently, the inner ear cannot re-
generate this portion of itself in a graft when the endolymphatic rudiment is
removed at the 43-somite stage.

Two grafts of the isolated pars inferior were recovered. Case A369-41 is a
graft of the pars inferior of an otocyst from a 41-somite donor. Its control age is
12 days. This labyrinth consists of two vesicular structures. A part of one of
the vesicles has differentiated as a lagena and shows a well developed papilla
basilaris. No other special epithelial differentiations were observed. The other
graft (A347-41) is not as completely differentiated as this one. It is of a pars
inferior from a 38-somite donor and has a control age of 10| days. A lagena and
sacculus are present but the only suggestion of a sensory area is some stratified
epithelium which shows no specific differentiation of hair and supporting cells.
These cases indicate that under the conditions of the experiment the pars inferior
is incapable of regenerating a pars superior and recessus labyrinthi by the stage
at which these transplants were made.

DISCUSSION

The transplanted labyrinths which have been studied show that cristae,
maculae and a papilla basilaris will differentiate in grafts of the inner ear rudiment,
although these sensory areas may not be innervated. An isolated labyrinth may
have more than one of these types of sensory areas, but it has been found that
generally a papilla basilaris does not occur in a graft when no other type of sen-
sory area is present. For the most part, these sensory areas are well developed
and, while not always comparable to the control in all respects, their differentia-

DIFFERENTIATION OF THE AVIAN EAR 261

tion indicates that histogenesis has been only slightly retarded. The complete-
ness of differentiation of individual sensory areas is not affected by the age of the
donor, although the otocysts which are older at the time of transplantation con-
tain more numerous sensory areas than do otocysts from younger donors. The
sensory areas may or may not be innervated.

The results reported here are in accord with those of Hoadley (1924) and of
Fell (1928). Their studies and the present results indicate that the sensory areas
of the inner ear of the chick possess a considerable capacity for independent dif-
ferentiation. When the nerves are prevented from reaching the otic epithelium,
the histological differentiation of the sensory areas is retarded only slightly if at
all. In connection with this study, some transplants were made which included
the myelencephalon and the otocyst in their usual relations. These labyrinths
do not show innervation of all of the sensory areas. At least one sensory area in
each labyrinth has no nerve fibers running into it. The cases where nerves pene-
trate the epithelium do not yield any more highly differentiated sensory areas
than do those grafts of isolated otocysts in which there are no nerves in the vicinity
of the sensory epithelium. Other sense organs beside the sensory areas of the
inner ear are capable of differentiating when isolated from the influence of the
nervous system. The studies of Harrison (1904) on the lateral line organs of
anurans show that these organs can develop and differentiate in the absence of
nerve fibers. Several more recent investigators have examined the capacity for
independent differentiation possessed by taste buds in a number of forms.

The results reported here present strong evidence that the sensory areas of
the avian inner ear are capable of independent differentiation from the otic epi-
thelium after the 12-somite stage, at least in so far as the nervous system is con-
cerned. Since in otocysts isolated from nerve elements, sensory areas appear
which are histologically comparable to those of the control, the nerves themselves
appear to play no part in the development of the sensory areas and may be only
passive elements which are attached to the sensory epithelium and follow its
divisions during the development of the sensory areas. If the nervous system
exerts any "initiating" influence upon the otic epithelium which results in the
formation of sensory areas, this effect probably occurs prior to the stages studied
here. That would place the time of action previous to the appearance of the first
demonstrable nerve fibers in either the central or the peripheral nervous system.

SUMMARY

The capacity for differentiation of the sensory areas of the avian inner ear
independent of innervation, has been studied by transplanting isolated primordia
of the inner ear to the chorioallantoic membrane. Maculae, cristae and a papilla
basilaris differentiated in the transplants. All three types of sensory areas are
seldom found in any one graft. The sensory areas of the transplanted labyrinths
are comparable to those of the control. The morphogenesis of the membranous
labyrinth was greatly suppressed in the grafts but the histogenesis of the sensory
components showed but little retardation.

Since the sensory areas of the inner ear undergo typical development when
isolated from their nerve supply, it is concluded that they are capable of inde-
pendent differentiation in so far as the nervous system is concerned.

262 HIRAM J. EVANS

LITERATURE CITED

BODIAX, DAVID, 1937. The staining of paraffin sections of nervous tissue with activated protargol.

Anal. Rec., 69: 153-162.
FELL, H. B., 1928. Development in vitro of the isolated otocyst of the embryonic fowl. Arch.

f. exp. Zellforsch., 7: 69-81.
HARRISON, R. G., 1904. Experimentelle L'ntersuchungen iiber die Entwicklung der Sinnes-organe

der Seiten-linie bei den Amphibien. Arch.f. mikros. Anat., 63: 35-149.
HOADLEY, L., 1924. The independent differentiation of isolated chick primordia in chorio-allan-

toic grafts. I. The eye, nasal region, otic region, and mesencephalon. Biol. Bull.,

46: 281-315.
WATERMAN, A. J., AND H. J. EVANS, 1940. Morphogenesis of the avian ear rudiment in chorio-

allantoic grafts. Jour. Exp. Zool., 84: 53-71.

THE UTILIZATION OF GLYCOGEN BY FLIES DURING FLIGHT

AND SOME ASPECTS OF THE PHYSIOLOGICAL

AGEING OF DROSOPHILA

CARROLL M. WILLIAMS,1 LEWIS A. BARNESS, AND WILBUR H. SAWYER

(The Biological Laboratories, Harvard University, Cambridge)

The frequencies with which many insects move their wings during flight are
unparalleled in the appendicular movements of any other animals. The extent
to which this is true may be judged from Table I where the maximum frequencies

TABLE I

Wing-beat frequency of Drosophila and the maximum frequencies yet recorded for the

muscular movements of other animals.

Animal

Activity

Frequency (cycles/sec.)

Authority

Drosophila

Rattlesnake

Humming-bird

Mouse

Man

Wing-beat during

flight
Movement of rattle

Wing-beat during

flight
Scratching reflex of

hind leg
Voluntary vibration

of the opponens

pollicis muscle of

the hand

100-300
17-100
60-70

20
10-13

Chadwick & Williams

(unpublished data)
Chadwick & Rahn

(unpublished data)
Edgerton & Killian

(1939)
Chadwick & Pearson

(unpublished data)
Schafer, Canney &

Tunstall (1886);

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "The Biological bulletin"

See other formats