Studies on the effect of parasitism upon the tissues. I. With special reference to certain gasteropod mollusks

STUDIES ON THE EFFECT OF PARASITISM UPON
THE TISSUES. I. WITH SPECIAL REFERENCE
TO CERTAIN GASTEROPOD MOLLUSKS

By

Helmer Pareli Von Wold Kjerschow Agersborg

Bes;. M. S., University of Washington, 1916
A. M., Columbia University, 1920 .

THE LIBRARY GE THE

24a/0 6 "ts a = 2 Pale :
UNIVERSITY OF WLINeRS

A DIGEST OF A ‘THESIS

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY IN THE
GRADUATE SCHOOL OF THE UNIVERSITY
OF ILLINOIS, 1922

Reprinted from ‘The Quarterly Journal of Microscopical Science,’
Vol. 68: 361-401, 10 double plates, ~
‘ October, 1924, Oxford, Enpland.

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Studies on the effect of Parasitism upon the
Tissues. I. With special reference to certain

Gasteropod Molluscs.!
By
H. P. Kjerschow Agersborg,

Professor of Biology, the James Millikin University,
Decatur, Illinois.

With Plates 12-21.

CONTENTS.

INTRODUCTION. Comments on the present status of the ee
ACKNOWLEDGEMENTS
MATERIAL AND TECHNIQUE
OBSERVATIONS
I. Condition of Normal Cytoplasm
II. Normal Tissue :
III. Parasitized Tissue
1. Avenue of Infection .
2. Effect of Infection
3. Glycogen
IV. The Liver
1. Anatomy of ule Diver
2. Physiology of the Liver

3. The Livers of parasitized Physa Att B L anor wy ists,

DISCUSSION
I. General Discussion .
Metabolism of Host ae ae
II. Is an Antidote formed ?

Antidote for Parasitic Toxin .
SUMMARY AND CONCLUSIONS
REFERENCES
EXPLANATION OF PLATES

PAGE
362
367
368

370
373
374
375
376
377
378
378
379
380

384
384
385
385
388
390
396

1 Contribution from the Zoological Laboratory of the University of

Illinois, under the direction of Henry B. Ward, No. 235.
No. 271 Bb

362 H. P. KJERSCHOW AGERSBORG

INTRODUCTION.

Tue cytological changes which take place in parasitized
tissue is one of the details of parasitology that now may be
undertaken without any suffering from lack of a proper founda-
tion; for many admirable works on parasitology have long
since appeared and are steadily being added to this important
branch of zoology. Histological and anatomical changes in
parasitized organisms have been studied in various ways and
by a number of workers. And yet detailed cytological studies
of the effect of parasitism upon the host is nearly a virgin field.
Perhaps this may be demonstrated, although necessarily only
briefly, by referring to the labours of certain workers in para-
sitology and pathology, to wit:

Schaper (1889) in his study on ‘ Die Leberegel-Krankheit der
Haussaugethiere ’ made a distinct addition to the development
of pathology of the subject. His pathological anatomical
descriptions of the host are perfectly clear, but one may be
justified in stating that he did not give an adequate treatment
of the subject since the cytological aspect was of significance.
This is true both as regards the text and illustrations.

Naumann (1892), commenting on the pathological anatomy
of the sheep, says: ‘ The first effect of the penetration of the
flukes into the bile-ducts is inflammation (period of traumatic
hepatisis, according to, Gerlach). The liver is enlarged, and
contains more blood than usual; it is friable in texture, and
its surface is smooth, or marked in places by openings the
size of a pin’s head to that of a millet-seed, from which exudes
a sanious fluid on pressure. ‘There are traces of local peri-
tonitis, or exudations, which cover the young flukes; and
small haemorrhagic centres exist in the parenchyma. The bile
is slightly reddened and the peritoneal serosity 1s more abun-
dant, and often contains small flukes. The faeces do not
yet contain ova’ (p. 535). ‘ The hepatic parenchyma is soft,
and the thickened connective tissue slightly grates on section ;
the surface of the latter is of a dirty-gray, yellowish-red, or
blood colour, and perforated by spaces the size of a pea, which

EFFECT OF PARASITES ON TISSUES 363

contain one or more young flukes in a blood-clot, or in a sanious
fluid formed of white and red corpuscles, hepatic cells which
have undergone fatty degeneration, and a finely granular
detritus. . . . Over the entire liver the cells are granular, and
infiltrated with fat; the connective tissue is in process of
proliferation ’ (p. 586). Neumann’s description indicates that
he made cytological studies of the livers of hosts suffering from
fluke-infection. Unfortunately, however, his methods are not
mentioned, nor are his statements quoted above supported
by illustrations.

Acland and Dugeon (1902: 1318), in their study on
Primary carcinoma of the liver, made a general histo-
logical examination to show the relative amount of connective
tissue, and the normal and abnormal liver cells of that infected
organ. Their purpose of study would certainly have been
advantaged by cytological studies. In such an event, however,
a different fixing agent than was used should have been
employed. The fixing agent used was alcohol and the stain
was Mayer’s haematoxylin. :

Leiper (1915: 177), in his study on Bilharzia, pointed
out the avenue of infection of the Bilharzian trematode larva
of its vertebrate host, and stated that the cerearia ‘ are able
to pierce the skin very rapidly’. He also referred to the

intermediate hosts by saying: ‘The glandular tissue of an
infected organ disappears apparently through pressure atrophy
(fig. 44).’ :

Fantham, Stephens, and Theobold (1916), in describing the
pathological anatomy of the sheep, give graphic pictures of
the general condition: * The bile ducts are conspicuous on the
surface of the liver. They are thickened and much dilated
and in part saccular, and considerable atrophy of the liver cells
accompanies the condition. Histologically there is immense
proliferation of the epithelium of the bile ducts leading to
‘““adenomata’”’’ (p. 241). * Anaemia through loss of blood to
worm ; enlarged spleen, toxic in origin (?) ; phlebitis, throm-
bosis, due to portal stasis; the eggs, however, cause the
greatest mischief. They are carried by the circulation to various

Bb 2

364 H. P. KJERSCHOW AGERSBORG

organs where they produce inflammation, granulation tissue,
and later connective tissue ’ (pp. 280-1).

Cawston (1918), Manson-Bahr, and Fairley (1920) may also
be mentioned, although they do not give any pathological facts.
about the tissues of either the primary, intermediary, or
secondary hosts. Walton (1918), on ‘ Liver Rot in Sheep’,
points out certain facts briefly and says: ‘ The liver of many
are found to be atrophied and hardened to the consistency of
leather’ (p. 287). And again: ‘... her liver was then found
to be “like a stone”’’ (p. 248). In his study on the effect.
of cercariae on the snails he says: ‘ The only conclusion arrived
at was that, while differences were observable in structure of
the liver of infected snails, yet no rediae or cercariae were
recognizable ’ (p. 264).

Noguchi (1918, 1918 o,.1918'b, 1918 ¢ 4919 F 1910 a) 191g)
demonstrated the yellow fever organism through his study
of the pathological changes of the tissues of the human host.
Up to 1918 the causative agent of yellow fever outside the
mosquitoes Aedes aegypti (Linnaeus) was thought to be
an ultra-microscopic virus. By painstaking and resourceful
methods and skilful technique he demonstrated definitely
the mysterious organism which, in more than one way, had
been an object of much speculation for a long time. Whether
the swamp fever virus may not be demonstrated also is a
question of some interest (Van Es, 1911; Swingle, 19138;
Van Hs and Schalk, 1917; Schalk, 1920).

The work of Faust (1920) is significant. To my knowledge
it is the first scientific treatise which contains a careful analysis
of the liver of gasteropods suffering from trematode infection.
Unfortunately the original coloured illustrations of this paper
were redrawn in order to reduce the cost. The author did not
see the paper after the figures were redrawn until the contribu-
tion appeared in print. The published article lacks some of the
original merit of the contribution. This explains why the illus-
trations of Faust’s paper do not quite support the text. This
contribution nevertheless is by far the most comprehensive
work on this subject up to the present time. It concerns

EFFECT OF PARASITES ON TISSUES - 365

itself, however, with the hepatic organ only. Further comments
will be made on this later.

Pirie (1921), in his important discourse on Carcinoma
and Cirrhosis of African natives, points out Schisto-
sOmiasis as the causal factor. This author’s comment
on the cytological condition of the organs involved is much
to his credit. Since, however, he does not describe his technique
one cannot tell whether the 3,900 specimens sent him for
‘general histological examination’ were favourable for cyto-
logical study. Unfortunately so many doctors who have
an excellent opportunity to secure important material of
immense biological importance do not realize the significance
of the fixing of the tissues for microscopic examination. A
twenty-four hours’ post-mortem examination cannot
possibly reveal the same cytological structures as existed
ante mortem or in articulo mortis (vide Hance,
1917, 1917 a). Every cytologist will agree with this state-
ment. In point of fact Pirie states : ‘ I have also been strongly
impressed by the variety of- structure to be met with in a single
case, so that where only a single piece of tissue is examined
a false idea of the variety of tumour might easily be given,
and it is possible that in some of the cases received a different
opinion might have been given, at all events of the type of
structure, had a large number of pieces been examined.’
Indeed, this variety of structure may be due in part to the
methods of fixing the tissue. And the variety may be exag-
gerated greatly by the lapse of time between death and the
preserving of the specimens in question. A great deal of credit
is due to Pirie for his care in analysing the cytological structure
of the tissues with which he dealt.

The labours of many other authors might have been cited.
Suffice it, however, to add: It is clear from the works reviewed
above, their meritorious nature notwithstanding, that the
pathological changes of the tissues of almost any organism
suffering from parasitic attack have not been studied by many
authors, neither in very much detail by any one. Of course,
in order to understand the pathological condition of any organ,

366 H. P. KJERSCHOW AGERSBORG

it is necessary first to know its normal condition. The writer,
therefore, has attempted to raise Physa gyrina (Say),
the most common species studied in this work, in the laboratory,
and studied a number of young while in different stages of
growth and age and before they became infected. This was
quite necessary since all the specimens collected in nature were
infected with flukes in various stages of development.

The works of human pathologists have hardly touched on
the cytological phase of the problem. Leading men in this
field, Cohnheim (1882), Sternberg (1893), Adami and Nichols
(1919), and MacCallum (1920), indeed, might have increased
the value of their published works considerably had they
considered their subject-matter from a cytological point of
view also and not only from a histological one. The work of
Faust (1920) deals with the liver only, and no consideration
is paid to any of the other organs. All the other students of
vertebrate and invertebrate hosts suffering from trematode
and other infections have not studied the tissues from a cyto-
logical point of view. Their technique, except in a few cases
(Noguchi, Faust, Ward, and Calkins), has not lead to new
discoveries. Merely some of the gross histo-morphological
changes of the host have been studied, and many of these
are recorded only briefly. As we shall see, then, ample excuse
exists for approaching to this subject seriously from a cyto-
logical standpoint.

The purpose, then, if this investigation has been to study
the cyto-physiological changes which take place in the hepatic
organ and all the tissues of certain organism affected by
parasites. This has not been attempted earlier. The aim of
this paper is to present a general survey of the subject and give
some of the data obtained. It is hoped that subsequent
papers dealing with further and more detailed phases of this
topic may be published.

For this investigation fresh-water snails were used ; partly
because several species (Physa gyrina (Say) and Plan-
orbis trivolvis (Say)) were easily obtainable in nature
all the year, and partly because of the direct application which

EFFECT OF PARASITES ON TISSUES 367

may be readily made of fresh-water gasteropod material of
this nature both from an economic and medical standpoint.
Some material was collected each month from September
1921 to May 1922, and also during August 1922, from the
Drainage Ditch of Crystal Lake at Urbana, Illinois. Some
material of Physa was raised in the laboratory from January
to August. Some of these and their progeny were kept alive
until September 1922, when most of them were killed and
preserved for later study. This Drainage Ditch has been
a collecting ground for several previous investigators (vide
Cort, 1914; Faust, 1919). It is remarkably suitable for this
purpose because of its considerably diversified fauna. The
Drainage Ditch is artificial, and was built to carry off the surplus
storm and seepage water. At times the streamlet may nearly
overflow its banks. At other times it decreases very much in
size, and filamentous algae, Elodea, et al., flourish abun-
dantly. ‘This offers a suitable breeding-place for animals.
In these aquatic plants are found representations of several
animal phyla: Amphibians—adult and larvae; Pisces—
Boleosoma nigrum (Raf.), Notropis anogenus
(Forbes), et al.; Pelecypoda and Gasteropoda;
Crustacea and Insecta, the latter both in adult and
larval stages; Polychaeta. Among the higher forms may
be mentioned the kingfisher, wading birds that feed on the
lower forms. I have not attempted to give a full list of the
fauna, but to show that the opportunity is good for parasites
to find primary and secondary hosts.

ACKNOWLEDGEMENTS.

The writer wishes to express his appreciation to the various
members of the Department of Zoology of the University of
Illinois for their ever-ready and kind disposition and sym-
pathetic interest in this work. Among these he wishes to thank
Professor Charles Zeleny in particular. Special mention
should be made of Professor Frank Smith and his Assistant,
Mr. J. L. Hyatt, for their friendly readiness to leave at the
writer’s disposal considerable material which they had collected ;

368 H. P. KJERSCHOW AGERSBORG

of Mr. F. C. Baker, Curator of the Museum of Natural History,
for identifying the species of snails upon which this work
is based. Special thanks are due to Professor Dr. Henry
B. Ward, under whose direction this work was done, for his
helpful suggestions and friendly criticism.

MATERIAL AND TECHNIQUE.

The material used for this study was three different species
of fresh-water snails: Lymnaea obrussa (Say), Physa
gyrina (Say), and Planorbis trivolvis (Say). Some
specimens were studied alive. The shell was first removed
and the animal placed on a slide with a few drops of water.
Very slight injury to the surface of the snail made it apparently
easy for the cercaria, if present, to come out of the host, for they
frequently came out in considerable number. For killing
purposes specimens were partly dissected and immersed in the
fixing mixture. The smaller specimens (8 to 7 mm.) were
placed whole in the killing fluid.

A peculiar difficulty developed in connexion with fixing the
specimens. A great deal of free gas developed in the body of
the specimens which were immersed in acid mixtures, so that
small bubbles of this gas covered the surface of these specimens
and prevented penetration of the fixatives. A modification
of Carnoy’s fixing agent caused a steady stream of gas to
flow from the fixing object for several seconds. The regular
standard acid-mixture-fixatives, which penetrate more slowly,
proved satisfactory, provided the fluids were constantly and
gently stirred. Because the gas liberated tended to be caught
in the mucus exuded by the snail. Hence good fixations
were often impaired. Hammersten (1885: 393) found grains
of CaCO, present in the mucus of the mantle of Helix
pomatia. Substances of this nature may be the cause of
some of the gas formed. Owing to some bad fixation, caused
by too slow a penetration of the fixative, it was necessary to
prepare a large number of duplicates of fixed specimens of both
infected and apparently uninfected individuals.

The fixing agents used were (1) F.W.A., a formula intro-

EFFECT OF PARASITES ON TISSUES 369

duced by Gatenby for Flemming’s mixture without acetic
acid. (2) Flemming’s strong mixture. (3) Lams’ fixative,
which consists of equal parts of the following: (a) saturated
aqueous solution of picric acid; (b) 2 per cent. osmis acid
aqueous solution; (c) 2 per cent. chromic acid aqueous solu-
tion ; (d) glacial acetic acid ; (e) 40 per cent. formaldehyde ;
(f) saturated aqueous solution of corrosive sublimate; and
(g) absolute alcohol. This fixing agent was used by Dr. Honoré
Lams (1910) on the ovotestis of Arion empiricorum
(Fér.) with very fine results. (4) Saturated aqueous solution
of corrosive sublimate with 3 per cent. to 5 per cent. glacial
acetic acid. (5) Corrosive sublimate saturated in normal
saline solution with 3 per cent. glacial acetic acid. (6) Bouin’s
picro-aceto-formol fluid. (7) Zenker’s solution. (8) Miiller’s
solution. (9) Hetherington’s modification of Carnoy’s fixing
agent ; this is made as follows: (a) absolute alcohol, 20 parts ;
(b) chloroform, 15 parts; glacial acetic acid, 5 parts; and
phenol crystals to raise the volume by 10 parts. The material
is handled thus: fix for half an hour to twenty-four hours or
until clear; add oil of wintergreen, little by little, until
transparent (the object becomes hard); rinse in wintergreen,
chloroform, cedar-wood oil, or clove oil, to get rid of the acid ;
imbed. (10) Saturated aqueous solution of corrosive sublimate
with glacial acetic acid, and from a few drops to equal parts
of normal urine at 87° C. (11) 10 per cent. formaldehyde.
(12) Alcohol. (13) Bowen’s modification of Weigl’s sublimate-
osmi¢ fixative ; fix in glass-stoppered bottles for twenty-four
to thirty-six hours in Mann’s sublimate-osmic made up of
equal parts of 1 per cent. osmic acid solution and corrosive
sublimate saturated in normal salt solution. (14) Kopsch’s
method for mitochondria and Golgi apparatus. (15) Mann-
Kopsch’s method for cell-organs. (16) Champy’s method for
mitochondria, &c. (17) Champy-Kull’s method for mito-
chondria, and Golgi apparatus and cytoplasmic granules.

A number of stains were used. (1) Tests for glycogen:
(a) the iodine method, (b) Best’s carmine stain. (2) Heiden-
hain’s iron haematoxylin. (38) Weigert’s iron haematoxylin.

370 H. P. KJERSCHOW AGERSBORG

(4) Delafield’s haematoxylin. (5) Mallory’s phosphotungstic
acid haematoxylin, and (6) safranin. For counter-stains were
used : (a) light green, (0) orange G, and (c) eosin. ;

Hetherington’s Carnoy-phenol penetrates very rapidly, and
the fixing is quite uniform throughout. The sublimate-aceto-
normal-urine-mixture is good as a cytoplasmic fixative. It
preserves a large number of cytoplasmic granules which cannot
be stained otherwise, e.g. if fixed in Flemming’s or Bouin's
fixing agents. The results obtained by the last four methods
cannot be discussed here. Fine results were obtained with
material fixed in Flemming’s solution and stained with safranin
and light green. Most sections were made less than 10 micra
in thickness. The thickness of the sections ranges from 4 to
15 micra. All specimens sectioned were mounted serially.

OBSERVATIONS.

I. Condition of Normal Cytoplasm.

The classical works of Altmann (1889, 1894, 1896, 1896 a),
Wilson (1895, 1899, 1904), and later of Schreiner (1916, 1918),
show that the cytoplasm is primarily granular in structure.
The ultra-microscope reveals objects ca. 2 to 5uy~—far below
the possible limit of vision. There are numerous kinds of
recognized cytoplasmic granules specified both in structure and
in origin: (1) microcomes, (2) mitochondria, (8) chromidia,
(4) metachromatic granules, (5) secretory granules (vide
Schreiner 1916, 1918), (6) pigment granules, (7) metaplasmic
and paraplasmic granules, such as fat, yolk, starch, leucocytes’
granules and many others. In addition to these may be men-
tioned the Golgi bodies. Gatenby’s treatises on ‘ The Cyto-
plasmic Inclusions of the Germ-cells’ (1917, 1917 a, 1917 },
1918, 1919, 1919 a, 1920, 1920 a, 1922), and Gatenby and
Woodger (1921) are in support of the workers who claim that
protoplasm is fundamentally granular in structure. But the
_works of Gatenby, &¢., need not be considered in detail in
this connexion as these treatises are concerned with the germ-
cells only, and the germ-cells will not be discussed in the present

EFFECT OF PARASITES ON TISSUES 371

paper. It is, however, the opinion of the foremost cytologists
of to-day that the protoplasm is fundamentally granular in
nature. ‘This is in agreement with Altmann’s contention that
the cytoplasmic granules exist in a constantly regressive size,
from larger granules to smaller ones, which finally become
ultra-microscopic. In my own studies at Wood’s Hole in 1920
(not yet published) I was able to demonstrate in the growing
ege of nudibranchs an apparent constant gradation in size of the
cytoplasmic granules from fairly large ones to the ultra range
of the microscope. These granules had in most cases affinity
for three different stains.

Schreiner (1916, 1918) builds on Altmann’s work. He
carries this phase of cytology to a climax. He shows better
than any other cytologist up to the present time the inter-
nucleo-cytoplasmic relationship. He found that cytoplasmic
granules of different nature had their origin in the nucleolus
and the nuclear ‘ Netzknoten’. From the works of Schreiner
it is seen that the nucleus plays an important role in the various
activities of the cell. Inmy study on Physa and Planorbis
T have come to the conclusion that the large amount of black
granules (which will be discussed later) among the tissues of
individuals newly infected by trematodes have their origin
in the nucleus also. A more detailed report on this phase of
this problem will follow later.

Miller (1896 : 321) also agrees with Altmann that the saliva
‘aus typischen Granula-stammt’. Indeed, Altmann claimed
that the granules originated from the homogeneous inter-
sranular substance. In his opinion both the granules and the
intergranular substance are living (1894: 51). Nussbaum
(1882), in his study on vertebrate glands, saw a special cyto-
logical activity in the vicinity of the nucleus. Lange (1902)
elucidated this point better than Nussbaum in his work on
the structure and function of the salivary glands of gasteropods.
He showed that the nucleus takes part in the secretory activity
of the salivary glands, in that the nuclear membrane dissolves
and the nuclear contents mixes with the cytoplasm, so that
the first part of the secretory activity of the gland is noticeable

STY H. P. KJERSCHOW AGERSBORG

on the nucleus. And Korschelt (1891), in his ‘ Morphologie
und Physiologie des Zellkernes’, brings out this point by
showing that the nucleus and cytoplasm are dependent on
each other in the life of the cell, and yet, ‘im Allgemeinen
erscheint der Kern als ein Theil der Zelle, der sich vom Zell-
plasma scharf sondert’. My findings (Agersborg, 1928) for
the nuclear activity of the nidomental gland of Melibe,
Lange’s for that of the salivary glands of certain gasteropod
molluses, and Korschelt’s for a variety of cells, agree with
Schreiner’s observations on Myxine. Dahlgren and Kepner’s
(1908: 8-9) secretion substances, secretion fibrils, and secre-
tion material which arise in the nuclear environs of the gland-
cells resemble very much the paranuclear bodies (PI. 18, fig. 34,
pnb) of the liver of Planorbis (vide infra). Fuchs (1902)
demonstrated some very interesting facts in regard to the
secretion activity of the epididymal epithelium of the mouse.
A certain ‘ Fadenknauel’ arises at the distal region of the
nucleus, and from it * Zellfaiden’ pass to the periphery or free
border of the cell. The argument brought forth by Fuchs
substantiates the works of Altmann and others. His illustra-
tions are strikingly similar to points found in my study of the
intestinal epithelium of Physa gyrina (Say) (PI. 21, figs. 53,
54), 1n which case excretory accumulations on the luminal side
of the nucleus appear as a nuclear cap (nuc), and from it may
be seen passing similar delicate strands as in Fuchs’s figures
to the border and beyond it into the lumen. In the liver of
Planorbis trivolvis (Say) paranuclear bodies are some-
times seen (Pl. 18, fig. 34, pnb). These resemble very closely
the nuclear caps (PI. 21, figs. 53, 54, nuc) and the * Fadenkniuel ’
of Fuchs. In this case, however, as may be noticed in Pl. 21,
figs. 53 and 54, no fibrillar stands (st) are seen. A fact which
seems to be peculiar to the entire hepatic organ. The para-
nuclear body (PI. 18, fig. 34, pnb) represents apparently some
nuclear products preparatory for secretory material, which of
course sooner or later leaves the cell. That this is most probable
is gathered from the vacuolar condition of the cell in the imme-
diate vicinity of the paranuclear body. This is also the opinion

EFFECT OF PARASITES ON TISSUES 873

of Fuchs. Schneider (1902: 570) recognized two kinds of
granules in liver cells of Helix pomatia. Both kinds
may be jointly voided into the lumen frequently in the
process of forming into spheres. ‘The excretory cells of
the liver have bubbles which contain a large excretory ball
of similar consistency as the excretory granules of the liver
cells. It appears thus that leading cytologists agree upon
a very essential point, viz. the cytoplasm is fundamentally
granular in structure, Wilson (1899: 28); even the astral
rays in the dividing sea-urchin egg are formed by linear arrange-
ments and fusion or close union of granules or microsomes of
the reticulum (Wilson, 1895: 467). It may be fair to say,
however, that Wilson is cautious in his statements, but he is
not far from supporting Altmann’s view altogether. Altmann
even thought that the secreted extracellular substance was
granular also. On this point his claim agrees with my observa-
tions on the pathological tissues of the aquatic pulmonates:
Physa and Planorbis. For, in this case, it is very evident
that there are in the intercellular (extracellular) substance an
abundance of granules which had their origin in the cells. The
presence of an extra amount of certain granules among the
tissues, located generally intercellularly, is the main secondary
feature of the tissues of the parasitized snails. This condition,
as will be pointed out presently, is most striking in the newly
infected individuals. After some time following an infection
the granules seem to decrease quantitatively. That is, the black
intercellular granules of parasitized Physa and Planorbis
are a temporary element which under a certain physiological
condition appears in the homogenous intercellular matrix,
and which origin is intracellular.

Tel NO 2. a EE 6s use 2

The anatomy and morphology of pulmonates in general
have been worked out by several investigators: Swammer-
damm (1737), Leydig (1850), Semper (1857), Leydig (1876),
Simroth (1885), Bronn (1896), and others. Leydig (1850)
points out that there are present black and yellow pigments

374 H. P. KJERSCHOW AGERSBORG

in the tissues, and in 1876 he calls them chromatophore
granules. But otherwise he does not record anything about
intercellular granules. In this second work this author worked
on four different genera of pulmonates: eight species of
Limax, two of Helix, one Physa fontinalis, and one
Arion. But he does not record anything about granules
other than those mentioned. Semper (1857) also pictures
pigment, but that is as far as he went on this point, although
he dealt with seven genera of pulmonates, containing nine
different species, one of whichwas Planorbis marginatus.

Since all the specimens I examined were infected at the time
of examination, it was impossible to ascertain from them the
exact nature of the normal tissues. But judging from many
cases with an early or mild infection, I believe that upon
infection the tissues become less responsive to ordinary stains.
Infected specimens are difficult to fix properly. It is difficult
to make good sections. The tissues have a tendency to crumble
before the edge of the knife. None of these difficulties were
met with in the uninfected material which was raised in the
laboratory. The normal tissue has less tendency to shrink
during the process of preparation. And the black intercellular
granules are absent.

Tilo Parasittiiged Taisane,

Since serial sections were made of all the specimens sectioned,
it was relatively easy to make a survey of the entire animal
from the anterior to the posterior and in this way ascertain
the degree of infection. At first a general survey of ca. 40,000
sections were made and notes recorded on points observed.
Then additional material was prepared, both from fresh speci-
mens collected in nature and from some reared in the laboratory.
After this mode of procedure a more intensive study ensued.
This was coupled with the preparation of drawings made to
scale as indicated below. Every specimen collected in the
Drainage Ditch was infected with trematode larvae. The
infection consisted of miracidia, rediae, and cercariae in various
stages of development. All of these stages were present every-

EFFECT OF PARASITES ON TISSUES 375

where in the body of the host (Pl. 12, figs. 1,2; Pl. 18, 3-14;
Pl. 16, figs. 20-3; Pl. 17, figs. 24-830; Pl. 18, figs. 31, 35;
Pl. 20, fig. 48 ; Pl. 21, fig. 55). In Pl. 12, fig. 1 (par), a parasite
(probably a cercaria) may be seen just below the ectoderm
of the foot.

1. Avenue of Infection.

Infection is apparently established by way of the blood
system. (1) Because the earliest stages of the parasite are
found in the sinuses all through the body. This is most mani-
fest as regards the finer connective tissue of the respiratory
organ. (2) Because later stages are found in the loose connective
tissue of any organ. By the time the cercaria stage is reached
the parasite has arrived in the hepatic sinuses. In fact the
hepatic sinuses seem to be a general collecting-place in so
much that they may be completely filled even to the extent
that the hepatic wall is distended and stretched until the
epithelium is practically obliterated (Pl. 13, figs. 3-14; Pl. 18,
fig. 34). On this pomt my observations agree with Leiper’s
findings, for this author claims that the epithelium disappears
apparently through pressure atrophy. And Faust (1920: 81)
verifies this when he says: ‘The food which the parthenita
takes in first of all is from the lymph.’ That is, the infection
takes place by way of the blood system. Faust also says:
‘ The parasite is always found in the connective tissues and the
interstices between tubules, while the portion of the epithelial
cell bordering the lumina are always intact except in the most
necrotic tissues.’ According to Leiper (1915: 177), if an
infected Planorbis boissyi is kept in tap-water which is
renewed daily, it may discharge large numbers of cercariae
daily for weeks. But the cercariae will die within thirty-six
hours if they do not find a definite host. Cort (1914: 74)
found specimens of Planorbis trivolvis infected with
encysted stages of Cercaria trivolvis (Cort), showing,
as this author claims, that Planorbis trivolvis (Say)
is able to serve both as intermediate and secondary-inter-
mediate hosts for this trematode. In this connexion it may be

376 H. P. KJERSCHOW AGERSBORG

noted that such a condition seems to be common for Physa
gyrina (Say) as well. Does this mean that the bilharzian
cercaria of Planorbis boissyi is more specialized than
Cercaria trivolvis?

2. Effect of Infection.

The first and immediate effect of infection as represented
by the changes in tissues of the host is that of a distorted and
disintegrated condition (Pl. 12, fig. 1). As the parasite passes
from one stage to another it migrates into all the parts of
the host’s body. Cercariae or rediae may be found in the
tentacles (Pl. 12, fig. 2), the foot (Pl. 12, fig. 1), and the pharyn-
geal wall.

The second noticeable change in the tissues of the secondary-
intermediate host infected with trematode larvae is an attempt
apparently by the tissues of the host to react to the presence
of the parasite. This is shown first by an increase of black
granules throughout the host. According to Schreiner (1916)
1918) the origin of certain cytoplasmic granules is in the nucleus.
Tividence as to the origin of the black granules so abundantly
present (Pl. 14, figs. 15-17 ; Pl. 15, figs. 18,19; Pl. 16, figs. 20-
23; Pl. 17, figs. 24-80, gr) in all the tissues of newly infected
Planorbis trivolvis and Physa gyrina, seems also
in this case to point toward the nucleus as a source. A detailed
discussion of this will be given in another paper. Heavily
infected specimens seem to have less of these granules, reverting,
as it seems, toward the normal even while parasites in all
stages are abundantly present. It does not seem that the
parasite is the source of this excessive pigmentation. But,
as pointed out by Ward (1920: 51-2), it is the product of the
tissues of the host. It is well, however, to note that Ward,
in the case of the cyst of the myxisporidian parasite Myxo-
bolus aureatus (Ward), finds that the cyst itself has
a pigmentation of its own. This pigmentation of the cyst
may be the product of Myxobolus. The later phase of this
stage of infection is noted by an apparent readjustment (in
some cases); this readjustment does not take place in every

EFFECT OF PARASITES ON TISSUES 377
specimen. When it does not the organism dies. After the
readjustment of the tissues of the host is accomplished there
is a diminution of pigmentation or granules, and the tissues
approach the normal condition. This is easily comparable
to the tissues of young uninfected individuals and adults with
only a few cercariae in the foot. The response of the tissues
of the host to the parasite may be partly illustrated by figs. 20-
30 (Pls. 16,17). The black granules (gr) are shown definitely
in the tissues, and there are no transitional stages of granules
passing from the parasite to the host. An acute case of maxi-
mum pigmentation 1s demonstrated in figs. 15 and 16 (PI. 14).
This case represents a condition of a newly infected young
Physa (Pl. 14, fig. 16) in which miracidia could be found.
Fig. 15 (Pl. 14) represents a conditionin Planorbis generally
infected. During the second or readjusting period the tissues
may proliferate considerably also, as in certain cases, e.g. in
the walls of the hepatic sinuses (Pl. 18, figs. 31, 33, 35, mfct,
mct). This is accompanied by a rapid growth of the parasite
(Pl. 18, fig. 35, par). ts |

The third stage of the parasitized tissues is a much distorted,
disintegrated, and shrunken condition., During this time the
host ‘ hangs between life and death’. In the liver the epithe-
lium may be nearly destroyed. Only small nuclei remain,
and these are in a highly concentrated condition giving the
effect of squamous epithelium instead of columnar (PI. 18,
figs. 32, 33).

The fourth period is marked by definite changes toward the
normal condition ; this may be called a period of regeneration.
During this time the parasite is less abundant and the tissues
are slowly recovering.

3. Glycogen.

Nearly forty years ago Barfurth demonstrated the presence
of glycogen in the various parts of the body of gasteropods
(Limax variegatus and Helix pomatia). He writes
(1885: 842): ‘ Bei der mikrochemischen Untersuchung fand |
sich in fast allen Organen reichlich Glycogen.’ That is, he

NO. 271 cc

378 H. P. KJERSCHOW AGERSBORG

found glycogen in all the organs save the tentacular retractor.
In the case of Physa gyrina I found that glycogen was
present in the muscles. But as a granular substance it is quite
different from the black granules mentioned above. Granules,
then, may be present everywhere in, and between, the tissues.
Shun Ichi (1920) finds that osmicated mitochondrial fixatives
preserve glycogen which can be stained in Best’s carmine and
iron haematoxylin. Hammersten (1885) substantiates the
findings of Blundstone and Barfurth. The works of Barfurth,
Blundstone, Hammersten, and Pfliger deal with normal tissues.
It is necessary, however, in such a work as the present one, to
have in mind the condition of the normal tissue as a compara-
tive basis for pathological tissue. Parasitized Physa has
glycogen in its muscles. In this connexion it may be well to
recall that Faust demonstrated the presence of glycogen in the
parasite. And, in his opinion, this glycogen was obtained from
the host. That is, the parasite which may be anywhere in the
body of the snail may not only obtain food from the blood-
stream but also from stored-up food of the body in general.

LV} the Liver:
1. The Anatomy of the Liver.

The liver offers an interesting topic of study because of its
important physiological activity in the life of the organism.
Several morphological regional differences of this organ are
noticeable. These differences may be exaggerated in parasi-
tized specimens. The anatomy of this organ has been worked
out by several workers as stated above (Swammerdamm,
Leydig, Semper, Simroth, Bronn), and also Faust (1920)
described it partly. It will therefore not be necessary to
go into any description of the anatomy here, although the
anatomy of Physa gyrina (Say) has not been described
before. It is not so very different, however, from other related
aquatic pulmonates. This also is true as regards the liver.
The molluscan digestive gland has also been the subject of
the attention of a number of other authors, who have studied

EFFECT OF PARASITES ON TISSUES 379

it both from the standpoint of its finer structure and from
the standpoint of physiology.

2. The Physiology of the Liver.

Before considering the livers of parasitized Physa and
Planorbis it is necessary, although briefly, first to point
out the normal condition in other gasteropods. De Quatre-
fages (1842) maintained that the liver in nudibranchs was of
a threefold function; hence his term ‘ plebenterism’ to
designate that species of gradation which consists in the union
of different function in one system of vessels. One unquestion-
able function of the hepatic tubules, as far as Aeolidia is
concerned, is as an exit of harmful indigestible parts taken in
with food (Alder and Hancock, 1845 ; Glaser, 1903 ; Hertwig,
1912). Frenzel (1886: 278) believed with Max Weber (1880)
and Barfurth (1883) that the liver of molluscs has a double
function: (1) as in Crustacea it is a digestive gland ;
(2) in addition this gland, according to Weber for the Crus-
tacea and Barfurth for the Mollusca, 1s of © excretorische
Funktion’. They think that the liver of these forms 1s analogous
to that of vertebrates. Frenzel described three kinds of epithe-
lial cells of the liver of the nudibranchiate mollusc Tethys
leporina (Linnaeus): (1) ‘ Kornerzellen’, (2) ‘ Keulenzellen’,
and (8) ‘ Kalkzellen’. ‘These are also described by Hecht
(1895: 671). Eliot and Evans (1908) think that some of the
liver cells in a doridiform ¢ladohepatic nudibranch are excretory
in function and are dropped into the follicle as they become
extended with excreted material. Eliot (1910: 39) attributes
to the liver the function which in the case of the nudibranchiate
molluse Melibe leonina (Gould) I have shown (Agersborg,
1923) to be that of the epithelium of the posterior chamber of
the stomach, e. g. the gizzard. The epithelium of the liver
of Melibe leonina (vide Agersborg, 1923) shows a similarity
to the ‘ Keulenzellen’ of Frenzel, or ‘ Cellules vacuolaires
excrétrices ’ of Hecht. The nucleus as a rule is basal in posi-
tion and contains one or two nucleoli. That part of the function
of the liver of Melibe is secretory and digestive may be

CG 2

380 H. P. KJERSCHOW AGERSBORG

judged from the fact that some of its product passes into the
stomach. This is readily shown by the fact that the surface
of the stomachal contents gives the same staining reaction
as the wall of the hepatic ducts which pass through the walls
of the gizzard. In fact, these are stained differently from any
other part of the organism treated chemically in the same way.
In looking at the hepatic epithelium one is impressed with the
nucleo-cytoplasmic relationship. There is a strong indication
that the nucleus takes an active part in the secretory activity
of this gland (Agersborg, 1923). Lange (1902) recorded a similar
phenomenon for the salivary glands among gasteropods, viz.
the nucleus seems to take an active part during the secretory
activity of the cells. This he could observe easily after feeding
and starving experiments on the snails with which he worked.
Boas (1916: 389) states that the liver of gasteropods is a large
compound acinous gland which secretion among certain snails
has been shown to have a strong dissolving effect on cellulose.
But besides being a secretory organ the liver also acts as an
absorption organ in that it takes up finely parted solid particles
of food (for example, starch granules), dissolves and absorbs
them. Further, it serves as_a storage chamber partly for
nutrition material (glycogen, fat), partly for calcium salts
which are stored in different cells of the liver. This is also
the opinion of Barfurth (1883: 332-4) and Bierdermann and
Moritz (1899: 61). But the liver is not the only organ in
which glycogen may be stored, for Barfurth (1885) and Blund-
stone (1885) demonstrated independently the presence of
glycogen in practically all parts of the body of Limax
variegatus and Helix pomatia. Using Best’s carmin
test, I found glycogen in the muscle-tissue of Physa gyrina
(Say). The liver in gasteropods, according to Barfurth (1883),
performs several activities which in higher animals may be
divided between several organs.

_ 8. The Livers of Parasitized Physa and Planorbis.

It was pointed out that the tissues of the host try to adjust
themselves to the presence of the parasite. In the liver this

EFFECT OF PARASITES ON TISSUES 381

may be manifested in various ways. The fluke, being first of
all in nearest contact with the interstitial cells of the sinus,
affects them first. The earliest change seems to be a quantita-
tive increase in cell material in the lumina of the sinuses (PI. 18,
fig. 31, mfct). The nucleus of the hepatic epithelium is uniformly
remarkably nearer the free luminar border than is usually
the case. Presumably the parasite uses up available food quite
quickly, which source of supply both for the hepatic wall and
the parasite is the blood-stream. The nearest supply for the
hepatic wall is in the hepatic epithelium itself. The nucleus
must migrate to the source of supply—the free luminar border—
a fact also observed by Korschelt (1891). I have reason to
think that the hepatic epithelium takes a more active part in
digestion than is generally accepted (Pl. 21, figs. 49, 58, 54).
If such be the case then digestive fluid may be available in
the hepatic lumen. Hence the position of the nucleus of the
hepatic epithelium. Be this as it may, the hepatic gland seems
to be an organ of secretion, which is also contended, among
other things, by several investigators. The position of the
nucleus near the free border, as seen in fig. 31 (Pl. 18), may be
initial to disintegration, since in this position-—at this level
within the sinus—may be found a parasite (par). The striking
condition of the basement membrane (bm) should be noted.
The basement membrane is not discernible in fig. 33 (Pl. 18)
(taken from Planorbis). But this is an exception to the
rule, as will be seen presently.

The nucleus of the atrophied hepatic epithelium does not
drop out of the cell altogether, as may be inferred from fig. 31
(Pl. 18). Because, in another follicle adjacent to it, the nucleus
is still present although the cytoplasmic reduction is exceed-
ingly great, the cytoplasm having practically disappeared
(Pl. 18, fig. 832). In other young Physa (5 mm. long) killed
in strong Flemming’s and F.W.A. fixing reagents (Pl. 20,
figs. 46 and 47) respectively, and in which the liver was not
infected, the nucleus was basally situated. And on the luminar
border are seen in many cells prominent secretion vacuoles
(vac) and granules (Pl. 20, fig. 47, sg). The secretory activity

382 H. P. KJERSCHOW AGERSBORG

is beautifully demonstrable in the liver of Planorbis
(Pl. 18, figs. 88-6; Pl. 19, figs. 87-8, 40-2). In fig. 33
(Pl. 18) nearly all the cells on this plane of the organ have
large secretion vacuoles (vac) with a secretion ball (para-
nuclear body) init. The free border in some of them is charac-
terized by fine granules. This vacuolization (vac) is much
more easily demonstrable in fig. 34 (Pl. 18). The paranuclear
body (pnb) is in closer proximity to the nucleus in this case
than in the previous figure, and the border is even more
oxyphil (ozb). The specimen was quite heavily infected.
The sinuses (s) are in many places crowded with developing
flukes in various stages (Pl. 18, fig. 35, par). In spite of such
high infection the hepatic epithelium (he) presents a normal
picture. This specimen was killed in strong Flemming’s fixing
fluid, and the figs. 88-5 (Pl. 18) were drawn from three different
places of the same individual in order to show the variable
condition of the liver. The nucleus in all these cases is located
basally, and shows quite a contrast to the condition found in
a heavily parasitized two-months old Physa (PI. 18, fig. 31)
also killed in F'lemming’s mixture. In the case of Planorbis
(Pl. 18, fig. 35) the interstitial cells (mct) of the sinus (s) are
greatly modified. This is no doubt a direct response on the
part of the tissue of the host to the presence of the parasite
(par). The globules (met) are nuclei located in a cyncitium.
Black granules as shown in the connective tissue (ct) imme-
diately below the basement membrane (bm) and in the epithe-
lium near the basal border are stained black by the osmic acid.
Quite a contrast to the condition as represented in the last
three figures (83-5) may be seen in fig. 36 (Pl. 18). This
drawing was made of a section of the liver of another individual
of Planorbis killed in Bouin’s fixative. The organism was
generally infected and showed the same general features as
demonstrated in figs. 18 and 19 (PI. 15). In fig. 36 (PI. 18)
the epithelium may be seen in the process of eliminating
secretion products into the lumen. The cells show vacuoliza-
tion and fibrillar formation, a condition common to normal
glandular activity.

EFFECT OF PARASITES ON TISSUES 383

As pointed out already by Barfurth, Boas, Faust, and others,
the hepatic gland has several functional properties, among
others that of absorption. Planorbis killed in F.W.A.
shows absorption substances (Pl. 19, fig. 88, sgr) in great
abundance in the epithelium, some of which seem to have
passed into the sinus (cts). In this connexion it is imperative
to note the simple condition of the basement membrane (bm)
and compare this fact with that shown in fig. 381 (Pl. 18).
The specimen upon which fig. 88 (Pl. 19) is based was very
heavily infected, also the specimen represented by fig. 31
(Pl. 18). Whether the condition of the basement membrane
is affected by the parasitic action on the host or by the absorp-
tion action on the part of the epithelium of the hepatic organ
of the host remains a question. This same condition is mani-
fested in fig. 41 (Pl. 19), taken from the same specimen as
fig. 88 (Pl. 19). But here the epithelium, besides having the
absorption substances well distributed within it, also exhibits
the sloughing off or discharging of products into the lumen (sp).

The nucleus, indeed, may not be situated basally as shown
in fig. 42 (Pl. 19), in which case it is located at various positions
within the cell. Previous to this I have stated that it has held
a basal position (PI. 18, figs. 33-6; Pl. 21, fig. 49). There is
an exception to this, as has already been pointed out and
demonstrated in fig. 31. The basement membrane even in
the same individual may fluctuate considerably (Pl. 19, figs. 38,
41; Pl. 20, fig. 43). Such a condition as represented in certain
parts of the gland (PI. 20, fig. 48) 1s quite extraordinary. But
it shows that the liver of the same individual is capable of
extensive variation (Pl. 18, figs. 88-5; Pl. 19, figs. 37, 38, 41 ;
PI. 20, fig.48). Planorbis killedin Bouin’s fluid may show the
basement membrane quite prominently (Pl. 20, fig. 45) and the
secretory activities of the epithelium as well. ‘The vacuolated
and fibrillated condition of the hepatic organ may be demon-
strated also in the epithelium of the genital duct (PI. 19, fig. 39).
An apparently non-infected Physa (except with a few mira-
cidia (?) in the foot) shows a highly vacuolated hepatic epithelium
(Pl. 20, fig. 44) with practically no basement membrane. _

384 H. P. KJERSCHOW AGERSBORG

DISCUSSION.
Il. General Discussion.

Metabolism of Host and Parasite.

As pointed out by Ward (1907), the effects of parasites on
the host may be mechanical, morphological, and physiological.
Mechanical changes are recognized as the local structural
disturbances ; morphological changes such as abnormal pro-
liferation of the tissues and cyst-formation ares common
phenomena; and physiological effects are expressed in the
limitation or modification of the normal physiological processes
of the host. The body of an organism, in order to live, must
carry on the necessary metabolic activities. It is evident that
the metabolism of an individual whose body is invaded by other
organisms becomes very greatly augmented. In addition to the
normal metabolic activities of the host, if it 1s to continue its
own existence, it must provide for itself and its guests. This
is done in various ways according to the nature of the para-
sitism. Metabolism being a process of taking in food, assimilat-
ing it, building up new material, and voiding material of no
longer use—a process alike in host and parasite—it 1s evident
that the host has to work harder if it 1s to continue its existence
successfully. Endoparasites do not only feed upon the host,
but their metabolic wastes must be taken care of by the host
as well as its own. This is often a greater difficulty than the
providing of board for the parasites. The parasitic excretions
become frequently poisonous to the host. That is, the meta-
bolism of the host may be impaired in various ways: either
by lack of sufficient food, owing to the drainage on the food-
supply of the parasite; or by the impaired function on the
part of certain organs of the host, owing to the particular
specificity of the parasite which is adapted to certain host-
organs only and the consequent overwork of these organs in
the attempt of the host to maintain metabolic equilibrium,
or by actual general or local weakening of the host by the
toxic parasitic excretions. The life of the host may be absolutely

EFFECT OF PARASITES ON TISSUES 385

shortened. This, of course, is a disadvantage for the parasite
inasmuch as the death of the host, indeed, may result in the
death of the parasite also. On the other hand, it is of advantage
to the parasite that a tolerable condition, or balance in relation-
ship between host and parasite, be reached. The continued
existence of the host may ensure the life and reproduction of
the parasite. Therefore it becomes of mutual importance both
to host and parasite whether an adjustment in the physio-
logical relationship between the two can be established quickly.
Since this may save the life of the host it may also secure the
propagation of the parasite and of the host without which
neither may long endure.

Ebeiseane Antidote tormed ?

Antidote for Parasitic Toxin.

As stated above, the first reaction of the tissues of Physa
and Planorbis infected with trematodes is to provide
an antidote against the parasitic excretions. In the case of
the monkey and of man the bilharzial excretions are very toxic
(Fairley, 1919: 299). This author states that pigmented cells
were not uncommonly found in the vicinity of bilharzial lesion.
He was not able to tell, however, whether the pigment originated
in the haemolytic action of the bilharzial toxin or was derived
from blood metabolized in the process of digestion by the
adult parasite. This’ pigment seems to resemble closely
malarial pigment, and it actually exists in the intestinal coeca
of the worm. Brown (1911: 299) found that melanin is the
product of the action of a proteolytic enzyme of the malarial
parasite upon the haemoglobin of the erythrocytes. The con-
dition of the liver in Schistosomiosis in man, as shown
by Phalen and Nichols (1908), is an increase of connective
tissue of the liver at the expense of the hepatic epithelium.
This, of course, leads to serious metabolic disturbances of the

1 | purposely avoid the term antibody, which has a very precise signifi-

cance, and which, so far as I know, no one has ever seen. I prefer also not
to coin a new term.

386 H. P. KJERSCHOW AGERSBORG

host, which ultimately is fatal. There is not the proper
adjustment between host and parasite. Fairley (1919: , 312)
showed that there is a definite relationship between the cellulo-
humeral response in experimentally infected monkeys (infected
with Bilharzia haematobia and Bilharzia man-
soni), and the prognosis. In hyperinfected monkeys dying
within few weeks he found that there was a constant leucopenia,
absence of eosinophilia, and a negative complement fixation
reaction. In monkeys surviving the sixth week of infection
there was constantly present an eosinophil leucocytosis asso-
ciated with a positive serological reaction. Finally, the death
of the hyperinfected monkeys prior to the deposition of the
ova, and the constant presence of positive serological reactions
in monkeys recovering from the initial stages of infection,
go far to prove the action of some toxic body elaborated
by the metabolic activities of these parasites, and the protec-
tive immunization of the definite host by antibody production.
From this excellent work of Fairley, one may well conjecture
that the molluscan host suffering from trematode infection
also has to solve the problem of immunization.

Faust (1920) states that melanin deposition in the connective
tissue 1s a matter of record in molluse and vertebrate liver,
as a result of trematode infection. I am not able to agree
yet on this point as far as the gasteropods which I have studied
are concerned. That ‘ the flukes work their way to the liver
through the blood sinuses (in molluscs) coming to reside in the
interstices between the liver tubules’ is substantiated by my
studies. Faust proved that a great deal of absorption of the
digested foodstuffs on the part of the fluke took place in the
hepatic sinus of the host. More than that, it may be stated that
the hepatic epithelium in some cases is actually greatly decreased
quantitatively by the presence of the parasite (compare Pl. 18,
figs. 32 and 35, he); also that flukes ‘empty a burdensome
amount of excretory wastes into the tissues and cause serious
cytological changes both in the epithelial cells of the (hepatic)
tubules and in the intertubular connective tissues’ (Pl. 18,
figs. 31, mfct ; 82, ct; 35, met). In other words, Faust then

EFFECT OF PARASITES ON TISSUES 387

anticipated me in my opinion relative to the problem which
the infected snail has to solve. That is, molluscan hosts
suffering from trematode infections have to solve the problem
of immunization.

That the parasites produce and liberate a toxin or toxins
into the system of the snail may not be doubted. That a
visible difference between non-infected, newly infected, and
long-infected snails exists is very apparent. Part of this
difference consists in the presence of a large amount of an inter-
cellular granular substance which may be blackened by osmic
acid. It seems to be of a lipoid nature. This granular substance
does not originate in the parasite. It originates in the cells
of the tissues of the host, e.g. in the muscles. Is the granular
substance something produced to counteract the parasitic
toxin ? Oris it the result of the parasitic waste upon the tissues
of the host ?. Why is it more abundant during an early infection
stage than at a later and even more heavily infected stage ?
Has the host-metabolism adapted itself to the extra task of
caring for the elimination of the parasitic wastes ? Are even
necrotic tissues of heavily infected snails better able to take
care of the parasitic toxins than normal tissues of lightly
and newly infected snails? These are some questions that
must be answered. It does not seem reasonable that tissues
which are highly abnormal morphologically can function better
than normal tissues. Therefore to conjecture that the gradual
decrease in intercellular granular substance proportionally with
the duration of the infection, irrespective of the continuous
increase of the infection and the consequent destruction of the
tissues, 18 an indication of a balance in reaction between a
tissue-reaction substance and the parasitic waste, does not
seem logical although it may be a helpful suggestion. However,
if this supposition is correct, do antibodies counteract the
parasitic toxins? What is the nature of the intercellular
granules so abundantly present during an early infection ?
Are they metabolic wastes of the host ? What becomes of them
later ? Are they voided by the host and are less and less formed
even during progressively increased infection? Without

388 H. P. KJERSCHOW AGERSBORG

designating any role to the intercellular granular substances
at present, I must call the attention to their presence. If they
represent metabolic waste of the host in its combat with the
parasitic toxins, then their consequent decrease may suggest
that the metabolic activities of the host becomes more effective
because of the antibodies which counteract the toxins, the
necrotic condition of the tissues notwithstanding.

SUMMARY AND CONCLUSIONS.

1. This work shows that the fresh-water snails Physa
gyrina (Say) and Planorbis trivolvis (Say), infected
with trematode larvae, respond to the infection by a cytological
secretion on the part of all the tissues.

2. This secretion probably originates in the nucleus and is
further developed in the cytoplasm before it is discharged into
the intercellular spaces.

3. The secretion remains intercellularly all the time during
an infection. It seems to be most abundant during the early
infection stage, and decreases toward the normal after the
infection has lasted for some time.

4, This cell-product, which possibly may have the function
of an antidote for the parasitic excretions, is represented in the
prepared specimens and in my drawings by fine black granules
situated everywhere in the tissues.

5. These granules may be blackened by osmic acid (OsOs).
They can be demonstrated also, but less easily, in material
fixed in Bouin’s fixative.

6. Physa gyrina (Say) raised in the laboratory became
infected in the aquarium containing several minnows?
(Notropis anogenus (Forbes)) collected in the same

1 Mr. R. E. Richardson, who kindly identified this species for me,
was quite unwilling to believe that it had been collected at Urbana,
Illinois, because he had hunted for it all through the state for twenty
years without any success of finding it. (See also 8. A. Forbes and R. E.
Richardson, ‘ The Fishes of Illinois ’, 1920.) However, this fish was present
in large numbers during the fall and winter of 1921-2 in the Drainage
Ditch of Crystal Lake, Urbana, [llinois.

EFFECT OF PARASITES ON TISSUES 38Y

place as the parents of the snail. The aquarium was balanced
with filamentous algae (Spirogyra, Cladophora) and
Elodea canadensis. They were very heavily infected
when three weeks old. At this time cercariae filled the hepatic
sinuses.

7. The liver may present a normal physiological aspect
although the organism may be heavily infected.

8. Several striking morphological differences in the liver of
the same individual may be demonstrable at one and the same
time.

9. The basement membrane seems to wax and wane accord-
ing to the physiological state of the liver (Pl. 18, figs. 31-6 ;
Pl, 19, figs. 37-8, 41-2; Pl. 20, figs. 48-5). This is in
strong contrast to the condition found in the ectoderm of
the foot in which the basement membrane is never present.
The ectoderm passes imperceptibly on to the underlying
connective tissue (Pl. 21, figs. 50-2). This condition is common
both for Physa and Planorbis in young as well as adult
states.

10. Absorption and secretion are demonstrable functions of
the liver of Physa and Planorbis. Conspicuous bodies,
which I have called paranuclear bodies, are present in the cells
of the hepatic epithelium of Planorbis trivolvis (Say).
They are usually situated between the nucleus and lumen ;
but sometimes this position may be altered. They are granular
in structure and resemble the Golgi bodies. Their variable
position seems to indicate that they are related to the different
functional states of the cell (Pl. 18, fig. 34, pmb). In the
intestinal epithelium of Physa gyrina (Say) there are
definite nuclear caps which break up and pass out of the cell
and into the lumen (PI. 21, figs. 58, 54, nue).

11. The adult cercaria having left the host (Planorbis
trivolvis and Physa gyrina) may re-enter and encyst.

12. Trematode larvae in various stages of development
may enter any part of the host.

13. A macerated condition of the tissues caused by. the
invasion of larval flukes may be overcome by subsequent

390 H. P. KJERSCHOW AGERSBORG

reaction on the part of the host. This reaction is of two kinds :
(1) the building up of an antibody ; (2) the regeneration of the
macerated tissues. ) |
14. Four infection stages may be recognized. (1) The
parasite invades the host. The tissues shrink and become more
and more difficult to handle for cytological purposes. The
tissues become friable. (2) The tissues secrete an intercellular
granular substance which becomes present everywhere in the
host. It is not present in the parasite. (3) During prolonged
heavy infection the tissues disintegrate. The epithelium of the
liver may be reduced from tall columnar to squamous. The
host may die. (4) If the host does not die, then there follows
after the third stage a gradual return to the normal. The
parasites also decrease in number within the host.

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jn2te

396 H. P. KJERSCHOW AGERSBORG

HXPLANATION OF PLATES 12-21.

N.B.—The drawings were made by the use of the camera
lucida and Leitz microscope. Tube length 117 mm. and 334 mm.

total length from the ocular to the drawing. Ocular no. 3;

objectives nos. 8, 5, 8, and ,, (75 =the oil-immersion). The

magnification is indicated in each case. Also the fixatives
and stains employed are mentioned. F.W.A.=Flemming’s
solution without acetic acid.

PLATE 12.

Fig. 1.—Section through the foot of Planorbis trivolvis (Say),
killed in Bouin’s picroformol-acetic solution ; stained with Heidenhain’s
haematoxylin and light green. Sections 8 micra thick. ct, connective
tissue ; ect, ectoderm; ics, intercellular secretions ; mus, muscle-fibres ;
par, parasites. In this specimen, though it was highly infected, pigment
granules are relatively scarce. Magnification: 50 mm. = 250 micra.

Fig. 2.—Longitudinal section of a tentacle of Planorbis trivolvis
(Say), to show encysted parasites (par). , optic nerve; eye. In this
instance, too, note the scarcity of intercellular granules. The section is
from the same specimen as fig. 1. Magnification also the same.

PuaTE 13. |

Figs. 3-14.—Serial sections of a hepatic sinus from two-months-old
Physa gyrina (Say), toshow: (1) the presence of two cercariae, a and b,
in the sinus, and (2) the distended condition of the hepatic wall. ¢, tail;
vs, ventral sucker; wlf, hepatic wall. Killed in Flemming’s strong solu-
tion; stained with safranin, light green. Sections 8 micra thick. Mira-
cidia are found in the foot ; pigmentation less abundant ; hepatic follicles
filled with cercariae. Magnification: 50 mm. = 100 micra.

PLATE 14.

Fig. 15.—Section of part of the body-wall covering the connective-tissue
capsule (tunica propria) of the liver, to show the intercellular secretion
granules among the muscle-fibres of Planorbis trivolvis (Say).
Killed in Flemming’s solution without acetic acid. Stained with Heiden-
hain’s haematoxylin. Infection general; intercellular secretion granules
abundant. Trematode larvae were noticeable during dissection. Sections
8 micra thick. m/f, muscle-fibres; nf, nerve-fibres; sgex, intercellular
secretion granules. Magnification: 50 mm. = 50 micra.

Fig. 16.—A section through the base of a tentacle'of Physa gyrina
(Say), to show the intercellular secretion granules (sgexv). Killed in

EFFECT OF PARASITES ON TISSUES 397

Hetherington’s carnoy-phenol ; stained with Mallory’s phosphotungstic acid
haematoxylin. Sections 5 micra thick. m/f, muscle-fibres. The specimen
was partly infected with rediae; pigmentation was quite prominent through-
out. The liver of this specimen shows a striking pathological picture :
many cells are sluffed off in part into the lumina. The magnification is
the same as in the previous figure.

Fig. 17.—A few muscle-fibres from Physa gyrina (Say), to show
the presence of black granules within the muscle-fibres; killed in Weigl’s
corrosive sublimate acetic mixture: stained with Heidenhain’s haema-
toxylin. Sections 10 micra thick. -mf, muscle-fibres; nu, nucleus ;
sgint, intracellular granules (mitochondria). Magnifications the same as
the last figure.

PLATE 15.

Fig. 18.—Horizontal section of the right jaw of Planorbis trivolvis
(Say), killed in Flemming’s solution; stained with safranin and light
green. Sections 10 micra thick. csmc, cross-sections of muscular columns ;
ct, connective tissue ; gr, intracellular cytoplasmic granules ; m/f, muscle-
fibres. The specimen showed a general infection throughout its body.
Magnification: 45 mm. = 30 micra. ;

Fig. 19.—Longitudinal vertical section of the jaw of Planorbis
trivolvis (Say), showing the muscle-columns (/smc) cut longitudinally.
epc, epithelial cover of the organ ; gr, intercellular granules ; mf, muscle-
fibres; nu, nucleus. Magnification the same as in fig. 18.

PLATE 16.

Figs. 20-3.—Serial sections of a parasite in the foot of Planorbis
trivolvis (Say), killed in Flemming’s strong mixture; stained with
safranin, light green. These sections show: (1) a scanty amount of
secretion granules (gr), and (2) these granules are not present in the
immediate proximity of the parasite, (3) the dark spherules in the parasite
are the nuclei which are all stained uniformly red by the safranin. cap,
cyst-wall; gr, secretion granules ; nu, nuclei; par, parasite ; su, sucker.
Sections 10 micra thick. Magnification: 50 mm. = 50 micra.

PLATE 17.

Figs. 24-9.—Serial sections from the foot of Planorbis trivolvis
(Say), to show the parasites imbedded in the tissues. Killed in Flemming’s
mixture. Stained with safranin, light green. Sections 10 micra thick.
cap, cyst-wall; gr, secretion granules; nu, nuclei; par, parasite; pg,
pedal gland ; spar, small parasite (micracidia ?). Magnification: 50 mm.
= 100 miera.

Fig. 30.—Section through the foot of Planorbis trivolvis (Say),
killed in Flemming’s fixative. (Same specimen as in figs. 24-9.) To show
the intercellular secretion granules (gr) of the host in relation to the

398 H. P. KJERSCHOW AGERSBORG

parasite (spar). The muscle-fibres have been omitted from the drawing.
Six encysted parasites are seen in the field. All the parasites stain faintly
oxyphil (green). Stained with safranin, light green. The granules are
black; muscles red; parasites green. These parasites are probably
developmental stages of rediae. Magnification: 50 mm. = 50 micra.

PuateE 18.

Fig. 31.—Cross-section of a hepatic follicle of Physa gyrina (Say),
about two months old. Killed in Flemming’s solution. Stained with
safranin, light green. Micracidia found in the foot; pigmentation less
abundant ; liver filled with cercaria. The cercariae are not in the lumen,
but in the sinus. Sections 8 micra thick. he, hepatic epithelium ; lu,
hepatic lumen; mfct, modified connective tissue; par, parasite within
sinus ; s, sinus. Magnifications: 50 mm. = 50 micra.

Fig. 32.—Cross-section of a hepatic follicular wall adjacent to the one
represented in fig. 31, to show the flattening of the hepatic epithelium (he)
and the secondary change of the connective tissue (ct) which borders on
the sinus (s); bhm, basement membrane of hepatic epithelium ; bms,
basement membrane of cells lining the sinus; ibmr, inter-basement-
membranal substance ; lu, hepatic lumen.

Fig. 33.—Cross-section of a hepatic villus showing a central sinus (s)
of Planorbis trivolvis (Say). Killed in Flemming’s solution without
acetic acid (F.W.A.); stained with Heidenhain’s haematoxylin and light
green. he, hepatic epithelium ; lu, lumen; s, hepatic sinus ; vac, vacuole.
Sections 8 micra thick. Magnification the same as in fig. 31.

Fig. 34.—Section of a liver tubule from the same specimen as in fig. 33.
The epithelium is highly vacuolated, and oxyphil in staining reaction.
Note the paranuclear bodies which are acid or oxyphil in staining reaction.
bm, basement membrane; he, hepatic epithelium ; Ju, lumen ; oxb. oxyphil
border ; pnb, paranuclear body; vac, vacuoles ; s, sinus. Sections 8 micra
thick. Stained with Heidenhain’s haematoxylin. Magnification as above.

Fig. 35.—Section of a liver tubule from lumen to lumen of the same
specimen as shown in fig. 33. The columnar hepatic epithelium shows
a striking condition of an actively functioning epithelium at the time of
death. Immediately below the interstitial connective tissue (ct) are a large
number of densely basic (red with safranin) bodies (mct) which in general
resemble nuclei of mesenchymous tissue. The granules of the epithelium
are black (from the osmic acid). bm, basement membrane ; ct, connective-
tissue cells of the interstitial cells ; he, hepatic epithelium ; mct, nuclei of
modified connective-tissue cells; nu, nucleus; par, parasite within the
hepatic lacuna; s, lacuna or sinus. Sections 8 micra thick. Magnifica-
tion as above.

Fig. 36.—Section through the hepatic wall of Planorbis trivolvis
(Say), killed in Bouin’s fixative. The specimen at the time of killing showed

EFFECT OF PARASITES ON TISSUES 399

the same general condition as the specimen represented in fig. 19. Neither
was apparently infected, but microscopic investigation revealed that both
were infected. 06m, hepatic epithelium. Stained with Heidenhain’s
haematoxylin and light green ; 10 micra in thickness, Magnification same
as above.

PuaTE 19,

Fig. 37.—Cross-section of the hepatic follicle of Planorbis trivolvis
(Say), killed with F.W.A.; stained with Heidenhain’s haematoxylin,
light green. chl, interhepatic lacunae; Jt, interstitial hepatic tissue ;
lu, lumen; « (vide fig. 38). Magnification: 50 mm. = 250 micra.

Fig. 38.—z2 from fig. 37 ; magnified 50 mm. = 50 micra. bm, basement
membrane; cts, hepatic sinus; he, hepatic epithelium loaded with fat
granules blackened with osmic acid ; sgr, absorption granules.

Fig. 39.—Section of oviduct of Planorbis trivolvis killed in Flem-
ming’s fixing agent; stained with safranin and light green. Sections
8 micra thick. ct, connective-tissue capsule ; e, epithelium ; nw, nucleus ;
vac, semi-vacuolar space. The lumen had considerable mucilaginous
substance which had stained with safranin. Magnification the same as
in fig. 38. |

Fig. 40.—Section through the tunica propria of Planorbis trivolvis
(Say), fixed in Bouin’s fixative. ct, connective tissue of tunica propria ;
hc, ectoderm; bm, basement membrane; he, hepatic epithelium; lu,
lumen hepaticum. Stained with safranin and light green. Magnification :
50 mm. = 100 micra.

Fig. 41.—Section of a liver-tubule of Planorbis trivolvis (Say),
fixed in F.W.A., and stained with safranin, light green. bm, basement
membrane; ct, interstitial connective tissue; cts, lacuna; he, hepatic
epithelium ; sp, cell-particles sluffed off into the lumen. Note the irregular
condition of the hepatic border; the marked basement membrane as
in fig. 38; also the granules of the epithelium. Sections 8 micra in thick-
ness. Magnification: 50 mm. = 50 micra.

Fig. 42.—Section of a liver tubule of Planorbis trivolvis (not
the same specimen as in fig. 41), fixed and stained in the same way as the
previous one (fig. 41). bm, basement membrane ; he, hepatic epithelium.
The liver shows a very variable condition of its epithelial lining. Sections
10 micra thick. Magnification as in fig. 41.

PLATE 20.

Fig. 43.—Section of the liver of Planorbis trivolvis (Say), fixed
in F.W.A. and stained with safranin, light green. Sections 8 micra thick.
bm, basement membrane; cic, connective-tissue capsule; he, hepatic
epithelium ; Ju, lumen; vac, vacuoles of the epithelium ; sp, secretion
particles sluffed off from the epithelium into the lumen of the organ.

400 H. P. KJERSCHOW AGERSBORG

The finely stippled border is oxyphil ; the remainder is basiphil. Magnifica-
tion the same as the foregoing.

Fig. 44.—Section of the liver of Physa gyrina (Say), fixed in four
parts absolute alcohol, one part glacial acetic acid ; stained in Best’s carmin
stain for glycogen. A few rediae were found in the posterior end of the
foot ; pigmentation of the mantle quite general, also in the anterior region
of the body. The liver was highly vacuolated. Sections 8 micra thick.
bm, basement membrane; lu, lumen; sgr, secretion granules; vac,
vacuoles. Magnification as above.

Fig. 45.—Section of a liver tubule of Planorbis trivolvis (Say)
(the same specimen as in fig. 36), killed in Bouin’s fixative; stained with
Heidenhain’s iron haematoxylin, light green. Note the general feature
as seen in fig. 36. This section was taken from the inner portion of the
hepatic organ. Sections 10 micra thick. Magnification as above.

Fig. 46.—Cross-section of the liverof Physa gyrina (Say), ca. 5 mm.
long, killed in Flemming’s solution and stained with Delafield’s haema-
toxylin. The organism was a little infected with miracidia (?). Note the
vacuoles between the nuclei and the hepatic lumen. nu, nucleus; s,
hepatic sinus. Section 7 micra in thickness. Magnification: 45 mm.
= 30 micra.

Fig. 47.—Section of the hepatic tubule of Physa gyrina (Say),
ca. 5 mm. long, killed in F.W.A. and stained with safranin, light green.
The organism was found to be a little infected with miracidia (?).
Sections 7 micra thick. bm, basement membrane ; Ju, lumen; nu, nucleus ;
s, hepatic sinus; sg, secretion granules; vac, vacuoles. Magnification :
the same as in fig. 46.

Fig. 48.—Section of the genital tubule of Planorbis trivolvis
(Say), killed in Bouin’s fixative and stained safranin, light green. Sections
10 micra thick. alc, alimentary canal; gd, genital duct; gh, hepatic
gland ; icto, inter-organ connective tissue ; par, rediae; spar, miracidia.
Magnification: 50 mm. = 250 micra.

PLATE 21.

Fig. 49.—Section of the stomach of Physa gyrina (Say), killed in
F.W.A., and stained with Heidenhain’s iron haematoxylin. Sections
9 micra thick. bgr, basal granules ; bm, basement membrane ; cil, cilia;
ctc, nucleus of connective-tissue capsule; nu, nucleus of entoderm.
Magnification: 45 mm. = 30 micra.

Fig. 50.—Cross-section of the foot of Planorbis trivolvis (Say),
showing ciliated ectoderm with pedal glands, both of which are inter-
spersed with black granules. The basement membrane is absent; the
ectoderm rests imperceptibly upon the connective tissue. Section 10 micra
in thickness. bgr, basal granules; cil, cilia; ect, ectoderm; ct, connec-
tive-tissue cells; sgr, secretion granules; pg, pedal glands. Magnifica-

EFFECT OF PARASITES ON TISSUES 401]

tion: 50 mm. =50 micra. (Specimen killed in strong Flemming’s fixative
and stained with safranin and light green.)

Fig. 51.—Cross-section of the foot of Physa gyrina (Say), ca. 5 mm.
long, raised in the laboratory ; killed in Flemming’s fixative ; stained with
safranin, light green. Section 7 micra thick. The organism was infected
with a few miracidia in the foot. A basement membrane is absent. bgr,
basal granules ; cil, cilia; ect, ectoderm of the foot ; nw, nucleus. Magni-
fication: 45 mm. = 30 micra.

Fig. 52.—Cross-section of the foot of Physa gyrina (Say), one day
old. Killed in Bouin’s fluid; stained with Delafield’s haematoxylin. This
demonstration stands in striking contrast with those represented by
figs. 50 and 51. The absence of the basement membrane in this part of the
body is apparently a common characteristic of both the young and adult
of Physaand Planorbis. Sections 8 micra thick. bgr, basal granules ;
cil, cilia; ct, connective tissue; ect, ectoderm; mgl, mucous gland ;
nu, connective-tissue nuclei. Magnification as above.

Fig. 53.—Cross-section of the intestine of Physa gyrina (Say),
2 mm. long; killed in Hetherington’s carnoy-phenol fixing agent ; stained
with Mallory’s phosphotungstic acid-haematoxylin. Sections 5 micra
thick. This drawing demonstrates an interesting phenomenon relative to
secretion. Note the nuclear caps (wc) and strands (st) which extend from
them and beyond the ciliated border. bgr, basal granules ; bm, basement
membrane; cil, cilia; ctc, connective-tissue capsule ; nu, nucleus; nue,
nuclear cap; sf, mucous strands. There are no miracidia in the foot ;
pigmentation general. Magnification: 50 mm. = 50 micra.

Fig. 54.—Cross-section of the intestine as in fig. 53, showing the same
phenomenon still better ; stained in Delafield’s haematoxylin. Section
5 micra thick. bm, basement membrane; nu, nucleus; nuc, nuclear
cap ; wsbi, unstained internal border. Magnification: 45 mm. = 30 micra.

Fig. 55.—Section of the foot of Planorbis trivolvis (Say), infected
with flukes in various stages. Killed in Bouin’s fixing agent; stained in
Delafield’s haematoxylin. Section 8 micra thick. This shows an encysted
cercaria in the foot ; the tissues around the parasite does not seem to have
any of the black secretion granules present. cap, capsule of the parasite ;
mf, muscle-fibres of the host ; nw, nuclei of the parasite ; par, parasite ;
spar, smaller parasites (miracidia ?); su, suckers. Magnification: 50 mm.
= 100 micra.

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VITA

HELMER PARELI VON WoLp KJerscHow AGERSBORG, born on the 26th of
October, 1881, at Gjersvik in Roédoy, Nordre Helgeland, Norway.

PARENTS

Hr. Albert Martin Petersen Agersborg, and fru Hansine Marie Zahl Agersborg
(Née: Hansine Marie Zahl Christensen Mechlenborg).

1887-1900.
1900-1906.
1906-1908.
1908-1912.
1912-1916.
1917

1918-1820.
1921-1922.

1913, 1914,

Loty, 120,

EDUCATION

Attended school in the District, and under private Tutor at home.
Lived on parental estate, and traveled in Norway.

Attended evening schools in Brooklyn, N. Y., and Seattle, Wash.
Attended Seattle Seminary (Seattle Pacific College) Seattle, Wash.
Attended the University of Washington, Seattle, Washington.
Attended the Universitas Regia Fredericiana, Oslo, Norway.
Attended Columbia University, New York, N. Y.

Attended the University of Illinois, Urbana, Illinois, and the Summer
Session of the University, 1922.

1921, Student and investigator at the Pugent Sound Biological Sta-
tion, Friday Harbor, Washington (summers).

Expeditions to the aretics of Norway, to collect littoral mollusks for
the purpose of a study of distribution, variation and evolution
(summers).

1918, 1919, 1920, 1923, Student and investigator at the Marine Biological Labora-

tory, Woods Hole, Massachusetts (summers).

DEGREES

1916. Bachelor of Science, University of Washington, Seattle, Washington.
1916. Master of Science, University of Washington, Seattle, Washington.
1917. Akademisk Borger, Universitas Regia Fredericiana,, Oslo, Norway.
1920. Master of Arts, Columbia University, New York, N. Y.

1923. Doctor of Philosophy, University of Illinois, Urbana, Illinois.

1916, 1914,
1914-1916.
1913-1916.

LOL -1 918;

1918-1919.

1919-1920.
lee. 019°

LEO -1 920:

1920-1921.

PosItTIONS HELD
Assistant -and Acting Curator, respectively, Pugent Sound Biological
Station, Friday Harbor, Washington (summers).
Charge of the Biological Supply Department, University of Washing-
ton, Seattle, Washington.
Student Assistant in Zodlogy (general invertebrate zoology), Uni-
versity of Washington, Seattle, Washington.
Tutor in Biology (botany, general zodlogy, and comparative vertebrate
anatomy), and in the Evening College (comparative anatomy, and
histology), College of the City of New York, New York, N. Y.
Assistant in Zodlogy (general biology, and vertebrate comparative
anatomy), Columbia University, New York, N. Y.
First Assistant in Zodlogy, Columbia University, New York, N. Y.
Demonstrator in Anatomy (histology and embryology), Long Island
College Hospital Medical College, Brooklyn, N. Y.
Instructor in Anatomy (histology and embryology), Long Island
College Hospital Medical College, Brooklyn, N. Y.
Assistant Professor of Zodlogy (general zodlogy, comparative verte-
brate anatomy, histology, and economic zodlogy), University of Wyom-
ing, Laramie, Wyoming.

1920-1921. Assistant Parasitologist, University of Wyoming Agricultural College,

Laramie, Wyoming.

1921-1922. Graduate Assistant in Zovdlogy (ontogeny and general zodlogy),

University of Illinois, Urbana, Illionis.

1922-1923. Instructor in Zodlogy (general z00logy ), University of Nebraska,

Lincoln, Nebraska. 4

1923-1924. Instructor in Biology (general invertebrate zodlogy), Williams Col-

1924-

1915-
1917-

1918-

1918 a-

1919-

1919a-

1920-
1921-

ot

vo)

RO RSS
Ia)

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LOLS:

1918a.

OED

1919a.

lege, Williamstown, Massachusetts.

Professor of Biology (and Head of the Department), The James
Millikin University, Decatur, Illinois.

POSITIONS DECLINED

Assistant in Anatomy, College of Physicians and Surgeons, Columbia
University, New York, N. Y.

Associate Professor of Anatomy, Emory University, College of Medicine,
Atlanta, Georgia.
Associate in Anatomy, University of Illinois College of Medicine, Chicago,
Illinois.

Konservator av den zodlogiske Avdeling, Tromsé6 Museum, Tromso, Nor-
way.

MEMBER OF SCIENTIFIC- AND OTHER LEARNED SOCIETIES, SINCE:

Pacific Fisheries Society, Seattle, Washington. -

Det kongelige norske Videnskabers Selskab (Trondhjems Museum),
Trondhjem, .Norway.

The Corporation of the Marine Biological Laboratory, Woods Hole,
Massachusetts.

The American Scandinavian Foundation, Fellow, New York, N. Y.

The American Association for the Advancement of Science, Washington,
DC.

The Ecological Society of America.

The American Society of Mammalogists.

The International Honorary Scientific Society of the Sigma Xi, (Illinois
Chapter).

The National Geographical Society, Washington, D. C.

- The Nebraska Academy of Science, Lincoln, Nebraska.

The American Society of Zoodlogists.

- The American Nature Association.

The American Tree Association.

American Association for Medical Progress, Inc., Boston, Massachusetts.
The Honorary Scientific Society of the Gamma Epsilon Tau, Alpha Chap-
ter, Decatur, Illinois.

The Illinois State Academy of Science.

The American Ornithologists’ Union.

CONTRIBUTIONS TO SCIENCE

‘¢Nematodes on marketable fishes.’’ Science, N. 8., 48: 493-495.
‘¢Bilateral tendencies and habits in the twenty-rayed starfish, Pycnopodia
helianthoides (Stimpson).’’ Biological Bulletin, 35: 232-254, 3 text
figures, and 1 plate.

‘‘The teaching of natural science in the Primary and Secondary Schools
of Norway.’’ School and Society, 9: 675-678.

‘¢Notes on the nudibranchiate mollusk, Melibe leonian (Gould).’? Publi-
cations Puget Sound Biological Station, 2: 264-277, 2 plates.

=P

1920, ‘The utilization of echinoderms and of gasteropod mollusks. ’’ American
Naturalist, 54: 414-426, 4 text figures.

1921. ‘Contribution to the knowledge of the nudibranchiate mollusk, Melibe
leonina (Gold).’’? American Naturalist, 55: 222-253, 12 text figures.

1921a. ‘“On the status of Chioraera (Gould).’’ Nautilus, 85: 50-57.

1922. ‘The relation of the madreporite to the physiological anterior end in the
twenty-rays starfish, Pycnopodia helianthoides (Stimpson).’’? Biological
Bulletin, 42: 202-216, 10 text figures. :

19229. “*Notes on the locomotion of the nudibranchiate mollusk, Dendronotus
giganteus O’Donoghue.’’ Biological Bulletin, 42: 257-266, 4 text figures.

1922b. “Some observations on qualitative chemical and physical stimulations in
nudibranchiate mollusks with special reference to the rédle of the
‘rhinophore’.’’? Journal Experimental Zodlogy, 36: 423-444, 4 tables and
2 text figures.

1923. <‘*A eritique on Professor Harold Heath’s Chioraera dalli, with special
reference to the use of the foot in the nudibranchiate mollusk, Melibe
leonina (Gould).’’ Nautilus, 36: 86-96, 3 plates.

1923a. ‘“Notes on a new cladohepatic nudibranch (Olea hansineénsis) from
Friday Harbor, Washington.’’ Nautilus, 36: 133-138, 1 plate.

1923b. ‘*Gymnosomatous Pteropoda (Clione kincaidi and Trichocyclus han-
sineénsis) from Friday Harbor, Washington.’’ Annales Sciences
Naturelles, Zodlogie, (10) 6: 391-402, 5 text figures.

1923e. ‘The morphology of the nudibranchiate molluse, Melibe leonina (Gould).’’
Quarterly Journal of Microscopical Science. 67: 507-592, 10 double plates.

1923d. ‘*The sex of the nudibranchiate Mollusca. I. With special reference to
Germ-cell secretions in Melibe leonina (Gould).’? An abstract in An-
atomical Records, 25: 346, (Read in part before the American Society of
Zoodlogists, Cincinnati, December 28, 1923).

1923 e. ‘*The sensory receptors and the structure of the oral tentacles of the
nudibranchiate mollusk, Hermissenda crassicornis (Eschocholtz 1831), syn.
Hermissenda opalescens Cooper 1862, 1863.’? An abstract in Anatom-
ical Records, 25: 347 (Read in part before the American Society of Zoolo-
gists, Cincinnati, December 28, 1923).

1924. ‘Studies on the effect of parasitism upon the tissues. I. With special
reference to certain gasteropod molluses.’’ Quarterly Journal of Mi-
croscopical Science, 68: 361-401, 10 double plates.

1924a. ‘‘The sex of the nudibranchiate Mollusca. I. With special reference to
Germ-cell secretions in Melibe leonina (Gould).’’ Archives de Biologie,
34: 215-233, 3 plates.

1924b. ‘‘A proposed biagraphical entomological dictionary.’’ Science. 60: 431.

1925. ‘The sensory receptors and the structure of the oral tentacles of the
nudibranchiate mollusk, Hermissenda crassicornis (Eschscholtz 1831),
syn. Hermissenda opalescens Cooper 1862, 1863.’’ Acta Zodlogica. (in
press), 23 figures.

1925a. ‘‘Studies on the effect of parasitism upon the tissues. II. With special
reference to a new diplostomous trematode found in the minnow,
Notropis anogenus (Forbes).’’ (In press) 3 plates. (Read in part be-
fore the parce State Academy of Science, Springfield, Hlinois, February
21, 1925).