FISHERIES RESEARCH BOARD OF CANADA ,-g pc,), ves Translation Series No. 2949
Studies on air-bladder Cocgidia of Gadus species (Eimeria gadi n. sp.)
by J. Fiebiger
Original title: Studien ueber die Schwimmblasencoccidien der Gadusarten (Eimeria gadi n. sp.)
From: .Archiv fuer Protistenkunde ('Archives of Protistology' .31 : 95-137, 1913
Translated-by the Translation Bureau(m)'' Nultilineal Services Division .Department of the Secretary of State of Canada
Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S.. 1974
72 Pages typescript DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT
TRANSLATION BUREAU BUREAU DES TRADUCTIONS
MULTILINGUAL SERVICES DIVISION DES SERVICES CANADA DIVISION MULTILINGUES F.e.ge_9 2/9
TRANSLATED FROM - TRADUCTION DE INTO - EN Germa.n English
AUTHOR - AUTEUR J. FIEBIGER
TITLE IN ENGLISH - TITRE ANGLAIS Studies on air-bladder Coccidia of Gadub species (Eimeria gadi n. sp.)
TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÈRE (TRANSCRIRE EN CARACTÉRES ROMAINS)
Studien ueber die Schwimmblasencoccidien der Gadusarten (Eimeria gadi n.sp.)
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Archiv fuer Protistenkunde
REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS
('Archives of Protistology t )
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"Studien ueber die Schwimmblasencoccidien der Gadusarten (Eimeria gadi n.sp.),"
Archly fuer Protistenkunde 31, 95 - 137, 1913
Studies on air-bladder Coccidia ••• of Gadus species (Eimeria gadi n. sA.)
by J. FIEDIGER From the Institute of Fish Pathology, Vienna. Veterinary University, Vienna, Austria
(With Table 10 and nine Figures in the text)
Introduction .
Our knowledge of the Coccidia is based chiefly on three species, viz.
Eimeria schubergi, E. stiedae and Cyclospbra caryolytica. These species
have been studied so thoroughly and--mith respect to their biologies and, in
part, also with respect to their morphologies—exhibit so many coinciding
features that we regard their properties as those of the Coccidia in general
and tend a priori to regard considerable deviations from the known pattern
as personal errors in observation.
UNEDITED DRAFT TRANSLATION Orey for information TRADUCTION NON REVISÉE Pasted-in pheocapies provided by Information seulement SOS-200-10-31 courtesy of freelance translator
■■••••/...,.■•■••••• 7530-21-029-S332 3
We should, however, not forget that life patterns must undergo modi- • fications once -new features enter into them.
The three afOrementioned species, now, are parasites of terrestrial animals. For that reason, these particular coccidians are exposed to the direct influence of the atmosphere during the most important stage of their life cycle, which takes place outside of the'host animal, viz. spore develop- ment. In the case of aquatic animals, on the other hand, the parasitic cocci- dians get into either fresh or marine water once they leave the host animal,.
.Since a certain influence has been ascribed to atmospheric oxygen in parti- • 96'. cular during the afore-mentioned stage of development, we are forced to regard differences of that type as being profound in character already for that reason, apart from the purely physical influences exerted by the water. The species under consideration in the present paper, which I, with a few interruptions, have been studying for several years, in fact does exhibit several significant deviations from the known life pattern of the Coccidia, and this, in particular, with regard to biological Characteristics.
The three species mentioned further above could be subjected to such detailed investigations because they are relatively readily accessible to experimontal research. This type of ready accessibility is not given in the case of the present species--a parasite of ocean fishes--and this, in 'parti- cular, not for a worker doing his research at an inland institute. Procurement of truly fresh or well preserved material involves great difficulties, and observation of the living parasite is entirely excluded under the latter conditions. - 4 -
The present study, thus, could not provide a complete picture of the life cycle of this parasite; and I was forced to fall back to make conclusions from analogy and propose hypotheses. Despite the fact that 19 for the reasons just mentioned, cannot consider the investigation of this parasite as being completed, I have decided to publish the-results so far obtained in my in- vestigations, first, because I have been able to make several observations worthy of note and, secondly,.because there is practically no hope at all that . we, in the foreseeable future, may have available either new methods or better material.
In 1906, I undertook a voyage into the Icelandic waters on board a . fish- ing trawler belonging to the Nordsee Fishing Company (Dampffischereigesellschaft
"Nordsee"). lithe course of the gutting of the fishes caught in abundant num- bers, I was able to collect a number of objects of interest from the points of view of both parasitology and pathology, and this, in particular, in the case of Gadus species. Amnng others, I observed rather frequently the presence • of a yellow, creamy mass in the air-bladders of fish, which mass reminded me of thickened:pus. Only after my return, I discovered under the microscope that this mass to the greater part consisted of coccidian spores.
On reviewing the pertinent literature, I found a paper published by
J. Mueller in 1842, which contains a description of a similar finding in the air-bladder of Gadus callarias G. morhua; Transi.], parts of which I will quote verbatim.
J. Mueller described . the mass found inside the air-bladder as "yellow matter," which frequently filled the rather large bladder,completely, adhering to the swollen inner surface of that structure. This material consisted -5 largely of capsules, which were arranged together in groups of three or four, with each capsule consisting of two halves (naviculae), which, in part, had • 97 already separated in the manner of the two halves of a pod. Between the two halves,.Mueller saw a little knob-like structure exhibiting vesicles; this little structure partly filled the cavities of the pod-like shells. "The little bodies (the spore capsules) are released, their contents undergo de- • velopment, and the bodies then divide in the longitudinal direction. For a short period of time, the two halves remain connected through their contents in contact across their center portions; finally, the halves separate com- pletely and the contents are released, perhaps, to form the base for a new developmental cycle." The infested fishes showed also other signs of being diseased viz. their tail parts were emaciated. The fishermen well realized the connection existing between the air-bladder infestation and the afore- mentioned changes in the external appearance of the fishes, and they regarded specimens of that type as being not fit for human consumption.
J. MUeller classified these structures among the "psorosperms," which term he had used several years earlier in connection with the protozoans oc- curring inside small tubules and nodules in the skin, the gills, the muscles and the urinary bladder of [certain.] fishes and frogs. Since the structures just mentioned, no doubt, represent myxosporidian nodules, the view appears to have evolved among workers that these air-bladder parasites must also have been Myxosporidia. That, at least, appears to be indicated in the his- torical review presented by Buetschli in Bronn's Klassen und Ordnungèn
[Classes and Orders]. 4r
6-.
At the present time, all we have to do is to take one look at the plate
presented by J. MUeller in his paper in order to realize that we are dealing
here With characteristic coccidian tetraspores. In any case, it must surprise
that this(coccidianjparasiticl, in such a massive and relatively freuent
manner, in an organism readily available could have avoided being subjected
to investigation, and this despite the fact that the Coccidia have become a
favorite subject of research these past fifteen years.
The fact that workers were concerned already at that time with "cancer-
inducing parasites" is made evident in the discussion part of J. Mueller's
paper, in which that author, however, argued against the existence of any
relations between these parasites and tumor formation.
It is very obviuus that-this coccidian species is also of historical
interest, since it represents one of the earliest coccidians discovered (only
Eimeria stiedae was discovered earlier). Due to the incompleteness of the
description, corresponding to the level of knowledge in sporozoan research
existing at that time, I believe that I have been justified in regarding this
particular coccidian species as a form newly discovered. On the basis of its • 98
characteristic features (opening of the spores along the longitudinal- suture,
and absence of residual bodies from the sriores), I, following Labbé's system
of classification, have assigned this coccidian in a preliminary communication
to the genus Goussia, and gave it the non-binding name Goussia gadi; in ac-
cordance with subsequent changes in the nomenclature, I have then in my text-
book of parasitology changed that name to Eimeria gadi.
Auerbach's paper, which also mentions this coccidian of the air-bladder of Gadus virens [probably Pollachius virens; Transi.] and identifies it as a Goussia species, reached me only after the publication of my first, pre- liminary communication. -7
According to Léger's system of classification, which has also been adopted by Doflein, the species under consideration must be assigned to the genus Eimeria belonging to the group Octozoica, which, in turn e belongs tb the Family Eimeridae. It should, however, be noted right from the start that the two filaments exhibited by the microgametes of other members of this
Family are missing in our species. With respect to further classification, I would assign this species most readily to the subgenus Goussia LABle, despite the fact that the se-pores, in part e open already inside of the fish's air- bladder.
Material and Methods
I based my preliminary communication on the examination of material,
which I , in addition to other interesting material, had collected aboard the fishing trawler and stored in a tin container in about 9% Formol solution.
Since Formol fixes nuclear features only in a deficient manner, I waS able at that time to sketch both the cytological details and the life cycle only in rough outlines. I have obtained new and relatively fresh material during the course of a stay at the Institut fuer Schiffs- und Tropenkrankheiten [mati- tu-te of Marine and Tropical Diseases], in - Hamburg. However, these fishes had been stored on ice already for four to five days. I have ueed that material for preparing both wet, mercuric chloride-fixed and dry, Giemsa -stained smears.
Material fixed either in Flemming's fluid or in a mixture of mercuruc chloride . and alcohol served later on in the preparation of sections. My friend Moroff assisted me in the latter procedures; unfortunately, the ylanned cooperative treatment of this material did not go beyond the preliminary stages. - 8 -
Additional material has been shipped to me from Hamburg through the friendly offices of Mr. Luebbert, Fisheries Director. However, infected • 99 .• air-bladders are relatively frequently available also on our [-Vienna] salt-water fish markets. Since the air-bladder is located retroperitoneally and, in the Gadidae, is attached to the ribs, that structure remains inside the fish body during gutting. When the fish on the market is then cut across into metameric pieces, one finds that the yellowish mass oozes Out of the opened air-bladder. In the case of inland cities one must, however, assume that the fish coming from the Icelandic waters have been stored on ice for about two weeks.
All this material was used to prepare both smears and sections, apart from the observations made by inspecting the native material. A mixture of . • mercuric chloride, alcohol and glacial - acetic acid was used almost exclusively
as fixation fluid. Flemming's mixture is less suited for these purposes, be- .
cause the chramatin elements upon staining after Heidenhain can be distinguished
only to an inadequate extent from the fatty depoSits, which also stain black.
Haemalum, Delafield's haeMatoxylin, iron haematoxylin, Boehmerls haema-
toxylin, safranine, borax carmine and methyl green served as nuclear stains, with the latter one not giving satisfactory results.
The modified method of staining after Giemsa was also used in the case of both sections and wet preparations—this, however, without obtaining any
advantages worth mentioning.
The occurrence of such great masses of protozoan bodies in a sealed ca-
vity originally filled with gas made this material appear to be highly suited
also for chemical investigation. Hitherto, investigations of that type have been successful in protozoan bodies only to a limited extent. Due to the microscopic minuteness of the single individual, we usually are forced to fall back to rather imperfect microchemical reactions. However, an extraordinarily large number of individual specimens is required in order to have available a mass adequate for chemical analysis. In the case of parasitic protozoans we find furthermore that separation of the material of interest from inter- fering admixtures (viz. the nutritive medium or the host tissues) can be made hardly at all. Only in oUr particular case do. the protozoans multiply into an empty container to an enormous extent, i.e. the air-bladder represents, as it were, a storage space for prOtozoan bodies. ProfessOr Panzer has subjected .
this mass to a detailed chemical investigation, which, indeed, led to results worthy of note. On the basis of his findings, I have then undertaken micro-
chemical evaluations of smears treated with osmic acid [osmuim tetroxide],
scarlet red and eudan black as well as studies using the polarizing microscope.
Occurrence and macroscopic features • 100
As already mentioned further above, the coccidian -under consideration
is found relatively frequently in the 5WiM -bladder of the various Gadidae.
I have found specimens in the pollock (Gadus virens [i.e. Pollachius virens;
Transi.]), in the Atlantic or common cod (Gadus morrhua or callarias) and in the haddock (Gadus [i.e. Melanogramus aeglefinus; Transi.]).
have found this coccidian most frequently in Gadus virens, which species
represents the main portion of the catch made by drag-net fishermen. On the
Hamburg fish market, where I have been ab1 to undertake systematic investi-
gations, I found this particular Ooccidian during the month of September in - 10- five percent of the cases. According to data provided by fish market officials, there exist periodic variations in the degree of infestation depending on the season--a finding which would correspond to a very rapid multiplication of the sporozoans. With regard to the proveniences of the infested fishes, I have been able to establish as definite catch areas: The most important fishing site along the south-easter edge of Iceland, the so-called Ingolfs Hoefde; furthermore, the area around the Faroe Islands or, to be more exact, latitude
600 north and longitude 2° east . (three pollocks and four haddocks), and, fur- thermore, the North Sea--exact sites not having been determined.
The fact that I, after more or less prolonged searching, always found infested specimens among the different catches, permits the conclusion that the distribution of this coccidian among these three Gadidae is a general one.
J. Mueller has suggested that other gadid species--like Gadus merlangus from the Mediterranean Seas [the whiting; a more recent German source gives Merlan- gius merlangus as name of this species; Trans1.3-may be infested by this coccidian; I have been unable to establish the correctness of that suggestion.
Before going on to discuss the mass present inside the swim-bladder, I - wish to outline briefly the anatomical and histological features of the WIViM- bladder of the gadid species.
In the gadid fishes the eNim-bladder is a simple, membranous sac, which is filled with gas[es], sealed all round, and tube-like in shape; it is located in the dorsal portion of the abdominal cavity. An air-duct [between the digestive tract and the swim-bladderl does not exist, i.e. the gadid fishes must be counted among the Physochisti. This membranous sac is separated, on the one hand, from the abdominal cavity by the peritoneum (at this site -11-
spotted with black) and, on the other one, from the vertebrate column by the
black,stripe-shaped kidney.
The anterior end of the swim-bladder is relatively spacious; the bladder
gradually tapers toward the posterior end. The anterior end.extends forward
into the cardial region. From that end, two lateral, blind and slietly curved .
horns are given off, in the cephalad direction, into the soft tissues along
both sides of the vertebral column. Cuvier erroneously regarded these horns
as air ducts.
- Toward the back, the swim-bladder ex-tends through the entire abdominal • 101
space and on further between the muscles and the hemal processes of the ver-
• tebral bodies, so that the tapered end of the bladder becomes visible only
following manual separation of the muscles and cutting of these osseous spicules.
In the swim-bladder wall we distinguish, on macroscopid inspection, two
individual layers, viz. a thick, tight external skin, which can be cut with
some difficulty only--the fibrous layer--and a thin, opalescent internal skin,
the mucous layer. The former is attached to theribs by ray-like structures'.
The swim-bladder can be removed from the fish only after all these ray-
like pillars or rods have been cut. The inner skin'is lobsely connected with
the fibrous layer and can be readily separated from the latter.
Viewed under the microscope, the fibrous layer appears to consist of
closely arranged bundles of connective tissue arranged in a parallel fashion.
These bundles contain greatly elongated, thin nuclei. Treatment of the material
with acetic acid results in the dissolution of these fibers and reveals the
presence of a network of elastic fibers, which can also be well demonstrated
with the aid of orcein. The mucous layer consists of a.network of connective- -12- tissue fibers, which, however, are modified in a peculiar manner. In teased- out preparations, these particular structures appear as very long e spindle- or needle-shaped, branched and rigid fibers. Numerous spherical nuclei are found in these fibers. In addition, there are flattened, cotpact and elongated- rectangular elastic leaflets present, which are amorphous in character, apart from the oval nucleus located at the center. A layer of smooth muscle fibers lines the inner surface of the compact fibrous membrane.
The anterior portion of the swim-bladder exhibits, on its ventral inner face, an extended, lawn-like structure, the red body. That structure consists . of an intricate . network of small vessels arranged in a parallel fashion. Its surface is lined with cuboid epithelial cells, which, in a glandular manner, . extend into the body between the.vascular coils; these cells actually form a kind of tubules. These tubules, which exhibit a distinct Itimen, reportedly produce a mucous secretion, which is not mucin and contains a conjugated nuc- leoprotein. The inner face of the swim-bladder is lined with squamaus epithel- ial cells, which, in the region of the red body, pass into the cuboid epi- . thelial cells.
Apart form the red body, there exists a vascular net between the two layers making up the swim-bladder wall.
In the case of fishes eXhibiting a sealed emim-bladder, the gases filling .102 that sac, reportedly, consists to between 69 and 87 percent of oxygen, with the rest of the gases consisting of nitrogen and carbon dioxide.
. In the case of infested swim-bladders, the mucous layer usually is dis- tinguished by a pronouncedly pink shade, which definitely must be attributed to the inflammatory reaction of the blood vessels. -13-
In cases where it fills the entire space of the swim-bladder, the
[coccidian] mass represents a rather considerable quantity of matter (viz. it weighs between 100 and 170 g). Its consistency is gelatinous, mucous Or viscous in character and can be compared best with cream. Along the walls of the swim-bladder, this mass is more compact. Its color varies between pure white and an intensively yellowish Shade of color. Immediately next to the mucous membrane there lies a greyish, transparent layer of mass, which con-
sists, to the greater part, of juvenile forms and, to the lesser one, of spores
Toward the interior of the swim-bladder, the mass becomes more opaque, in
correspondence with the abundant presence of fatty globules. The optical
difference between these two species of mass is brought about in a manner
similar to the optical difference existing between colostrum and milk.
The center part --a rather striking finding --consists of brown, rather
crumbly or friable masses, which under the microscope reVealed themselves to
consist of agglomerations of crystalline filaments and spores. I have found
brown, crumbly masses of that type in particular in the region of the red body.
In cases where the swim-bladder was completely engorged was matter, I
found that the horns, too, were filled with spores. The surface of the red
body is permeated by a certain kind of white stripes, which, too, consist of
tetraspores. Occasionally one finds that the cavity is not completely filled
with this particular mass e and the parasites form a tube-like coat lining
the Wall of the swim-bladder.
A certain case, which I have regarded as representing the early stage
of infestation, deserves more detailed discussion. In thàt specimen I found - 14 - that only the anterior end of the swim-bladder was coMpletely,filled with mass. The horns were empty..Behind the red body, I. found that only the ventral wall - of the swim-bladder was covered by a yellow, viscous layer of baltei., which layer dècreased in thickness toward the back and finally disappeared completely. The latter observation would indicate that the infection proceeds on from the anterior end of the swim-bladder, i.e. probably from the'red.body. From there, the infection spreads gradually toward the back over the sloping parts.
The spores produced in abundant masses, thus, fill first the anterior part of the swim-bladder.
Microscepic features • 103 .
If we view particles of the coccidian mass in a crush preparatioh under the microscope, we are immediately struck by the presence of thick-walled large spores representing the most characteristic feature seen. -
These large spores frequently are arranged in groups of four (tetraspores) and are then held together by a thin, translucent investing membrane. However, these spores may also be seen in the free state distributed over the entire field seen under the microscope, and this, in particular, in cases where the central parts of the swim-bladder are completely engorged. The capsules (or investing membranes) either are closed and then reveal in the interior a mass interspersed with highly refractive vesicles or are open. In the latter case, these capsùles reveal themselves to consist of two, entirely identical shell halves, which, by means of a longitudinal suture, are united in the manner of a pod (J. Muellerls naviculae). In the case of complet'ely intact spores we will find no trace of the latter feature. -15-
The two halves either gape openly only at one pole, while they are still united at the other pole, or have moved apart along the longitudinal axis •
and are united only at the middle by means of their protoplasmic substance
contents, or they exhibit a longitudinal line between themselves as the sign.
indicating the beginning of opening. In many instances, we will see these
shell halves distributed in a random manner through . the mass, in'which case .
they are empty.
In addition to the . spores, we see numerous oocysts exhibiting either
spherical sporoblasts or sporoblast just undergoing cleavage and segmentation.
The fully developed spores exhibit only a few, strongly refractive vesicles, •
while the younger stages show uniform, coarsè granulation.
Between these structures, we find varying quantities of both granules
and droplets of different sizes as well as masses, which can only be described
as detritus. Both the granules and the droplets are highly refractive in
character > and their oPtical behavior indicates that they consist of fatty
material (appearance of a light dot on high microscope setting; staining with
eudan black, scarlet red, and osmic acid). Many inclusions present in the
formed elements exhibit a similar behavior. We may, furthermore, find fre-
quently that the entire field seen under the microscope is floaded with pointed
needles--no doubt, representing the co-called margarine needles. The latter
may also occur as isolated features. Their occurrence in relatively large
numbers in the crumbly, greyish-brown masses of matter found in the center
of the swim-bladder contents has already been mentioned further above. We • 104
may furthermore observe characteristic cholesterol platelets, and this either
in isolated numbers or in relatively large quantities. -16-
Some parts consist almost entirely of spores; at other sites, however, we will find that the intermediate mass predominates and spores are present
only in sparse numbers.
Other developmental forms are found particularly frequently in the
transparent layer located next to the mucous membrane. The latter forms are
represented by coarsely granulated spheres,which are separated from the sur-
rounding matter by a light and relatively wide zone. The latter zone frequently
contains also highly refractive granules. A light zone of that type, by the way, also surrounds the oocysts and, occasionally, also the tetraspore groups.
It is absent from the central portions. The native preparation does not re-
veal any indications with respect to the nature of these stages. Information regarding these aspects can be obtained only in sectioned preparations or in emears.
Proper movements of the parasites—ehich otherwise are exhibited among
the coccidians by their sporozoites, merozoites and, in particular l . their
microgamets--could never be observed by me in fresh preparations, although
my investigations were carried out in part also at low temperatures in order
to imitate in that respect the conditions prevailing near the bottom of the
ocean. The latter finding must be regarded as a striking one, since the
viability of the sporozoans, required inconnection with the invasion of a new host, would not lead us to expect the occurrence of such a rapid dis-
continuation of the vital functions during the course of preservation on ice.
In this particular connection we must take into consideration the following
aspects. - 17 -
The host fishes are pulled up in the drag-nets from a depth of 80 to.
100 meters, i.e. in their natural environment, these fishes are exposed to a pressure of eight to nine atmospheres; at the surface of the water, that pressure is reduced by seven to eight atmospheres. The consequence of that difference in pressure ie reflected in a state of lethargy, which induces the captured fish to lie on deck of the trawler in a strikingly 'motionless marner, apart from a few reflex beats. The gases dissolved in the blood are released. In the transparent vessels of the abdominal organs, the blood can be seen inside in the form of disrupted columns. At sites where loose subdermal cell tissues are present, we find that the bases have invaded these regions lifting the skin up in a cushion-like manner, as may be seen, for instance, on the inner face of the gill covers. In.same fishes--like the .rosefishes, for instance--we find that a part of the expanded swim-bladder is forced out of the animal's mouth or even, along the sides, forced out through the gill slits and on further underneath the gill covers--a phenomenon, which, by the . 105 way, can be observed also in the case of the bottom fishes in aur inland lakes following capture ("Trommelsucht" - 'ballooning sickness 1 ).
It is self-evident that this sudden decrease in pressure must be of material importance also for the vital functions of the protozoan protoplasm.
Furtheluore, after gutting, the fishes are stored immediately in crushed ice and remain there until marketed. The gutted fishes are cooled in that ice to 0° 0, and ice needles are formed also in the protozoan colonies, which undergo thawing only when exposed to room temperature.
The latter events are also suited to àestroy the life of the protozoans. - 18 -
We must, furthermore, remember that we ascribe partiaularly great vitality only to the spores, which is ensured by the presence of the pro- tective capsule. In particular the forms possessing great motility (viz, the microgametes, sporozoites and merozoites) would, in the free state, exhibit a resistance not much greater than that of the tissue cells.
The ice.water crossing the swim-bladder wal by means of diffusion, finally, would have an injurious effect of the type produced by anisotonic solutions
on the red blood cells, which structures are particularly sensitive in the
case of fishes.
It, thus, becomes clear that the material available to me was not really
suited for the purposes of the highly important vital studies. Investigations
on these aspects would have, to be carried out either directly aborad the traw- lers or shortly after the catch had been landed in a harbor city not far from
the area where the fish were captured.
Stained strieras and sectioned preparations, on the other hand, have pro- vided findings regarding the relationships existing between the parasites and
the swim-bladder wall as well as regarding the different developmental stages
and their morphological structure--and this in a fashion better than native
preparations could. When viewing the stained material under the microscope, the worker is surprised . by.the abundance of features revealed in a single
preparation and, occasionally, even in a single viewing field. .Vie may find forms corresponding to all developmental stages of the known coccidians.
Only in the detailed study of the cytological features, there arise con-
siderable difficulties, due to which reason a number of questions must be left unanswered for the time being. As already mentioned further above, the - 19 - causes of these difficulties, in part, are associated with the deficiencies exhibited by the material available for study.
Sections through the wall of the swim-bladder reveal distinct stratifi- cation as was indicated already in the case of the native preparations. The .106. juvenile stages are located on the outer portions of the mass next to the mucous layer of theswim-bladder wall. The more advanced developmental forms and, above all, the spores are located further toward the interior of the mass, and they are released into the lumen of the swim-bladder. In accordance with that pattern, we must, to a certain degree, equate the coexistence of these forms with a sequence of forms, so that a description of the histological features provides already certain conclusions regarding the course of the developmental process.
The sectioned preparation (Plate 10, Figure 1) reveals the following pattern:
The coccidians are encountered first inside of the swim-bladder wall on the inner face of the vascular layer within the meshes of the mucous layer.
Proceeding from the outside toward the interior, we first ineet a layer of juvenile developmental stages showing varying degrees of maturation; with re- gard to the life cycle, these forms chiefly represent schizogony: Completely developed rosettes as well as merozoites distributed inside the tissue, with some of these merozoites still exhibiting club shape, while others are already more spherical or oval in shape. Occasionally, the merozoites are still clustered inside of nests filling elongated cavities, while other ones are found in the isolated state. Between the merozoites, on the one hand, and the mature schizonts, on the other one, we will find all imaginable transitional forms: Schizonts exhibiting only two nuclei as well as those exhibiting a multitude of nuclei. - 20 -
The nuclei are arranged in a circular manner around an imaginary center.
In other forms, we will find that the protoplasm has already undergone divi- sion and is concentrated around the individual nuclei.
Further categories of structures are represented by the microgametes and the macrogametes, respectively. The former can be readily recognized as such, in cases where very many spindle-shaped nuclei are arranged along the periphery of these bodies. Very striking in character, furthermore7 are the frequently rather numerous elongated mature microgametocytes with their pili- form microgametes. The macrogametes are distinguished by their uniform karyo- some, by their distinct karyolymph zone, by the presence of chromatin granules in the plasm, as well as by their size. These features distinguish them also from the schizonts, which-having reached a similar size-would exhibit already a great number of nuclei.
Further toward the interior of the swim-bladder, we then encounter the
oocysts: Oocysts exhibiting chromatin-deficient and apparently anuclear proto-
plasm, but nevertheless still uniform in character; furthermore, oocysts exhi-
biting the first indications of cleavage and fission; and, finally, oocysts having completed fission into four spherical sporoblasts. Next in development,
there are oocysts with four spores, with the latter exhibiting a doubly con-
tured capsule and two vacuolized sporozoites in the inside.
These oocysts are found in relatively thick layers in gaps in the connec-
tive tissue, which gaps resemble those filled by the early developmental stages.
Next to that layer-toward the interior of the swim-bladder--we find a layer,
which is deficient in formed elements and consists of detritus, small plaque- .107.
like structures and, as demonstrated by the results obtained in studies using - 21 - fresh material, of a virtual accumulation of fat droplets of varying sizes.
Still further toward the center of the svùim-bladder we then, among similar detritus, encounter again tetraspôres as well as individual spores (the latter being occasionally open) and also individual spore halves. The greatest part by far of the mass inside the swim-bladder consists of 'these particular com- ponents.
In sections through the swim-bladder wall with adhering protozoan bodies
as well as in the investigation of both crush and teased-out preparations from various.sites, I have occasionally missed the juvenile stages in the mucous membrane. That finding would indicate that not all parts of the wall are
equally 'prbductive,' i.e. that multiplication of the parasites is bound to
certain ''territories.' We must imagine that the merozoites settle on these
territories in order to proceed with multiplication by first initiating agamous reproduction. The spores produced in the course of sporogony beginning with
that event drop into the lumen of the swim-bladder and gradually fill it.
There can be little doubt that the thiclness of the mucous layer of the
swim-bladder wall increases due to expansion of the meshy spaces due to the
accumulation of protozoan bodies. It appears that the number of connective
tissue fibers also increases in the coursd of that accumulation, so that an.
increase in volume also takes place.
Schizogony
Despite the expenditure of much effort, my observations on the schizogony
of the species under consideration unfortunately are not as exhaustive in nature
as those made in the paradigmatic species. Above all, the cytological details - 22 - leave much to be desires, so that I have been unable to arrive at a complete
elucidation of that phase of the parasite' s. life cycle.
Both stained • smears and sectioned preparations show very frequently and in a striking manner the forms of completed schizogony, in which the merozoites are already formed. For that reason I will start my outline of these aspects with the desciption of these forms.
At this point in the life cycle we encounter arrangement of forms in rosettes of a type similar to that described in the case of E. schubergi
(Plate 10, Figure 6; Text Figures A and B).
In the case of the latter eimeriid coccidian We always find a residual
body, while that body is missing ad a rule in the case of the specieSunder
consideration, i.e. the entire substance of the schizont is utilized in the .
formation of the merozoites. The merozoites are only in exceptiànal cases
associated with a small, central and undifferentiated body.
The number of merozoites present usually is rather great. I have counted .108.
more than 40 merozoites in smears as well as in sectioned material. Frequently
there are several rosettes present. Since these rosettes are located in a
common cavity and are invested by a common membrane, we are permitted to assume
that they have been derived from a single schizont e which, in the course of
fission, gave rise to several centers.
The individual merozoites are club-shaped, i.e. one end gradually tapers
off, while the other one is rounded and forms the widest part of the merozoite.
The length of the merozoites usually amounts to 8 u, and their width, to 2.5 4.
In the distal parts, the protoplasm occasionally exhibits,a coarse honey-comb-
like structure. The nucleus consists of a halo located_more closely toward the • -23-
-,'
•,-,„ •
,
I
rY4
Figure À
\ 41 \, • '
(-•
Figure B
central end of the merozoite arranged within a rosette; -the halo surrounds
several chromatin particles. Occasionally, one may find that the chramatin
is united representing a uniform structure (karyosome?). It appears that this
consolidation of the chramatin precedes the event of fission taking place dur-
ing schizogeny. In sectioned preparations, the mature schizonts frequently
appear as round vesicles, inside of which the spherical merozoites are dis-
tributed at random with absence of any rosette-like arrangement (Plate 10 e
Figure 7); in these cases, we are, no doubt, dealing with tangential sections. - 24 -
A membrane can be clearly distinguished in almost all cases; between that membrane and the merozoites there exists a fissure of varying width.
In correspondence with the multiple rosette formation, the shape of the schizonts is an elongated oval one. Their size can be rather considerable; in the case illustrated in the text,- the length amounted to 53 g, and the width, to 20 g.
In a more advanced stage, we will find that the merozoites have been released from their unit and are distributed over gaps in the tissue.
In accordance with the life cycle pattern of the coccidians, subsequent development may proceed in three different directions: Formation of schizonts, .• of macrogametes, or of nicrogametocytes. Let us first give consideration to the formation of schizonts to the extent that our material provides findings in that regard.
The multiplication of nuclei (Plate 10, Figures 2-ro 4) begins already in very young individuals (measuring 9.5 g in length). Thamerozoite has been .109. transformed into an oval structure. The chramatin particles have unted to form a uniform interior body, which participates in fission. The karyosoum acquires an oval shape and takes up a transverse position. The two poles move apart, and the connecting piece becomes indistinct and disappears.
Mitotic figures eXhibiting an 'intermediate body' and a 'nucleolocentro- same'--as described by Schaudinn in the case of E. schubergi--could not be observed by me. I also saw neither a karyolymph cavity nor an outer nucleus.
The subsequent nuclear divisions appear to proceed in a very rapid nanner, since we, in fact, do find numerous multinuclear stages, but only a few forms exhibiting small numbers of nuclei. Apart from dumbbell forms, there are no transitional forms present. - 25 -
The-daughter nuclei are of approximately equal size. Already in the two-nucleus stage, we find vacuoles in the sectioned preparations, which are due to fat droplets. The schizont increases in size with the increasing number of nuclei present. The daughter nuclei aggregate in a circular zone.
We furthermore find stages, in which the daughter nuclei exhibit a dis- aggregation (Plate 10, Figure 5) similar to the one described by Reich in the case of E. stiedae and by Jollos in that of Adelea ovata. In these cases, the daughter nuclei then consist of chromatin granules, which are interconnected by a network of filaments.
It appears that apposition of chromatin elements takes place again later on in the course of the cycle, since we encounter already a more simplified situation in the nucleus of the merozoites. I do not understand the signifi- cance of this particular process. At this point we find that a distinct mem- brane has been differentiated on the surface of the schizont. Since schizogony takes place in the tissue meshes, that membrane can be nothing but a product of secretion of the parasites. The separation of the merozoites takes place in a manner similar to that seen in the case of E. schubergi, viz. a light halo àppears around each one of the daughter nuclei, which separates each nuc- leus together with a portion of protoplasm from the surrounding matter.
Merozoite formation can take place in successive steps, so that mature merozoites may already be present in one part of the schizont, while no changes can be distinguished in other parts of that structure.
Nuclear fission frequently takes place also in another manner. Relati- vely frequently we may encounter rod-shaped ('stab') daughter nuclei (Text
Figure C), which, in part, are arranged in a wheel-spoke-like manner around -26-
Figure O
a center. Apart from the 'stab' nuclei we also find Chromatin granules toward .110.-
the center. I have been unable to establish whether these granules are asso-
elated with subsequent divisions. It may be possible that they are involved
in a modification of merozoite formation, in which numerous small msrozoites
are formed, whose ends right from the start protrude at the surface in a hump-
like -manner--a feature not observed in the mode described first. The pronounced
staining of that structure is striking in character, and perhaps indicates -
the existence of a certain association . with microgametocyte formation. The stab
or rod-like chromation portions then would have to undergo disintegration in
the transverse direction and form the nuclei of the ricrogametes. On the other hand, it may be possible that we are dealing here with an aspect of Sexual
differentiation of the agametes, in analogy to the events taking place in the
case of Cyclospora caryolytica.
Even more interesting are certain patterns found in smears prepared in
Hamburg. The preparationsin question were derived from portions of the swim-
bladder content, which exhibited a peculiar reddish color. - 27 -
In these particular preparations I found numerous oocysts, which, in .
The latter were still located part, had already released their sporozaites. inside the oocystic investment, so that any confusion with merozoites would appear to have been excluded. It is striking that these sporozoites frequently exhibit multiple nuclei already when leaving the spore membrane. Numerous transitional forms lead to stages, which, no doubt, must be regarded as stages being part of schizogony. The more detailed situation is as follows:
The sporozoites of our form are distinguished by the fact that they possess a spherical interior body of chromatin--a karyosome--inside a nuclear cavity. Initially, they are elongated; later on e they become shorter and thick- set.
At this point in the cycle we see vigorous nuclear fission activity, i.e. we see repeatedly that small daughter nuclei separate along the sides of the structures under consideration. In individual cases, the two nuclei are con- nected by means of a centrodesmose (Plate 10, Figures 8 and 9). The subsequent events of nuclear multiplication involve mitotic processes, which cannot be elucidated. The schizont--the name we must assign to this particular stage-- grows and becomes more and more bulky. Its flask-shaped form e i.e. its pointed anterior end, however, reveals its derivation fram a sporozoite. The formation of merozoites takes place with separation of protoplasmic masses fitted around the daughter cells, so that the schizont finally is completely filled with • 111 numerous daughter nuclei surrounded by a light halo.
In the same preparation we furthermore find certain peculiar forms, which suggest that numerous degenerative processes are taking place. For instance, the sporozoites frequently show uniform thickening in the transverse direction - 28 -
Figure D
(Text Figure D). The chromatin is distributed in an irregular fashion; oc-
casionally, there are longitudinal rows of upright rods and granules in
evidence, so that one could think of the beginning of longitudinal fission.
the sporozoites are enorm- . Occasionally we may, however, find also that ously extended in the longitudinal direction and then are very thin (measuring
32 g in length, and 3.2 g in width, for instance). These sporozoites may either
exhibit only one nucleus or contain an entire number of nuclei, which are ar-
ranged in series and exhibit a distinct halo (Plate 10, Figure 10). The space
between the nuclei eXhibits coarse vacuoles. The proposition that merozoites
develop from structures of that type appears to me improbable, since there
really exists nô evidence to suggest that formation of sporozoites takes place
by means of transverse division. There also exist no illustrations that would
justify that assumption. For that reason we are forced to assume that the
sporozoites, in fact, possess the ability to reproduce in agamuus fashion in-
side a given swim-bladder, but that the process of division very frequently
- is not completed, and that the structures, in part, degenerate and die. It -2g- appears, by the way, that a large number of developmental forms dies. The great quantities of detritus present can hardly be regarded as anything but the sum total of the produçts of degeneration and parasite wastes. This pronounced tendency to undergo degeneration really cannot surprise us once we consider the extraordinarily vigorous reproductive activity of this parasite. Degene- rative processes and death of individuals have been observed, by the way, also in other coccidians in different stages of the'life cycle.
Microgamete formation
In this instance, we will again begin with the description of the mature microgametocyte. Microgametocytes are elongated oval to spherical structures exhibiting rather pronounced stainability; their protoplasm does not show any delicate vacuolization. Occasionally the body exhibits indentations, and the interior frequently contains even more markedly stainable particles.
The microgamets are very delicate, slightly wavy (spirochaete-like) structures. They resemble the microgametes of the malaria plasmodia far more than those of the other coccidians (Plate 10, Figures 14 and 15). They are . 112 rooted by means of condensed chromatin in the body of the microgametocyte.
At first the microgametes are tightly fitted against the surface of the micro-
-gamtocyte. With their parallel arrangement and their slightly curved course,
they produce the picture of a tuft of hair or of a distaff. Later on in de- velopment, they move away from the substrate and form an investment consisting
of a maze of the most delicate filaments. However, these structures may also
stretch out completely and then resemble very delicate, bizt rigid bristles, - 30 - which are arranged around the large residual body pointing vertically in all directions. Their length was found to be 17 VII i.e. they exceed the length of all other microgametes.
I have been unable to detect flagella in either stained smears and sec- , tioned preparations or native preparations.
In general, the microgametes exhibited no differentiation apart fram the basal granule; their stainability was only moderate. During the course of separation the basal granule appeared to remain in the residual body. At least,
I have been unable to establish the presence of chromatin accumulations on the free microgametes.
The apparent absence of nuclei, in any case, is highly worthy of note, but can be readily understood if we regard the entire microgamete as a structure, in which the nuclear substance is distributed in a diffused manner.*The simi- larity with the spirochaetes, thus, would hot be a purely external one.
I have estimated the number of microgametes to exceed one hundred in one preparations, i.e. that number is rather considerable.
Following their separation from the formative body, which subsequently remains in evidence as large residual body, the microgametes in the host tissue cannot always be readily distinguished from the connective-tissue fibrils, which, too, are delicate in character. The microgametes frequently are dis- tributed in a diffused manner over the entire tissue in the form of curved filaments. Occasionally, however, they are found in accumulations at well defined sites. I have been able to observe them in the latter state also in fresh preparations. - 31 -
In the stage ahead of the latter one we find a large number of spindle-
shaped nuclei, which are mainly arranged in a tangential plane. Transitional
forms reveal that these spindles gradually stretch in the longitudinal direction
and undergo transformation into microgametes, which, thus also on the basis
of these findings appear to consist of pure chromatin 'substance (Plate 10,
Figure 13).
I regard certain structures, in which numerous small daughter nuclei are
the periphery (Plate 10, Figure 12) as the stage ahead of the arranged along
latter stage of development. These daughter nuclei, however, are composed of
. small chromatin particles; these particular particles freqUently exhibit a
certain regularity with respect to arrangement to the extent that a small, . 113
relatively elongated particle is arranged in a tangential fashion e while a
second chromatin granule is visible--in the middle and toward the interior--
arranged in the vertical direction with respect to the latter particle. The
second one of these particlesi perhaps, would have to be regarded as reduction
body.
I regard the following forms as the initial stages of microgametocyte
formation:
In ovoid coccidians exhibiting a length of 13 g and a width of 10 g
(which forms e without doubt, have been derived from merozoites) there appears
pronounced stainability of the protoplasm in preparations stained after Heiden-
hain. The protoplasm furthermore contains numerous chromatin granules of vary-
ing sizes and dhapes. The karyosome, which is purely spherical in the early
forms and exhibits a relatively constant diameter of 2.5 4 1 has grown into a
strongly staining e transversely oval body eXhibiting a maximal diameter- of
5.5 g (Plate 10, Figure 11). -32-
Subsequent development--which was revealed in part in my preparations-- is accompanied by growth of the entire structure. The karyosome acquires an irregular appearance, expels numerous chromatin granules, and finally dis- integrates into small granules, which, initially, are distributed over the entire cell body and, later on in development, accumulate along the surface in. the form of groups of granules. These granules are observed also in star formations, as in the case of E. schubergi. Consolidation of the granule groups . and stretching of the daughter nuclei theh result in those forms, which we, further above, have met already as unquestionable microgametes.
. At first I regarded the forms just described as initial stages as arte- facts. The frequency of their appearance also at Sites where staining of the surrounding area is distinct and well differentiated then led me to assign a position to these forms in the life cycle of the parasite. Since the pro- duction of microgametocytes is both controlled and initiated by. a considerable increase in nuclear substance--in particular, of chramatin, it would appear that these forms fit only the position autlined above. The pronounced parti- cipation of the karyosome in chromatin multiplication is striking in character.
In the course of that process, the karyosome increases in size to an extraor- dinary extent; but it appears that it, at the saine tima, discharges chromatin in the liquid state into the surrounding area, leading to the pronounced stain- ability of the protoplasm. The karyosome disintegrates only after it has reached a certain size. It is not certain whether the stage presented in Text Figure C may be considered in this connection.
In comparison with other coccidial species I have found significant differences; we know, for instance, that the chromatin of the outer nucleus - 33 -
plays a main role in the case of E. schubergi, while the karyosome ocdùpies .114.
the forefront in the events in that of Cyclosrora caryolytica, but does not
grow and instead undergoes successive divisions. Our present form corresponds most readily to E. stiedae, when we compare it with Reich's'results, and this,
in particular, with regard:to the illustrations.
Macrogametes
The macrogametes represent those forms, which, in addition to the tetra-
spàres, are the most striking structures seen in the native preparations. •
Macrogametes are mostly large, strongly granulated spheres, which, due to their
strong granulation, largely hide their nucleus in the unstained state. Only . -
occasionally will we find that a more translucent spot indicates the position
of the nucleus (Plate 10, Figures 27 to 29; Text Figiare E). This particular granulation is produced, on the one hand, by large t highly refractive droplets,
which prove themselves to consist of fat, and, on the other one, by many small
granules also refractive, but this to a lesser extent, measuring 0.8 4 in dia-
meter, which must be compared to the reserve-substance-granules or plastic
granules of the other coccidial species. Like those of the other species, these
particular granules stain yellow with iodine, but they are considerably smaller.
The macrogametes are surrounded by a homogenous zone consisting of an apparently
gelatinous substance—i.e. they exhibit a certain resistance when the cover-
glass is shifted over the preparation with application of some pressure. That
zone contains relatively large droplets in suspension; toward the outside it
is limited by a distinct membrane. -34-
-:1r,y, - _: .- . , b .t,r• ^ ^C^('^.i^..è.^,'`•7c 8^ii^•.-^ -, i^ F'd+• ^^.: . ^ R., w ^; ^^ ).° i•4 _^^ " cij E. ÿ":r9 • . ;_,. -' r,• ;é,.f;e^ '^ r`'
" ^ c^Y;,.^^.`^^' ..ç,• •.t-^ -. ,. ? da, _.•. Lr 'h^^• w . r :4ay d .9 •^a.`'^K. .^^. ( ^•'y'. , . r^v B •: . ^'•^^ •' b^ u ,.6 ,.^. q4^ t:s,'" 7 . ^^• _ „ F,.,
^ ., c•a \ ^
/•_,,
Figure E
The diameter of the macrogametesq on average, amounts to 20 y, and, when we include the zone, to 26 ^1.
The macrogametes acquire their spherical shape only during the course of development from the more elongated juvenile forms.
In the stained state, the nucleus of the macrogametes exhibits the pro- perties of a karyosomal nucleus. In preparations stained after Heidenhain, the
chromatin appears to be. stacked almost completely inside of a homogenous, droplet-shaped, well . defined and spherical interior body measuring 2 4 in . 115
diameter. The nuclear-sap zone usually is distinctly visible, being set off
against the protoplasm by a wide and sharply defined border. An outer nucleus
cannot be demonstrated. In preparations stained with hematoxylin and safranine, -35-
I was occasionally able to perceive a more darkly stained granule (centriole?) in the area of the karyosome (Plate 10, Figure 29). It is questionable whether the pale zone surrounding the most intensively stained interior body shown on
Plate 10, Figure 18, should be regarded as being associated with the karyosome
(plastin). This particular karyosome, thus, would correspond to the lowest' form (or the most simple form) of the group of karyosomal nuclei established by Hartmann in his Classification system, i.e. to the form found in amebae.
Thé centriole l to be sure, cannot be demonstrated in a conclusive manner.
The nuclear-sap zone usually does not exhibit any particular structure, and only occasionally will we find filaments to run from the karyosame toward the protoplasm. Occasionally there are sharply defined chromatin granules visible in the region of that zone. One of these granules appeared to meto be highly constant in appearance. I have been unable to determine whether that granule possesses any particular significance. Occasionally, the nuclear-sap
zone gives off lobe-like extensions or processes into the surrounding area, • i.e. it gives an ameba-like impression.
The protoplasm does not exhibit an alveolar structure even in stained
preparations. The granules, packed together rather tightly, fill the entire
cell body as far as it is not occupied by fat droplets. In preparations treated with either volatile oils or ether, these fat droplets appeared as vacuoles.
In dry smears we see, following staining after Giemsa, a regular, very delicate
and pale netNork, whose round, unstained meshes are formed by the plastic
granules. The latter, thus, appear to be held together by a plasmatic cement-
ing substance. Further below we shall discuss the presence of fat in some detail. - 36 -
A distinct cellular membrane along the periphery of the cytoplasm cannot be
demonstrated. In smears vie rather frequently see—+no doubt, due to the pressure
applied to smear preparations--that plastic granules haire emerged from the
protoplasm of the macrogametes without, however, any tears being visible in
- a covering membrane.
In addition to theSe two forms of inclusions, there occur numerous chro-
matin granules in the cytoplasm. These granules are almost always very delicate
and they are particularly abundant in the area surrounding the nucleus (Plate 10,
Figure 11). However, they are never as numerous as in the case of E. stiedae.
Occasionally one sees in the sectioned preparations . macrogametes inside
of star-shaped, finely granulated structures, which resemble connective-tissue .116
cells (Plate 10, Figure .16). However, inclusion in the hyaline substance des-
cribed above is far more frequently seen; this substance then surrounds the
macrogamete in a coat-like manner. This coat, by the way, is found hot only
in the case of Macrogametes, but also in that of other developmental stages
(in that of schizonts, for instance). The fact that this coat is visible also •
in fresh preparations--and in them, indeed, in a particularly perfect manner--
excludes the suggestion that we may be dealing here with an artefact due to
shrinking in the course of fixation of the material. I regard that coat as a
structure produced by the parasite and imagine that the latter organism excretes
a liquid, which congeals into a membrane at the interface in contact with the
host tissue fluid. The outer limiting membrane or coat appears entirely smooth -
only in the case of fresh preparations; it is wrinkled in that of fixed ma-
terial. In preparations obtained from material of the latter type one frequently
sees strands running between the limiting membrane and the macrogamete surface; . S^.
-37-
Figure F
these strands are interconnected. They probably represent artefacts created in the course of preservation. A coarsely alveolar structure can also be demon-
strated occasionally (Text Figure F). In material stained with eosint that
zone is red; in material stained with iron hematoxylin, it is grey. It appears obvious to ascribe an important role in the metabolism of this coccidian to the intermediate ;ayer created by means of secretion produced by the parasite.
The occurrence of droplets in this particular layer is highly worthy of note. These droplets behave toward nuclear stains like chromatin. They stain - 38 - particularly intensively with hemalum stain (Plate 10, Figures 17 and 18).
One would therefore like to regard them as structures ejected by the nucleus.
Schaudinn has described emergence of chramatin in droplet form from the karyo - some also in the case of E. schubergi, and this in the course of maturation.
The same process, however, can hardly be involved in the present case, since these droplets, on the one hand, are in part larger than the karyosone itself and, on the other one, appear along the periphery at a time where the karyo
some apparently has lost none of its substance. It is, thus, not yet clear to me where this particular substance is actually formed.
Maturation and fertilization of the macrogametes . 131
The development of the macrogamete into the oocyst requires profound
changes, which are governed by maturation and fertilization. The former process is characterized by a reduction in and ejection of, chromatin. In the case of •
E. schubergi, we find that the entire karyosame is ejected and leaves the coc-
cidian body in the form of a droplet. In the case of Cyclospora caryolytica, we find that the karyosome undergoes fission, as is also the case, in a similar manner, in Adelea ovate.; in the case of Adelea zonula, we find that the chro- matin leaves the karyosomj in the dissolved state.
Ejection of chramatin substance takes place also in the form presently under consideration; in this forai, however, ejection of that substance goes
so far that the macrogametes ready to undergo division into sporoblasts, i.e.
structures which must already be regarded as oocysts, exhibit neither chromatin
Hartmann, by the way, has regarded the structure termed karyosome by Moroff as representing a simple nucleolus, and the 'nucleolocentrosame, as karyosome. - 39 - particles worth mentioning nor any structure resembling a nucleus. .
As in the case of Cyclospora caryolytica, we find also in the present form that the karyosome dissolves into particles. However, the separation of the latter takes place by means of different modes (Plate 10, Figures 19 to
23). We may observe the following patterns:
(a)The caryosame loses its uniform enclosing, which is replaced by a bud-like protrusion above the surface, which separates by means of segmentation.
(b) The karyosome is surrounded by an entire zone of chromatin granules.
(c) The karyosame has undergone division into two portions, one of which has undergone further dissolution into granules.
(d)A part of the chromatin elements has undergone apposition to form a transverse plate.
(e) The chromatin granules have arranged themselves in two rows in a manner similar to . that seen in the case of chromosomes. In almost all forms of that type, we will find that the nuclear-sap cavity is elongated in the longitudinal direction, with its autline becaming indistinct. It is not clear which ones of the elements finally remain present. At least the idiochromatin must remain in existence is some form (centriole).
Full elucidation also could not be obtained with respect to the process of fertilizatiOn. In fresh preparations, I was struck by macrogametes, in which the granulated contents terminate in a teat-like, granule-deficient process, which may extend through the hyaline zone up to the surface. The • 118 suggestion arises to regard that formation as a fertilization cone (Plate 10,
Figure 28). In fixed and stained preparations one frequeritly sees patterns resembling those illustrated by Reich and by Metzner, respectively, in the - 40 - case of E. stiedae, viz.a pseudopodium-like elongation of the nucleus toward the surface, and the presence of chromatin granules inside of this tube-like structure. I have been unable to observe either the entry of microgametes or the presence of.male nuclear elements inside of macrogametes. It is possible that the delicate structure of the microgametes makes investigation diffi- cult. In preserved and stained preparations one can occasionally see forms, in which a spindle-shaped differentiated structure stretches laterally through the substance of the macrogamete, which structure is set off against the granulated surrounding area (nuclear spindle?). On the other hand, there occur occasionally forms, whose surface exhibits an indentation at some site, i.e. umbilicated forms. In one case I saw a thin filament-possibly a microgamete- pointing toward that site from the surrounding area.
Spore formation sporogoE Spore formation or sporogony can be readily observed in its rough out-
lines in native preparations, since approriate specimens of the different
developmental stages can be found in abundance at the proper sites in the
peripheral portions of the coccidian mass.
Disintegration into the four sporoblasts is evidenced by the appearance
of semi-spherical protrusions on the surface [of the oocyst] ; in the interior,
pale stripes separate the densely granulated areas. As a rule, a residual body
cannot by observed; we rather find that the entire material is utilized, and,
at best, we will find that individual granules are excluded from the divisions
along the surface at the points where the sporoblasts are in mutual contact.
Only once did I observe a fifth, small protoplasmic globule in addition to - 41 - to the four sporoblasts (Plate 10, Figure 30). This particular inconstancy would deprive the formation of the residual body of its taxonomie significance.
The sporoblasts, thus, possess even faces at the sites of contact and turn a pointed end toward the center. •
The pyramidal stage of the sporoblasts described by Metzner in the case of E. stiedae, in which hyaline tips are pointed outward, could . be observed in the present species just as little as Schneiderts stieda body.
In the subsequent course of development, the sporoblasts acquires a purely spherical shape. Sporoblasts at that point exhibit a diameter of 9.6 g (Text .119.
Figure G). The sporoblasts then separate from one another and lie freely inside . the hyaline investment. The latter finding also permits us to conclude that the macrogametes, in fact, do not possess an investment of their own. In the stage presently under consideration we are able, already in the native pre- paration, to see a distinct roundish area in the interior of the granulated spheres, which, no doubt, corresponds to the nucleus. Chromatin elements of • the nucleus, i.e. indistinct nuclei with a very small karyosome (Plate 10,
Figure 30), can be seen at this point dnside of the sporoblasts also in stained preparations. Maturation does nnt in all cases lead tà the formation of four sporoblasts. Occasionally, there are only two of-them present, which either are of equal size or the one is very large and the other one is much smaller.
On the whole, it appears that sporoblasts of the latter type are destined for destruction, since I found that the spores always numbered four at the site of release.
The transformation of sporoblasts into spores is evidenced in the native preparation by increased translucency of the contents and the formation of a -42-
//-*
•
Figure G Figure H capsule. The uniform granulation of the sporoblast disappears. Instead there appears a small number of strongly refractive spheres of varying sizes, which consist of fat (Text Figure H). I am unable to decide whether these fat droplets . correspond to the eight large, strongly refractive droplets inside the oocyst described in the case of E. schubergi. We can hardly believe that a worker as qualified as Schaudinn would have missed their fatty nature. Along the circum- ference, there arises a doubly contured, distinct thick membrane, viz. the spore capsule. The latter finding already permits the conclusion that the granules are used in the construction of the capsule. Following staining .120. after Heidenhain, the strongly stained particles appear arranged along the periphery in a manner suggesting that they intend to spread out to form the capsule. The pronounced stainability of the formed capsule with iron hematoxylin is worthy of note; that stain makes the spores appear as black bodies, per- mitting no insight into their contents. - 43 -
Transformation into spores does not always take place simultaneously
in all sporoblasts. Occasionally we will find that one or two sporoblasts are
still granulated and without capsule, while the other ones already possess
spore nature. Initially the spores are still spherical, as àre the sporoblasts;
later on in development, and at the latest on formation of the sporozoites,
the spores adopt the shape of an ellipsoid of rotation. At that point in de-
velopment-they then exhibit a length of 11 to 12.5 g and a width of 7.5 to 9.5 u.
Initially they are still surrounded by a :frail membrane—the oocyst investment-- which is so thin that it is tightly fitted against the outlines of the spores'
and can be distinguished only with difficulty. This particular - membrane dis-
appears eventually, and the spores then rest freely. Forms of that type.are
found chiefly in the center of the eNim-bladder Mass.
The spores are resting in different ways inside of the membrane. In one
case, the spores may be arranged in the form of a double Cross, and in the
other one, they may be arranged in the form of a triangle, with their tips
being in contact. The fourth spore is then resting on top of the other three
(Plate 10, Figure 25).
Transformation of the spore contents into the two sporozoites could not
be followed under the microscope, since,the stains penetrate that structure
with difficulty only. It appears, however, that this particular transformation
takes place very rapidly, since we encounter sporoblasts and spores containing
formed sporozoites side by side. In sectioned preparations we may see the
development of a longitudinal fissure, which separates the protoplasm in the
interior in the longitudinal direction, leaving both ends, still in contact.
I have also in this case never been able to observe a residual body. The - 44 - sporozoites measure 16 u in length and 4 u in width, i.e. they are consider- ably larger than the spore. Both ends of the sporozoite taper off, but the • anterior end is more slender and pointed, while the posterior end is thickset and blunt. In order to find space inside the spore, the anterior portion of the sporozoite is bent..The sporozoites may be intercalated in the following ways: The posterior end of the one sporozoite is located in the bend of the other one, or the one sporozoite is located in the longitudinal axis of the spore, while the other one is partially coiled around the former in the trans- • verse axis of the spore (Plate 10 5 Figures 26a and 26b). The residual body is missing in most coccidian parasites of fish according to the findings presented . by various authors. That absence has been pointed out as representing the cardinal characteristic of the genus Goussia. The fact that the residUal body is not missing in all coccidian parasites of fish could be confirmed by me in the course of a second scanning under the microscope of preparations of • 121 .
E. subepithelialis.
The sporozoites stretch after leaving the spore membrane. Following that event we are able to distinguish the structural relationships inside the sporo- zoites in a satisfactory' manner in stained preparations. Incidentally, the sporozoites exhibit a nuclear-sap cavity in their middle, with a karyosome located in the center of that cavity. Occasionally, we may find an oval struc- ture surrounded by a translucent halo in the center, with the karyosume attached to its anterior end. My sketch (Text Figure J) resembles the one Jonas has presented in the case of male schizonts of Adelea ovata (his Figure 21). The entire structure probably must be regarded as a karyosome, which contains chromatin only in its anterior part, while the rest of the body consists of. - 45 -
Figure J
plastin. A striking finding is the one showing the presence ofsnultiple vacu- oles in the protoplasm; these vacuoles occupy the entire width of the sporo- zoite, and, indeed, may even push the limiting outline of that body outward.
Very frequently there are one to two vacuoles present in front of the nucleus and one behind the nucleus. The vacuoles contain fat. The possession of a karyosome represents a peculiarity of these particular sporozoites. Schaudinn has specifically stressed the absence of karyosomes from sporozoites of E. schubergi and Çyclospora car,yolytica, respectively. The presence of a karyo- some in the sporozoite of the species under consideration, perhaps, is asso- ciated with a certain property mentioned further above, viz. the performance of asexual reproduction in the same swim-bladder immediately after release from the spore.
The spores open along a longitudinal suture in the manner of pods. This longitudinal suture initially is visible only in the form of a fissure, which runs along the line connecting the two poles. Although that suture is not prominent in the intact spore, we may assume that it is already preformed in the capsule substance. - 46 -
On opening of the suture, the sporozoites leave the gaping spore halves and move outside. They do that occasionally at a time when the oocyst membrane is still intact, and they then accumulat in a fissure between that membrane
and the spores (Plate 10, Figure 25). Cases of that type are of importance be- cause there can then be no doubt that we are in fact dealing with sporozoites and not with merozoites (apart from the fact that these two bodies also differ in size). The 'hatching' of the sporozoites probably takes place by active
.means, since otherwise no driving force is present, which could transport the
sporozoites outside away from their resting site-at least in the case of spores
still located inside the oocyst membrane.
In the latter case, the sporozoites then must furthermore pass through
the oocyst membrane. However, considering its frailty, passage through that' . 122
membrane would represent no difficulty.
The factors inducing the spore capsule to open inside the swim-bladder
are as yet not clear, since the effects of the digestive juices, which bring
about that event in the other coccidian parasites, are not in force in the
present case. This opening of the spore capsule inside the swim-bladder repre-
sents a noteworthy peculiarity of this species. We all lnZow how difficult it
is to induce opening of coccidian and myxosporidian spores by artificial means:
Measurements of the different developmental stages
Merozoite: 8 a long, 2.5 p, wide Mature schizont: 53 it long, 20 4 wide Juvenile schizont: 9.5 p, long Mature microgametocyte: 22.5 4 long, 6W, wide Microgamete: 17 ^4 long Mature macrogamete: 204 in diam.; 26 ^t in diam. incl. the hyaline zone Sporoblast: 9.6 ^.t in diameter Spore: 12 p, long, 9tG wide Sporozoite: 16 ^t long, 4 ► h wide. -47-
Spore number
• In order to obtain an idea regarding the number of spores present in a given swim-bladder, I proceeded with counts carried out in a manner similar to that employed in counting blood cells in a Thoma chamber (manufactured by
Zeiss). I took 0.1 g of fresh swim-bladder content and placed it onto a slide.
I put a coverslip on top of that mass making certain that the mass did not go beyond the edges of that coverslip. With the aid of fine lines drawn with ink
I divided the coverslip into squares, and I also drew squares with ink on the glass insert of. the eyepiece micrometer. The latter squares wàre then calibrated with the aid of the mechanical stage, so that I, in the end, arrived at the surface value of one square of the eyepiece micrometer.
Next I carried out counts on material obtained from different sites of the preparation. For 0.1 g of mass I obtained a number of approximately 250,000 , tetraspores, i.e. one million spores. If we take the quanity of mass present in a fully filled gwim-bladder to amount to about 100 g, we arrive at a number of one billion spores.
Even if some parts within that mass may be less rich in spores and that number, thus, may represent an estimate somewhat too high, we nevertheless arrive at a rough ides of the enormous quanity of spores present in a single . 123 swim-bladder. The vigorous multiplication observed by me at all sites studied, of course, permitted me to expect that result of my counts.
We do not yet know hos intensive the parasitic infection is as a rule, nor do we know whether infection is multiple in character. In any case, it is clear, however, that the reproductive ability of this speCies is impressive. - 48 -
Chemical composition of the swim-bladder content
. We have mentioned already further above that the occurrence of such a , large number of protozoan bodies in one 'container' offers a unique occasion for chemical analysis. I wish to presently - briefly the results Professor Panzer has obtained in his laborious investigations.
The first investigation was made using 516 g of material, which had been obtained from five pollocks; individual swim-bladders contained 170, 160 or
100 g of mass.
The chemical analysis yielded the following values, in percent:
Water 89.9e Solid substances -14.07% • Organic substances 12.87% . Nitrogen 1.25% Inorganic substances
The second investigation was made on the swim-bladder contents from two pollocks and four haddocks. • Above all, the large amount of lipids was striking; in fact, the second investigation of the ether extract yielded the following numerical values:
Fats: 3.5e of the coccidial mass Non-saponifiable residue: 35.7e of the fats Cholesterol-free fraction: 2.87% of the fats Esterified cholesterol: 26.30% of the fats Other higher alcohols: 6.60% of the fats
These numerical data also reveal that a large percentage of the lipids is represented by cholesterol esters. In addition to the latter, there is also a small amount of free cholesterol present. The fats -are furthermore
distinguished by a particular abundance of free acids. That abundance, right -49- from the start, represents a striking difference compared to the body fat . 124 of the host fish. Panzer, however, has carriedout still other comparisons.
In that connection, he used the fats obtained from the livers of various
cod species, viz. cod-liver oil, as well as the muscular fats of haddock.
Detailed data on the former are available in the pertinent literature; the numerical data reqùired in the case of the latter for the purpose of compari-
son were determined.
The following data then were available for comparison:
Cholesterol- Muscular fat free from - Cod-liver oil coccidial fats haddock.
(a) The fats Acid number 55.14 70.59 Saponification number 223.68 190.13 175 -.189 Iodine number 118.3 102.8 139.6 - 168.4 - - - .. ------
(b) The fatty acids Molecular weight 234.4 287.8 - 292.5 Iodine number 116.2 164.9 - 170.1
This compilation reveals the existence of significant differences bet-
ween the composition of the fatty acids and the glycerides of the coccidial
fats, on the one hand, and the composition of the fats of the host animals,
on the other one.
Among the other data obtained by Panzer in his investigations, I wish
to draw attention to the following ones: - 50 -
The spore capsules dissolved in 2% potassium hydroxide solution apart from a small, colorless residue. That residue consisted without exception of colorless granules. The capsular substance must be assigned to the group of albuminoids, and this to those particular keratin- and elastin-like substances, which participate in the formation of egg shells of some higher animals. The complete absence of sùlfur represents a striking finding.
The sporozoites contain albumoses. Conjugated nucleoproteins as well as their products of cleavage (viz. nucleic acid, xanthine bases, reduced sugars and histones) could be demonstrated neither in the spores nor in the whole .
The albumoses detected apparently represent material submitted to analysis. only products of degradation of the protoplasm. The nature of the undegraded • proteinaceous substances is completely unclear to me. I assume that coaguable proteins do not have to be considered in this connection.
The coccidian mass furthermore contained: O • 125
(1) a phosphorus-free glycoprotein, and
(2) a gelatinous substance. •
"On the whole the investigations described have demonstrated that the chemistry of these one-celled organisme differs in some aspects to a signifi- cant degree from that of the cells of animals higher on the phyletic scale.
This finding then would represent the most recent warning against certain trends to apply in an uncritical manner the findings obtained in organisms low on the phyletic scale to the cells of higher animals; this trend has became more pronounced at the present time, where experimentation using free- living protozoans (infusoria) is the vogueè" -51-
The investigations carried out by Panzer have provided an overall picture of the chemical composition of the swim-bladder contents of infested fish. A separation of certain form elements for the purposes of chemical analysis was possible only with respect to the spores. It is obvious -that these data would gain in value to a considerable extent, if we are able to localize the individual, i.e. chemically different components in both the different deve- lopmental stages and the different parts of the protozoan body. In this con- nection we must furthermore take into consideration the fact that the mass, apart from the protozoan bodies, contains varying quantities of crystals, droplets and unformed elements (detritus). Localization of this type is possible only with the aid of microscôpic means. In the first instance, I made an attempt to elucidate the distribution of the fats.
Hitherto fats have only rarely been found in the body of parasitic pro- tozoans. Schaudinn, in fact, has described a fatty degeneration of the in- testinal epithelial cells infested by coccidians, but he denied any production of fats as well as--and this in particular--the presence of fats in the proto- zoan body.
However, Siedlecki has reported the presence of fats in Caryotropha mes- nili. In the case of that coccidian, there arises a dense protoplasmic strand between the nucleus of the parasite and the nucleus of the host cell, in which strand takes place formation of fatty granules, which must be regarded as re- serve substances.
Thélohan, furthermore, has observed both fats and fat-like substances in myxosporidians. •
According to Puerth, only a fraction of the long-chained fatty acids is present in the form of fats (glycerides); a far greater portion of these acids is present in the form of P] calcium soaps [tt]• -52-
V'rith regard to the coccidians, we find that a certain communication by
Moroff is worthy of note. In the case of the macrogaznetocytes of Adelea zonula
that author has describec.i theformation_of peculiar, highly lustrous spherical
structures (reserve substances), which stained black with osmic acid, "a find- .126
ing which would i.ndicate their fat-like character; they measure up to 2 p in
diameter". Moroff has provided further data on these aspects.in his large
Aggregata paper (page 9):
"Fat-like inclusions in the protoplasm are stained strongly black with
Flemming's mixture, and the study of the nuclear details is greatly impeded
due to that black staining. However, is section treated in. that way are placed
for about 24 hours into oil of turpentine, we find that all bodies stained
black by osmic acid [contained in Flemming's mixture] undergo complete dis-
solution, and we obtain then extremely delicate protoplasmic structures."
Finally, Borgert has found fatty inclusions in radiolarians; however, he iden-
tified these inclusions as products-of degeneration.
The usual smears and sectioned preparations are not suited for investi-
gations regarding the presence of fats, since they are treated routinely with
absolute alcohol, ether and xylene, i.e. with fat-dissolving or lipolytic
reagents. These reagents dissolve fats, and there appear vacuoles in their
places.
For that reason, I have either added the appropriate stains to the fresh
preparations or avoided using the afore-mentioned reagents in the processing
of the fixed smears.
Osmic acid, sudan black III and scarlet red are suited as fat stains.
Many workers use hematoxylin or safranine to counterstain the nuclear substances. - 53 -
In preparations fixed with Flemming's mixture we find that the black osmium precipitates are preserved also when using xylene. We are thus able to obtain both excellent results with regard to nuclear staining and fat inclusions also in sections embedded in paraffin using safranine as nuclear col:Interstain.
In the description of the morphological aspects presented further above,
I have repeatedly drawn attention to the presence of fats, and I new wish to summarize these particular findings. Fats are found free in large quantities in the swim-bladder contents in the form of droplets of varying sizes. Occa- sionally the fat droplets predominate over the spores; fats are furthermore - present in the form of fatty-acid needles, and this also in large quantities.
I:furthermore draw attention to the cholesterol platelets occasionally present in large numbers.
Fats, finally, are found also in the protozoan bodies (Plate 10, Figu- res 27 to 31). The schizonts contain fats in relatively large droplets next to the daughter karyosomes. I have been unable to establish whether each mero- zoite is provided with fat on fission. Fats are furthermore found already in relatively young macrogametes in the.form of large droplets, so that a large part of the protoplasm occasionally appears to be filled with that matter
(Plate 10, Figure 27). In adult macrogametes, the fatty inclusions frequently .127 occupy the background. Bather frequently we will find fat distributed over the entire protoplasm in the form of dust-like particles. Each microdroplet is then surrounded by a halo. Haloes of that type, by the way, may be observed also in the case of large droplets. Fat droplets are located not only in the protoplasm, but also in the nuclear-sap cairity, where they are fitted tightly against the karyosome. Fatty inclusions, however, do not occilr inside of the - 54 - latter structure. The sporoblasts, too, exhibit fatty inclusions. Such in- clusiOns are very distinct 'in the sporozoites, and this in vacuoles both'in front of, and behind, the nucleus. It appears that conflux of droplets takes place--a process promoted by the disappearance of the plastic granules. Fre- quently I was able to observe fat droplets also outside of the sporozoites, which droplets thus had not been included in the process of fission. They would then have to be equated with a residual body.
Staining of the sporozoites succeed better once the spore capsule gapes slightly. In these cases, of course, we cannot readily establish whether a given component may have left the spore -space or elements may have penetrated into that space from the outside.
In the examination of native preparations using a polarizing microscope, we find, with polarizer and analyser crossed, that only the cholesterol plate- ' lets and the spore capsules appear bright, while the lipid inclusions and the free fat droplets remain dark. Considering the large quantity of cholesterol esters demonstrated in the chemical analysis, that finding is rather striking, since cholesterol esters, in fact, may be birefringent.
Considering the sparse data available in the pertinent literature on the occurrence of fats in the bodies of parasitic protozoans, the regular finding of relatively large quantities of fats represents a fairly striking phenomenon.
Degenerative processes-which would otherwise have to be considered in the first instance--probably can be excluded considering both the regularity of the occurrence of fats in the different developmental stages and the intact state of the nucleus. A further finding speaking against the latter suggestion is the one showing that juvenile bacrogametes, in particular l 'contain considerable -55- quantities of fatty inclusions. Degenerative processes would be more readily expected in adult individuals. Furthermore, it is difficult to imagine repro- ductive activities of such importance in degenerating, i.e. pathological forms.
We now are faced with the following questions: At which site(s) is the fat produced, and what is its function? The resolution of that question l -in .128. part, is associated with the same difficulties existing in connection with both the elucidation of the absorption of fats by the vertebrate body and the question of the incorporation of the absorbed fats into the cells.
The body of codS is extraordinarily rich in fats, and the deposits of fats are abundant to such-a degree in some organs-and, above ail, in the liver
--that the latter organ consists to the larger part by far of fats, and we have difficulties understanding how the liver cells are able to function under these conditions. Already a short time after death, the liver fats—called liver-oil in the case of the gadid species--emerge by themselves from the organ in the form of an oily liquid. I have even observed entire toil cystst in the liver of these fish. I have also been able to demonstrate the presence of fat droplets in the meshes of the mucous layer in teased-out preparations. On the basis of the latter findings, we really cannot be surprised to find that the parasites, which depend on the body fluids of the host animal, take up also fats.
In this connection we must take the following aspects into consideration:
The coccidians obtain their nutrients by means of osmosis. However, fats are not accessible to the type of osmosis involved in nutrient uptake. There is no evidence indicating that the coccidihns, in analogy- to the amebae, take up fat droplets into their body by means of engulfment. The chemical analyses - 56 - mentioned further above, furthermore, have demonstrated the existence of differences between the properties of the fat of the coccidian mass and those of the fat of the host animal.
We could, in fact, imagine that the host fats would find entry into the coccidian body in the form of very sma11 droplets, undergo certain modifica- tions in that body, and then flow together to form larger droplets. However, it appears more probable that the fats of the host tissue are, first, degraded on the surface of the protozoan with active involvement of that protozoan and, then, split up into diffusible components, which, in turn, are used in the.synthesis of lipids inside the coccidi7..n body, which finally flow together to form droplets. The process in question, thus, would involve both degradation and synthesis in a manner similar to that demonstrated by Abderhalden in the case of proteins in the intestinal tract of mammals.
Since we, as a rule, find no fat droplets in the hyaline zone of the macrogametes, there arises the suggestion that the process of cleavage is located on the outer limiting membrane. Finally, we must also take into con- sideration the possibility that not the fats of the host body, but rather pro- teins and/or carbohydrates taken up by the coccidian represent the substrate' for the synthesis of lipids by this protozoan speçies.
The second question is the one after the physiological importance of the fats for the coccidian. In the form of droplets demonstrable by microchemical means, the fats probably can be regarded only as reserve substances. As a formative element, fats could be of significance for the lipid membrane, which, .121 at the present time, is generally believed to exist as ad organelle having very important functions also at sites on the cellular surface where we can- not demonstrate its presence by microscopic means. - 57 -
Other elements could also be constructed with use of that material-- elements like plastic granules, for instance. UtilizationHof that type, to - be sure, would require a process of transformation involving enzymic activi- ties of the protozoan cell. In that regard, I felt that the' halo around the fat droplets--which halo produces a structure similar to a food vacuole-- would be of importance. Finally, one could imagine that the fats serve as a source of energy. In this connection we do not have to consider particular movements of the entire coccidian body, but rather mechanical processes taking place in the interior of the cell, like the transportation of nuclear chromatin elements during the course of division, for instance.
Among the other substances demonstrated by chemical means to be present in the eoccidian mass, we must stress the detection of keratin-like substances in the spore capsule. Keratin has been reported to be a component also of
*other investing membranes of protozoans; hitherto, however, that fibrous protein had not yet been detected by chemical means in the case of coccidians.
The conjugated glycoprotein, as a mucoid, indicates a mucous substance.
The consistency of the mass under consideration would readily agree with the demonstration of such a substance. The'assumption that this substance wOuld be present also in the hyaline zone of the different developmental stages probably is justified.
The presence of animal gelatin is striking, since that substance is associated with collagenous fibers, i.e. with connective tissues, which, of course, are not present in the case of protozoans. -58-
Following staining after Mallory (which stain is used to demonstrate the presence of collagenous fibers) I found that the fiber network, which forms the meshes located along the periphery, stained intensively blue.
That network, no doubt, corresponds to the extensive and, pérhaps, proliferated fiber network of the mucous layer of the swim-bladder. However, I do not lnow whether this fiber amount, which is minimal relative to the coccidian mass, is sufficient to explain the quantity of gelatin demonstrated. The layer of detritus along the inner face of the mucous layer also stains blue in sectioned preparations treated after Mallory. It is possible that that detritus also consists of gelatinous substances. — 59 —
Summary review . 130 .
The following coccidian species have hitherto been described in fishes:
- --- Fish 1 Coccidian species Organ !species
31ustelus canis Murenmr. Dann (Spiraldarm) Goussia lucida LA13131:: Lainua cornubica Gm. Darm Pffifferella gigantea LABBÉ COMM i2tM, gigan felon LA.1313É Scylliunt stellare L. Goussiet lucida LABBE Acanthias acanthias L. Clupect harengs L. Leber c/upearton THÉ „ «pilchard us WALL. n . Tioden Coccidiunt sardinac TiiÉ r. :E1t/i n1W/8 Olen/Sidi OlitS L. Leber Gollesia clapcar fun 11 0É1,. Gobi() gobio L. Dann COccidium metschvikotri LAVERAN Tincit tinca L. Niue, Leber, Milz Goussia minuta r;1711ÉL. Motel/a tricirrata Daim, ntotellac LABBli , • Pylornsanbânge .11 in mod ytes tob hoots L. D arm bigemina LABBÉ .Labrus Si). Leber thelohani LADBÉ CM/ i/a br US sp. Darin variabilis ; Lepadogaster • gown? i LAC. GasIerostcus aculentas L. Leber Coceidium, iptsferostei Tnim. Gobius lutganellus L. D arm Goussia rariabilis THÉL. Cottus bubalis Eurim. &amber scombrus L. Cliyearitnt TH.ÉL. Trach arus &adieu us L. • Leber cruciata Cj/».in us ca)7)io L. Darla Coccidiumwierzejskii 1.1ovER Eintcria subepithelialis Monon, et FIEB.
Key:, Dam, intestine(s); Leber, liver; Hoden, testes; Niere, kidney; Milz, spleen; Pylorusanhânge, pyloric appendages. -60-
We then realize that twenty fish species•harbor a total of fifteen • coccidian species. The list presented above has been largely taken from Labbé i s wOrk on Sporozoa, i.e. it was compiled at a time before the more.recent ad- .
However, as far as I know, our know- vances in the field of coccidian research. ledge of the coccidians parasitizing fish has not been enriched since that time.
The descriptions of the different species, thus, is incomplete in almost all cases. In most instances we know only the spores. Pfeifferella gigantea apparently is part of the life cycle of Coccidium giganteum (schizogony and gamogony?)--as has already been suggested by Labbé.. In the case of E. sub- epithelialis we also have been able to observe only the final stage of schizo- .131. gony in addition to sporogony. E. gadi, thus, represents the first coccidian parasitizing fish, in which the entire life cycle has been studied.
It appears that all coccidians parasitizing fish have in common that sporogony takes place already in the host animal, while that event, in the case of coccidians parastic to terrestrial animals, takes place as a rule out- side of the host. In the case of E. stiedae both Metzner and Wasielewsky have found that the development of the spores is promoted by the oxygen of the atmosphere. Since water contains only a few milliliters of dissolved oxygen per liter, i.e. only a fraction of the quantity of oxygen present in the air, it appears that favorable conditions of that type would not be encountered by the spores on amergence into the water.
In the case of E. subepithelialis, it appears that the localization of spore formation in the submucous layer indicates a definite oxygen requirement; that layer is supplied at least with oxygen by way of diffusion from the blood capillaries (as in the case of internal respiration), while the intestinal lumen
--as has also been stressed by Fuerth--is deficient in oxygen. - 61 -
In the case of fishes having no tubular connection between the swim- bladder and the digestive tract and, in particular, in that a marine fishes
of that type, it has been reported that oxygen represents the predominating gas in the swim-bladder. The swim-bladder gases reportedly çonsist to 69 to
87 percent of oxygen. That fact perhaps represents the explanation for the
enormous multiplication of this coccidian inside the swim-bladder. In this
highly exceptional proximity of oxygen and nutritive substrate as well as in
the possibility of passing over from the host tissue into anox,ygen storage
space, we perhaps also find the explanation for the unique phenomenon show-
ing that this coccidian species not only completes its entire life cycle in
a single organ, but also immediately starts a new one in the same organ.
These particular spores then serve not solely in propagative reproduction
as postulated by Doflein, with infection of new host animals, but also in
multiplicative reproduction, with new territories in the same organ being
infested. From the point of view of biology, this particular species represents
an exceptional cases similar to that of Taenia murina of rats and mice, which
lives already in the intestinal wall of the afore-mentioned rodents as cysti-
cercus, and subsequently emerges as cysticercus into the intestinal lumen, in
order to undergo development into the tapeworm.
However, this resumption of schizogony cannot represent the sole task
to be fulfilled by the spores. Considering the economy, which we find in the
morphology of all living organisms, possession of the resistant capsule by
the spores of this species would make no sense. Furthermore, resumption of
schizogony in the infested swim-bladder does not contribute to the survival
of the species. In this connection, we are now faced with the following question: -62 -
How do these parasites get into their host, and how do they emerge again from .132. that host in order to infect a new one?
That question cannot be answered readily since the swim-bladder of the gadid species is sealed all round, and only the blood-vascialar system re- presents a means for communication with the intestines.
This particular parasite, then, would be in a position similar to that faced by, let us say, the cysticercus and the larva of Trichinella spiralis, i.e. the parasite presently under consideration can emerge into the open and reach a new host animal only if it is liberated from its prison by mechanical means; that means could be that the host or the organ containing the parasite is ingested by anôther host, i.e is dismembered and then dissolved chemically by digestive juices, or it could be that the host dies, is macerated by the water, and is eventually disintegrated and ingested-by bacteria and other or- ganisms, bringing about liberation of the coccidian spores. Despite their size, pollocks, no doubt, can become prey of other fish. For instance, I myself have witnessed once how an enormously-large monkfish [Lophius piscatorius] was pulled aboard, with the tail parts of two pollocks, measuring one meter in length, hanging out from its mouth. Haddocks would represent prey even more readily.
However, we must furthermore assume that these predatory fish play only the role of liberators and that the spores leave the intestines of the preda- tor without undergoing any change, as is the case with the spores of E. schu- bergi inside of arthropods. Assumption of the existence of an intermediate host would not facilitate the resolution of our question. No evidence is • available pointing in that direction. - 63 -
The most probable suggestion is that the infested gadid fish die, that both the abdominal wall and the swim-bladder wall undergo disintegration, and that the millions of spores are liberated. I imagine that the subsequent course of the life cycle proceeds in the following mariner: The spores are ingested by cods. The spore capsules open inside their intestinal tract; the sporozoites are liberated, bore through the intestinal wall, reach the blood-vascular system, and.are carried to the tissues of the hostts body. They find suitable conditions for both existence and development only in the eNim-bladder, In the connective-tissue meshes of the mucous layer, the parasite multiplies first by agamous means and then by means of sporogony.
The sporozoites infect large territories and start a new life cycle.
The swile-bladder gradually is filled with spores and products of disintegration.
The gases are gradually displaced and have completely disappeared once the swim-bladder is filled with parasites to overflowing. The function of the swim- bladder as static organ is impaired by that state; the weight of the fish is .133 increased in correspondence to the parasitic mass. In the most favorable case, the parasitic colony would act'as a foreign body exerting pressure on the surrounding space, i.e. on the red body and on the blood vessels. It is indeed surprising that the fish survive the loss of an organ as important as the swim- bladder. In some cases, I have actually found that the external appearance of the infested fishes was entirely normal. However, the fact that infested fish are injured is reflected in the state of emaciation reported by J. Mueller as well as in the dermal defects observed by me.
The gadid fish are bottom dwellers. Existing at the bottom of the ocean,
they are well able to move about and to overcome small differences in the level - 64 - by means of muscular power despite the loss of swim-bladder function. During the period of spawning, these fish migrate toward the shores and deposit their spawn, which then floats upward to the surface of the ocean. It is highly questionable that fish having no functioning swim-bladder are able to under- take that migration. Perhaps, the infested individual die just during that period.
In passing I wish to touch upon still another possibility. Since deve- lopmental forms doubtlessly do reach the blood-vascular system, as we have already suggested further above, one could perhaps imagine that blood-sucking parasites---I am thinking in this connection of the copepods parasitic on fish i.e. the Caligus species--ingest small developmental forms (i.e. sporozoites or merozoites) and then transmit them to another animal when they change hosts, in analogy to the situation existing in the case of Trypanosoma. I myself, by the way, have found myxosporidian spores in the blood of carps. It is practi- cally certain that the developmental forms of the species under consideration are present in the hostts blood for a certain period of time, since we other- wise would hardly be able to explain their appearance in the swim-bladder.
Occurrence of developmental forms in the blood has been demonstrated in the case of some coccidians (Isopora lieberkuehni, for instance) and has been proposed in that of other ones. Doflein believes that occurrence in the host's
blood cells is possible.
I well realize that all the afore-outlined suggestions are highly hypo-
thetical in nature; however, I have outlined these possibilities in order to
provide points of attack for future studies. -65-
The fact showing that this coccidian Proliferates in such great numbers
in the swim-bladder, but is absent from all other organs (intestines, liver,
spleen, kidneys) permits the conclusion that its organization is adapted to
the unique conditions prevailing inside of that organ. This'coccidian then .
represents an example of highly adapted organ parasitism...However, the respec-
tive adaptive features required in this connection cannot be acquired newly .134.
by each generation, i.e. these features must be transmitted by heredity. There
can be DO doubt that coccidians parasitic on swim-bladders of this type are
the offdpring of coccidians parastic on swim-bladders. This excludes the possible
objection that the residence in the swim-bladder of gadid fish is purely facul-
tative in nature, i.e. that we are dealing here with an accidental or casual
visitor.
The pronounced frequency of occurrence also would speak against the
latter suggestion. We are thus forced, in principle, to regard the observed
• phenomena as normal ones belonging to the life cycle of this parasite.
A further peculiarity of the parasite under consideration is found in
the behavior of the host tissue. In correSpondence with the occurrence of the
three well researched coccidian species, we are used to regard coccidians as
parasites of the intestinal epithelial cells and of the cells of the pyloric
appendages. The intestines; in fact, do represent the site of entry. That
statement is applicable in the great majority of the coccidians. It appears
to be applicable, in part, also in the case of coccidians parasitic on fish.
We have found, for instance, in the case of E. subepithelialis that the
juvenile stages parasitize the intestinal epithelial cells, while sporulation,
on the other hand, takes place in the submucous connective tissue. However, -66- there exist etill other exceptions. Schaudinn has observed penetration of .
Cyclospora caryolytica into the - nuclei of connective-tissue cells. Sporu- • lation of both E. stiedae and E. schubergi occasionally takes place in the intestinal wall. E. truncata parasitizes in the renal tubules of domestic geese.
E. mitraria undergoes development chiefly in the extracellular manner viz. in the intestinal lumen of Damonia reevesi (quoted after Doflein). In the case
of Isospora lieberkuehni, the sporozoites invade the blood-vascular system • frum the intestinal epithelial cells, produce a general infection, and may be found in the endothelial cells of the blood vessels of the ltIngs, the liver,
the large intestine, the fat body, and the kidneys. Caryotropha mesnili
parasitizes in the abdominal cavity of the polychaete Polymnia nebulosa.
Klossiella muris parasitizes in the renal glomeruli of the house mouse.
Adelea mesnili parasitizes in the fat-body cells of the moth Tincola biseliella:
Adelea zonula MOROFF parasitizes in the fat body of the larvae of Blaps mortisaga. Legerella nova paraSitizes in the nalpighian vessels of centi-
pedes. Klossia helicina parasitizes in the kidneys of snails. Orcheobius
herpobdellae parasitizes in the testes of Herpobdella atomaria. Among the coc-
cidians parasitic on fish mentioned further above, we may refer to the occur-
rence of Coccidium sardinae in the testes of Clupea pilchardus.
The situation existing in the species under consideration is a highly
peculiar one. Although epithelial cells are, in fact, present in the swim-
bladder and this in a glandular arrangement as the layer lining the villi .135.
of the red body, I found in one infested swim-bladder that these particular
cells were free from coccidians. However esthe connective-tissue cells, too, were free from parasites as a rule, apart from a few doubtful instances, - 67 - where I believed to have seen the outlines of a connective-tissue aell around the parasites. The merozoites, no doubt, as a rule do not penetrate into cells; they are distributed rather over the meshes of the connective tissue and there undergo development into schizonts and the other stages. The merozoites, thus, do not live at the expense of the cellular protoplasm of the host cells, but utilize the tissue fluid present in the meshes. The other developmental stages are also able to lead an extracellular life.
The main points of our results can be summarized as follows:
(l) The entire life cycle of the coccidian species described in the pre- sent paper takes place in the same organ of the same host animal.
(2) The sporozoites are able to reproduce again asexually in the same organ.
(3) The sporozoites possess a karyosome.
(4) The microgametes do not possess cilia. They resemble the microgam- etes of the Plasmodidae.
(5) This coccidian, on the whole, does not penetrate into cells, i.e. it exists outside of cells during its entire life cycle.
(6) Pats represent an important component of its body; these fats differ from the fats of the host body. - 68 -
Legends to Plate 10
With the exception of Figure 1, all Figures have been drawn with the aid of a drawing apparatus using 1/12 immersion and compensatory eyepiece No. 12; the .drawings were made at the object stage level and reduced to three quarters.of the original sketch size. Figure 1 - Transection of the wall of an infected air-bladde'r. (a) Fibrous layer; (b) smooth muscle fibers; (c) vascular layer; (d) mucous layer with juvenile stages; (e) tetraspores in the meshes of the mucous layer; (f) spores with detritus. Figure 2 - Juvenile schizont with two daughter nuclei; length 9.6 g.
Figure 3 - Schizont with four daughter nuclei; length 11.2 pi. Figure Multinuclear schizont; 22.5 x 14.5 4. Figure 5 Disaggregation of daughter caryosomes. Figure 6 - Merozoite formation with two rosettes; 53 x 20 g. Figure 7 - Section through a schizont, with merozoites undergoing detachment; apparently spherical in shape. Sporozoite, at the onset of a bew schizogonic cycle. Division of caryosomes. Centrodesmosis. Figure 9 - SchiZont, released by a sporozoite, with two nuclei. Figure 10 - Elongated sporozoite with numerous nuclei arranged in series, apparently undergoing degeneration. Figure 11 - Juvenile microgametocyte (?). Figure 12 - Microgametocyte with multiple small nuclei at the surface. .Figure 13 - Elongation of the daughter nuclei. Figure 14 - Mature microgametocyte with hyaline zone. Figure 15 - Mature microgametocyte with erected microgametes. Figure 16 - Macrogamete inside a star-shaped fibroblast (?). Figure 17 - Macrogamete. Figure 18 - Macrogamete with centriole. Figures 19 to 23 - Macrbgametes exhibiting signs of maturation. Figure 24 - Sporoblast formation. Figure 25 - Tetraspores with released sporozoites; diameter 28 u. Figures 26a and 26b- Spores with sporozoites. Figures 27 to 31 - Staining of fatty material. Figures 27 to 29 - Smears fixed in a mercuAc chloride-alcohol-glacial acetic acid system and stained with haemalum and Sudan III. - 69 -
Figures 30 and 31 - Fixation in Flemming's mixture e safranine staining (in these preparations fat appears black and chromatin, red). Figure 27 - Juvenile macrogamete. Figure 28 - Adult macrogamete with teat-like process (fertilization cone?). Figure 29 - Macrogamete with centriole (?) inside the caryosome. Halo around the fatty spheres. Figure 30 - Sporoblast with residual body. Oaryosome visible as a very small, grain-like structure. Figure 31 - Spore with one sporozoite (the other one is not visible). ^r
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• - 71 - Bibliography
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18. Multiple division and reduction in Adelea ovata. • 19. Introduction to the physiology of one-celled organisms (Protozoa). 20. Notes on Myxosporidia. 21. Constitution of protistan nuclei. 22. Contribution to the biochemistry of Protozoa. 23. Textbook of protozoology. Third Edition. 24. The rabbit-infesting coccidian Eimeria stiedae, etc. 25. Outline. 26. Chemical physiology of lower animals. 27.Aspects of fatty degeneration in triphylan Radiolaria. 28. Handbook of ichthyology. 29.Animal parasites of man. Fourth Edition. •
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