The excretory system of Anisakis
simplex
' L H V NEMAT.
(
Justus F. Mueller
With 4 figures in the text
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Saint-Hilaire, K., Histo-physiologische Studien über die Spinndüsen der
Tenthredinidenlarven. Mit 89 Textabbildungen
449
Mueller, Justus F., The excretory system of Anisakis simplex. "With
4 figures in the text
495
Wedekind, Georg, Zytologische Untersuchungen an Barrouxia Schneideri.
(Gametenbildung, Befruchtung und Sporogonie), zugleich ein Beitrag
zum Reduktionsproblem. (CoccidienuntersuchuDgen I ) Mit 8 Textabbildungen und Tafel VII—X
.505
Duyff, J. W., und Bouman, H. I)., Über die Kapillarisation einiger Kaninchenmuskeln. Mit 4 Textabbildungen
596
(Contributions from the Zoological Laboratory of the University of Illinois
under the direction of HENKY B. Ward, No. 295.)
T H E EXCRETORY SYSTEM OF ANISAKIS SIMPLEX 1 ).
By
JUSTUS P. MUELLER.
With 4figures in the text.
(Eingegangen am 24.Januar 1927.)
f
Introduction.
Although the usual forked type of nematode excretory system, such
as is found in Ascaris megalocephala for example, has received careful
consideration from a number of workers, there is to date no adequate
account of the peculiarly divergent t y p e which is found in members of
the family Anisakinae. Instead of tubes in the lateral lines, these forms
possess a ventral, unpaired, longitudinal, unicellular gland, t h r u which
passes the excretory duct, sending numerous side branches into the gland
substance, and opening on the head in the angle of the two ventral lips.
Previous work on this structure can be considered in few words
M E H L I S (1831) observing t h e gland for the first time in Ascaris spiculigera, described its general anatomical relations, the main duct and its
side branches, and the peculiar nuclear body, the nature of which was
not clear to him. Later workers have added nothing of importance t o
this early description, v. SIEBOLD (1838) described a similar structure
in the ambiguous Filaria piscium. ANTON SCHNEIDER (1866) erroneously
describes the cytoplasm of the gland as multinucleate. BASTIAN (1866)
and v. DRÄSCHE (1881) also describe the organ, b u t fail to go further
t h a n M E H L I S . All of these authors find the main duct and its branches
ramifying in the gland tissue, b u t are unable to make out how these
ultimate branches terminate or connect. They also interpret the curious
and dubious nucleus as being a simple cavity or bladder. NASSONOW
(1900), mainly interested in the phagocytic organs and their relation t o
the excretory apparatus, seems to regard without reservation this above
mentioned structure as a nucleus, b u t is otherwise not concerned with
the gland. However, the gland incidentally appears in some of his illu-1
) Presented by title before the Philadelphia meeting of the American Society
of Parasitologists, December 1926.
496
I
J. F. Mueller:
strations. GOLDSCHMIDT (1906), in his account of the excretory systems
of Ascaris lumbricoides, and A. megalocephala, believes t h a t the fundamental p a r t of the excretory apparatus of these forms is found in certain
specialized glandular areas, „Drüsenstränge", in the lateral lines adjacent
to the familiar tubes. To substantiate this conclusion he tries t o draw
a n homology between these structures and t h e excretory gland of Anisahis decipiens, basing his comparison on t h e above mentioned figures of
NASSONOW, for want of personal experience with this form. The validity
of this comparison will be considered later. H e concludes t h a t „Eine
vergleichende Untersuchung würde zweifellos manches interessante zutage fördern".
Through the kindness of Dr. H E N B Y B. W A B D , who placed a t m y
disposal the material for this study, I was recently enabled to reinvestigate this problem. To him I am also indebted for much valuable
criticism during the preparation of this paper. This material consisted
of parasitic nematodes t a k e n from a Sei Whale at Cachalot, B. C. b y
M B . I B A E. COENWALL, in J u l y 1925, and presented to t h e WABD-Collection of Parasites a t Urbana. They were here identified as Anisakis
simplex (RTTD. 1809).
After formol fixation the worms h a d been kept in 85% alcohol, a n d
came to m y hands in an excellent state of preservation. I n the course of
this work twelve specimens were used, six males and six females. Recourse was h a d to gross dissection, study of the glands in toto, a n d
serial cross sections of the entire worm. For the study of finer details
however the gland was always dissected out first, and carefully drawn
with the camera lucida ; it was then cut in small pieces which were also
carefully drawn, and located on the big drawing, and then kept in separ a t e numbered vials. Upon sectioning these pieces, it was very easy t o
know exactly the region t h e y represented, a n d to recall the appearance
of t h a t region as seen in toto b y referring to the drawings. The tissue
was dehydrated, cleared in cedar oil, embedded in paraffin, a n d sectioned
serially in ribbons from 2 to 7/.i thick. Whether due t o the formol fixation
or not I am unable to say, b u t the tissue proved unexpectedly brittle
so t h a t good sections were somewhat difficult to secure. Sections were
stained in HEIDENHAINS iron hematoxylin and orange G; DELAFIELDS
hematoxylin and eosin; orcein; and MALLOBYS triple and double connective tissue stains. Of all these, MALLOBYS double stain was b y far the
most useful, and gave contrast between structures t h a t could not be
differentiated a t all with other dyes. No advantage was found in the
use of the acid fuchsin solution of the triple stain. My preparations
include over one hundred slides of serial sections, b u t after careful
examination of all of these, only a few slides, and indeed only a few
sections presented clear pictures of the finer structure.
The excretory system o{ Anisakis simplex.
497
To t h e general description of this gland given b y others I have little
to add. I n A. simplex it lies in the body cavity along the ventral side
of the alimentary canal, and attached b y its left edge
t o t h e lower lobe of t h e left lateral line. I t is a b o u t
one third of the body length. Beginning in a narrow
point a short distance behind the head it is soon
joined on its free edge b y t h e excretory d u c t which,
passing backward from t h e excretory pore through
the cephalic commissure, joins the gland a t this
place. F r o m here t h e gland extends caudad in t h e
form of a narrow neck of almost even width for a
third or fourth of its length. At this point it abruptly
widens out to a ribbonlike form, which covers half of
the remaining distance, and then abruptly narrows
down to a long slender tapering region which disappears in a fine point (fig. 1).
>
I n toto preparations a lighter area can always be
seen corresponding to the posterior seventh of the expanded portion, where the gland seems to be thinner
and more transparent. The nucleus appears as a
light band of one third the t o t a l width of the gland,
and equidistant from its edges, passing from a point
just behind the anterior constriction to within a
short distance of the posterior constriction. On either
side of this band a series of darker patches occur,
which when viewed from the side are seen to be
thickenings on the ventral surface, which rise gradually from front to back, and then drop suddenly, forming low posteriorly directed cusps. The excretory
duct is easily seen passing along the length of the
gland near its free edge, and giving off numerous
side branches which ramify in the surrounding tissue.
These branches are most numerous in the expanded
middle region of the gland. They are practically absent in the anterior narrow region, and are few and
small in the posterior tail-like extremity. Although
the delicate subdivision and ramification of these
branches have beenknown to other writers, these have
been unable to determine t h e ultimate fate of these tu,
,
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Fig.1. Highly diagram-
bules. However theyhave givenlittle or noattention to maticrepresentationof
the character of the gland substance proper. I n this gJ,alld- .s™a.u cir(;le3
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F
showextentorgranules.
article I propose to consider certain minute features of Nucleus with broken
f. ZellJorschung
mikr. Anatomie
33 x^o.
thisZ.gland
whichu.have
escapedBd.
previous notice.
shaded,
498
J. F. Mueller:
I. The tubules.
In successful sections, with good staining and high magnification the
cytoplasm of the gland is observed to have a spongy texture, being
extensively perforated with small cavities which present approximately
the same appearance in frontal, longitudinal, or transverse section, and
accordingly might easily be mistaken for vacuoles. On further study of
thick sections and thin serial sections however these holes are found to
beinrealitythe cross-sectional appearance ofvery numerous and tortuous
minute passages, which interconnect in a most complex fashion to form
a three dimensional porous network, and which are quite distinct from
the branches of the excretory duct. The branches of the excretory duct,
no m a t t e r how fine, are always comparatively straight, and have a
distinct though delicate membranous wall, whereas the cavities of this
network are without a limiting membrane, being simple excavations of
the cytoplasm of the gland. In one specimen of Anisakis, and I have
observed it extensively in Contracaecum also, small areas of the gland
presented a highly vacuolated foamy appearance, b u t this is unusual in
the species under consideration and we are not concerned with it here.
The cavities referred to are fundamental features of the cytoplasmic
structure, and form a series of continuous irregular chambers. I n their
lumina can be seen a distinct core of solid material, the precipitated
contents, which always stains similar to the substance of the gland.
This core often adheres to one side of the passage so closely t h a t in consequence the lumen appears crescentic. These passages vary from 1,5 t o
3,0 [A, in diameter, and pass with equal frequency in all directions, except
near the outer edges of the gland, where t h e y tend to follow a more
longitudinal course. As will be clear later, these m a y be regarded as the
primary, or secretory passages of the gland.
The well known excretory duct and its branches m a y in contrast be
designated as the secondary or drainage tubules. The main duct has a
conspicuous hyaline wall which in diminishing thickness passes out to
line the side branches, and is distinctly visible as far as their finest
ramifications. The tubes of this system also have a solid core in t h e m
somewhat shrunken away from the walls, b u t always equally, and never
adherent to one side as in the case of the secretory passages. I t s substance is clear and glassy, and in no case does it have the flocculent
character of the core of the primary tubules except as will be explained
later. I t usually stains a light blue, but frequantly takes on a bright
yellow with MALLOBYS double stain, and in all cases is clearly different
from the surrounding wall and tissue. This core is present to the minute
branches of the drainage tubules, b u t here it takes on more of the flocculent character of the core of t h e secretory passages. These details can
The excretory system of Anisakis simplex.
499
be seen in most thin sections, although in some specimens the primarypassages are obscure, a condition probably due to some accident of fixation or the physiological state of the gland.
These d a t a suggest some connection between the two sets of passages,
b u t with most careful and continued searching it seemed impossible to
discover such. Finally however in
a preparation stained with D E L A FIELDS hematoxylin and eosin a
number of such connections were
found (fig. 2). Here several fine
branches of the drainage tubules
were seen to pass from larger drainage tubules, also in the plane of
section, and terminate by opening
into secretory passages much larger t h a n themselves. The diameter of the secretory passages a t
such points is about 3 i_i, t h a t of
the drainage tubules about 1 ft.
The presumable difficulty in disFig. 2. Connection of a drainage tubule with
a secretory passage. X 1800.
covering these connections more
frequently is the failure of the core of the finer secondary tubules to
shrink away from the wall sufficiently to allow the course of the
tubule to be followed. If my observations are correct it is obvious
t h a t the excretory product is secreted in the primary passages, and
passes to the exterior t h r u the secondary.
II. The n u c l e u s .
The single nucleus of this unicellular gland in A. simplex is so variable
as practically to defy description. I n general it extends along the greater
length of t h e expanded mid portion (fig. 1), b u t only occasionally can
its outline be clearly seen in toto>preparations. Its appearance differs
widely in young and old specimens. I n smaller individuals it usually
has the form of a cavity without distinct membranous walls, filled with
a smudgy coagulum having a distinct though not marked affinity for
nuclear stains (fig. 3a). This condition corresponds to what BASTIAN and
v. DRÄSCHE have described, and explains their interpretation of the
structure as a bladder. On the other hand it is regarded b y later workers
as the single nucleus of the excretory system. As a nucleus it has undergone most radical changes. I n older specimens, and occasionally in young
ones, the bladder-like form and likewise the sharp boundary tend to
disappear. The walls of the cavity come to lie almost in apposition over
most of their area, and while there is still no distinct nuclear membrane,
33*
500
J. P. Mueller:
there is nevertheless a deeper staining margin, which looks somewhat
tougher and denser t h a n the cytoplasm of the gland (fig. 3b). On the
ventral surface of the nucleus this wall is frequently lacking, and for a
short space the nuclear contents invade a n d blend with the cytoplasm
clear to the ventral wall of the gland, forming a nebulous darker staining
region in cross section (fig. 3c). I n other cases the walls of the nucleus
bend downward and outward, limiting this area and fusing with the
ventral wall of the gland. Always, however, this condition alternates
with stretches where the nuclear wall is complete, and the nucleus and
its contents limited to the center of the organ. In spite of this invasion
J?ig. 3. Aspect of nucleus in cross section, a in young specimen ; b older specimen, nuclear wall
complete : c nuclear wall lacking on ventral side. X 50.
of the cytoplasm by the contents of the nucleus, the anatomical features
of the cytoplasm are still preserved in such situations, and the primary,
and occasionally secondary tubules can be clearly seen, which fact proves
clearly t h a t this strikingly unusual condition cannot be attributed t o
deterioration of the material prior to fixation, or to other artifact. Frequently also there are present small outwardly directed folds in the
dorsal nuclear wall, from the points of which proceed outward darkly
staining fibrous streamers of nuclear material which frequently divide
and presumably penetrate to all portions of the gland.
I have been able to examine a t least one other gland of this type
from a species of Contracaecum, and here too I have noted t h a t the
ventral wall of the, in this case, small disc-shaped nucleus is not well
The excretory system of Anisakis simplex.
501
defined and the fibrous. streamers are very numerous and branching,
permeating far out into the cytoplasm. Here also the secretory chambers
are very distinct and numerous in the wall of the nucleus, whereas they
are difficult to see elsewhere.
Aside from this curious body there are no other nuclei in the gland.
If this be really a nucleus, it is certainly most highly modified. I t s
true n a t u r e can be ascertained only b y more comparative work or b y
studying its transformations in ontogeny. Such studies would throw
light on the nature of other elements of this gland.
III. S e c r e t o r y g r a n u l e s .
I n preparations stained in MALLOEYS double connective tissue stain
(methylene blue and orange G), in the regions of the anterior and posterior constrictions, is found a zone of conspicuous large spherical granules
(fig. 1). I t is true t h a t among others A. SCHNEIDBB and v. DEASCHB have
described the appearance of this gland as granular when seen in toto,
b u t t h e y refer to no definite regions, and it is evident t h a t they are
using the term in a vague sense, as is so frequently done when a heterogeneous substance whose structure is not easily defined requires
description; and when some more remote term would be more appropriate. The intimate penetration of the gland
b y the primary tubules breaks u p its substance in such a manner as to cause precisely t h a t appearance which is so often
called granular. B u t the granules here under
discussion are usually invisible in toto preparations, altho at times when their number
was very great, the corresponding locus
appeared somewhat darker t h a n the rest
of the gland. With most stains these cannot
be differentiated from the cytoplasm, b u t
i
,i
, .
- T -, T -i
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i,i
Fig.4. Appearanceoforangestaining
when the stam indicated above is used they g r a n u l e s £ s seen in s e c t £ n . x 800B
appear as clear bright orange spherules from
0,5 to 8,0,« in diameter (fig. 4). Usually they are surrounded by a small
space separating them from the cytoplasm, as tho they lay in vacuoles.
At times, however, this space is not seen, and the cytoplasm isin intimate
contact with the granules, or more rarely their outline is indistinct, the
orange color blending into the blue background around their edges. All
sizes and all conditions of these spherules m a y be seen in one preparation,
or again a specimen m a y show all of nearly the same size and appearance.
I n the anterior zone of their occurrence especially they are very densely
concentrated a t the right and left margins of the gland. In eight of the
twelve specimens examined these granules were found in both of the
J. F. Mueller:
designated regions ; three showed t h e m in t h e anterior region only, a n d
one, a young individual failed to show t h e m a t all. Likewise their number
varies greatly. They m a y also be found a t times extending a little farther
forward or backward t h a n usual. Apparently they bore no relation to
either set of tubules, and in one case t h e y occurred embedded in the
outer wall of the gland, and even in the wall of the main duct.
From the various degrees of separation of these granules from- the
cytoplasm, as well as their conspicuous variation in size, it is evident
t h a t they m a y be deposited, enlarged, and again resorbed. Although it
m a y be merely coincidence, it is curious t o note t h a t t h e regions of their
occurrence are the precise regions of the anterior and posterior terminations of the nucleus. Within t h e nucleus itself I have found a small
number of closely similar granules, b u t only in the regions where the
nuclear contents blended with the cytoplasm, so t h a t it was difficult to
tell whether t h e y should really be regarded as p a r t of the nucleus. Only
a few specimens however showed granules in this situation. When
stained in H E I D E N H A I N S iron hematoxylin these granules are barely visible to one already looking for them. I n this stain they take on a pale
grayish purple cast, b u t certainly not t h e deeper values characteristic
of basichromatin. They were also left unchanged after treatment with
strong acids or weak alkalis, and failed to give any characteristic color
with iodine, but since m y material was not fresh, chemical studies were
pursued no further.
The gland of Contracaecum sp. was also examined for these granules,
b u t here only a very few similar structures were seen in t h e vicinity of
the small nucleus. Within t h e vague nuclear wall on the other h a n d a
considerable number of similar granules were found varying from 3 to
5 /{ in diameter. Unfortunately none of t h e more nearly related forms
of the genus Anisakis were to be h a d for comparison.
Discussion.
A. simplex is one of the forms possessing a "ventriculus", a simple
modification of t h e alimentary canal between t h e esophagus a n d the
intestine. BAYLIS (1920) writing on these forms says: "Although no
special attention as yet has been devoted t o t h e ventriculus of Ascarids
from the histological or physiological standpoints, it is interesting to
observe t h a t in a nematode of another family, Camallanus, in which a
very similar organ exists, a recent writer (MAGATH 1919) has suggested
t h a t it is concerned with the excretory processes. I n the species dealt
with it seems t o have some structural connection with the excretory
apparatus. Now in some of the Ascarids in which a ventriculus occurs
we find it associated with a peculiar t y p e of excretory apparatus, terminating in a long unpaired unicellular "gland", opening close to the lips.
The excretory system of Anisakis simplex.
503
This — will very possibly be found to be the case in all forms in which
there is a ventriculus".
.G. americanus, worked on b y MAGATH has a typical paired excretory
system, b u t the bridge seems to be anatomically connected with the
esophagus b y means of an enveloping tissue. I n this region of the esophagus there appears a granular component in the form of two giant
cells,,which MAGATH considers to be a functional p a r t of the excretory
system.
I n A. simplex there are no such granular elements in the ventriculus,
b u t in the intestine there are very numerous granules, rather similar in
shape and size to those described in the excretory gland. However, these
seem suspended in a very light foamy substance filling large cavities in
t h e walls of t h e intestine, a n d t h e y stain a weak m u d d y yellow, quite
unlike the brilliant orange of those of the excretory gland. I have
furthermore been unable to detect any anatomical connection between
the excretory gland and any p a r t of the alimentary canal. While therefore the suggestion of BAYLIS m a y hold true, evidence is lacking to
show a n y positive relation between the presence of a ventriculus and
this type of excretory system.
GOLDSCHMIDT (1906), who bases his opinion on the incidental figures
of this gland given b y NASSONOW, says : „Die Darstellung und besonders
auch die Abbildungen NASSONOWS zeigen nun, daß zweifellos genau der
gleiche histologische Aufbau vorliegt wie bei den exkretorischen Drüsensträngen von A. lumbricoides. Der Unterschied besteht nur in seiner unpaaren Beschaffenheit und der Unabhängigkeit von der Seitenlinie, —
doch möchte ich aus Mangel eigener Erfahrungen dies nicht weiter ausführen."
This comparison, however, does n o t hold, for GOLDSCHMIDTS glandular strands are multinuclear structures quite different from this large
single cell. I t is also impossible to compare GOLDSCHMIDTS fig. 12
representing the finer structure of these strands with t h a t of this cell.
I n these strands on the one hand there are no cavities, tubules, or
granules, and on the other t h e y contain fibres not found in the unicellular gland of Anisakis. The masses of glassy secretion which GOLDSCHMIDT represents are not obviously comparable b y their shape, appearance, or position, t o anything in Anisakis. Furthermore, it is difficult
t o compare the branching duct of this t y p e with its single layered wall,
and t h e paired unbranched ducts of Ascaris, with their inner hyaline
and outer striated layers. I t is evident therefore t h a t whatever the true
relations of this structure m a y be, GOLDSCHMIDTS comparison cannot be
sustained. A comparative s t u d y which m a y disclose affinities with other
types of excretory systems in nematodes is now under way.
504
i%
J. F . Mueller: The excretory system of Anisakis simplex.
Bibliography.
At-'
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bis 193. 1882. — 4. Goldschmidt, B . : Mitteilungen zur Histologie von Ascaris%
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Monographie der Nematoden. Berlin 1866. — 9. v. Sicbold, C. Th.: Helminthologische Beiträge über geschlechtslose Nematoidien. Arch. f. Naturgesch.
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• M
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