aT

ON THE PHYSIOLOGY
OF DIGESTION, RESPIRA-
TION AND EXCRETION
~_ IN ECHINODERMS

H. C. VAN DER HEYDE

sae a

Cornell University

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http:/Awww.archive.org/details/cu31924003408188

ON THE PHYSIOLOGY OF
DIGESTION, RESPIRATION
AND EXCRETION IN
ECHINODERMS

ACADEMISCH PROEFSCHRIFT

TOT HET VERKRIJGEN VAN DEN GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE
AAN DE UNIVERSITEIT VAN AMSTERDAM
OP GEZAG VAN DEN RECTOR-MAGNIFICUS,
Dr. J. K. A. WERTHEIM-SALOMONSON, HOOG-
LEERAAR IN DE FACULTEIT DER GENEES-
KUNDE, IN HET OPENBAAR TE VERDEDIGEN
OP VRIJDAG 5 MEI 1922, DES NAMIDDAGS
TE DRIE URE PRECIES, DOOR

HENRI CHRISTIAAN VAN DER HEYDE,
GEBOREN TE HEM. 7

GEDRUKT BIJ C. DE BOER JR. ~ DEN HELDER ~— 1922

by

QL
2es\
Gi

Selyo
/)Y

AAN MIJNE MOEDER EN
AAN DE NAGEDACHTENIS
VAN MIJN VADER.

De gelegenheid, in dit proefschrift openlijk mijn dank te
brengen aan allen, die in mijn academischen studietijd hebben
bijgedragen tot mijn wetenschappelijke vorming, grijp ik vol-
gaarne aan.

Hooggeleerde Weber, Sluiter, De Meyere, Dubois,
Verschaffelt en Stomps, gij allen hebt mij de wetenschap
van het levende doen zien vanuit uw standpunt en veel heb
ik uit uw lessen geleerd, wat mij in de toekomst van
onschatbare waarde zal zijn. Dat ik tenslotte het voetspoor
van geen uwer ben gevolgd, was niet uw schuld, maar de mijne.

Hooggeleerde Sissingh, Holleman, Smits en Korteweg,
uw lessen hebben het tekort mijner gymnasiale opleiding aan-
gevuld. De moderne biologie, ontgroeid aan het beschrijvend
en ontledend stadium, voelt meer en meer een dringende
behoefte aan contact met uwe wetenschappen. Te betreuren
is het daarom, dat de nieuwe Hooger-Onderwijs-wet het
mogelijk maakt, dat een geslacht van biologen gaat opgroeien,
dat niet voldoende vertrouwd is met de beginselen van de
»exacte’’ wetenschappen. De woorden van den grooten planten-
physioloog Osterhout, tot een mijner vrienden gericht:
First study mathematics, then physics and chemistry, finally
some botany and I'll make you a plant-physiologist’, hebben
mij intuitief voor den geest gestaan toen ik onder uw _ leiding
mij voor het candidaatsexamen in de scheikunde bekwaamde.

Hooggeleerde Buytendijk, gij zijt het geweest, die een
grooten invloed op mijn wetenschappelijke ontwikkeling hebt
gehad. Was mijn neiging naar dier-physiologie en -psychologie
al in den aanleg aanwezig, toen ik u vroeg, onder uw leiding
te mogen werken, gij hebt aan dien onbewusten drang vorm
en bewustheid gegeven. Onder uw leiding heb ik mijn eerste
wetenschappelijk werk mogen verrichten en uw felle kritiek
— vaak misschien te fel — op de fundamenten van het gebouw,
waarin de biologen zich veilig wanen, heeft mij de betrekkelijk-
heid van vele hunner waarheden leeren beseffen.

Gij, hooggeleerde Jordan, hebt in uw voordrachten mij
doen verstaan, wat vergelijkende physiologie is en hebt mij
vervuld met bewondering voor deze groeiende, jonge wetenschap.

My dear Dr. Morse, the time I spent in your department
as an instructor in physiology and physiological chemistry and
our subsequent journey through the ‘East’, will remain one
of my most pleasant memories, even if I do not return to
your country. Your valuable help and advise, the time you
spent in trying to enable me to carry out the work I had
hoped to do, have always been very much appreciated by
me and I will never forget them.

Het werk, waarvan dit proefschrift een verslag is, werd
gedaan in het Marine Biological Laboratory te Woods Hole. Mass.,
voor een gering deel ook in het Bermuda Biological Station
for Research op de Bermuda eilanden. Dank ben ik den
directeuren van deze zoologische stations, Dr. Frank R. Lillie
en Dr. E. L. Mark, verschuldigd voor de edelmoedige wijze,
waarop zij mijn werk mogelijk maakten. Het werd neerge-
schreven in een atmospheer, die doodend is voor dingen des
geestes, dank zij de in ons land helaas nog altijd noodzakelijk
geachte ,opkomst in werkelijken dienst’. Dit moge strekken
tot verontschuldiging voor vele tekortkomingen. Dank ben ik
daarom ook verschuldigd aan de velen, die mij in staat hebben
gesteld, toch mijn werk door te zetten, in het bijzonder aan u,
hooggeleerde Van Rijnberk, voor het openstellen van uwe
bibliotheek en aan u, Mej. G. A. Jonges, omdat ge in uw
qualiteit van bibliothecaresse van Artis u de moeite hebt
getroost mij de omvangrijke literatuur te verschaffen.

Hooggeleerde Sluiter, hooggeachte promotor, u ben ik
dankbaar, dat gij dit werk, al is het niet onder uw leiding tot
stand gekomen en al ligt het niet in uw lijn van onderzoek,
toch hebt willen aanvaarden als proefschrift. Uw colleges
hebben mij in het begin van mijn studententijd, toen ik nog
geheel onder den invloed verkeerde van Hackel’s Anthropogenie
en Weltratsel, vervuld met de grootste bewondering. Ook
later heb ik altijd uw morphologische lessen ten zeerste
gewaardeerd; geen zoologische physiologie is mogelijk zonder
een goeden morphologischen grondslag, zooals gij dien weet
te leggen.

Be we No

25.
26.
27.

TABLE OF CONTENTS.

Introduction. . « « & A + # e & * * ee we L
Species used. . . . . ‘ sole gee Bs
Anatomical details on the digestive tTAChg gio es a Gees WR
Biology. Prey . . pais “GR (Be Fo Bo Hu veo de PIO
Toxicity of the stacilal momach; ; Sa Re Bo we AA
The enzymes. a. proteolytic . : a aatn wo a Os
bs suctolytie: eu ee A oe ke DI
c. lipolytic . . . « 22.
Are the enzymes free? Phagocytosis i in Echinedeins boo 22s
Autolysis in Echinoderm guts . ........ . 26.
Contents of the gut of Thyone. . . . : bis sth Geis
Composition of the faeces of Arbacia. ee a ee ee
. Py of the gut contents. . ... . ee 30.
The digestive fluid . . . . sive | Bas
Transportation of the food. The peityisceral fluid bi omy: ae WO
Are there enzymes in the perivisceral fluid?. . . . oo El
Réle of the corpuscles . . . . 42,
The digestion and utilisation of fatty siibsrantes: Benwllstfyaiiy
enzymes?. . . Seah nes SDs
Lactic acid development in - aitelygates of the — 2 cas on OOO
Lipolytic enzyme in the amibocytes of the Asteroidea?. . 52.
The nitrogen metabolism of the Holothurians. . . . . 54.
The resorption in Echinoidea and Holothuroidea. . . 56.
. The resorption in Asteroidea. Function of their ,,liver”. . 65.
. The resorption from the perivisceral fluid . . . . OAR
Excretion in Echinoderms. Function of the rectal coeca
of the Asteroidea . . 2. 1. 2... ww ee e807
Respiration. a. General . . . . : 88.
b. Are there catalases tid pensipinees in ‘the
perivisceral fluid?. . . . . 90.

c. Contents of the Polian moaitdlin é Hse.
Importance of the water vascular system

in respiration . ...... .. . 92,
Reserve substances in Echinoderms. . . sow ae ve oe OBS
General metabolism . ........2.2.~%. . 97,

Bibliography. . . . ea ee a + « 104;

La morphologie constitue la base des sciences biologiques,
mais on ne peut la considérer comme leur aboutissement;
la_,,science de la vie’ a pour objet l'étude des fonctions,
l'exposé de leurs relations réciproques dans un méme étre et

l'histoire de leur évolution passée.

Victor Willem in Arch. Néerl. de Physiol. T. II.

INTRODUCTION.

Trotz der bedauerlichen Unvollstandigkeit unserer
Kenntnisse iiber die Ernahrung der Echinodermen bietet
doch schon das wenige was bisjetzt dari:ber festgestellt
ist, ungewdhnliches Interesse dar und ldszt weitere
Untersuchungen héchst wiinschenswert erscheinen.

Biedermann in Winterstein’s Handbuch.

More than anything else perhaps the young science of com-
parative physiology needs systematic work, in which the old
literature is taken into consideration, but where the results are
controled and verified by newer and often times better methods.
This literature is on account of the fact that in the last 35 years
biologists have almost exclusively been specialising on compara-
tive anatomy and related sciences, in most cases either very
old or very recent. The old literature, for a large part French,
contains unknown treasures, but is frequently difficult to find.
Many of the older papers will prove to be completely untrust-
worthy, be it because the methods are no more up to date,
be it because the writer, not thoroughly acquainted either with
chemistry or physics or with the anatomy of the group studied
— the latter is often the case with work of medical physiolo-
gists — has made more or less fundamental mistakes.

This work must not only consist of a ,.Nachpriifung” of the
work of previous authors and its appreciation, but also in a
supplementing of those parts in which our knowledge is still in-
complete. If all comparative physiologists work together in this
way, we will in a short time have a basis from where to start,
we will have formulated our problems and will be able to
explore new fields more fruitfully than this has been done till now.

Ideas like these have induced me to study a problem like:
Digestion in Echinoderms, a little more closely. 1 do not claim
that my work contains an answer to burning questions or is
concerned with such problems as occupy the modern biological
mind, but as ,,clearing work” it may have some value.

The study of one definite group more in detail is especially
desirable because other relations and other functions can be
studied at the same time, so as to give a clear picture of the
whole process of food intake, digestion, metabolism, excretion
and respiration, a view of a definite complex of functions of
the animal machinery. The whole literature contains an abundance
of single facts, but real problems as there are hundreds, have
seldom been attacked.

2

For a phylogenetic physiology the Echinoderms are one of
the most interesting groups. The great importance they have
for general physiology has already been realised clearly by
von Uexkiill, Jordan and other authors. Their study throws
a light on many burning questions of comparative physiology-
not to speak of the rdle their eggs play in genetics-because one
organ has many functions and one function many organs —
see f.i. the chapter on respiration — ; specialisation and inte-
gration take place before our eyes. Their absolutely indepen-
dent anatomical structure complicates this study still more.

Many of the older investigators have made more or less
serious mistakes. One of those workers who by their work and
by the authority they had, have done more evil than good, is
Krukenberg. This author who by his astonishing working
capacities, has been quoted by a great many authors, worked
without a sufficient knowledge of the histology and anatomy
of the animals he studied. As an example I may mention one
of his experiments in which he calls the well-known yellow
genital organs of Cucumaria: liver — no gut appendices are
found in Holothurians —. Starting from this preconcieved idea
he finds enzymes in them. He also finds these in the waterlungs,
which as we can find in every text-book, are organs of respi-
ratory and perhaps excretory function. In a glycerin extract of
these organs he finds a peptic enzyme, but no trypsin and no
amylase. One page before this statement the same author says
emphatically that ,,alle Wersuche, aus den Polischen Blasen, den
Cuvierschen Organen, den Wasserlungen und dem Blute der
Holothurien, Fermente zu extrahieren, nur zu negativen Resul-
taten fiihrten’’.

He finds proteolytic enzymes in the digestive juice, but is
unable to detect them in a glycerin extract of the intestine.
He concludes that the enzyme present must be derived from
the food, from which: it gets free by autodigestion, which is
the more improbable because the larger part of the food of
the Holothurians are plant-tissues. Remarkable is his result that
glandular cells are present in the blood vessels of the Holo-
thurians, which secrete the enzymes. He does not try to explain
how these enzymes get through the wall of the gut, but the
work of Enriques 37) has elucidated this question sufficiently.

Krukenberg’s result that a peptic and a tryptic enzyme are
present simultaneously in Echinoderms as in many other groups
he studied, will be discussed in chapter 6. a.

The unreliability of Krukenberg’s work isa pity, because of -
the great influence he had in his time and because his work
contains so many precious data, which are of not much value
in that way.

Another author whose conceptions the present author is not
prepared to follow, is Otto Cohnheim. His plan, the study

3

of invertebrate groups just for the sake of clearing questions
of mammalian physiology, is wrong to my opinion. The many
errors in his work will be discussed in various chapters, it is
of no use to discuss them here.

These examples may serve to show the desirability of a
careful re-testing of all the work of older investigators. Many
mistakes have passed unnoticed and came into the text-~books.

A summary of the literature which generally appears in the
beginning of a somewhat larger paper like the present one,
will not be given in this introduction, because there is practi-
-cally no important literature and because the data which can
be found scattered in the different journals, have been summa-
rised in an excellent way by Biedermann in Winter-
stein’s Handbuch 135) and by Jordan in the first part of
his: Vergleichende Physiologie 142).

The writer does not flatter himself to have collected the
whole literature and to some important papers little attention
has been paid. This was necessitated by his desire not to drown
the personal observations in the immense literature.

The groups of the Crinoids and the Ophiurids have been left
out for practical reasons, because no appropriate"material could
be obtained.

2. SPECIES USED.

As a representative of the group of the Asteroidea I have
used Asterias Forbesii (Desor) Verrill, a starfish, very common
around Woods Hole. It is as far as appearance is concerned,
practically identical with our A. vulgaris, but for the madre-
poric plate which is more clearly visible in the American
species by its brilliant orange color. ;

The sea-urchin which I used, Arbacia punctulata (Lm.) Gray,
is also very common in the neighbourhood of Woods Hole.
It is a rather small form of a dark violet color with long
spines. Its shell is semi-spherical in form, rather thick and
provided with a so-called epistroma. The periproct is oval, the
place of opening of the anus, covered by numerous sceletal
elements in other urchins, is in the family of the Arbaciidae
covered with four triangular plates. The interradia are open
in the neighbourhood of the apex.

The spines are flattened — in the way of a spatula — on
one side at their top. The tuberculae are not perforated, secon-
dary tuberculae are not present.

Thyone briareus. Lesueur (= Anaperus br. Pourtalés =
Sclerodactyla br. Ayres, Verrill) is the dendrochirote Holo-
thurian, which I studied in Woods Hole. It is dark brown-
greenish in color, almost black, ascidian-like ; its size depends
largely on the state of contraction of the musculature of the

4

body wall, animals of 15 c.M. eventually are as short as
6~7 c.M. Numerous small tube feet are found scattered all
over the surface of the body. Except for the enddiscs of the
tube-feet no calcareous bodies are found, according to Selenka.
Ayres finds numerous long plates with a kind of crown in
the middle. The members of the calcareous ring are fused
together completely. Five calcareous teeth are found in the
anus. The behavior of this species has been described in detail
by Pearse 98).

In Bermuda I had occasion to study Stichopus moebii.
Semper, a flat and very large aspidochirote Holothurian. It
has 18 gray tentacles; on the back only very few tube-feet
are found. One Polian vesicle and one stone-canal are present.
Its color is reddish-grey with black spots in the West-Indian
specimens — one on a hundred specimens in the Bermuda-islands
also has this coloration —, absolutely black in most of the
Bermuda specimens. Very strong calcareous elements are found
in the tube-feet.

3. ANATOMICAL DETAILS ON THE
DIGESTIVE TRACT ').

Related as the Echinoderms may be in anatomical respect
(,, This is one of the best characterised and most distinct Phyla
of the Animal Kingdom.” Ray Lankester) by their penta-
gonal symmetry, by the structure of their body cavities and
by their larval forms, physiologically there is a big difference.
The feeding habits are widely divergent in the different groups,
as will appear from the short descriptions, given in the next chapter.
Stichopus eats calcareous sand; Thyone, other cucumbers and
the crinoids catch small planctonts; the starfishes are the vora-
cious carnivores of the group; the urchins the rodents. Such
differences must give rise to big differences in morphological
respect and in fact the digestive tract is, at least as far as the
macroscopical anatomy is concerned, almost entirely different
in the various groups.

In the starfishes the organs of digestion have a very peculiar
structure. This is on one hand due to their ,,external digestion”,
which will be described in the next chapter, on the other hand
to their peculiar star-like shape. It consists chiefly of a large,
strongly muscular ,,stomach” in the disc and of the _,,radial
sacs’ or ,,pyloric coeca’, which usually lie in pairs?) in the

‘) This chapter does not pretend to be complete. It is beyond the scope of
the present paper to give such account; the interested reader can find enough
details in the numerous papers of Hamann 50), 51), and 514), of Tiede
mann 127), Frenzel 41), Jourdan 71) and 72), Koehler 74), Prouho
103) and others. A good key to this literature is found in 140).

*) In some cases — probably on account of mutilations — I found three of
these organs present in one arm.

5

five arms'). The mouth situated at the ventral side of the
animal, leads by a narrow neck, called the ,,oesophagus”’ 2),
into a voluminous sac, sometimes divided in two parts by a
circular sphincter, called the ,,stomach”’. In front of the mouth
we find the proximal members of the adambulacral series of
sceletal parts, the so-called mouth-spines. The stomach is
produced into five ,,stomach pouches’, sometimes forked, pro-
jecting into the arms. These pouches and therewith the whole
stomach can be retracted by means of two very thin and long
muscles, lying ventrally and lengthwise on the ambulacral
groove and fixed to the median ridge of the ray. The stomach
pouches are continued by one or two ducts — in the place of
bifurcation there is considerable variation — running out into the
arms. Here they form the so-called pyloric coeca, accumula-
tions of small pouches the little ducts of which all come out
on the chief duct. These radial sacs are dark greenish yellow
in color and attached to the dorsal wall of the coelomic cavity
by suspensory bands of membrane, called mesenteries, simple
continuations of the peritoneal lining of the coelom. Beyond
the pyloric coeca the alimentary tract is continued as a slender
»rectum”, ending in the anus. Both are thin, relatively small
and not of great importance; they are absent in the Astropec-
tinidae and the Porcellanasteridae. The rectum gives off 2—5
pouches, sometimes forked, which are called rectal or interradial
coeca. They mostly contain a dark, brown fluid; their function
will be discussed in the chapter on excretion, since they pro-
bably have excretory function. The radial coeca of the stomach
are very constant in structure and number; the rectal coeca
vary largely. In Asterias we find two simple fans, in the
Pentacerotidae there are five forked coeca, in Asterina five
simple sacs and in the Echinasteridae and Astropectinidae one
five-lobed coecum.

The histology of the digestive tract of the starfishes is in
its general features identical with that of all Echinoderms. We
can distinguish (from outside to inside): 1. an endothelial layer,
a simple continuation of the peritoneal lining of the coelomic
cavity, showing a. mucus cells and b. simple ciliated endothelial
cells, 2. a thin layer of connective tissue, 3. a layer of muscles
and 4. the epithelium which consists of long cylindrical cells,
ciliated in most parts, with nerve fibres in their basal part.
The mouth is opened and shut by means of an internal circular
sphincter and of external radial dilatators; in other parts the
longitudinal muscles lie at the inside, the circular ones are
external. In the stomach we find a very strong musculature ;

‘) If more arms are present (their number can be as high as 25, e. g. in
the Heliasteridae and Brisingidae), their number of course follows that of the arms.

2) Sometimes (in Echinaster and Cribrella f. i.) the oesophagus shows ten
pouches, five radial and five interradial ones.

6

in the radial sacs there is no mucle layer (Cuénot) or avery
thin one (Hamann). The epithelium of the oesophagus shows
three types of cells, all of which are shorter here than in other
parts: a. simple epidermal cells, ressembling those of the skin,
b. mucus cells with one to three cilia, but without cuticula
and c. granular cells, with brownish or yellow grains,
K. C. Schneider 141) in his comparative histology of animals
distinguishes these three groups as ,,Nahrzellen”, ,,Scheimzellen”’
and ,,Eiweiszzellen”. The cells of group a., the ,,N&hrzellen’”
of Schneider, are ciliated (in Echinaster spinosus f.i.), they
are very slender and cylindrical, have a thin membrane and a
, Stiitzfibrille’’, connecting flagellum and nucleus. The membrane
forms a kind of collar, a ,,Kragen”, sticking out from the
epithelium, reminding weakly of the choanocytes of the sponges.
They seem to play a role in resorption (Cuénot). Those of
group c. have the same structure and cilia as those of group a.,
but their cytoplasm is reticular and their secretory activity is
betrayed by the numerous granulae which they contain. The
granular cells are much longer'and much more typical in stomach
and radial sacs where the epithelium almost exclusively consists
of such ,,glandular cells’. Their granulae can be seen lying in
two or three regular lines and are very different in the different
stages of secretion (Schneider).

In the sea-urchins the digestive system is altogether different.
The mouth is situated at the centre of a peristomial membrane
which forms the so-called lips. Generally we distinguish five
of these lying between the five teeth. These are simple folds of
the skin; if the lantern is retracted they can close together so
as to make the whole lantern invisible. The gut commences
with a short vertical tube, a stomodaeum or pharynx, which
is surrounded by the upper ends of the teeth and their sup-
porting ossicles, the whole of which is called Aristotle's lantern,
a masticating apparatus of extreme complexity. Its mechanism
has been studied in detail by von Uexkill 128). The gut
has pentagonal form as long as it runs through the lantern and
on the corners of this pentagon we find columns of connective
tissue. A small piece between this stomodaeum and the stomach
is frequently called oesophagus. It is separated from the sto-
modaeum by a small muscular ring which narrows the lumen at
that place. It is cylindrical in shape.

The gut itself first turns around clockwise in one direction,
then bends sharply and comes back anti-clockwise. The first
part is a baggy, flat tube, sometimes called the stomach, running
horizontally round the animal supported by strings of tissue
from the coelomic wall. These mesenteries connect it at the
same time with the gonads, so that the whole hangs down in
a series of festoons, five in number. The second part, frequently

7

called the intestine, runs around in opposite direction and parallel
to the first. Its festoons alternate with those of the stomach ;
in Arbacia they are usually full of faeces. The gut ends by a
short rectum, opening into the dorsal anus on a space, called
the periproct. Parallel to the stomach and opening into it at
both ends runs the so-called ,,siphon” or ,,accessory intestine”
(Nebendarm), discovered in 1825 by Delle Chiaje. This struc-
ture is in some species (e.g. in Dorocidaris papillata, see Prouho
103)) merely a gutter, comparable to the endostyle of the Tuni-
cates, in others a narrow, cylindrical tube lined by cilia. A
blood-lacune runs lengthside of this tube (Hamann), In the
Regulares it follows the stomach, in the Clypeastroidea it makes
a short-cut. Its function is completely unknown, several possi-
bilities will be discussed in chapter 21.

The histological picture of the oesophagus ressembles that
of the starfishes very much. Outside of the layer of circular
muscles which is also present in tbe pharynx, we find here
longitudinal fibres and the ciliated peritoneum, both derived
from the lining of the coelomic cavity. The inside layer of
connective tissue and circular fibres is often called the visceral
or splanchnic part; the longitudinal fibres plus endothelium the
parietal peritoneum. The layer of connective tissue is separated
from the epithelium by a very thin, hyaline basal membrane
(Hamann). The epithelium of pharynx and oesophagus contains
two types of cells: 1. glandular cells, full of granulae and 2.
,otiitzzellen”, simple supporting epithelial cells. It is about ten
to fifteen times as high as all the other layers together.

Both types of cells are ciliated. The glandular cells-are espe-
cially frequent in the five corners of the pharynx and in the
neighbourhood of the mouth. At their base we find a layer of
nerve fibres. The granulae have been studied with much detail
by Hamann 51°), the same author gives an accurate picture
of the act of secretion. In Arbacia we find a pigment present
at the outer rim of the cells.

The first or direct curvature, as the stomach is frequently
and more rightly called, shows the same picture except for two
peculiarities: 1. between the layer of connective tissue and the
peritoneal lining we find a whole system of lacunar spaces, full
of ,lymph cells’; 2. the cells of the epithelium have almost
exclusively the glandular type, all cells are high, ciliated and
loaded with granulae of all colors. The first curvature is usually
much darker than the intestine; this is on one hand due to the
presence of pigment and secretory granulae in its epithelium;
on the other hand to the blood-vessels which are especially
frequent here. This indicates that this part of the gut plays an
important role in enzyme secretion and absorption. It also shows
many folds, in these folds the glandular cells are especially
accumulated (Hamann).

8

In the second or recurrent curvature the mucosa is much
thinner again and the epithelial cells do not have the highly
specialised type of those of the ,,stomach”’. ')

In the siphon the ciliated cells do not show any special
granulation. : ‘

The rectum is characterised by a thick layer of circular
muscles, its histology is the same as that of the rest of the gut.

The alimentary tube of the Holothurians shows four distinct
parts: 1. a short oesophagus, passing through the calcareous
ring (= Aristotle’s lantern), frequently folded longitudinally ;
2. a so-called ,,muscular stomach” (J. Miller), very short {less
than 2 c.M. in Thyone), initiated by a constriction and characte-
rised by its musculature; 3. the so-called intestine (Frenzel
also calls it ,,Chylusdarm”), very long — the whole gut is
frequently longer than 75 cM. in our small Thyone —; 4. the
cloaca” or ,rectum’’, a widened part, connected to the body-
wall by muscular bands transversing the coelom. The whole
intestine is suspended by bands of membrane, called ,,mesenteries”’
and shows the same general curvatures and clockwise turning
as in all Echinoderms, as a study of cross-sections shows. Only
in the Synaptids it runs nearly straight from mouth to anus.

In the stomach the food is thoroughly mixed with the visceral
fluid, kept there for some time and passed through in small
quantities. This is the case in Thyone where the food is swimming
about here and there in little clumps in the large quantities of
digestive juice. In Stichopus however, we find the whole gut
always full of sand, the process of digestion seems to be con-
tinuous here. The extreme thinness of the intestinal wall is
very striking and is common to many animals which eat mud
and sand for the sake of organic matter which they contain
(e. g. Sipunculus, Spatangus). Peristaltic movements, stronger
perhaps than those in Vertebrates, are characteristic for the
Holothurian intestine.

The histology of the gut of the Holothurians has been studied
by Jourdan 71). Externally we find the peritoneal lining of
the body cavity, a pavement endothelium. It has the same two
types of cells which are present in the other groups. Most of
them are simple endothelial cells which lie in a single layer:
they have cylindrical shape and carry vibrating cilia. In the
second place we find mucus cells, which have first been described
by Semper.

The second layer is the fibro-muscular one; it has the same
structure as in the other groups. It contains internal longitudinal

1) A special gland or coecumfoccurs in the digestive tract of the Spatangoidea:;
it is situated on the boarder of oesophagus and first curvature. This coecum
contains a clear brownish yellow fluid, slightly acid, which contains proteins
since a precipitate is formed by heat and alcohol (3—4 c.c.;V. Henry. 53)).

9

and external circular fibres with on both sides a layer of con-
nective tissue. In the outside layer we find the so-called mar-
ginal vessels, at the inside the general lacunar system and many
nerve fibres.

In some places of the intestinal wall the peritoneum leaves
an open space of ellipsoid form, bringing the external connec-
tive tissue (and with it the marginal vessels?) in contact with
the coelom, a so-called ,,button” (Hérouard). By them the
marginal vessels communicate with the general lacunar system
and here the circular musculature is perforated.

The cells of the epithelium are very long and have cylindrical
shape. They are covered with a fairly thick cuticula and show
two types. Many cells shaw a fine granulation and appear to
be secreting cells; others have the character of mucus cells. In
pharynx and cloaca we find normal epidermis cells.

Note. Of special interest is the presence of amiboid migra-
ting cells probably of mesodermal origin-though Frenzel denies
this- in the visceral epithelia of all Echinoderms. Biedermann
in Winterstein’s Handbuch 136) emphasises this point particu-
larly. Frenzel 41) already found numerous red migrating
cells in the normal epithelia of Toxopneustes and Spatangus,
which he calls ,,rothe Wanderzellen”. The color of their guts
is very different in different circumstances and physiological
conditions. In the same species of Toxopneustes f.i.. Frenzel
found the gut in one case black, at another time pink with black
spots, sometimes again the whole gut reddish-brown. This
partly accounts for the difference in color of the gut as a whole.
St. Hilaire 112)') also observed them and found the same,
cells present in the perivisceral fluid. Cohnheim and Cué-
not found the same thing and found the protein crystalloids,
studied bij List 81) and a fatty substance present in them.
Are they carriers of food? Most probably not, because they
only move towards the lumen, as Frenzel himself states.
Possibly they carry excretion-products to the lumen of the gut ;
this is rather probable from their movements which can even
be observed in vivo according to Frenzel. Enriques 37)
brings them in connection with the peculiar enzyme secretion
described by him in Holothuria tubulosa. According to him

1) Saint Hilaire makes a sharp distinction between the ,,granular cells”
and the ,,phagocytes’”. The granular cells according to him do not have any
phagocytical activities; they are so to say ,,unicellular glands’, formed in the
peritoneal epithelium and dying and dissolving in the intestinal lumen. They
do not participate in the clotting process and do not play a rdle in digestion:
they are neither ,,Fermentzellen”, nor phagocytes, neither do they contain any
reserve substances. They should take care of the excretion by taking up foreign
substances from the surrounding fluids, storing them as granulae and carrying
them through the gut-wall.

10

we have to do with something also found in other forms; in the
duodenum of the frog he described analogous cells 36). Frenzel
also calls them ,,Sekretzellen’’.

The argument of Hérouard that these groups of cor-
puscles do not have any duct and consequently can not have
excretory function, is of course valueless. His opinion that they
would serve for the transport of food, is not proved by any
experiments.

4. BIOLOGY. PREY.

The old story of the starfish devouring a bivalve, is too old
to be repeated again in full detail. As early as 1826 Eudes-
Deslongchamps 38) already described the process in the
charming, primitive way of the observing biologist. He found
starfishes on groups of Mactra stultorum. L. in the act of
devouring this prey. ,,Je remarquai qu’elles avaient introduit
entre ces valves de grosses vésicules arrondies, 4 parois trés
minces et remplis d'un liquide transparent.’’ Here he means the
stomach-edge of course, which is inserted between the valves
and applied directly to the soft parts of the prey. He takes
them for sacs in which there is a little opening through which
the liquid content, a ’’*humeur engourdissante’’, drips out gradually.

The opening of such bivalves is still always a problem when
we take in consideration the enormous force of the adductor
muscles of these animals. Many authors believe that a toxic
substance of some kind helps here — this problem will be
discussed in a separate chapter —, Schiemenz (Mitt. d.
deutschen Seefischereivereins. XII. 1896. p. 102. Quoted from
Mc. Bride in: Cambridge Natural Hist.) has figured out the
dynamics of the process. He showed, 1. that while a bivalve
can resist a sudden pull of 4000 grams, it yields to a long
continued pull of 900 grams; 2. that a starfisch can exert a pull
of 1350 grams; 3. that a starfish is unable to open a bivalve
unless it can raise itself into a hump so that the pull of the
central tube feet is at right angles to the prey. To prove this
he mentions the interesting case of a starfish, kept between
two glass-plates, walking around all day dragging about a bi-
valve, but unable to open it.

Mc. Andrew and Barret 2) soon found that the ,,vési-
cules” observed by Deslongchamps, are nothing else but the
stomach, protruded as it is filled with the perivisceral fluid. It
has considerable motility and can even penetrate to way up in
the shell of a Littorina, as I could observe myself in one case.

About the completeness of this ,,extra-intestinal digestion
(Jordan)”, practically nothing is known. Whether it is complete
as in Carabus 67) and Dytiscus-larvae or incomplete as in the
case of the mesenteric filaments, ,,Saeptalrdnder’’ of the Actinians,

11

where only a desintegration takes place, is not sufficiently known.
I did not get a chance to study this part of the problem, be-
cause only a few times I succeeded in securing animals in
the act of eating.

An important réle in the capturing of the prey is played by
the pedicellariae the mechanism of which has been studied in
detail by Perrier, von Uexkiill 129) and Jennings 62).
The latter author gives a photograph of a starfisch which has
captured five little crabs (Hippa analoga) in this way and keeps
them fixed on its back, Many smaller animals, as annelids,
copepods and others are captured in that same way, also by
the sea-urchins.

Even the seemingly completely inaccessible urchins, can be
captured by a starfiish. A very interesting picture of a fight
between these two has been given by Prouho 104). The
starfish allowed the gemmiform pedicellariae of its prey to get
hold of its body, then wrenched them off, and so on, till the
urchin was a helpless prey.

Even fishes can occasionally be captured by starfishes, as
Jennings decribes in his famous monograph on the behavior
of Asterias Forreri. Dead prey is also occasionally taken
according to Delage and Hérouard 136) p. 65. Cases of
cannibalism have been described by von Uexkill 130) in
brittle-stars, by Cuénot in Strongylocentrotus and by Prouho
103) in Dorocidaris.

Small animals (snails), like Littorina, Terebra, Strombus,
Murex etc. are frequently digested inside of the stomach. The
shell is then removed by the mouth, not by the anus. Shells
up to 3 c.M. in width can pass through the mouth of an
Astropecten.

Tremendous destroyers as the starfishes are, it sounds strange,
that they have almost no enemies, at least not in their adult
form. This may be due to the toxicity of their skin, most star-
fishes have moreover the premonitory color which is typical
for animals protected by chemical means (Heliconia, Ebolis,
catterpillars and insects, the Gasteracanthidae among the spiders).
The only enemies capable of attacking them with success, that
I know of, are the acid-secreting sea-snails, as Dolium and
Tritonium. Semon 119) reports that these attack starfishes,
urchins and cucumbers with much success.

Starfishes have considerable negative economical value.
Collins £i. (Bull. U.S. Fish. Commission. Vol. 9, 1889. Quoted
after Ludwig-Hamann) estimates the damage done by star-
fishes in one year (1888) on § 631.500.—. Cuénot 21) saw
that a natural oyster-bed of 10—12 K.M. length was completely
destroyed by starfishes, incidentally introduced by fishers in their
nets. Hamann found 10 specimens of Pecten, 6 Tellina, some
Conus and 5 Dentalium in the stomach of one Astropecten.

12

The food of sea urchins is very different in different species.
The only food that I actually observed our Arbacia eating,
was Clione sulfurea, a yellow calcisponge. The study of the
faeces however, will reveal us many other preys. The European
species, Echinus esculentus, chiefly feeds on the brown fronds
of Laminaria and other sea weeds (Mc. Bride in: Cambridge
Natural Hist.) and its small inhabitants, chewing them all up
with its teeth. According to Scott 117) it eats sea-weeds and
sand. Chadwick 13) considers this species as carnivorous and
found Balanus-shells in their guts. If some specimens are put
into an aquarium, in which some Balani are present, they will
immediately attack them and chew them up.

Not only such peaceful prey is takeri however. The Neapo-
litan species, Spaerechinus and Toxopneustes, even capture such
alert crustaceans as Squilla mantis, as Dohrn 32) reports.
They cover themselves up with mussels and other harmless
objects and move around camouflaged in that way. Suddenly
they attack their prey which can not escape any more. If a
dozen Toxopneustes were put into one bassin with a dozen
Squilla, it did not take more than eight or ten days for all the
crustaceans to disappear. The remarkable coordination appa-
rently present in these sluggish animals is very interesting from
the point of view of animal behavior and has been described
in detail by Dohrn.

These crustaceans are caught by means of the ambulacral
feet: as soon as one of them gets hold of a prey, all others
are loosened. Some smaller animals are caught by the pedicel-
lariae, some by the spines. H. Eisig (Kosmos. 8. 1883. p. 28)
describes the capturing of a little annelid by Echinus lividus
by means of its spines.

Sponges, Gorgonidae, fishes, Crustaceans etc. are also prey
of some urchins (Prouho 103) for Dorocidaris papillata). Even
calcareous algae are sometimes digested. Echinus miliaris bores
holes in rocks, covered with crusts of Lithothamnium poly-
morphum, for the sake of food, according to Hesse 58), maybe
for the sake of Ca and of protection against the waves, according
to Jordan 142).

Quite a different food is taken by the Spatangoidea and
the Clypeastroidea, They live in the sand and eat them-
selves through it. Since no representative of these groups is
studied in the present paper — the common sand-dollar, Echi-
narachneus parma, is too small for most purposes — I do not
consider it desirable to describe their very interesting habits
here; the reader can find them described in a paper by Hor-
nyold 61) and in von Uexkiill’s Umwelt und Innenwelt.

Holothurians usually eat mud. It may be of some interest to
describe the feeding habits of our Thyone a little more in detail.

13

Whereas our Bermudian species, Stichopus moebii, eats mud,
or rather the calcareous sand of the coral rifs and monotonously
continues eating it day after day, this species shows a little
more complicated behavior.

In its natural habitat this species lives in mud in shallow
places. This mud is of the darkest and richest kind, almost blue
in color and ‘full of detritus. If one puts some specimens in an
aquarium the bottom of which is covered with this mud, one
can observe that after a little while part of the animals have
buried themselves in the mud and on dissection the gut will
be found full of this material. After some time however the
majority of the animals is found partly buried in the sand, but
with their ring of tree-like tentacles sticking out of the mud.
These tentacula arborescentia are in constant motion and a
closer study of their behavior reveals a most interesting game.

Like nets these little arms move through the water ina fan-
like and slow movement, apparently for the purpose of fishing
small planktonts and débris out of the water. As soon as one
of these bumps against one of the tentacles, the organ slowly
bends downward and disappears into the mouth.

Of the ten tentacles the two ventral ones appear to be very
much shorter. Shortly after one of the large tentacles has disap-
peared into the mouth, it comes back, and the small tentacles
now functionate as ,,scratchers’ and whipe the returning tentacle
off. Another tentacle again disappears and the same rhythmic
game goes on for hours and hours at a stretch.

Has a Thyone once found a suitable place, it can stay there
for weeks and weeks, fishing in uninterrupted rhythm, once
about every 7'/, minutes (Pearse 98)), a most interesting and
fascinating spectacle. Cucumaria planci according to Dohrn 32),
has preference for thick bushes of algae in which they hang
for months.

From these observations it will be evident that Thyone is
by no means an exclusive mud-eater, and it can not astonish
us any more that, as we will see in chapter 19, the dried
contents of the stomach contain much more nitrogen pro gram
of substance than the mud in which the animals live.

Many other Holothurians live only on mud or sand, i.e. on
the organic material contained in it. The importance of this
process, the amount of bottom material ,,worked through” by
Stichopus moebii, has been estimated by Crozier 18).

Stichopus moebii. Semper, a very large species, such as are
eaten by the Chinese as ,,trepang”, occurs in great abundance
in the Bermuda islands on the shallow littoral bottom. Its sits
on the sand and eats it by means of its 18—20 shovel-like
tentacula peltata. Crozier determines the amount of sand present
in the gut when it is filled completely. Then he observes how
many times a day the gut is filled. This is possible because

14

there appeared to be a peculiar synchronism in the feeding
habits of different specimens. On certain moments of the day
almost all the animals had their gut either completely full or
halfway full or empty. In this way he could observe that as
a rule the contents of the gut are changed about three times
a day. By determining the number of animals present in dif-
ferent localities around the Bermuda’s — this is very casy
since the big animals are clearly visible through the transparent
water of the tropical sea —, he could figure out the amount
of sand displaced in a certain amount of time. Restricting him-
self as far as necessary in his assumptions, he came to the
conclusion, that in areas frequented by this species, roughly 6
to 7 kilo’s (dry weight) of sand passes through Holothurian
guts per year and per square meter. In the enclosed sink,
Harrington Sound, he finds that the quantity eaten annually,
is something like 500 to 1000 tons.

These figures illustrate more clearly than speculation could
the tremendous importance of these animals and show _,,that
the feeding activities of these animals may have an effect on
the sea-bottom not unlike that, so carefully described by
Darwin, which earthworms produce in the soil.’’ This is the
more probable, because as we will see later on, the gut con-
tents of this species are fairly acid, so that calcium carbonate
is brought into solution.

5. TOXICITY OF THE STARFISH STOMACH.

In almost every popular description of the attack of a starfish
on bivalve molluscs, we are told that the starfish after having
opened the shell, drops a few drops of ,,poison”” on the prey
and facilitates his work in that way. It seemed to me to be
worthwhile to investigate this question a little more closely
from the experimental side. The question, formulated more strictly,
is: Does the stomach of the starfish secrete a substance which
is poisonous to the muscles or in general, to the contractile
tissues of the prey? In-that way the secretion of the stomach
would have a double function: 1. to kill the prey or at least
to abolish the tonus of the adductor muscle, 2. to dissolve the
tissues more or less completely in order to make them fit to
enter into the stomach or the radial sacs.

The fact that as Eudes-Deslongchamps reports if an
oyster is taken away soon enough, it is dead even if it has not been
dissolved, pleads in favor of this hypothesis. If it had been
dead previously, it would smell as strongly as all decaying sea
material does; this is not the case. W. Hesz 59) also believes
in the presence of a toxic substance. Cuénot 22) speaks ofa
toxic mucus (,,glaire’”). The same mucus is according to him

15

secreted by the skin (p. 381; uric acid?). Schiemenz 115) does
not believe in any toxic action.

A sea-watery extract was made of a large number of starfish-
stomachs. Since the substance to be studied might be destroyed
by heat, the extract was made in the cold by vigorous shaking.
In order to avoid decay the extraction was not continued longer
than about four hours. The mass was now filtered and the
filtrate used for the following experiments. :

In the first place the action of this extract on the heart of
the bivalve Pecten was studied. This proved to be a very
typical one.

The heart of Pecten can be isolated very easily. If one
pulls the two shells which the animal always keeps open in
its natural environment, apart, one easily succeeds in tearing
the adductor muscle into two. If this is done carefully, the
heart is found in absolutely uninjured condition right under the
muscle. It is found beating in its pericard which contains a
fairly large quantity of ,,coelomic’ liquid. If left alone the
heart will continue to beat for a considerable length of time,
as contro! experiments showed.

By means of a small injection-syringe the stomach extract
was now dripped on the beating heart in a fine stream. The
first beats are perfectly normal and do not show any effect of
the fluid on the beating. But by and by the beats come at shorter
intervals and finally a highly accelerated and irregular beating
results. Though no counts were made, I dare say that the heart beats
more than three times as fast as usually. If the irrigation is conti-
nued, the reverse happens after some time. Gradually the rate
of heart-beats slows down, till it falls below the normal rate
and finally the heart stops in a contracted condition. The
whole process takes place in only a few minutes and strongly
impresses one that it is due to a toxic action of some kind of
the extract. The poison would in that way have a stimulating
effect at first and finally cause the stopping of the heart in
contracted condition.

Experiments of the same kind were made on the adductor
muscle of Pecten. Here however I did not succeed in obtaining
satisfactory results, though I was impressed of the fact that
the muscle weakened down after an initially vigorous reaction
to the fluid. Several trials for a graphical registration of this
fact gave disappointing results. By means of a light pulley the
movements of one free shell — the other shell was clamped
in a stand, while the open side of the shell was pointed
upwards — were transmitted to a counterbalanced lever,
writing on a drum, but the quite irregular behavior of the
muscle made these experiments a failure.

Better results were however obtained with a frog’s gastrocne-
mius. At regular intervals of 30 seconds the muscle was stimulated

16

and the twitches registered on a drum. From above the muscle
was slowly irrigated with an extract of the starfish stomach in
frog's saline. In a control experiment the same thing was done
with saline only.

The results of these experiments are clearly visible in fig. 1
and fig. 2. In the case of the extract we see how the muscle
_after a short time falls into a series of almost tetanic contrac-
tions, allthough the signal indicates that I only stimulated once.
The muscle remains in this semi-contracted condition for a long
time, regularly responding to electrical stimulation. The response
however, becomes weaker and is frequently irregular, after

a)

Fig. 1.
Poisonous action of extract of starfish stomach on frog's gastrocnemius.

about half an hour no response whatsoever is obtained for
several minutes. After the experiment is stopped, the muscle
does not even respond to direct stimulation any more and is
apparently exhausted or ,,killed’’.

A control muscle did not show as irregular a behavior, gives
the same response after half an hour as in the beginning, could
be stimulated both directly and indirectly after this time and
did not show the peculiar contracting phenomenon,

From this evidence the writer is inclined to believe that a
toxic action of some kind can be attributed to the stomach of
the starfish. Time for a closer study of this problem could not

17

be found: by the nature of the work in a summer laboratory one
can scarcely expect to exhaust a subject like the one taken up here.

I must mention however that the phenomena observed re-
semble closely those caused by the
toxic action of the ,,poison-glands”’ of
the Cephalopodes,

R. Krause (Zentralbl. £. Phy- [a |
siol. 9. 1895. S. 276), who was the
first to study these, observed ,,zuc-
kende Bewegungen der Extremitaten”’
in poisoned animals, followed by a
gradual weakening till death. The
same initial stimulation is indicated
here.

Lo Bianco (Mitt. Zool. Stat.
Neapel. Bd. 13. 1899. S. 530) describes
a ,incertezza della locomozione, sub-
entrando poco dopo movimenti con-
vulsivi, con un tremito rapidissimo
in tutti i piedi toracici,” which is
also followed by death. |

A. Briot (C. R. Soc. Biol. 1905
(T. I), p. 315) also mentions the ,,tré-
mulations des membres”.

These few indications will serve
to show that there is a certain ana-
logy between these poisons, at least
as far as their action is concerned.
Frogs seem to be sensitive to the Fig. 2.
cephalopode-poison, according to Control-muscle.
Krause. Briot however denies this, Baglioni 3) solves
this controversy by showing that the poison can only act here
after 10—20 minutes, when true typical attacks of clonic con-
tractions occur. In our experiments we also find contractions
occurring after a considerable length of time.

The poison of the cephalopodes, is according to Baglioni,
a poison of the C. N. S., which first produces clonic contrac-
tions, followed by a ,,Lahmung’’ of the central functions
afterwards. It seems to be of phenolic nature and this is the
more probable since crabs are very sensitive to phenoles.

The analogy between these two poisons has also been seen
by Piéron 100), who gives as a possible explanation ,,une
égale action chimique provenant de sécrétions, liées a l'appareil
digestif, sécrétion de l’estomac chez 1’ Astérie, sécrétion de la
glande salivaire chez le Poulpe.”

A closer investigation of the relation of this poison and the
,omnipresent” substance in these animals, which is colored blue
by Folin-Wu’s uric-acid reagent, seems most promising.

2

18

6. THE ENZYMES IN THE DIFFERENT GROUPS.

a. Proteolytic enzymes.

In my introduction I said that the work presented in this
paper would not only consist of the study of some new aspects,
but also of a checking up of the old data in the literature.
This is especially true of the present chapter; much was known
about enzymes in our group already. Nevertheless I have tried
to study some as yet obscure points and have come to results
different from those of previous authors in one case.

That there is a proteolytic enzyme present in the starfish,
is evident already from its biology. It has more over been
found repeatedly already, f.i. by Frédéricgq 40), Cohnheim
17), Chapeaux 15) and Griffiths 48).

Not clear however is the exact relation of radial sac and
stomach as far as the secretion is concerned: this question is
to be discussed in the chapter on the resorption in the starfishes.

About the chemical nature of the proteolytic enzyme we are
not very well informed either. Here as in almost all groups of
invertebrates, we find Krukenberg, who assumes that two
enzymes are present simultaneously, i.e. pepsin and trypsin. Roaf
and Bourquelot came to the same conclusion. How this is
possible, why he assumes the presence of pepsin when the
reaction of the digestive juice is on the alcaline side of the
neutral point, is not clear. Neither how these two enzymes can
act simultaneously while they require an absolutely different
hydrogen-ion concentration. All such assumptions of Kruken-
berg have been critisised severely by several authors, among
which Jordan 65), his ,,Helicopepsin”, ,,[sotrypsin” and ,,Ho-
maropepsin” have been shown to be fictions.

His ideas on the simultaneous presence of pepsin and trypsin
at the same spot and in the same organ have also been critisised
from several sides. In Protozoa, where an acid reaction in the
vacuoles preceeds a later stage of alcalinity ~ as first discovered
by Metalnikof 83) ~, he assumed the same thing. Later investi-
gations, like those of Greenwood 47) and Nirenstein 94),
have shown clearly, that the acid period only serves for the
»killing’” and preparing of the prey, but that no digestion takes
place during this time. The digestion and resorption take
place in the alcaline period.

In order to study the nature of the proteolytic enzyme present
here a little more in detail, several digests were made. Some
of them were acidified with 1.3 pro mille HCl, another part
was kept alcaline by means of sodium carbonate. As a sub-
strate either gelatin or egg-white — raw — were added. Of
lateral sacs and stomach equal quantities were used; 100 c.c.
of water were added in each case and some toluene.

After four days the digests were taken out of the 37° in-

19

cubator and in the protein-free filtrates tests for peptones —
biuret — and for amino-acids — ninhydrin — were made. The
proteins were precipitated by means of an excess of trichloracetic

acid and filtered off.
The results of these tests are given in the following table:
Table 1.

Peptone and amino-acid tests in several digests of
starfish material.

; 1
. Reaction. Material. Biuret. | Ninhydrin.
|
Egg-white.| Gelatin. | Egg-white.| Gelatin.
Alcaline {Stomach. _ + ae +
digest. | )Radial sac. ~ + _ as
Acid digest. Stomach. +7 + - a
Radial sac. +7 of = a

From this table three things are evident. In the first place
we see that qualitatively at least there is no difference between
the activity of the stomach digest and that of the radial sacs.
We will come back on this point in the chapter on the resorp-
tion in the starfishes.

Secondly we see that gelatin is much better as a substrate
than egg-white, which does not astonish us when we remember
from human physiology, how relatively indigestible raw egg-
white is.

In the third place we find that either the enzyme present
acts just as well in an acid medium as in an alcaline one, or
that Krukenberg is right in assuming the simultaneous pre-
sence of the two proteolytic enzymes.

Therefore we must try to put the question on a more quan-
titative basis, which I did in the following way:

11'/, gr. of radial sac and of stomach material were ground
up with sand. Equal quantities of gelatin, toluene and 100 c.c.
of water were added to each digest, one of which was acidified
as described above, the other one kept alcaline. After 24 days
they were removed from the incubator, the proteins pecipitated
with an excess of 5°/, trichloracetic acid and filtered off.

In the protein-free filtrate total-nitrogen determinations were
made on an aliquot, according to Folin’s method. The Nessleri-
sation was direct.

In the acid digest 25.4 mgr. of non-protein nitrogen were
present, in the alcaline one 144 mgr.

This proves clearly that only one enzyme is present, i. e.
trypsin, which as we know, works over a relatively wide range.
If pepsin were present, we would expect much more non-protein

20

N and not about one sixth of what we find in the alcaline
digest. Not improbable however is that the trypsin present
here should work over a wider range than the trypsin of
mammals.

Another fact which proves that trypsin is at work here, is
the regular occurrence of amino-acids in the acid digests:
pepsin, as we know, does not carry the hydrolysis any further
than up to the peptones.

In order to make sure that trypsin can work in this degree
of acidity, I ran a control in which pure trypsin (Merck)
was used. After three days already I could demonstrate the
presence of peptones and amino-acids, after two weeks J found
16 mgr. of non-protein N.

It does not seem improbable though, that in these lower
forms we may have to do with enzymes which do not fit into
the classical division of trypsin and pepsin. I have the impres-
sion that probably there is a whole series of enzymes, working
at very different optima of Py, of temperature etc.

An interesting paper by Rakoczky 106) has given.some
experimental evidence for this view. He compares the pepsin
of the dog and of the pike. Both had been extracted in exactly
the same way, yet there were very decided differences in their
activities. Some proteins are attacked more easily by one, some
by the other enzyme. On the whole the dog’s pepsin has. a
greater latitude of possibilities. The optimal acidity is higher
for the dog’s pepsin. Pike pepsin is destroyed much more easily
by higher temperatures, on the other hand it stands freezing
much better.

All these properties are extremely purposeful (,,zweckmassig’’),
from the biological standpoint. It seems as though everywhere
such enzymes are present as are necessary or desirable in the
conditions. A detailed investigation of the lower forms from
this point of view seems very worthwhile.

In Echinoidea the same proteolytic enzyme is also present,
but the enzyme appears very weak here when we compare
its action to that of the strong enzyme of the starfish. After
eight days no amino-acids could yet be demonstrated, though
a peptone-biuret was positive. Later on, especially when dialys-
ing tubes were used, they could be demonstrated very easily.
Roaf 108), Henry 53) and Krukenberg (Toxopneustes)
also found a protease in the species they studied. ;

In Holothurians the presence of proteolytic enzymes has
repeatedly been denied. One of the authors who were not able
to find it, in Holothuria tubulosa, is Cohnheim 17). He tried
to find it in several different ways, but came to the conclusion
that no such enzyme is present. In other chapters — that on

21

autolysis and that on the nitrogen metabolism of Holothurians —
we will discuss his arguments more in detail; here I only
mention my own experiments.
Several digests were made in which either egg-white or
gelatin, Na,CO, or HCl were used as described before.
After one week and a half the following tests were made:

Table 2.
Reaction. Substrate. Biuret. Ninhydrin.
Na,CO, gelatin . -b —_—
egg-white. -+- sw
HCl gelatin. + =
egg-white. + | je

The amino-acids which could not be demonstrated in the
protein-free filtrates soon appeared to be present in dialysates.

Summarising our results on the proteolytic enzymes, we may
say, that only one enzyme is present, i. e. trypsin, which possibly
works over a wider range of Py than the same enzyme in
mammals, but that no pepsin is found. The enzyme is present
in all groups studied.

b. Sucrolytic enzymes.

Invertases are also present in the three groups. Experiments
of exactly the same nature as those described above, were
made, in which cane-sugar (glucose free, the so-called rock-candy)
was used as a substrate. The enzyme is much stronger in the
starfish and Thyone, than in Arbacia in which form it is
very weak. The same observation was made by Cohnheim in
Sphaerechinus granularis. This author considers the invertase
in the starfishes as an adaptation to a sweet, but not reducing
sugar of unknown nature present in bivalves around Naples.

As far as the amylolytic enzymes are concerned our experi-
ments were complete failures. 1 do not know whether I must
blame this on my method, because I used the organs themselves,
be it after having crushed them with sand, instead of extracts,
as they have mostly been used by previous authors'). At any
rate, I did not get any positive Fehling’s tests in the protein-

1) Frédéricq 40), Griffiths 48), Chapeaux 15) and Clerc 16a)
found an amylase present in starfishes; Krukenberg, Cohnheim 17) and
Henry 53) in sea-urchins; Clerc 162) and Cohnheim 17) in cucumbers.

22

free filtrates of any of the digests. In many cases I could convince
myself, that starch which was added as such or after it had
been boiled for a few minutes, was still present. The blue
roan which the digest assumed with J in KJ proved this
clearly.

Though Fehling’s test was entirely negative in all cases,
Benedict's reagent gave results which were neither positive
nor negative. In some of the digests I found that the blue
coloration had disappeared and in its stead a red color was
seen. Maybe this is to be explained in the following way:
Biedermann 8) found that ,,durch das Mitteldarmsecret einer
Raupe aus Starke fast nur Erythrodextrin und nur sehr wenig
Zucker gebildet wird.” He concludes that we have to do here
with an amylolytic enzyme entirely different from that in snails
and other animals. This might account for my observations
which greatly puzzled me during my stay in Woods Hole.Since
however Biedermann’s paper came too late to my attention,
unfortunately I could not study this problem more in detail.

c. Lipolytic enzymes.
For these I refer to chapter 16.

7. ARE THE ENZYMES PRESENT IN THE FREE FORM?
PHAGOCYTOSIS IN ECHINODERMS.

A question of great importance is whether the enzymes on
which we have reported in the preceding chapter, are present
in the free form.

We know that in unicellular organisms the whole process
of digestion is an intracellular one. In a single food-vacuole
everything is digested, all enzymes are secreted and even diffe-
rent degrees of acidity are realised here successively.

In pluricellular organisms, in their most primitive forms, the
same .state of affairs persists. This is especially true of the
lower worms, the coelenterates and the sponges. The entoderm-
cells of these animals can easily be compared with protozoa
of different groups, which take care of one of the functions in
the service of the organism as a whole. The chemical destruction
of the food which prepares the same for the assimilation by
the living substance, is in that way originally a purely cellular one.

The excellent study of Jordan 66) on digestion in Actinians,
gives a clear idea of phagocytical processes of this kind. And
yet the process is in some way complicated here; the large
prey of these animals necessitates a device of some kind for
breaking them down. Mesnil’s hypothesis (Ann. Inst. Pasteur.
T. 15. 1901 p. 352—397), that a kind of dissection and frag-
mentation by the rims of the saepta would take place, aided
by a small quantity of proteolytic enzyme, secreted on contact,

23

seems to be wrong. Jordan could show this by folding a prey
in blotting paper; this prey was broken down just as well
though less vigorously.

‘One of the first authors who clearly realised the importance
of this way of digestion and understood it as the phylogenetic
precursor of the process in higher organisms, was Elie
Metschnikoff 84). in his paper on intracellular digestion in
coelenterates. The epithelium of the coelenteric cavity of these
animals may be called an amiboid epithelium, in some species
(e. g. Praya diphyes, a Siphonophore) a true plasmodium is
even formed. In Ctenophores the food is carried further into
the’ body by mesodermal cells, as in Sponges.

An excellent monograph on the intracellular digestion in the
flat-worms has been given by Saint-Hilaire 113). The food
taken by these animals is found as such in the cells of the
lining of the gut. Blood-corpuscles f.i. keep their characteristic
shape and only later they are gradually broken down. The
products of digestion are collected in special vacuoles and
deposited there in the form of crystalline structures. Numerous
cell-inclusions appear to grow at the expense of the food as
it is broken down. ')~

In the ,,higher’’ animals conditions are totally different.
Apart from the Gastropods (Enriques, Biedermann and
Moritz) digestive phagocytosis has been observed in no group
of the higher animals. Digestion has become an extracellular
act: it takes place in the ,,interior exterior’ and it is doubtful
whether in the resorption the separate protoplast plays a réle
as such. Here the free enzymes are not, as in the Actinians,
a preliminary adaptation, a ,,Hilfsmechanismus’, but the very
essential thing in digestion.

In snails we find a kind of transitory stage. The so-called
nliver” of these animals (,,Hepatopankreas’’), has become an
organ of special interest since the investigations of Bieder-
mann and Moritz. In the digestive juice of the represen-
tatives of this group all enzymes — up to a cytase studied
specially by Alexandrovicz 1) —are found free except for the
proteolytic one. The proteolysis is localised in the liver. Protein
is actually digested, as a careful analysis of the faeces and the
food-N shows; fibrin is not digested however, if treated with
the stomach fluid or with a watery extract of the liver. A free
acid is formed, as we will mention in chapter 17, the whole
mass becomes gelatinous, but no _ ,,digestion” takes place. Com-
pletely different is the result however if the shreds are placed
between two cut surfaces of the liver. In less than no time it

1) 1. Even a vacuole is not always necessary, as le Dantec showed
for Gromia and Pfeffer for Myxomycetes in the case of crystals of asparagin.
2. Excretion also takes place by the same epithelium in many of these forms.

24

is digested and melts away to a greyish mass which can scarcely
be discerned from the surrounding liver material. This problem
has also been studied by Stiibel 125). These experiments seem
to show that the protein can only be dissolved by living liver-
cells and shows something of the nature of the activity of
the mesenterial filaments of the Actinians, as it was pictured
by Mesnil. Jordan 69) made moreover phagocytosis in
these animals probable.

From this point of view an investigation of the Echinoderms
in this respect seemed most promising. It appeared however
that all the enzymes are found free here; this group ranks
high as far as the physiology of its digestion is concerned.

In order to get some definite information on this question, I
made a series of experiments on the digestive juice, as secured
from the intestine of fresh specimens. In Thyone and Arbacia
this is very easy, one simply punctures an isolated loop of
intestine, washed in sea water, which as a rule are full of
liquid. In Asterias the liquid was collected by pressing on the
outside of the stomach, after the arms had been cut off. The
few drops of liquids which appeared on the oral disc were then
collected. In this way I did not succeed of course in separating
the digestive juice completely from the perivisceral fluid. The
method however seems to be the only practicable one.

To these liquids a certain quantity of substrate was added
in a deep-depression slide and a droplet of toluene. The whole
was covered with a cover-glass and as a rule evaporation was
prevented by the use of vaseline. The slides were then put
into the incubator at 37° and inspected after different intervals
of time.

It soon became evident that all the enzymes are present in
the free form. Our trypsin-like enzyme is present in a free
form in the digestive juices of every one of our three species.
Small shreds of fibrin were dissolved within 24 hours, the same
happened to pieces of gelatin. A positive ninhydrin — which
test was negative in controls — demonstrated the presence of
the products of enzymatic action. In some cases a positive
biuret for peptones could be obtained.

No evidence whatsoever could be obtained of an amylolytic
action of the visceral fluid. A microscopical examination of
starch grains which had been digested with the digestive juices
for one, two or three days, did not reveal any changes. A
slight swelling takes place in the grains, probably due to the
alcalinity of the medium, but no trace of corrosion can be
observed. In one case I found a reduction of F ehling, but
a control showed that the starch used here was not entirely
trustworthy.

These results are the same in starfish, urchin and cucumber

25

and even continued digestion does not give any evidence of
the presence of amylolytic enzymes in free form.

Strange enough the same thing is true in many cases if cane
sugar is used as a substrate. In the previous chapter we have
seen that an invertase is certainly present in the representatives
of this group. It could however not be proved so readily that
this enzyme is present in free form, in Thyone I only got one
positive result on six tests, in Arbacia one on five '). In Asterias
no positive evidence was obtained.

I am at a loss for an explanation of this fact. Maybe it
means that there is a certain periodicity in the enzyme secretion.

For experiments of the same nature on fat I refer to the
chapter on the digestion of fat.

When we look over the general result of these experiments,
we see that those enzymes which are present in the watery
digests, are (occasionally) found in free form.

On the other hand: the enzymes present are rather weak;
we will be more impressed of this fact when we come to
consider the gut-contents of Thyone and the faeces of Arbacia.

The mucous membrane in which the food is included in the
digestive juice of the sea-urchins according to Scott 117) and
Roaf 108), is other evidence in favor of the occurrence of
free enzymes. These membranes are sometimes formed already
in the first part of the gut, mostly however later on. In such
conditions there is no possibility of phagocytosis or anything
of the kind; digestion and resorption must take place in and
from this food-vacuole.

Whether or not phagocytosis plays a réle in the digestive
processes of our group, is hard to decide. I started the present
investigation with the idea of finding very primitive conditions.
For this reason I made many experiments in which I fed
urchins or starfishes on suspensions of carmin or bone-black.
In the large amount of material collected from such animals
I found but one section in which the grains of carmin could
be seen lying inside of the cells (Fig. 3 of our colored plate).
One positive experiment of this kind of course does not prove
much; a closer investigation of the importance of phagocytosis
in the digestion of the Echinoderms seems very well worthwhile
however.

The fact that carmin is found in granular form inside of
the cells, does not always prove phagocytosis. H. Eisig 35>)
in the case of Capitella assumes, that carmin is absorbed in
dissolved form and reprecipitated in the cell-body. In fact no
other evidence whatsoever of phagocytosis was obtained.

1) Roaf also failed to find any evidence of the presence of asaccharase in
the Echinoidea.

26
8. AUTOLYSIS IN ECHINODERM GUTS.

Discussing the presence or absence of proteolytic enzymes
in Holothuria tubulosa Cohnheim 17) states that he could not
observe a trace of autolysis in their guts, if they were kept
under aseptic conditions. This observation is doubtlessly right;
I could observe something ‘similar in our Thyone, its gut can
be kept for an almost indefinite length of time in-sea water
under toluene without showing signs of profound changes.
Is this however an argument in favor of his contention that
Holothurians should have no proteolytic enzyme in their gut
and that the N-metabolism of this group does not amount to
anything? -

I do not believe it. In the first place we must remember
that the gut of these animals is strongly muscular and that
muscle-tissue autolyses much less completely and rapidly than
any other kind of tissue. It is very well possible that the
epithelium of these guts autolyses away while the muscular
wall keeps its coherency and gives the impression of being the.
whole gut. As a matter of fact we can observe a change in
color and other changes in these guts allthough they do not
fall to pieces. oe

To settle this argument and to find out whether Cohnheim
is right in his conclusions, I decided to- study autolysis of the
guts of Asterias and Thyone a little more in detail. In Asterias
nobody will doubt the presence of a proteolytic enzyme and
yet autolysis proves to be exceedingly slow even in this case.

For this research I ground up a large quantity of radial
sacs and of stomachs of Asterias with sand. Some water was
added to these masses, a large quantity of toluene and a trace
of Na,CO, in order to keep the reaction of the digests in the
neighbourhood of neutrality.

The digests were then put into the 37° incubator and left
there to autolyse. At different intervals I made tests for pep-
tones and amino-acids by means of the biuret reaction and
ninhydrin on the protein-free filtrate. The proteins were as a
rule precipitated with 5 °/, trichloracetic acid. The liquid: as
it came through the filter was clear, but not completely color-
less, perhaps on account of the fact that a small quantity of
a water soluble pigment which proved to be very troublesome
in all these experiments, was not precipitated out.

After six days all these tests gave completely negative results.
After nine days I got a positive peptone-biuret in the radial
sac sample, but no evidence whatsoever was seen of autolysis
in the stomach sample.

The radial sac sample became one homogeneous mass, but
the stomachs did not seem to be liquified, they could still
be seen separately.

27

The same result was invariably obtained for a period of
about two months; in the protein-free filtrates I never got
positive results with ninhydrin tests.

The digests had a slightly acid reaction which is optimal
for autolysis. Yet autolysis seemed to proceed exceedingly
slowly. This may of course have different reasons: it is possible
that the proteins of these tissues have a great resistance
against proteolytic enzymes. We saw in the chapter on the
enzymes that other proteins are digested much more rapidly,
this might be an indication in this direction. It is also possible
that the conditions of reaction were very unfavorable; from
what we know about autolysis in other forms, this does not
seem very probable however. In the third place it is very
well possible that the quantity of enzyme at hand is very
small,

Autolysis occurs however, and this was more evident yet
when I dialysed a small quantity of the. digest through a thin
collodion sac. A strongly positive ninhydrin test in the outside
liquid showed that amino-acids though they did not show up
in the protein-free filtrate are surely present.

The same thing proved to be the case in autodigests of
Thyone guts. No trace whatsoever of autolysis could be
obtained in the protein-free filtrates. The guts remained in
ualiquified condition for a long time and nothing indicated
that autolysis were it even very slight, was going on. Yet
when I dialysed part of the liquid, I obtained a positive
ninhydrin reaction in the outside liquid.

From this evidence I have concluded that autolysis goes on
very slowly in the guts of Asterias as well in those of Thyone.
There is no doubt however as to whether it actually takes place.
And there can in that way be no doubt that a proteolytic
enzyme must be present in their guts.

But one thing is very evident on the other hand, i. e. that
in these form this process does not take place with the same
intensity and rapidity as in the higher forms on which bioche-
mists have been working up to the present day. The solution
of the problem why ‘this is the case, must be left open for a
more detailed investigation.

9. CONTENTS OF THE GUT OF THYONE.

In studying the contents of the gut of a particular species,
we can on one hand get some information about the natural
food of the species, on the other hand we may get an idea
about the enzyme activity in their gut.

For the present study I used animals which had been brought
into the laboratory on the same day on which the observation
was made. Their gut-contents were put ona slide and examined
through the microscope.

28

Apart from numerous sand-grains many pieces of decaying
plant-tissue were found, recognisable by the cell-walls and by
their brownish appearance. On the other hand however |
could frequently see different species of unicellular green algae,
which were perfectly intact and still in the possession of green
chloroplasts, a nucleus and all other structural details. . Many
empty diatom shells were present, also a flagellate species,
empty, with cirri sticking out. Many diatoms however were
perfectly intact just as the green algae. Encysted Protozoa (?),
perfectly round bodies, also frequently occurred. A complete
copepod was seen, not moving, but very complete up to the
smallest brittles, the eye full of pigment. Nothing gave the
impression of being dissolved or dissolving, the heart was not
beating. Pieces of other Crustaceans were also found, some-
times nothing but the chitinous sceleton.

Large quantities of detritus were present in another animal
of which I studied the contents of the rectum. Here also
complete green algae, uni- as well as pluricellular forms were
found. Diatoms, either empty shells or in full possession of
all the cell constituents, green algae, thread-like and with
contracted cytoplasm — as if in a hypertonic solution ~—,
were also very abundant. Pluricellular red algae with contracted
cytoplasm, but uninjured and in full possession of the natural
colors were also found occasionally.

To this list Pearse 98) still adds nematodes and an ostracode.
In every animal I dissected many alive Protozoans were found
in the rectum, moving around with great speed in the visceral
fluid. According to Pearse they belong to the genera Gym-
nodinium and Lichnophora.

Ulva-leaves digested with the digestive juices of Arbacia
and Thyone did not seem to be very much attacked. Some-
times the cells at the outer margin had a slight brown color,
the whole leaf, even after digestion at 37° for one or two
days, remained green however and kept all its structures.

10. COMPOSITION OF THE FAECES OF ARBACIA.

A study of the faeces of Arbacia was made with a double
purpose. In the first place it may give us an idea as to the
intensity of action of the enzymes in the intestine, secondly it
may give us some information about the feeding habits of this
species.

The faeces of this species are most easily secured. Shortly
after the animals have been brought into the laboratory, their
back is as a rule covered all over with small, oval bodies of
a white color which prove to be their faeces. Even after 5—6
days the gut is still always not empty and faeces occasionally
produced. According to von Uexkiill 128) defecation takes

29

place by the pressure caused by retraction of the mouth-mem-~
brane.

Several samples were studied a little more closely. Some
contained practically nothing but sand and some detritus, others
however were full of interesting substances, most of which
could easily be identified. Some were full of calcareous material,
as a foaming with hydrochloric acid proved; others did not
contain anything of the kind.

As examples of materials which I could easily identify, ] may
mention the following constituents.

Several species of diatoms were seen. Some of these moved
around very actively. Though I tried to avoid all contamination
with the sea water of the aquarium, some planctonts may have
come into the material; for this reason I am not inclined to
consider this observation as very important.

Otherwise it would be a very convincing demonstration of
the. weakness of the action of the digestive juice. Some unat-
tacked unicellular algae were also found. Sand-grains are present
in nearly every sample. Brittles, maybe of Bryozoa, are abundant.
A hydroid was found (Sertularia?). In some of the compart-
ments a: seemingly intact, but contracted individual was seen.
Spiculae were seen, probably derived from sponges. Red algae,
also seemingly intact, frequently occurred. The granulation of
the cell-contents and the chromatophores were clearly visible.
It’ was a pluricellular branch: it did not seem to have been
attacked severely.

Peculiar honey-comb-like structures, like minute plant-tissue
(Cyanophyceae?), could not be identified. A leg of a copepod
seemed to be attacked and the flesh dissolved away.

‘From this evidence we are once more impressed of the relative
weakness of the enzymes present in the free form in this group.
Of the large amounts of material which pass through the gut
only a very small-part can be attacked succesfully, some sub-
stances pass without serious changes. If really alive organisms
are present, this is the most convincing proof; this does not
seem to: be very probable to me, though. ')

As far as the-feeding habits of the species are concerned,
we see.that it is practically omnivorous. Most probably it eats
nearly everything, chews algae, also calcareous species, sponges,
Bryozoa etc., by means of its strong masticating apparatus, and
in this way also gets hold of its microscopical inhabitants and
some other materials. The only thing which J actually observed
an Arbacia eating, was Clione sulfurea, a calcisponge.

1) The same incompleteness of the digestion has been found by Plateau
(Recherches sur les phénoménes de la digestion chez les insectes. Bruxelles.
1874) in catterpillars and by Knauthe (Untersuchungen iiber den Stoffwechsel
der Fische. Zeitschr. f. Fischerei..5. 1897. 189) in fishes.

30

11. HYDROGEN-ION CONCENTRATION
OF THE GUT CONTENTS.

The enormous importance of the concentration of the
hydrogen-ions in almost every biological process, so clearly
realised by the numerous investigators who followed the
example of Sérensen, justifies a closer investigation of the
digestive liquid of the Echinoderms in this respect and a
comparison with the very scarce studies on other invertebrates.

One reason why these studies have always been so very
scarce, is the fact that methodologically biologists were so very
poorly equipped. It took a long time before the Pu work
penetrated through all the branches of biology, so that allthough
Sérensen’s work appeared already in 1902, even biologists
with the physiological attitude, have been going on using the
old fashioned methods, chiefly the mere coloration of indicators,
for the determination of acidities of media to be investigated.

Plateau (Mém. de l’Acad. Roy. de Belgique t. 41. 1874)
studied a series of myriapods, arachnids and crustaceans as to
the reaction of their digestive juices in this way. He found
that in all articulates the gut contents are alcaline, sometimes
near neutrality, but never acid. In a later paper he states that
frequently a weakly acid reaction is found in carnivores and
omnivores, but that the digestive juice of herbivorous species
is always alcaline.

Other authors came to similar results, mostly the contents
of the fore-gut are more acid than those of the other parts.
On this point I will come back presently.

The only investigator who ever studied the reaction in
Echinoderm guts, was Roaf 108). Though his method was
relatively up to date in his days, it does not give us as
complete information as one might desire. The argument of
his investigation was the question which we have discussed in
the chapter on the enzymes, whether it is possible to have a
tryptic and a peptic enzyme present, working at the same
time. He succeeded in accomplishing what I never could get
done, his starfishes and urchins took fibrin colored with diffe-
rent indicators. He now determined at the changing point of
which indicator the Py of the intestinal juice lies. The follo-
wing indicators were chiefly used: dimethyl-amido-azo-benzene,
congo-red, litmus, neutral-red, phenolphthalein.

In the case of the sea-urchin whose feeding reactions this
author describes in detail, he found by comparing the results
obtained with different indicators, that the hydrogen-ion con-
centration of their intestinal juice is about that of the changing
point of neutral red, i.e. Py = 8. This indicator which is
colored yellow by sea water, turns red in the gut of the urchins.
The intestinal juice is alcaline to litmus however.

31

The indicator diffused out of the food masses, so that no

definite information could be obtained as to the reaction’ of
the middle- and end-gut.
. Asterias was fed on fibrin which had been colored with
congo-red. The initial reaction proved to be on the alcaline
side of this indicator, that is Py >) 4. The rectal coeca are
blue from the beginning and remain blue. This is interesting
in connection with their function as uric acid excreting organs,
which will be discussed in chapter 23. On treatment with
alcali however they contract and squeeze out a liquid which
instantaneously changes the indicator into red. After their
initial alcalinity the food masses are transported into the
interior of the radial organs, which show a distinctly blue
color, ,,as if containing a blue solution’. This would mean a
very high acidity, which Roaf does not quite seem to realise,
since the changing point of congo-red is at Py = 4. This
acidity has however also been found by other authors. ,,Das
Sekret reagiert schwach saver”. Schneider 141). p. 653. ,,Les
coecums pyloriques sécrétent un liquide 4 réaction acide.” Delage
et Hérouard 136). p. 66. Also Stone 124) '). Chapeaux
finds the secretion alcaline.

The same strongly acid reaction was observed in the small
species, Porania pulvillus, which was fed on vesuvian-brown
(changing point also at Py = 4). Roaf concludes that the
reaction of the pyloric coeca is about that of a decimolar
solution of sodium di-hydrogen phosphate. Here also the rectal
coéca appeared to have a brown color, which shows an acid
reaction.

Of very recent date is a paper of Jameson and Atkins 614)
on the physiology of the silk-worm. These authors used the more
recent method of Clark and Lubs ”), — see for a description
of these methods the excellent book of Clark on the deter-
mination of hydrogen-ion concentration 134) —, comparing the
color of a drop of digestive juice added with some indicator
to the colored plates of Clark 134). A decided acidity could
be observed in the gut-contents of the imagines, the Py = 5.2 ~
5.8. In the larvae however the digestive secretion proved to be
alcaline (Py == 9.0 — 9.8); in the hind-gut somewhat less strongly
alcaline (Py = 8.4). The acid mulberry leaf is apparently ,,over-
neutralised” in the intestine.

My own experiments were made with the same indicator
method. I had the privilege of using a set of Sdrensen-
standards, made up by Dr. J.B. Collip of the U.o. Toronto

1) Stone found the reaction of the secretion of the pyloric coeca slightly
acid: they turned litmus blue. This acidity must be due to some organic acid
since tropaeolin OOO which is extremely sensitive to mineral acids showed
no change of color. Lactic acid? v. d. H.

2 J. Bact. 2. 1917. 1,109,191.

32

and used by him in his work in the M. B. L. The standards
were kept for about a month in Jena Erlenmeyers closed
tightly with rubber stoppers.

Juice of the different parts of the gut of Thyone and of
Arbacia was secured. One drop was put on a white porcelain
plate on which a series of standard drops had been laid out
previously. Equal quantities of indicator were added to the
different samples—I used a narrow capillary tube for this purpose —
and the color comparison made. As an indicator I used phenol-red.

On Thyone four sets of determinations were made. A series
of figures was secured for the different parts of the gut. This
is especially easy in these animals on account of the length of
their gut and because their intestine is nearly always full of
liquid. The results obtained in this way are represented in
table 3.

Table 3.

Hydrogen-ion concentration in Thyone gut.

: 1, 2. 3. |Average.

Stomach. 7.4 7.8 7.5 7.6
Intestine

7.0 7.6 7.1 7.2

7.8 7.8 7.4 7.7

8.0 8.0 7.7 7.9

¥ 8.2 —_ = 8.2
Rectum

In the urchins it proved to be very difficult to secure liquid
from definite parts of the gut, partly on account of the more
complicated anatomical relations, partly because these guts
usually do not contain much liquid. I obtained two series of
figures, but I do not give them any more value than just
figures of the Py of an arbitrary drop of digestive juice. Their
values are represented in table 4.

Table 4.
Hydrogen-ion concentration in Arbacia gut.

33

In the starfishes it is most difficult to secure any digestive
juice. Since, as mentioned before, I did not succeed in feeding
these animals artificially, I had to be content with a drop of
liquid eventually falling out of the organ to be tested. Consi-
dering the roughness of such procedure, it is rather remarkable
that even these drops had a Py very much different from that
of the perivisceral fluid by which, though the utmost care was
taken to avoid this, it might have been contaminated. The Py
of the radial sac fluid appeared to be 7.3; that of the contents
of the stomach 7.1, 7.6 and 7.7 in three samples.

One fact was very striking in all tests. After the color
comparison had been made, the apparatus was frequently left
at the same place for some time, before it was cleaned. Then
a comparison of the colors would have given entirely different
results: the digestive juices which at first were rather far on
the acid side of the sea water, more and more turned over
to alcalinity. I do not know definitively how to explain this
remarkable phenomenon, since the standards were exposed to
the same amount of evaporation etc. Maybe it is due to a
loss of CO, which might be the cause of acidity, since phos-
phates are not present in quantities large enough to account
for the acidity, as we will see in the next chapter. Gas-bubbles
present in the intestine of Tenebrio, have been supposed by
Biedermann 8) to consist of CO.

It will be noted that all these Py’s are on the acid side of
that of sea-water, which in Woods Hole appeared to have
a Py of 8.2—8.3. In the experiments of Roaf mentioned above
we have seen the same thing in his experiments with neutral-
red. For such acidity there must be an explanation and in the
next chapter we will endeavor to find such reason.

First I want to mention another interesting phenomenon, i.e.
the relatively high acidity of the true intestine, just behind the
stomach in Thyone. In Stichopus we even find real acidity
according to Crozier (on p. 388 of 18)), where he says: ,, The
yellow fluid, contained in the stomach of an ,,empty” Stichopus
gives with indicators an apparent acidity of 5.0—6.5. Fluid
obtained by centrifuging the stomach contents of animals engaged
in feeding showed acidities varying from 4.8—5.5, the latter
being the most common.” Here of course, we have to do with
a specific adaptation to the calcareous sand, taken as food by
these animals, which as we pointed out in the chapter on the
biology of our group, may have considerable economical im-
portance, because the calcium present in nature in undissolved
form, may be brought into solution in this way. Rain-water
present everywhere in nature as dissolving medium, only has
a Py of 6.0; the water dripping from the tip of stalactites
one of 7.9—8.0 (Crozier).

But something of the same nature is also the case in our

3

34

Thyone which also eventually feeds on calcareous matter. The
aquarium in which the fresh Thyone’s were put, frequently
contained an immense amount of small and very characteristic
calcareous bodies on the next day. I have not been able to
identify these, but according to some morphologists who were
especially acquainted with the local fauna, they ressemble very
much the calcareous sceletal elements of Alcyonarians.

The same phenomenon, an acidity in the beginning of the
middle gut has also been observed by other authors in other forms.
Basch ') found it in the digestive tract of Blatta orientalis,
The secretion of the salivary glands, the contents of oesophagus
and fore-gut are acid. In the middle-gut (,,Chylusmagen’’) the
reaction is neutral in the first part, alcaline further on. Basch
explains it by an alcaline secretion of the crypts of the middle-
gut, Jousset de Bellesme assumes that an acid liquid, con-
taining pepsin, is poured into the beginning of the middle-gut by
the small coeca, found right behind the ,,Kaumagen’”’.

Biedermann 8) fed Tenebrio larvae on flour, added with
litmus. About two thirds (the first part) were colored red, the
rest blue. Similar results were obtained by Kovalevsky in
Muscidae, Blattidae and Tenebrionidae.

These few references will serve to show, that an original
acidity, gradually passing over into alcalinity is by no means
rare in lower animals. The alcalinity in the very first part
of the gut of our Thyone is of course a complication due to
the fact that we have to do with a marine form here. The
sea water taken in with the food accounts for this alcalinity.

In the next chapter we will report on our trials to find the
cause of the relative acidity in the present case. It does not
seem to be due to free acid in most cases; congo-red which
indicates free mineral acid, was never colored blue in Bieder-
mann’s experiments — this might be due to the low Py (4)
of the changing point (v. d. H.) —~; Giinzburg’s reagent
(2 gr. phloroglucinol, 1 gr. vanillin and 30 gr. absolute alcohol),
also indicating free mineral acid, especially hydrochloric acid,
gave the same result. It is not due to butyric or lactic acid
fermentation either, since lack of carbohydrates in the food
does not change the hydrogen-ion concentration.

12. THE DIGESTIVE FLUID.

Studies on the composition of the digestive juice in inverte-
brates are not so very abundant; the only ones that have come
to my knowledge, are those of Biedermann (and Moritz)
on the intestinal contents of snails and of the larva of Tenebrio.

The first question of importance is: ,, What causes the relative
acidity of these juices?’ Though not absolutely acid, they are

1) Sitzungsberichte Akad, Wien. 33. No. 25. 1859. p. 234.

35

more so than the sea-water and more so than the perivisceral
fluid, which both have a Py of 8.2-8.3. This acidity is strange
is so far as the secretion of free acid has never been shown
to occur in lower organisms. This is true, of course, as far as
the digestive juice is concerned; the secretion of acid by sea-
snails etc. (Semon 119)) and analogous phenomena are not to
be discussed here. Even in animals as high in the scale of
phylogenesis as the dogfishes, it has not yet definitely been
settled whether free acid actually is present or not. In a
controversy between Weinland and Miss van Herwerden
— see van Herwerden 56), Weinland 132), and van
Herwerden and Ringer 57) — the former author has been
critisising the Sj6qvist method for determination of free acid,
used by Miss van Herwerden, as not being reliable if
earth-alcalies are present. Van Herwerden and Ringer
have answerred that the Py = 1.69 which is the same as that
of 0.02 N acid and given many other arguments in favor of
the presence of free acid.

Yet the acidity of the digestive juices in our group must have
a cause of some sort and maybe a chemical investigation of the
digestive fluid will furnish us some information. Since the ma~-
terial was not available in large quantities, ] had to run my
tests microchemically. A certain quantity of the digestive juice
was secured; separate drops were used for the tests to which
small quantities of the reagents were added from capillary tubes.

All the tests gave the same result in the digestive juices of
Arbacia and Thyone. For this reason I do not treat these two
species separately.

The first possibility to be thought of, is the presence of phosphates.
They are present in large quantities in the gut of Tenebrio,
where a weakly acid reaction has also been found'). These
acid (dihydrogen-) phosphates can, if magnesia is present at the
same time, be demonstrated very easily by a white crystalline
precipitate of ,,tripel-phosphate’’ formed after the addition of am-
monia. They are absent however in the digestive fluid of the
snails and do not seem to be present either in our Echinoderms.

Ammonia did in fact give a precipitate. But this precipitate
was probably nothing but simply calcium hydroxyde since it did
not have the typical appearance of the ,, Tripelphosphat’’. Other
tests were equally negative, Mg-mixture did not give any preci-
pitate, with Ur-nitrate a very slight precipitate was obtained.
This might indicate that a trace of phosphates is present, but
at the same time, that this quantity must be very small.

An ammoniacal solution of ammonium oxalate gave a heavy

1) A fluid behaving chemically just like the digestive juice of these animals,
could be obtained by adding a trace of ammonia to a solution of mono-
sodium phosphate.

36

precipitate. This proves that Ca must certainly be present.
This is not strange since sea water also contains a fairly large
quantity of calcium.

Carbonates are not present in large quantities, since if a small
quantity of the liquid is evaporated, the residue when treated
with hydrochloric acid, does not foam,

Chlorides of course are present in large quantities, silver nitrate
gives a heavy precipitate.

In the digestive fluids of Gastropods a large quantity of
proteins seems to be present. The same thing is true of the
larva of Tenebrio, even when the animals have been starving
for some time. It does not seem to be the case with the Echi-
noderms. On heating no precipitate is formed before evapo-
ration takes place. A xanthoproteic- and biuret-test gave ne-
gative results. Since the biuret-test is negative, no peptones
are present either. A ninhydrin is negative, no amino-acids
are present in that way, though the animals from which the
juice had been obtained, apparently were in full digestion.

Di-sodium phosphate gives a precipitate in an ammoniacal
solution; this may indicate the presence of Mg, but since Ca
is present, it may be due to this metal.

The precipitate with sodium carbonate, also indicates the
presence of Ca. Bichromate does not give a precipitate, Ba is
not present.

The results of my own experiments and those of Bieder-
mann have been represented in table 5.

Table 5.

Composition of digestive fluids of different species from personal
observations and data of Biedermann.

Species. | Proteins. Ca. Mg. | CO;. PO,. Cl.
Helix pomatia L. Large na Trace? + — —_
quantities.
Tenebrio molitor. L. | Rich. 2 + ? + a
Larva. Globulins?
Arbacia None. + Trace? - a +
punctulata. Gray.
Thyone None. + Trace? - — +
briareus. Les.

All constituents mentioned above do not explain the ob-
served acidity satisfactorily. The very small quantity of phos-
phates, if they are present at all, does not account for an
acidity which as Roaf 108) states may be as high of that of
a decimolar solution of di-hydrogen phosphate.

In the chapter on excretion we will discuss the importance
of the excretion of uric acid into the lumen of the intestine.
In connection with the results reported on in that chapter it is

37

interesting to mention the work of Sitovski 121). This author
found an acid reaction in the rectum of the catterpillars of the
moth, Tinea biselliellaj which he fed on wool which had been
permeated with a solution of litmus. In the last two segments
a distinctly acid reaction was found. Sitovski supposed that
this acidity is due to uric acid.

The question is whether we may make the same hypothesis
for the Echinoderms. This does not seem very probable. In its
favor pleads for instance the acid reaction which Roaf found
in the rectal coeca of the Asterids. In chapter 23 we will see
that these organs probably are the place of uric acid excretion
in the starfishes. My own findings on the Py of these organs
do not quite agree with his, I never found it lower than 7.3,
but my method is doubtlessly much more crude. The same
thing appears to be true for the urchin and the cucumbers
hqwever; we saw that the acidity is highest in the middle-gut
and decreases towards the end-gut. This does not plead in
favor of his hypothesis, since we will see that the acid is
chiefly excreted into the rectum. Moreover we must remember
that uric acid is an very weak acid so that it is scarcely
possible that in a ,,buffer-mixture’’ like this fluid — carbon dioxide
and a trace of phosphates are present! — it should have any
noticeable influence on the Pu.

The only possible explanation of the low Py which seems
left in this way, is the excretion of CO, into the gut. As a
matter of fact the samples described in the previous chapter
changed in reaction if left alone. They appeared to become
more and more alcaline which would prove the presence of
a volatile acid.

13. TRANSPORTATION OF THE FOOD.
THE PERIVISCERAL FLUID.

A definite blood system is not present in Echinoderms and
a special propulsatory organ is certainly lacking. Two systems
represent this one in this group: 1. a so-called blood-lacunar
system and 2. the perivisceral fluid. Opinions are different as
to whether these two systems communicate. Cuvier believed
in their communication, more recently Vogt and Yung in
their Lehrbuch der vergl. Anatomie (1880. Braunschweig. p. 523)
have defended the same opinion. Hamann denies it, he does
not consider injection experiments as convincing since the holes
may be caused by the force of the injection. Many other
investigators found elements present in the blood system which
are absent in the perivisceral fluid, Enriques 37) f.i. finds
green-red dichroitic granulae of enzymatic nature in the blood
system of Holothuria tubulosa, partly carried by amibocytes and
transported to the gut-wall by {the oscillations of the fluid;

38

H. Ludwig reports on the presence of ,,clotting” substances
in the blood” of cucumbers.

One of the most important characteristics of a blood system
is completely absent in Echinoderms. A propulsatory apparatus
has never been demonstrated, neither valves, though the axial
organ has for a long time been called ,,heart’. Tiedemann
127) was the first to see rhythmical contractions in the blood-
vessels of Holothuria; Cohnheim also saw them in those of
Cucumaria, but as he says, no definite movement in either
direction was seen if one corpuscle was spotted. Enriques
37) discusses his observations in detail and comes to the con-~
clusion that a definite movement actually exists (see the chapter
on resorption in Echinoidea and Holothureoidea).

At any rate, these movements are exceedingly slow and not
to be compared to those in vertebrates and higher invertebrates.

The importance of this blood system in resorption is to be
discussed in chapter 20. As far as its respiratory function is
concerned, Ludwig in Bronn’s Klassen und Ordnungen etc.
in the case of the Holothurians is very sceptical on this point.
The wonder-nets (retia mirabilia, Wundernetze, réseaux admi-
rables) found around that left respiratory tree, are so loosely
attached to the lung, the one can scarcely expect them to have
any such importance. In the chapter on respiration I hope to
demonstrate that the perivisceral fluid actually plays the réle
of internal respiratory medium, maybe also the water-vascular
system. Perhaps it plays a rdle in excretion; the excretion by
means of phagocytes -after physiological injections and perhaps
also in normal conditions- takes place through the water-lungs ').

The perivisceral fluid is of much more importance; 175 c.c.
of the contents of a Sphaerechinus, measuring in toto 225 c.c.,
is according to Cohnheim perivisceral fluid. Physiologically the
contents of the coelomic cavity must play the rdle of blood,
in as far as it is a fluid medium, interposed between intestine
and consuming tissue and tissue and organs of excretion. A
little closer investigation of the chemistry of this medium shows
that apart from the corpuscles which are present in abundance
in all groups and which show a phenomenon of clotting,
entirely different from that in mammals-yet leading to the same
end result (see f. i. the paper of Geddes 42) on this point)-, it has
practically the same composition as sea water.C ohnheim already
found that this is the case. It contains the same percentage of
chlorides and the same inorganic constituents. In as far as it
is physiologically ,,blood” we must investigate if it carries any
food- or waste-constituents and whether it plays a réle in
respiration. The latter topic will be discussed in chapter 24,
for the present will test for food and waste constituents.

1) See the references in the chapter on the respiration.

39

Though no special organs are present for this purpose, the
perivisceral fluid is by no means stagnant and without currents.
The whole peritoneum is ciliated and these cilia of course cause
currents. That such ciliar currents can take care very well of
the distribution of dissolved substances, was shown by W id-
mark 132) for jelly-fishes in a detailed investigation.

A ninhydrin test for amino-acids in the perivisceral fluid
invariably has a negative result in animals which have been in
the aquarium for more thana day. In some very fresh specimens
one occasionally .gets a slight ‘pinky color; in one starfish that
was caught while it was eating a Littorina, a strongly positive
result was obtained. Fehlingh’s test is, as Cohnheim found
already, always negative, no monoses appear to be present.
A biuret test invariably gives negative evidence; neither the
violet color which indicates the presence of proteins, nor the
pink of peptones was ever seen. This proves that no proteins
are present in the filtered perivisceral fluid, as moreover a nega~-
tive xanthoproteic reaction showed. This result is in contra-
diction with the assumption of many of the older investigators:
Cuénot 23)'), Semper 120) Geddes 42), Mourson and
Schlagdenhauffen 90) and Williams (Philos. Transact.
1852). No free fat is present; the Py is in most cases the same
as that of sea-water (see chapter 24). Its specific gravity is also
the same as that of sea-water (Geddes). An analysis of the
ash compounds has been given bij Griffiths 49) and by
Mourson and Schlagdenhauffen 90). Griffiths how-
ever finds 0.042—0.049 °/, fibrin (!) and Mourson and
Schlagdenhauffen 0.010~—0.013°/, urea, both of which are
now known to be absent. More CO, and less O, is of course
found in the perivisceral fluid as compared to the sea water (M.&S.)

As far as the waste constituents are concerned, no more am-
monia is found in the perivisceral fluid than in sea water.
Nessler’s reagent gives a white precipitate of Ca-~hydroxyde
with sea water. If however a trace of ammonium sulfate has
been added, the precipitate has a deep orange color.

The precipitate is just as pure white in the perivisceral fluid
as in sea water. No creatinine is present as Jaffé’s test, which
demonstrates the presence of this substance even in a dilution
of 1:5000, is negative.

Thus far it seems that none of the constituents which are
present in most bloods, occur here. One of the waste consti-
tuents however is present, i. e. uric acid. Folin-Wu’s phos-
photungstic-phosphomolybdic acid gives a blue coloration in the
alcalinised liquid. The intensity of this color is very different
in different cases, but it always occurs. It is not due to poly-
phenoles, since ether extraction does not take away the color,

1) This author changed his opinion later on,

40

Cohnheim discovered that the only substance present in
the perivisceral fluid and more than in the sea water, is a sub-
stance which gives a precipitate with phosphotungstic acid. It
is not arginin or any other amino-acid (no precipitate with AgNO)),
since as we saw above none of these are present. Is this perhaps
the same substance? Phosphotungstic acid in fact precipitates
uric acid (Carl Oppenheimer. Handbuch der Biochemie.
Jena. Gustav Fischer. 1909. Bd. I. p. 625).

He also found slightly more N in the perivisceral fluid of
our animals than in the sea water, as the following table shows :

Table 6. After Cohnheim.

100 c..c. sea water (Kjeldahl). . . . 0.4 mgr. N.
100 c.c. Holothuria ,blood”. . . . . 2.5 mgr. N.
100 c.c. urchin , blood” . . . . .. 5.7 mgr. N.

He was unable to explain such excess of N; possibly it is,
at least partly, due to uric acid.

Mourson and Schlagdenhauffen found urea in the
perivisceral fluid of Strongylocentrotus lividus. Brandt and
Toxopneustes lividus. Lacken in a concentration of 0.010 °/,—
0.013 °/). I have not been able to detect any by means of
Nessler's reagent after hydrolysis (see also the results of
Griffiths in chapter 23 and remember the scarcety of urea in
lower animals). They also find a ,,ptomaine ')’’ present which
has a toxic action on frogs. The same ptomaine is also found
between the shells of bivalves. Whether or not our substance
is meant here, the present writer does not dare to decide.

The occurrence of uric acid in the perivisceral fluid of the
Echinoderms is very interesting, because it may throw a light
on the dark problem of their excretion. I refer to chapter 23
for this topic.

All the tests mentioned above have been made on Asterias,
Arbacia and Thyone with the same result. We see that in
these representatives of the chief groups of the Echinoderms, the
coelomic fluid is really nothing much more than sea water in
which almost constantly a small quantity of uric acid is present
and occasionally some of the products of hydrolysis of the
food, depending on the feeding conditions.

A fairly constant blood picture is only characteristic for th

) I do not understand what these authors mean here. Ptomaines are as
far as my knowledge goes, basic substances, products of bacterial decomposition
of protein, such as cadaverin and putrescein. I can not concieve how such
substances present only in decaying material and highly toxic for every living
organism, should be found in the ,,blood” of the representatives of this group.
Since however uric acid has some reactions in common with these substances —
the authors do not indicate which test they have used — it is possible that
this might be the explanation.

41

Vertebrates; in these lower animals there is a very strong
variation. As yet unpublished experiments of Dr. Morgulis
who was working in the same laboratory last summer, showed
that the blood picture of many invertebrates of various groups
is not constant at all, but shows a wide variation, due most
probably to the moment's feeding condition.

Note. The perivisceral fluid of Strongylocentrotus and of
Toxopneustes is said by Mourson and Schlagdenhauffen
to be used in the Midi of France as a tonic. What might
account for such activity, I do not know.

14. ARE THERE ENZYMES PRESENT IN THE
PERIVISCERAL FLUID?

»Bei frischgefangenen, also in Verdauung begriffenen Seeigeln
fand ich mehrmals in der Leibeshdhle ein diastatisches, bei
Holothurien ein diastatisches und ein invertierendes Ferment”.
(Cohnheim 17) p. 42).

Bacterial action was excluded, because toluene was present;
no hydrolysis took place if the liquid was boiled. These experi-
ments according to Cohnheim, show the presence of enzymes
in a solution which except for some inorganic salts, does not
contain any other substances. ,,Dieser Befund ist chemisch recht
interessant, weil er das Vorhandensein von Fermenten in einer
Lésung zeigt, die weder Eiweisz noch Kohlehydraten und
iiberhaupt organische Substanz nur in Spuren enthaélt und so
einen Beweis mehr fiir die Nichteiweisznatur dieser Kérper
liefert.’” He compares them to the ,,Fermentschlacken”, found
more or less regularly in the blood and the urine of mammals.

As a matter of fact, such result would be exceedingly inte-
resting, but I believe that Cohnheim in wrong in the inter-
pretation of his observations. I am more inclined to believe
that a small amount of corpuscles or remainders of corpuscles
present in the liquid may account for his result.

In order to ascertain whether this could be the case I made
some experiments of my own on this question.

Two large samples of perivisceral fluid of Thyone and Arbacia
were secured. One of each. was centrifuged repeatedly with a
strong centrifuge in order to get rid of the corpuscles. This is
as a matter of fact, not as easy as one would suppose it to be.
One is almost never sure that no more corpuscles are present.
A part of the corpuscles seems to be crushed or ,,laked”’ which
is especially clear in Arbacia where the liquid is never colorless.
This is what made me think that an error of this kind might
have been made by Cohnheim.

Cohnheim reports that he could observe this enzymatic
, activity only in freshly caught specimens. For this reason I used

42

Arbacia’s which had been brought into the laboratory on the
same morning. The Thyone’s I used, however, were in the
aquarium already for a week.

The corpuscle samples and those which had been freed from
corpuscles, were added with liberal quantities of cane-sugar
and starch. Some toluene was added to each of them; after that
they were placed into the incubator at 40° and kept there for
some days. In case either an amylolytic enzyme or an invertase
were present in the liquid, one might in that way expect a
reduction of Fehling’s solution.

After two days a Fehling’s test was made on an aliquot
of all the samples. All were negative at first sight, on standing
however a precipitate was seen which was much heavier in the
corpuscle samples in both cases.

Two days later a complete reduction was seen in the corpuscle
sample of Thyone. In the others the reduction was incomplete
and the precipitate only became visible on standing. In the
urchin this precipitate was at least five times as heavy — estimating
this roughly from the quantity of precipitate — in the corpuscle
sample as in the otherone. On the next day the difference was still
more striking, now at least ten times as much was present in
the corpuscle sample.

This experiment shows clearly that the corpuscles must explain
the remarkable observation of Cohnheim. Since this author
does not mention whether or not he filtered his samples, this
seems to be very well possible.

15. ROLE OF THE CORPUSCLES.

Several assumptions have been made with regard to the func-
tions of the amibocytes of the Echinoderms. Some of these do
not have any direct bearing on the problems studied in the
present paper. Some authors have described special calcigenous
cells, carrying reserve-calcium for the formation of sceletal ele-
ments. Others have emphasised their function as phagocytes.
The original experiments of Metschnikoff 84*) on phago-
cytosis which gave rise to his very illuminating conceptions of
this process were conducted on this very group. As a matter
of fact they seem to play a very important réle in the meta-
morphosis of this group. Secondly he could show that foreign
bodies, bacteria, dying mesodermal cells, etc. can be swallowed
by these corpuscles. Mammalian corpuscles injected into these
animals were surrounded by a kind of plasmodium of amibocytes
and digested; I myself could observe something of the same
nature in Stichopus after bone-black injection (see chapter 23). The
role which the corpuscles play in excretion will be discussed in
in chapter 23; that they may carry waste has been proved
by Durham 34) and 35) and Saint Hilaire 112).

43

Their rdle in the metabolic processes has doubtlessly been
exagerated, especially by Cuénot 23) who assumed that they
take care of the transportation of the food to the tissues.
Chapeaux 15) also has exagerated their réle in the assimilation
of fat; his hypothesis that fat would be infiltrated by the gut-
epithelium, passed unchanged, given off to the coelomic liquid
and be digested there by the corpuscles, will be discussed in
the next chapters. They doubtlessly contain enzymes; see chapter
14. But this is the case just as well with mammalian leucocytes.
Miiller and Jachmann (Miinch. med. Wochenschr. 1906.
1393, 1507 and 2002) demonstrated the presence of a proteolytic
enzyme in leucocytes; Haberlandt (Pfliiger’s Arch. 132. 1910.
175) and Mancini (Biochemische Zschr. 26. 1910. 140) found
a diastase.

All data concerning movements of the corpuscles unanimously
prove that they only move from inside to outside (see chapter 3).
I myself never succeeded in finding any amibocytes in the gut-
contents samples studied for the experiments in chapter 9 etc.
From such considerations it is not probable that the amibocytes
would participate to any extent in the transportation of food;
on the contrary, their life-circle goes the inverse way; born in
the lymph-glands they pass into the coelomic fluid and leave
the body by different ways (see the chapter on excretion) loaded
with waste.

Possibly they also play a réle as store of reserve substances
(Cuénot). Echinochrome and other fats are found in them, the
protein-crystalloids will be discussed in chapter 25. ] can not
concieve however how the two functions of excretion and
storing of reserves can be located in the same organ however.

16. THE DIGESTION AND UTILISATION OF FATTY
SUBSTANCES. EMULSIFYING ENZYMES?

The digestion of fats is one of the most difficult and obscure
chapters of biochemistry and up to the present time the question
has not yet been settled whether or not the fat is hydrolysed
before it passes into the cells of the intestinal epithelium. There
is about no subject in which so many controversies exist or
have existed.

It is beyond the scope of the present paper to discuss the
problems which have stirred the minds of the medical physio-
logists. But it may be justified to discuss briefly some of the
discrepancies and differences of opinion in invertebrate physiology.

The comparatively simple problem whether or not Protozoa
digest fat, has been studied by many authors and almost every one
of them came to different conclusions. Nirenstein 95) in his
paper on fat-digestion and -storing in infusoria, is of opinion
that fat is digested easily and eagerly by these animals. In the

44

first place he finds that fat is normally present in them, as a
simple test with Sudan III in 80°/, alcohol shows. This fact in
itself does not prove anything however; we know that fat may
be derived just as well from carbohydrates or proteins as from
fatty food: see fi. Chanievski 14). Moreover, Nirenstein
himself shows that the quantity of fat in Paramaecium increases
just as well after a prolonged feeding on rice-starch and on
albumin from eggs (Albumin aus Eiern. Merck), as by a fine
oil-suspension.

Nirenstein however states most emphatically that he has
observed the digestion of fat in his animals. He studied this
process with egg-yolk. As long as the reaction of the vacuole
remains acid, the fat droplets remain unchanged; as soon as
alcalinity is established, the fat droplets gradually form a homo-
geneous solution with the ,,Vacuolenschleim”. No emulsification
is observed, but Nirenstein believes in a hydrolysis and
recombination within the cell-body. Animals fed on sodium
oleate and glycerin deposit fat in their cells, the same thing
happens if Na-oleate alone is used. Saint-Hilaire 113) came
to conclusions of the same kind for the planarians, Mesnil is
also one of the advocates of the hydrolysis-hypothesis in inver-
tebrate physiology.

Biitschli however already saw, that fat-droplets are thrown
off just as they come in. The same observation has been made
also by Stanievicz 122); A. Drz.(Drzewina? v. d. H_) 33)
in the Revue Scientifique, abstracting this paper, seems to be of
the same opinion. Stanievicz has extended his experiments
over a large quantity of animal and vegetable oils and seems
to be quite careful in drawing his conclusions. He completely
agrees with Nirenstein as far as the ingestion of fats is
concerned. While however bacteria and algae, taken up at the
same time, are digested pretty soon, the fat-droplets remain
unchanged, no emulsification occurs and no hydrolysis.

,Aprés un certain temps’, he says, ,,la membrane se dissout,
les bactéries et les algues sont partiellement digérées, par contre,
les globules de graisse ne sont point altérés et aprés plusieurs
heures sont éliminées avec des parcelles non digérées.” After
feeding olive oil to Paramaecia, he does not find any increase
in fats!

The fact that Sudan III is decolorised in the vacuoles, does
not prove hydrolysis according to this author. It is probably
due to a reduction of the dye, some of these dyes are even
dissolved out of the fat and eliminated by way of the pulsating
vacuole. Moreover Mesnil and Mouton have shown experi-
mentally that a lipase is not present in Protozoa.

After feeding of egg-yolk and of carbohydrates, he finds fat.
His negative results with regard to the digestion of fats and
these findings, force him to conclude, that the fat which is natu-

45

rally present in these infusoria, is built up from carbohydrates
or proteins.

This example may illustrate the extreme difficulty of drawing
conclusions in problems of this kind. Biedermann in his
studies on the digestion of Helix and of the lar'va of Tenebrio,
is more definite in his statements. Yung already found that
fat can be emulsified by the contents of the gut of snails,
Biedermann and Moritz show that a steapsin is present.
This enzyme is more essential to their opinion.

Milk to which the digestive fluid of these animals is added,
turns acid and coagulates. The residue, treated with osmic acid,
does not turn black. Fatty acids can actually be demonstrated
by ether extraction, addition of KOH or NaOH gives a turbid
solution or a jelly of the soaps. The free acids can be precipi-
tated from the solution by means of HC1 (What about the
lactic acid which is known to occur in these digestive juices?
v. d. H. — see the chapter on this topic —).In the cells fat-
droplets can be seen ,,in zierlichen Reihen”’.

»Man kommt so zu der Anschauung dass es sich bei der
Fettresorption nicht sowohl um eine Einlagerung von reinem
Fett in die betreffenden Zellen, sondern vielmehr um eine Art
von Infiltration von neugebildetem Fett in eine andersartige
Grundsubstanz handelt, eine Vorstellung, die freilich mit der
herkémmlichen einer direkten Aufnahme von genuinem, fein
verteiltem (emulgierten) Fett nicht wohl in Uebereinstimmung
zu bringen ist’’.

These arguments sound rather convincing, the last word ho-
wever has not yet been spoken. I'll pass the numerous data
in the literature on the subject ~ in nearly every order some
authors have reported the presence of emulsifying enzymes,
others that of a steapsin, while a third group denies its capacity
of digesting fats —, but first report on some experiments of my
own on Echinoderms.

As a substrate I have used a neutral olive oil. I believe that
this substrate is very much better for the study of steatolytic
enzymes than commercial esters, like ethyl butyrate, which have
frequently been used for this purpose, because the latter sub-
strates only give us the right to draw conclusions about esterases,
not about a lipase.

Fat is either taken up in hydrolysed form or as such. In
the latter case emulsifying enzymes must be present enabling
the cells of the wall of the gut to take hold of the small fat-
globules.

For this reason it is in the first place necessary to make sure
whether such emulsification can be demonstrated. For this purpose
I secured samples of the intestinal juice of Asterias, Thyone

46

and Arbacia in the way described in chapter 7. A deep-depres-
sion slide was now filled with this fluid and a drop of olive
oil added. The sample was covered, with a cover-glass and
put into the incubator. At different intervals these tests were
inspected.

In none of these tests I could see a dispersion or emulsification
of the oil amounting to anything. Occassionally the drop was
found to be split into two, sometimes even more (5 or 6) parts.
A true emulsification which would have a biological importance
could never be seen however. Shaking of the samples was of
course avoided as much as possible.

Another possibility is the assumption of the hydrolytic disso-
ciation of the fats. Griffiths 48),Stone 124), and Clerc 16*)
in fact report that they found steatolytic enzymes. I studied
this problem on digests of the same kind as those described
in the chapter on the enzymes, using olive oil as a substrate.

A large quantity of starfish radial sac material was ground
up with sand and put into an incubator after olive oil and an
ample quantity of toluene had been added. At regular intervals
the mass was shaken vigorously and 20 c.c. samples of the
homogeneous mass taken. These were titrated with 0.05 N KOH.
The results obtained are represented in the following little table:

Table 7.
Titrable acidity of digests of radial sacs and fat.

= ona

Moment. c.c. 0.05 N KOH.

Control on initial sample. Monday

evening. 1.20
Tuesday, 6 p.m. 6.35
Wednesday, 6 p.m. 8.05
Thursday, 7 p.m. 8.40
Friday, 7 p.m. 8.36

These figures show a regular increase in acidity and super-
ficially one might consider them as a proof for the presence of a
lipolytic enzyme. The fact that the process goes on rapidly at
first, then slows down, would be explained by the assumption
that the enzyme had been destroyed by the accumulation of the pro-
ducts of its action. It is strange however, that the process takes
place with such surprising rapidity, practically the whole acidi-
fication has taken place on the first day.

For such reasons it seemed advisable to me to run a control.
A similar digest was made and no olive oil added this time.
Toluene was added as always and some water. The titrations
were not made every day, since I was only interested. in the
end-result. The results of this experiment are given in table 8.

47

Table 8.
Control on the experiments of table 8.
Moment. c.c. 0.05 N KOH.
Saturday evening, control. 2.10
Next Tuesday morning. 11.77

In all these titrations I used phenolphthalein as an indicator.

This control experiment shows clearly that the acidity develop-
ped, may be due to other than fatty acids, resulting may be
from autolytical phenomena. Remarkably enough Chapeaux
found such digests alcaline.

The next thing to do was to try separating out the olive
oil plus formed fatty acids from the material. This was done
by extraction with liberal quantities of ether; the ether was
evaporated away from the extract and a titration was made. A
control-titration was also made on the same amount of olive oil.

The result of these titrations gave me more courage again
and made it probable that at least part of the acidity might be
caused by fatty acids.

Table 9.
Titrations of ether extracts.
Material. c.c. 0.05 N KOH.
Ether extract of the material studied. 6.12
Control: same amount of olive oil. 3.60

One more control was necessary however. It should be proved
‘that the ,,other acid’’ was insoluble in ether, in other words,
the same proceedure should be repeated while no olive oil had
been added. For this purpose I used the material which had
been used for the autolysis experiments of p. 26. A large amount
of this material, containing many more radial sacs than the other
digests, was extracted with ether in exactly the same way. The
final titration gave 12.80 c.c. of twentieth normal alcali.

From these critical experiments one can see how careful we
must be in judging about the presence of lipolytic enzymes in
our group. These difficulties have not been seen by other authors
who have been working on our group; for this reason I am
inclined not to take their results too seriously. The formation
of an acid of some kind in these digests of the radial sacs,

48 _

makes the analysis of this problem especially hard. In a special
chapter I will discuss briefly the importance of this development
of acidity in dying tissues; for the present I will confine myself
to a report on analogous experiments on Arbacia and Thyone.

Experiments of the same kind as the ones described above,
have been made on the guts of these two species. The guts
were crushed with sand, taken up in some water and added
with toluene, olive oil and some water.

In the case of Arbacia 8.5 gr. gut was used — wet weight —.
After two weeks the etheric extract of the material appeared to
need 11.34 c.c. of twentieth normal KOH for its neutralisation
whereas a control sample of the same amount of olive oil
only required 1.80 c.c.

The same thing proved to be true for the cucumber. The
amount of gut used here was 19 gr. — wet weight and including
the ,,calcareous ring” ~—. After 1'/, week this digest required
16.3 c.c. of 0.05 N KOH for neutralisation, while for a control
8.0 c.c. were needed.

Whereas this series of experiments gave rather undefinite
evidence, there are others which have given me the absolute
proof that a digestion of fats takes place in Echinoderms.
Starfishes and sea-urchins were fed on olive oil. The methodo-
logy of such experiments will be described in chapter 20 and
21; it is rather difficult to inject a syrupy fluid like olive oil,
but after some practice one easily succeeds in forcing the animals
to take it.

At different intervals the animals were dissected and their
guts taken out. They were fixated in Flemming’s fluid. The
usual imbedding proceedure in paraffin could not be used be-
cause I wished to avoid the passing through the alcohols, which
might dissolve out the fats. For this reason I used a very old
method, recently very much improved by Mc. Junkin 729),

This method is based on the principle of imbedding the
material in soap. This can take place by passing the material
right away from water or even from a fixating fluid which is
volatile (fi. formaldehyde) into the soap. The method has been
described with much detail by Mc. Junkin; the great difficulty
is the choice of the right kind of soap. The author claims that
if one has once succeeded in finding a suitable kind, sections may
be cut thinner even than in paraffin. The soap is hardened
in a saturated salt-solution, before the sections can be cut; the
ribbon is dissolved away in water and the sections floated on
slides. This procedure takes a lot of practice, which is a
decided disadvantage of the whole method.

In sections of the starfish radial sacs enormous quantities of
fat could be seen. The sections showed a typical greyish, almost
black color, which on closer examination appeared to be due
to numerous little fatdroplets in the epithelium. A similar obser-

49

vation has been made by Biedermann and Moritz onthe
liver of Helix 9): ,,So erschienen etwas dickere Schnitte einer mit
Osmium behandelten Schneckenleber fast gleichmassig schwarz
und undurchsichtig”; only in very thin sections could they see
the separate droplets. After about 10—20 hours this loading of the
epithelium with fat has reached its maximum, I obtained the best
results in specimens which had been injected in the late afternoon of
one day and dissected early in the morning of the next. The
staining of the fatby Flemming’s fluid — one day ~ was very
incomplete in my sections, under high power the droplets were
perfectly clear and not black. The soap sections freed from
the soap, could easily be used for a test with Sudan III and I
have used this reagent in order to make sure that the droplets
which I had observed, actually were of fatty nature. Sections
thus treated gave a most splendid and enchanting sight, the
hundreds of droplets all colored reddish could be seen everywhere
and in every cell of the epithelium. In some of the sections of
later stages, the cell-border turned towards the intestinal lumen,
was fairly clear and free of globules. The fat then began to
accumulate in the sub-epithelial connective tissue in the same
way as many other substances — see the chapter on resorp-
tion in starfishes —.

Some of the guts of Arbacia which had been treated in exactly
the same way, but which were imbedded in paraffin ~ so that the
alcohols through which they passed, might have dissolved away the
fats —, gave very nice sections. The fat was stained very comple-
tely here and the sections immediately showed the presence of fat
by the many pitch-dark globules they contained. A piece of a
section of this kind has been drawn by Mr. H. van Laar,
who also made the other pictures for me '). (Fig. 4 of our plate).

These results seem to indicate with a fair degree of certainty,
that fat is actually used and digested, but they do not give
us any evidence as to whether it is ingested as such after
having been emulsified (phagocytosis?) or whether it is hydro-
lysed first. Our titration figures would prove that the latter
took place were it not for the formation of acid even in digests
without olive oil. Since however no emulsification takes place,
I am inclined to believe that hydrolysis actually occurs.

There are some facts, however, which deserve a more general
attention in connection with the whole problem of the digestion of-
fat. I mean the experiments of Churchill 16) on the absorption
of fat by fresh-water mussels.

Mussels were kept in soap solutions, prepared from olive oil,
both unstained and stained with Sudan III. Histological exami-
nation of such mussels and of controls revealed the fact that
fat is absorbed abundantly by all epithelia and carried all over

} I am very much obliged to him for this service.

50

the body by the blood-corpuscles. Sections of mussels kept in
such solutions for short periods, e.g. 18 hours, showed such
heavy loading of fat in the epithelium of gills, mantle and foot,
that it seemed very probable, that the cells of these epithelia
absorbed the fat directly from the solution. This could in fact
be demonstrated experimentally. Mussels with the valves wedged
open and suspended so that the mouth did not come down to
the surface of the liquid, showed the same absorption.

Such results are surely remarkable, for no steatolytic enzyme
can be present here making the fats ,,fit’’ for resorption. They
indicate that at least some epithelia can absorb fat without
previous hydrolysis. Perhaps this is a general phenomenon,
perhaps there is some complicated colloid-chemical procedure
which forces fatty substances to be absorbed by every epithe-
lium. In that case our experiments would show nothing with
regard to the actual utilisation of fats by Echinoderms.

The presence of fats in itself of course never proves the
presence of lipolytic enzymes. They may have been built up
just as well from carbohydrates, proteins, fatty acids and many
other substances as from fats. Biedermann found this true
for the liver of snails e.g.; after a heavy meal of flour he always
found the liver cells full of' fat droplets.

There is still another thing which might favor this view,
namely the abnormally high respiratory quotient which Piitter
observed in his Cucumaria grubei. He explains it by the as-
sumption of a butyric acid and methene fermentation '), in chapter
26 we shall will hear of another possible, indeed, a more probable
explanation, in the third place it may also be caused by trans-
formation of carbohydrate into fat. Cane-sugar f.i. can yield one
third of its weight in fat and at the same time 2/; of the total
quantity of its energy (Moore 87), etc.), If such processes take
place, much more CO, is produced than corresponds with the
oxygen-intake. The R.Q. consequently is ) 1.

17. LACTIC ACID DEVELOPMENT IN AUTOLYSATES
OF THE LIVER.

In the preceding chapter we saw that an acid of some kind
was formed in our digests which made it hard to judge whether
fatty acids were actually produced. or not. This spontaneous
development of acid seems to me to be of particular interest,
since | believe that we have to do here with a phenomenon
of a more general importance than one might expect.

*) Such fermentations are not very frequent and I am not impressed by the
validity of his arguments. To quote Frankel 138): ,,Sumpfgas (Methan, CH4)
entsteht nur bei Pflanzenfressern und Omnivoren im Darm, nicht aber in dem
der Fleischfresser. ..."" .,Es entsteht wohl nur aus Cellulose...’ (p. 207).

51

Dr. Withrow Morse and the writer 89) studied the
changes in reaction in dying tissue, in connection with the
senior author's work on autolysis. The liver of guinea pigs was
taken out immediately after they had been killed, and ground
up in a mortar. Sometimes they were first frozen by means of an
ethyl-chloride spray, sometimes used without freezing. They were
taken up in a small quantity of destilled water and the whole
mass was then put in the cup of a Leeds and Northrup
potentiometer, type K. Py readings were made at different
intervals, It appeared that very soon after death occurred, a
relatively tremendous acidity was established, even up to Py = 4,
but that afterwards this acidity gradually diminished to near
the neutral point. No trial could be made to study more in
detail the nature of this acid on account of our poor equipment ;
we supposed that we might have to do with lactic acid.

Lactic acid is also formed in autolysates of the snail’s liver.
Biedermann and Moritz 9) found that if the digestive fluid
of Helix was left alone, even with shreds of fibrin in it, no
decay could be observed. If decaying pieces of fibrin were put
into that liquid, the decay stopped in a very short time. A
strongly acid reaction which he found in these samples solved
the problem. Whereas e.g. in crabs the digestive fluid decays
at once here the acidity prevented all bacterial growth. He
show showed that lactic acid was the cause of this acidity.

A digest of crushed liver substance also appeared to contain
a large quantity of this acid. It did not digest proteins, but
a coagulation of the material was observed caused by the acidity.
That lactic acid was also present here, could be shown by
means of Uffelmann’s reagent, a 2°/) phenol treated with
dilute ferric chloride till of an amethyst violet color. This reagent
is decolorised by mineral acids, but colored yellowish by lactic
acid and some other organic acids. The development of this
acid is due to bacteria according to Biedermann: chloroform
and thymol prevent its formation.

Other experiments showing this same phenomenon came to
my attention in a paper of Lindemann 80). This author
finds in 100 gr. autolysed liver of a rabbit an acidity titrable
with 7.6 c.c. decinormal NaOH. Estimated as butyric acid this
would mean 66.9 mgr.

As we see, this phenomenon occurs rather regularly in the
most divergent groups. In our digests of starfish ,,liver’ we
found, as mentioned above, an acid of some kind to be present.
This acid is soluble in ether — this isin fact the case with lactic acid.
From a small quantity of material I extracted the acid by means
of ether; Uffelmann’s reagent lost its violet color and changed
into yellow. These facts make it rather probable that here we
have to do with lactic acid, though they do not prove it defini-
tively, since many other organic acids may give the same reaction.

52

There is still another remarkable thing to be mentioned in this
connection; it has been observed by Enriques 37) (p.11),
where he says: ,,Del resto, il succo gastrico non putrefa o
putrefa soltanto dopo molto tempo, anche senza nessuna precau-~
zione, certamente a causa della sua acidita’. The same thing
proved to be true for extracts of the retia mirabilia.

This fact shows a remarkable analogy with the results of
Biedermann on the gastric juice of the snails. J have made
the same observation repeatedly on samples of digestive juice,
as used for the experiments described on p. 30 and 34. They
never had that hideous smell of decaying marine material, the
remainder of these samples were left standing in the room for
more than a week, they were yet fresh at the end. As the
paper of Enriques, who states that it is due to acidity, came too
late to my attention I could not test for lactic acid in my material.

In one point I came to conclusions different from those of
Biedermann, The acid mentioned in the preceding chapter
was formed in an autolysate in the presence of toluene. The
same was the case in Lindemann’s experiments. Bacterial action
seems to be excluded in that way; most probably we have
to do here with a spontaneous development of acid of as yet
obscure nature.

These few remarks do not pretend to be built on a very
strong basis. They only indicate a problem on the solution
of which I hope to be able to work more carefully in the future.

18. IS THERE A LIPOLYTIC ENZYME IN THE
CORPUSCLES OF THE STARFISHES?

In the chapter on the resorption in the urchins and the
cucumbers I shall mention the conceptions of Chapeaux 15)
as to the réle of the corpuscles in the digestion and transpor-
tation of fats. In this chapter I only want to mention one of
his findings because I came to different results.

Chapeaux studied the réle which the corpuscles play in
the digestion of fatty substances. He found that fat-droplets
are taken up eagerly by the corpuscles and digested in the
cell-body. He finds a lipolytic enzyme present in them and
observes the development of acidity in plasmodia to which olive
oil has been added. This acid reaction was not found in a
control.

In order to amplify my own experiments on the digestion
and utilisation of fatty substances in our group, I made some
experiments of my own on this question.

A large quantity of starfish amibocytes was separated out
by means of a strong centrifuge; they were taken up in 100
c.c. of destilled water and a large quantity of neutral olive oil
and toluene were added. The whole was put into an incubator

53

at 37° and at certain intervals titrations were made on 20 c.c.
samples. On 20 c.c. of the original liquid a control titration
was made.

The results of this series of titrations are given in table 10.
Table 10.

Titrable acidity of a digest of olive oil and corpuscles.

Day. Hour, c. c. 0.05 N KOH.
Control. Td. 6 p.m. 0.80
Wad. 6 p.m. 0.80
Thd. 7 p.m. 0.97
Fd, 7 p.m. 1.01
Td. 3 p.m. 0.98

These figures indicate very clearly that if there is any in-
crease in acidity, which does not fall within the limits of
error, that this increase is too slight to have any biological
meaning. A priori it is not improbable that in these amibocytes
which are primitive and independent cells without any special
function, a lipase should be found, just as well as in an Amoeba.
More important, however, is the question, whether this enzyme
plays a réle in the digestive processes of the animal as a whole.
This seems to be very improbable, considering our figures.

I have also tried to study the same problem in two other.
ways. Two fairly large quantities of coelomic fluid of Arbacia
were secured. One was centrifuged in order to remove the
corpuscles, the other one was left as it was. To both an equal
quantity of .olive oil was added and some toluene. Then
they were placed for two days in an incubator at 37°. After
that time both gave exactly the same color with a piece of
litmus paper, only to neutral litmus paper they showed a very,
very slight acidity. If there was any difference at all, it was
too slight to be worthwhile mentioning.

After this I repeated the experiment in a way similar to
that which Chapeaux employed. A large quantity of amibo-
cytes, collected by means of a centrifuge, was put into a deep-
depression slide, a drop of olive oil was added, the whole
covered with a cover-glass and put into the incubator. A very
distinctly acid reaction was found after 24 hours. After my
previous results I was rather surprised to find this and therefore
I decided to run a control without olive oil. The same acid
reaction appeared after a short time. Most probably the de-
caying material had produced an acid of some kind, which had
escaped my attention in the titration experiment on account of
its great dilution or which did not appear there on account
of the presence of toluene. If toluene was added to the samples,

54

the acidity did not appear; for this reason I am inclined to
believe that this is the source of error in Chapeaux’s
experiments.

19. THE NITROGEN METABOLISM OF THE
HOLOTHURIANS.

In his paper on resorption, digestion and metabolism in
Echinoderms Otto Cohnheim 17) discusses the nitrogen
metabolism of the holothurians. He found no proteolytic en-
zyme present in holothurian guts. ,,Holothuriendarme geben
auch nach wochenlangem Stehen an Meereswasser kein Ferment
ab, das rohes Fibrin zu lésen imstande ist’’. Fibrin was not dis-
solved by such digests in. one day at 37°, if toluene was added.
Neither were pieces of mussel changed. Since no glands are
present which could secrete a proteolytic enzyme, Cohnheim
concludes that there is no such enzyme. Another proof in favor
of his view is the negative result of autolysis experiments with
Holothuria-guts. This result, namely that autolysis proceeds
almost imperceptibly and that such guts can remain unchanged
for weeks at a stretch, the present author is prepared to affirm,
as mentioned before.

Other negative results were obtained in experiments on resorp-
tion of nitrogenous materials. Ammonia was not resorbed; in
the case of proteins, no protein could be demonstrated at the
outside and the total Kjeldahi-N did not increase.

From these experiments Cohnheim draws the conclusion
that ,,die Resorbtion N-haltiger Kérper sehr gering ist, im Ver-
gleich mit der reichlichen Resorbtion und Verbrennung von
Kohlehydraten, zu gering jedenfalls als dass ich sie erfolgreich
untersuchen konnte. Wie und in welcher Form die Holothurien
Stickstoffhaltige Nahrung aufnehmen, vermag ich danach nicht
anzugeben”’.

In our chapter on the enzymes we have seen that a proteo-
lytic enzyme is surely present in Thyone. And in the following
chapter we have demonstrated that it even occurs in a free
form.

It is clear that these results are in the most evident contra-
diction with those of Cohnheim. Theoretically the possibility
of course exists, that there may be a specific difference between
the two species. Considering the fact, however, that in many
other instances we will see that this author has made more or
less serious mistakes, I am inclined to have my doubts about
his results, the more because Enriques 37) found a proteo-
lytic enzyme present in the same species on which Cohnheim
worked.

Assuming at first that Cohnheim was right in his experiments,
but not satisfied with his attitude of resignation, I studied the

55

whole question over again. Cohnheim himself states that all
nitrogenous excretion takes place through the gut. How is it
possible that nitrogen is excreted, if it is not assimilated? This
would mean a constant loss of nitrogen, which we can not
understand. That an organism as highly organised as a Holo-
thurian should have no nitrogen metabolism seems to be exclu-
ded a priori. Growth and formation of proteins are not possi-
ble then and yet these substances are present.

There is still one possibility of error left in Cohnheim's
experimental procedure. The work of Enriques has shown
that the vasular reticula play a very important réle in the enzyme
secretion (see chapter 20). One of the enzymes secreted by them
is a proteolytic one. If Cohnheim has removed these plexus-
ses, he may have missed the opportunity of finding the enzyme.

In our last chapter we shall discuss the application of Piit-
ter's doctrine to Holothurians. Thinking of Piitter’s work
and assuming that actually no proteolytic enzyme is present, I
made some experiments on the resorption of ammonia and
nitrates by Thyone guts. The possibility of the binding of such
substances to carbohydrates f.i. as a source of nitrogenous body
constituents, should be studied.

For this purpose I secured Thyone guts. These show the
same active movements which mammalian intestine shows in
the living organism and move over the table like worms. Pieces
were cut out, tied off at one end with a soft, but strong thread,
then filled by means of a hypodermic syringe with the solution
to be tested. It was then tied off at the other end with a knot
made previously. The loop was now washed sea water and
then put into a watch glass in some sea water.

In this way it became evident that at various concentrations
ammonium sulfate diffuses through easily. At the end of the
experiment I tested for ammonia in the liquid at the outside
with Nessler’s reagent and invariably got positive results.
The gut was not dead, for very frequently it could still be stimu-
lated by pinching it with a forceps. Neither were there
leaks since, the liquid did not disappear from the gut though
the muscles were in constant tension.

The same thing holds true for nitrates, for which I tested
with the ferrosulfate-sulfuric acid ring reaction. The rate of
this resorption is somewhat slower than in the case of ammonia,
but most assuredly resorption took place.

However, we do not have to take refuge in such more
or less probable speculations, since there is a proteolytic enzyme
and since it is even:present in the free form, Amino-acids do
diffuse through the intestinal wall, as experiments of the nature
of those mentioned above showed. Such experiments were made
with a digest of gelatin by means of trypsin (without toluene).
A strongly positive ninhydrin-test in the outside liquid took away

56

all doubt. This shows that the products of hydrolysis are
also actually resorbed, so that the evidence in favor of my opinion
was complete.

There is still another way of demonstrating the actual resorp-
tion and this way is the more biological one. I collected (1) a
certain amount of the mud in which the animals are found in
nature, (2) a sample of the contents of the fore-gut, -chiefly
from the stomach- and (3) one from the end-gut and rectum.
These materials were dried for some weeks in an _ exic-
cator, ground up, dried again and finally weighed. After that
a total-nitrogen determination was made, based on the principle
of the method of Folin. The digestion which could not be
carried to completionwith Folin-Wu’s digestion mixture, became
complete after the addition of a little crystal of copper sulfate.
In a preliminary experiment I found that direct Nesslerisation
was impossible because a heavy precipitate was formed. For
this reason the distillation method was used.

Knowing the amount of material used and the resulting color,
it was very easy to figure out the amount of total N in one
gram of the three samples. In one gram of the mud as it is
found in nature, 3.1 mgr. of total-nitrogen were present, in one
gram of a composite sample of each of fore-gut contents as much as
21.1 mgr. were present, and in the end gut 1.0 mgr. These
figures of course represent samples and do not pretend to have
an absolute value, though the samples were of course obtained
from many animals. Nevertheless they show very clearly that,
at least in certain periods, withdrawal of nitrogenous substances
from the mud takes place. The samples 2 and 3 were secured
from the same animals, whose fore-gut and rectum were emptied
at the same time.

This withdrawal can be observed even with the naked eye.
As long as the material was wet, all samples had the same color.
The dried substances showed great differences in color: it was
clear, that almost everything present in the stomach sample had
been dissolved away except for the sand.

It will be noted that the quantity of N in the contents of the
fore-gut is much higher than that of the mud found in the
animal’s natural environment. This fact has been explained in
chapter 4.

20. THE RESORPTION IN ECHINOIDEA
AND HOLOTHUROIDEA.

In the chapter on the perivisceral fluid we have compared
this liquid to the ,,blood’” in higher animals. The question is
whether it actually has the function of transmitting the resorpta
from the intestine to the consuming tissue or whether the
corpuscles have this function. Cuénot 23) considers the cor-

57

puscles as the chief transporters of food, other authors: also
attribute considerable importance to their activity in the service
of digestion. Among these authors is Chapeaux 15), who
considers them as especially important in the digestion of fats.
In chapter 16 we saw that in some respects he exagerated their
importance and that I have never been able to observe the
emulsification of fats by the intestinal juices which he found.
He states that the emulsified fat passes through the gut-wall
without any chemical change but gives no experimental proof
for his contention.

»La dissolution s’opére dans la cavité générale’; hydrolysis
takes place here by the corpuscles, as the change in reaction —
which I have critisised in chapter 18 — indicates. If
olive oil is injected into the perivisceral fluid, the amibocytes
,eat’” it. That such experiments do not prove much, will be
discussed in the chapter on excretion.

There is one small problem, however, which he does not
attack. Is there any increase in fat in the corpuscles after fat-
feeding ? in other words, can we actually prove his hypothesis that
the fat would not be changed chemically during its passage through
the intestinal wall, that it would then enter into the perivisceral
fluid and finally be ,,eaten up’’ and hydrolysed by the amibo-
cytes? I have made experiments of this kind repeatedly (on
Arbacia), but always with negative results. Except in two cases
in which the coelomic cavity contained very large drops of oil,
probably on account of the breaking of the intestinal wall, I never
found a trace of free oil. Neither could I observe an increase of
fat in the corpuscles. Centrifuged corpuscles of injected animals
were put on a slide and treated with Flemming’s fluid, as was a
control sample. I never could see any difference between two
such samples though they were withdrawn at various periods of
time after the feeding. For this reason J am rather inclined
to believe that Chapeaux is wrong in his hypothesis.

In the chapter on the perivisceral fluid we saw that this
liquid is by no means absolutely stagnant and motionless. This
makes it possible that such functions as the distribution of food
could be taken care of by this medium.

To study this problem I made some very simple experiments
on Arbacia. Food solutions (e.g. solutions of cane-sugar or of
casein as the most simple carbohydrate and protein) were in-
jected into the digestive tract of the animal.

Feeding experiments never had any success, because the
animals, urchins, starfishes and cucumbers refused to take any
food in captivity, even if their natural food was presented to
them. Starfishes, put into one basin with either mussels or small
sea snails (e.g. Littorina), never ate any of them.

In this respect I was very unfortunate. All plans to feed
animals stained with some vital stain to our Echinoderms for

58

the study of resorption or for the hydrogen-ion concentration
work, could not be carried out.

Twice only I did obtain an animal feeding in its natural way.
They were both brought in with their prey. Once I got a
starfish, in the act of devouring a little snail, in its perivisceral fluid
aminoacids appeared by a positive ninhydrin-test to be present.
The other case was an Arbacia in the act of chewing up a
Clione sulfurea, a calcisponge. A ninhydrin-test again gave
positive results, no monoses could be demonstrated by means of
Fehling.

Apart from these two cases I had to feed the animals artifi-
cially. Several substances were injected in that way for the
study of the resorption and in order to find out whether phago-
cytosis takes place in these forms.

The injection of such liquids is very easy in the case of the
sea-urchins. If one lays an Arbacia on a table with the oral side up,
the lantern will start to perform rhythmical movements up and
down, accompanied by a circular movement of the distal part
of the lantern in a vertical plane. When the lantern is in its
most distal position, the teeth are closed tightly. When it moves
down again, i.e. towards the body, the jaws open up widely.
This is the moment to slip in the needle of an injection syringe.
With some practice one easily succeeds in bringing it down
just as far as the masticating apparatus reaches, without injuring
the tender gut behind it. The animal now tries to bite this
intruder, sometimes also brings it out of the centre of the five
teeth, pushing it down between two of them, so that it
leans against the circular ,,lip’’ and easily falls down.

These movements resemble closely those described by Ge m-
mill 43) and 44) in his papers on the locomotor function of the
lantern. He saw animals moving around by lurches on the
table — the same thing can also frequently be observed in our
Arbacia — and studied this method of locomotion a little more
in detail. The five little teeth, brought down in closed position,
form a powerful central stilt, which lifts the body. This body
of course capsises in a certain direction, which direction is
determined by the circular movements in a vertical plane, described
above. This method of locomotion seems to be rather common in
urchins; not only Echinus esculentus. L. and Echinus miliaris.
Gmel., studied by Gemmill, show it, but also our Arbacia.
They move in that way in a rather surprisingly quick way;
in my work I was sometimes suddenly scared by one of the
animals dropping down from the table, from the centre of which
it had reached the rim. Even loaded — up to half a pound —
and on an inclined plane, they seém to be able to execute
these movements (Gemmill). In protruding the lantern, the
animal relaxes its posterior retractors and contracts the anterior
protractors. These same protractors serve to close the teeth.

59

The contrary of course happens when the lantern is pulled inward.

It looks to me as if there is a great analogy between the

movements which I described above and those described by

Gemmill. It seems to be a general reflex of the urchins if

taken out of the water; perfectly purposeful in case the

ventral side is down, but senseless if the animal lies on its back.

Whether or not these same movements play a réle in respiration, when

the animal is under water, has not been proved experimentally, but it

seems probable from the description of von Uexkill. In the chapter

on the respiration I shall describe the so-called internal gills of the urchins.

These baggy prolongations of the peristomium are filled with water when

the lantern is raised and the water is expelled, when the lantern is lowered.

One indication in favor of their respiratory importance is that in water with

low oxygen-concentration the animals move their lantern up and down

without changing their position. Interesting from the general physiological

standpoint is the fact that these movements seem to follow van ‘t Hoff's
rule with regard to temperature, as Roaf has shown 108).

Returning to our injection experiments, we may say that
in the case of proteins a strongly positive ninhydrin-test after
a little more than an hour showed that they have actually been
broken down .and resorbed and that the perivisceral fluid has
the function of conveying them to the tissues.

In the case of the sugar injections I have never yet succeeded
in demonstrating glucose in the perivisceral fluid by means of
Fehling’s test. This may be due to several circumstances.
Possibly the larger part is ,,burned up” in its passage through
the intestinal wall, as Cohnheim assumes for the case of
Holothuria ; it may also be due to the quick resorption of this
substance from the perivisceral fluid, which we will discuss in
chapter 22.

About the resorption in Holothurians we have two studies
at hand, and strange enough the two authors contradict each
other in nearly every detail Cohnheim 17) was the
first to attack this problem, Enriques 37) in the Archivio.
zoologico discusses his results and comes to absolutely different
conclusions, notwithstanding the fact that both authors worked
on exactly the same species.

Enriques’ paper came too late to my attention: in that
way I was not able to perform any but very superficial ex-
periments on this question; since however it seems to me that
this problem has enough importance, I may be justified in giving
here a short summary of their controversy.

One of the chief points of their discrepancy is the question
of the function and importance of the ,,blood (i. e. the lacunar)’
system. Cohnheim does not pay any attention to it and
simply states, ,dass bei den Seeigeln und Holothurien jede
Circulation iiberhaupt und imbesondere jede circulatorische Ver-
bindung zwischen dem Verdauungskanal und dem iibrigen Korper

60

fehlt. Wasser und in ihm geléste feste Stoffe, die bei der Ver-
dauung entstehen, haben keine andere Méglichkeit zu den iibrigen
Organen, die sie ernahren sollen, zu gelangen als dass sie aus
dem Darm in die Leibeshdhle eintreten und durch diese hindurch
zu den andern Organen gelangen (p. 18)’.

Enriques has made a very extensive study of the anatomy and function
of this system, which consists of a dorsal mesenterial and a ventral anti-mesen-
terial complex of vessels. On the dorsal side we find a dorsal marginal
vessel over the whole length of the gut, adhering so firmly to its wall
that it can not be torn off. Between the ,,loop’ of the intestine, it forms
a so-called transversal vessel. In the ,,oesophageal ring” there is a blood-
ring by which it communicates with the ventral vessel. From this ring
five vessels radiate out, following the five ambulacrae.: There is also present a
ventral marginal vessel which splits further on into a left and right ventral mar-
ginal vessel. These communicate by means of the anterior and posterior trans-
verse vessels. From the right ventral marginal vessel the rete mirabile runs
over the whole gut and mesenteries.

Other vessels go the water-lungs and to some other organs; the genital
organs are also richly vasculated.

Something of the same nature is also found in the sea-urchins and
starfishes. Here the whole system is much less complicated; in the starfishes
it took a long time before it was discovered by Cuénot 25).

Enriques first mentions that in studying the movements of
a body suspended in a liquid, one does not get a clear idea
of the real movements of such liquid (inertia). He succeeded by
means of a small syringe in bringing into one of the vessels
an oil drop which filled the whole vessel so that no water
could flow between the wall and the drop. He then registered the
movements of these drops photographically. He finds that the
oscillating movements of the blood, the pulsating of the vessels,
finally causes a gradual movement in one direction, moreover,
that the drop is broken down into two parts as soon as the
vessel branches into two. This process goes on and in this
way the fat is finally distributed all trough the blood system. This
is another proof for the effectiveness of the mixing in the blood
system.

The vessels pulsate especially when they are full of liquid as
he could demonstrate by experiments in which he injected a
small quantity of sea water into a vessel. This is the case
during the period of resorption.

A very interesting process of secretion (of enzymes?) is described by
Enriques. Greenish dichroitic granulae showing much similarity to the
liver secretion of Molluscs (Enriques) and whose enzymatic nature is
made probable by the fact that they stain brownish with osmic acid, are
formed in the cells of the wall of the vessels; they are carried to the
gut-wall, sometimes by amibocytes, and penetrate into the lumen. Their
number increases during inanition.

Let us now proceed to discuss Cohnheim’s experiments
on absorption. A remarkable thing in all his experiments is that
he nearly always fills the intestine with 20—30 c.c. of liquid. Now,

61

according to Enriques, the gut of very large animals does
not contain much more than 10 c.c. In that way Cohnheim
must have made his experiments on distended guts. This is ac-
cording to Enriques one of the chief sources of error in his
experiments: the epithelium after death has allowed the contents
to be pressed out through smail holes by the surviving muscu-
lature. He himself seems to be conscious of this pressure, for
he says (p. 24): ,,Ich fiillte ihn (i.e. den Darm) unter geringem
Druck aus einer Biirette’’.

Cohnheim finds that if sea water is put both inside and out-
side the gut, it is resorbed almost completely within 24 hours.
Enriques does not fill the guts with as much liquid, and
does not find any such resorption.

This discrepancy is rather striking and surely proves that one
of the authors must have made more or less serious mistakes.
Enriques warns against using the guts for 24 hours or lon-
ger, they frequently fall to pieces after that length of time.

Other experiments have been made both by Cohnheim
and by Enriques on the resorption of various substances
‘dissolved in the sea water. Some of Cohnheim’'s experiments
are very unnatural: e.g. he injects a 1°/) solution of sodium
iodide in sea water and a ?/;°/, solution of sodium phosphate.
Experiments of this kind can not possibly elucidate as impor-
tant a question as the present one. His results are nevertheless
remarkable, he finds that from such hypertonic solutions the
dissolved substance as well as the solving medium passes to
the outside.

This is almost pure resorption as we find it in vertebrates
and of substances which, if not toxic, are certainly indifferent.
On the other hand these salts are used in concentrations which
are much too high and their toxic action may easily have kil-
led or harmed the gut wall.

The experiments of both authors on the resorption of
sugar etc. also contradict each other in many points and it is hardly
possible to get a clear and unprejudiced picture of the process.

Enriques severely critisises Cohnheim’s work and shows
many contradictions in his experiments. In some of these ex-
periments Cohnheim uses solutions the osmotic pressure of
which is much too high. Sugar solutions in sea water are used
e.g. of 3—18°/,. Such solutions are beyond the range of phy-
siology and results obtained therewith only give an insight in

a , pathological physiology”, as Enriques calls it. Against such
osmotic potentials no semipermeability of the gut-wall can resist.

In the experiments on sea urchins too serious operations are made;
since they gave the same result of those on the cucumbers, | will not
discuss them here.

The outcome of the differences in their experimental results is

62

an entirely different conception of the process of resorption.
Cohnheim considers the gut wall merely as a diffusion-mem-
brane through which substances pass equally well in both
directions. There is no ,,polarity’; there are no substances
which are resorbed against an osmotic difference. On the
other hand there is an active water-resorption, ,,die sich
aus den osmotischen Kraften allein nicht erklaren lasst’’. The
diffusion of dissolved substances takes place just as in every
diffusion membrane, without any ,,orientation”, but water is
resorbed quite independently and simultaneously.

Enriques pictures the process of resorption in an entirely
different way, very much as it takes place in mammals where
this remarkable, and as yet physically unintelligible process was
discovered by Cohnheim (Zschr. f. Biol. 36. 1898. 129 and
37. 1893, 443) and by Waymouth Reid (Philos. Transact. Ser.
B. 192. 1900. 211).

He makes the very interesting observation that during the
period of digestion the Cl-percentage in the visceral fluid is
lower than in sea water. This phenomenon is most pronounced
during the actual digestion, after resorption has taken place
the difference is only very slight. I could myself verify the latter
stratement, but at the same time I observed that it stillalways exists,
in some hasty experiments which ] made as soonas Enriques’
paper came into my hands. On the same day on which the
animals were brought into the laboratory, he eventually found as
little as I. 84 gr.°/) of Cl,in older specimens kept in captivity
for some time, the concentration of the chlorides sometimes was
the same as that in sea water, i.e. 2.11 °/). The same thing holds
true for the sulfates which he titrates with barium chloride.
He explains this peculiar phenomenon thus: during the period
of digestion when the larger molecules are broken up into
smaller ones by hydrolysis, the osmotic pressure inside the gut-
wall increases. Consequently water diffuses out into the gut,
diluting the sea water which was present at first. Consequently
the percentage of chlorides in this liquid, decreases. It does not
increase in the coelomic liquid, however, as experiments show.
The assumption is therefore made, that regulatory processes
take place either through the water-lungs or through the many
other places where the perivisceral fluid comes in close contact
with the surrounding medium, the sea.

Gradually the products of hydrolysis are resorbed now and
the contents of the gut become hypotonic. Practically no chlo-
rides can enter into the gut, but the water is resorbed back
with the food substances in it.

The blood system becomes full of liquid, consequently the
vessels begin to pulsate — see above — and distribute the
food all over the larger part of the body. Part of it is given

63.

of to the perivisceral fluid, which also takes care of the distri-
bution of the food.

Real resorption though in its kind primitive takes place here
in that way. ,,L’assorbimento e dunque necessariamente nelle
Oloturie un processo fisiologico.”’ This is still more evident
from the following set of experiments. Enriques demon-
strated that sugar can be resorbed from solutions of equal
osmotic pressure or even lower. For this purpose he prepared
artificial salt-sugar solutions, the osmotic pressure of which he
controlled by freezing point determinations. The osmotic exchange
takes place chiefly by means of water passage, the chlorides do
not pass as quickly. In one of Enriques’ experiments as much
as 2.3 c.c. of water had passed over to the outside and only
0.004 gr. of NaCl into the gut. As a rule such osmotic diffe-
rencesas are used in the experiments will not occur in nature.
Thus we can conclude that the Holothurian intestine possesses
as Enriques calls it, an ,, absolute semipermeability”, and is not
capable of allowing either salts or food substances to pass
through by ,,diosmosis’”. To quote his own words: ,,La mem-
brana intestinale presenta dunque in modo palese due proprieta
apparentemente contradittorie: impermeabilita per diffusione,
permeabilita per assorbimento.” A true ,,physiological’’ resorp-
tion occurs the mechanism of which, as in Vertebrates, is
as yet completely unknown.

This resorption question is one of the big problems of general
physiology and it is to be hoped that in the next decennia com-
parative physiologists will study the genesis of this remarkable
property of the living gut wall more in detail. It is in the various
groups of invertebrates that we must observe it at its origin. It is
by no means primitive: in the lower forms (Sponges, Coelen-
terates, Plathelminthes, etc.) the ,,assimilation”’ of the food occurs by
means for phagocytosis, later on we find special organs developped
for resorption (as the ,, Mitteldarmdriise’’ of Crustaceans and Mol-
luscs, the ,,liver’’ of the starfishes — see the next chapter ~,
the coeca of Aphrodite, etc. etc.) and only in the higher invertebra-
tes do the first traces of resorption over the entire gut wall occur.
In the lower forms the gut is just as long as is necessary for a safe
conduction of the food to the organ of enzyme secretion and resorp-
tion and for a rapid elimination of the waste. Frequently it is even
covered by completely impermeable substances, such as chitin,

Only in animals in which the gut begins to be much
longer, may we expect to find the solution of this big problem.
And even in many of these three characteristics of this function
are absent: 1. the polarity of the intestinal membrane, 2. the
resorption of fluid together with the food substances and 3. the
rapidity of the process, as Jordan states for the case of Helix.
The study of the gradual development of these three properties
in the different groups — in Helix e.g. sugar can be taken up

64

- from an isotonic solution by the intestinal wall (Jordan 69)) —
may give us the key to the explanation of this phenomenon as
yet unexplained on a physico-chemical basis. Such instances
as Helix may be especially instructive, their gut epithelium does
not take either iron, carmine or fat. These properties have
also been considered as characteristic for resorbing epithelia
(Cuénot. Arch. de Zool. exp. et gén. (3). 25. 1899. 7;
Biedermann and Moritz 9)). ~

Among the many, perhaps unreliable, results of Cohnheim,
one is very interesting. If sugar is injected into the perivisceral
fluid, he finds it back inside of the gut. I have made similar
experiments, using cane-sugar for the injection. Two interesting
facts could be observed in such animals: 1. If the gut contents
were secured after about eight hours and hydrolysed, a strongly
positive Fehling’s test indicated the presence of sugar;
this proves that Cohnheim did not make a mistake in his
observation, 2. Some of these animals showed a peculiar swelling,
as though they had taken up water to compensate for the exces-
sively high osmotic pressure of their coelomic fluid. This phe-
nomenon was not observed regularly, but was very striking.
It even attracted the attention of some visitors to the laboratory.

The first result is interpreted by Cohnheim as proof in
favor of his conception of the gut wall as diffusion-membrane.
I am inclined however to believe that another explanation is
more probable and I was glad to find that Enriques had made
a remark of the same kind. In our chapter on excretion
we shall see that uric acid, a normal excretion product of the
Echinoderms, is excreted into the lumen of the gut. Probably,
as we shall there see, this is the normal way of excretion in
this group, which is certainly true in many other invertebrate
groups. The same thing may hold true for sugar, the excess
of non-utilisable sugar — see the chapter on enzymes in the
perivisceral fluid — being possibly excreted here, just as in mam-
mals in cases of alimentary glycosuria.

The swelling phenomenon then might be explained by the
assumption that the animal is not capable of ,,working through”
so much sugar at once but is capable of taking up water by means
of its many water-permeable membranes. In that way a pre-
liminary dilution would make the high osmotic pressure less
harmful. Since I could not study this problem more in detail,
I shall leave this explanation in a very hypothetical form; the
recent work of Dekhuyzen 30), however, on the Sipuncu-
lidae makes such hypothesis rather probable.

Note. Enrigques has kept some of his Holothurians in a
vessel filled with sea water containing milk. After some time
he finds fat droplets in the animals blood-vessels and later on
in its perivisceral fluid. Still later the quantity of fat in the

65

coelomic cavity has increased, but not so that in the blood system.
To him this is a proof in favor of his conception of the impor-
tance of the blood system in resorption, as one can easily
understand. I mention it in connection with my own experiments
on sea urchins.

Enriques finds the fat in free form in the coelomic cavity.
It is not taken up by the corpuscles, according to him. This
agrees very well with my own experiments on sea urchins;
there also I did not find any increase in fat in the corpuscles
after fat feeding, it disagrees however with the experiments of
Chapeaux, mentioned previously. I did not find any free fat
in the coelomic cavity of my urchins however.

21. THE. RESORPTION IN ASTEROIDEA.
FUNCTION OF THE THEIR ,,LIVER”.

There is an old question in comparative physiology which
Jordan 68) calls the ,,Leberfrage’. The numerous appendages
of the middle-gut of invertebrates have usually been called
»liver’’ by the older, especially by the morphological writers.
This can not astonish us, since in most groups they are located
exactly where the liver is found in higher animals. Furthermore,
their secretion frequently contains a yellow or brown pigment
and tastes bitter. One of the things which chiefly accounts for this
assumption is the fact that they frequently contain glycogen. Their
secretion also contains sugar by which even Claude Bernard
6) and 7) was misled.

Later on it appeared that the secretion of these organs
resembled more that of the pancreas of the Vertebrates; in
order to recognize this fact the name ,,Hepatopankreas (Max
Weber)” was accepted and is used up to the present day in
the leading text-books of zoology.

One of the chief reasons why this belief was not sooner aban~
doned is the fact that with the word gut one generally associ-
ates the notion of a tube, such as is found in vertebrates. A
ramified gut was known to occur in many of the lower groups, e. g.
the flatworms and the coelenterates, but the idea that these ,,livers”’
are nothing much else but ramified middle-guts, did not at once
succeed in getting a foot-hold. Yet, just as in many of these
lower groups the respiratory organs branch out all over the
body, so to a certain extent, does the digestive tract. In many
instances we have to do here with an adaptation to the lack of
circulation, the intestine follows the form of the body in order
the assure an equal distribution of the food.

Now in this connection it is very remarkable that organs
of the same nature are found in the starfishes, just where the
problem of food distribution is most difficult owing to the
strange shape of the body.

5

66

In the Holothurians we find that the body wall is simply
a sac in which the gut is folded many times. The urchins are
also simple, round containers and even here the gut shows two
curvatures and winds around in every direction. But in the
starfishes it would not be possible for sufficiently large quan-
tities of food to reach the tips of the arms on account of the
lack of circulation, if the gut did not extend into these arms.
The ,,Mitteldarmdivertikel’’ are a logical postulate here.

These middle-gut sacs are on the one hand a place of enzyme
secretion, but on the other hand, chiefly organs of resorption.
The first authors who clearly realised this important fact, were
Biedermann and Moritz 9) in their almost classical monograph
on the function of the so-called ,liver’ in molluscs. They
showed that the anatomical relations are such that the food
is forced to enter into the ,liver’’ up into the smallest rami-
fications. This can easily be shown, if e. g. the animals are fed
on flour and the iodine test is applied to microscopical sections.

Numerous structures which have always been a puzzle to
anatomists and on which a very particular light is thrown by
this discovery, have since been explained by it. Two very inte-
resting cases have been described by Jordan 64).

In case this ,liver’’ is not present, i.e. in those groups to
which the collective name of ,,injecurata’’ has been given, other
devices take their place in such groups as have an insufficient
circulation. In this way the ramifications of the gut of the
flatworms, the coeca of Aphrodite, the curvatures in the digestive
tract of sea urchins and sea cucumbers etc. may be explained.

It has frequently been stated that bile pigments are found
in these livers. This is not true and later authors (Hoppe-
Seyler, Frenzel, Plateau, Voit, Frédéricg et al.)
always failed to find them.

Summarising this brief introduction we may say that these
middle-gut sacs and the liver of the vertebrates have no specific
function in common. These so-called livers are nothing but
extensions of the middle-guts for the function of resorption, a
system of blind-guts.

From the very beginning of this work I suspected that the
radial sacs of the starfishes would appear to have that same
function. To get some more definite information on this question
I used different procedures partly of a positive, partly of a
negative nature.

Two different views have been held in the past; they were
either considered to be organs of secretion or organs of resorption.
The first view is the one held by nearly all authors f.i. Grif-
fiths, Frenzel, Frédéricg, Krukenberg, Cuénot (in
1887) and others. Biedermann in Winterstein’s Handbuch,
summarises the earlier views thus: ,,dasz die Radialanhange des

67

Magensackes bei den Seesternen in erster Linie der Absonderung
des wirksamen Verdauungssaftes dienen, w&hrend der riesige
Magen selbst lediglich als Behalter der aufgenommenen Nah-
rungskérper dient und nur insoweit eine active Rolle spielt, als
er sich vorstiilpend, die oft sehr groszen Beutetiere noch auszer-
halb des K6rpers umschlieszt ..... -

Krukenberg, about whom we have given our opinion
already in the introduction, denies emphatically that food
ever penetrates into the ,,Leberga&nge” of the starfishes. He
fed fibrin stained with different dyes to these animals and the
result of his observations was, ,,dasz die sogenannten Radial-
anhange des Asteridenmagens reine Ausfiihrgange der Leberdriise
sind’. In some cases he found parts of the water vascular
system colored even out to the tips of the arms, but no dye
present in the ,,liver’’.

Similar observations were made by Frenzel and Frédéricq
who also believed that the radial sacs only have secretory and
no resorbing activity or that they are the only secreting organ.

Chapeaux, Hamann and Cohnheim have come to a
different conclusion. Chapeaux found that after the ingestion
of fat the cells of the radial sacs were all fullof it, Cohnheim
proved by autodigestion-experiments that enzymes are present
just as well in the stomach as in the radial sacs.

If the radial sacs are the exclusive or even the chief organ of
enzyme secretion, it must be possible to demonstrate a difference
in enzymatic activity between equal quantities of stomach and
radial sac material. On the contrary, if such difference is found
not to exist, if both prove to be about equally active, it is not
very probable, though yet concievable — on account of the
relatively enormous quantity of radial sac material present in
one animal —, that a difference can be shown.

An equal quantity of stomach and of radial sac material was
secured. Equal quantities were taken rather than proportional
parts, because there is in reality about 7 or more times as much
radial sac as stomach material which would not give an unfair
comparison. Both were ground up with sand, to both an equal
quantity of gelatin, water, sodium carbonate — to keep the reaction
slightly alcaline — and toluene were added. The digests were
now placed in the incubator at 37° and kept there for 24 days.
After they had been taken out of the incubator the amount of
total non-protein nitrogen present in them was determined.
The proteins were precipitated with an excess of 5°/, trichlor-
acetic acid. In an aliquot the amount of total nitrogen was
determined by means of the method of Folin. The diluted
digestion mixture was used and direct Nesslerisation.

In both digests in which 11.5 gr. of tissue had been used,
almost exactly the same amount of nitrogen proved to be present.
In the digest of the lateral sacs. I found 144 mgr. of nitrogen, in the

68

other 138 mgr. The difference between these two figures is most
probably within the limits of error. If not, it is sufficiently
explained by the fact that proportionally more epithelial tissue
is present in the same quantity of radial sacs, since the muscular
wall of the stomach of course has also been included.

These figures certainly do not favor of the assumption that
the radial sacs are the organs of enzyme secretion; on the
contrary they prove that relatively just as much enzyme is
secreted by the stomach. In the chapter on the histology of the
two organs we have seen that the ,,granular’’ cells are present
in the stomach just as well as in the radial sacs; this affords
other evidence in the same direction ').

There are, however, in the literature certain statements which
seem to make the other view more probable. One of these we
find in Cohnheim’s paper. Cohnheim observed that if one
puts small pieces of mussel flesh or fibrin into the stomach of
an Asteropecten aurantiacus, ,,so wird es nicht verdaut da die
Fermente nicht in den Magen gelangen. Fiir die Thatigkeit des
Seesternmagens ist ein complicirter Bewegungsmechanismus nétig
die das von dem iibrigen Tiere losgeléste Organ nicht mehr
leisten kann’. He states that with some practice it is easy to
prepare the stomach free. I never succeeded in accomplishing
it without tearing the wall. I do not understand either how
one can bring a solid particle into these stomachs which con-
tract their sphincter as soon as the animal is treated roughly.

For these reasons I have made similar experiments in a some-
what different way. The arms of a starfish were cut off at a
little distance from the disc. Ventrally each arm was now opened
carefully near the centre of the disc. The ducts of the radial
organs were located and prepared free from the dorsal mesen-
terium. This operation is exceedingly delicate and I had to
eliminate several animals before I succeeded in accomplishing
it. The ducts are very fragile and break at the least
touch. A ligature was now passed around the ducts after which
they were tied off firmly. The ducts were now cut distally and
the remainder of the arm was eliminated which is very easy
since they naturally break off at the articulation of the arm
with the disc (see p. 70).

After all rays had been treated in this way, the stomach
was filled with a food substance in liquid form, be it a sugar
solution or a solution of some protein for which I usually took
casein — dialysed carefully ~ or K.L. I. M., a commercial milk
powder. The injection was made by means of a small hypodermic.

These experiments had a double purpose. In the first place
I wanted to find out whether, as Cohnheim found, the isolated

1) Chapeaux could extract enzymes even from oesophagus and rectum.

69

stomach is incapable of digesting food, secondly whether it
is capable of resorbing the products of hydrolysis.

The preparation after having been filled with the solution to
be studied, was put into a shallow watch-glass in some sea water
with the oral side up — in order by gravitation to keep the
liquid in the stomach ~. After various periods of time tests
were made for glucose or amino-acids in the surrounding liquid.

As far as the digestion is concerned, completely positive evidence
was obtained. A Fehling’s test and a ninhydrin both gave
positive results in so many cases that there does not seem to
be any reason for doubt as to the capacity of the isolated
stomach for digesting food substances.

As far as the resorption is concerned, I believe that we are not
entitled to draw sufficiently justified conclusions from a few experi-
ments like these. There are three important possible sources of
error. In the first place we do not know how long the stomach
actually remains alive; its wall might finally become a dead
membrane through which everything passes; in the second
place the products of hydrolysis may have escaped through
the mouth and thirdly the stomach pouches and the remnants
of the ducts may have taken care of the resorption. One fact
especially makes me doubtful, i.e. that it always took a long
time for the substances to make their appearance. '

The anatomy and histology of the radial ducts does not
give us much information either. In the snails the anatomical
relations are such that the food masses are forced to
enter into the ,,liver’’ (see Biedermann and Moritz” 9)),
The same thing is true for the crawfish (see Jordan 64)).
Here we do not find anything of the kind. I have fixed some
of these ducts including a little piece of the stomach and of the
radial sacs in acetic-alcohol and cut them up in a serial section.
Though these sections were very poor, partly maybe on account
of the fixing fluid, they showed at least this, that there is no
particular device for forcing the food into the stomach neither
is there a filtering apparatus (,,Reusenapparat”) The ducts are
simply hollow tubes, leading streight from the stomach into the
radial sacs. The lining epithelium is ciliated everywhere : possibly.
these cilia by their movements account for the transport of the
food from stomach to radial sac. In the sections moreover
small pieces of organic material, stained with the eosin employed
to color the sections, were seen lying within the tubes. To me
they looked like food particles on their way from the stomach
to the radial sacs.

If all these data do not give us any positive evidence whether
or not the radial sacs are organs of resorption, there are others
however which to me are absolute proof for my thesis that
they have this function.

I have fed starfishes on various substances and I could observe

70

that all these substances actually penetrated into the radial sacs.
Since the animals did not take any food voluntarily, I had to
feed them artificially. The animal was laid down on the table
with its mouth turned upwards. The needle of an injection
syringe was now introduced into its stomach. As the animals
on such rough mechanical treatment frequently withdraw their
oral sphincter, I was often obliged to remove some of the
spines surrounding the oral cavity. The fluid was then injected,
in some experiments the animal was put into the water
again soon after the injection, sometimes the mouth was closed
with recently melted paraffin of a low melting point. The
animal frequently succeeded in soon getting rid of the piece of
paraffin, but yet, the paraffin did prevent the immediate squeezing
out of the injected substances.

In some of these experiments the animals autotomised their
arms, probably on account of the unusual stimulation by injec-
tion and unnatural food. This peculiar autotomy takes place in
starfishes along the interradial saepta which divide the coelom
of the disc in five parts. It takes place very easily in some
species; Luidia ciliaris, according to Cuénot 22), is almost never
caught in intact condition. They are like folds projecting
inwards from the interradial spaces; they are stiffened by a calca-
reous deposit and are the areas of lateral adhesion of the arms.
In them the stone-canal and the axial sinus are found. If all the
arms are removed in this way, only an exceedingly small part
of the disc is left as central part.

It needs not to be stated that ,abnormal” animals of this
kind were always excluded.

One of the injected substances was the saccharate of iron.
This. substance has frequently been used in experiments on
resorption, on account of the fact that it is so very easy
to make iron visible in microscopical sections. As examples from
invertebrate physiology, I might quote the papers of Jordan 69)
on the resorptive function of the middle-gut gland of Helix
pomatia and that of Steudel 123) on absorption and secretion
in the gut of insects.

The substance was prepared according to the U.S. Pharma-
copea (carbonas ferrosum saccharatum). In injecting such prepara-
tions one must be very careful to use a very small dosis, since
even traces of iron are toxic (see also Jordan 63)). A very
dilute solution of the preparation was used for this reason, which
has a light brown color.

At different intervals after the injection, the animals were
opened, inspected and, if they looked promising, the radial sacs were
fixed. It was most interesting to observe how the material entered
the radial sacs through the ducts. The small iron particles which
had not gone into solution could clearly be seen through the wall
of the ducts and eventually one could even see them move.

71

The material penetrated into the very tip of the ,,livers” ; a small
piece of the tip of one of these organs was dissolved in nitric
acid and gave a strongly positive prussian-blue reaction. When
one has actually seen an experiment of this kind, one imme-
diately realises the importance of the radial sacs as organs of
resorption.

The fixed material was cut up in Amsterdam where Mr.
K. Boedyn obliged me very much by his valuable and expe-
rienced help. It appeared that up into the smallest ramifications
of the radial sacs little iron particles could be seen, which can
easily be made visible as blue grains -prussian-blue- by a
treatment of the sections with ferro-cyanide and hydrochloric
acid, described by Tartakowsky 126) '). But this is not the
only thing visible in these sections. In fig.5 of our plate, we
see one of these sections in its natural colors, resulting from a
combined treatment with eosin and the prussian-blue procedure.
In this figure we also see that the material is actually resorbed.
The cuticula of many cells shows a dark blue coloration,
the iron seeming to diffuse out into the gut epithelium. Then we
can not follow it any more till we see it again in the pavement
endothelium of the coelomic cavity. This layer has in some
sections a very dark blue color. No considerable quantities of
iron are found in the layer of connective tissue, but the coelomic
endothelium, the ,,stain-layer’’, seems to store the materials which
have been resorbed, before giving them off to the perivisceral
fluid.

Something of the same nature could be observed in some
experiments in which a solution of ammonium carminate was
injected. On dissection the radial sacs of such animals appeared
to be absolutely red. They were fixed in alcohol, in which,
as we know, ammonium carminate is insoluble. They could in
that way be passed through xylol and paraffin without fear
that the stain would be dissolved. In these sections one of which
has been pictured in fig.6 of our plate, we again see something
of the same nature. The carminate has been resorbed by the
epithelium and passed over to the ,,membrane nutritive’, as
Cuénot has called an analogous layer in other forms. It is
seen here not only in the coelomic endothelium, but also in the
layer of connective tissue. Unfortunately these radial sacs were
taken out at a late stage of the resorption. The actual
resorption process can not be observed as in the iron
sections, but the result is as clear as it could possibly be. The
presence of the carminate everywhere in the coelomic lining
investing the radial sacs, is the most conclusive proof that
these organs have resorbed it.

1) The sections, after having been freed from paraffin and xylol, were

put intoa 1*/,% solution of potassium ferro-cyanide for 15 minutes, then for 10
min. into a 0.45, HCI solution. ~

72

Another series of injectioas was made with olive oil. This
substance also was seen to enter the radial sacs, which
looked like a kind of oily gland shortly after the injection
had taken place. For fear that I might dissolve away the fats
by passing the fixed radial sacs through the alcohols and xylol,
they were fixed in Flemming, washed and imbedded in soap.
The recent method of Mc.Junkin described in chapter 16
was used for this purpose.

Also in these fat preparations the same thing could be observed
again. The whole epithelium appeared to be full of fat in drops
of all sizes. It was so full that the sections, notwithstanding the
fact that the Flemming had stained the fat only incompletely,
appeared to be dark greyish even on superficial examination.
The addition of Sudan III] gave a most brilliant picture as
described in chapter 16 which one could not possibly reproduce
in print. The whole epithelium appeared like one mass of reddish
droplets.

From all this evidence we see that without any doubt this
liver’ like so many other invertebrate livers is primarily and
chiefly an organ of resorption. It is rather remarkable that
Krukenberg 78) came to results which are absolutely con-
tradicting mine in experiments of the same kind. He fed Astro-
pecten pentacanthus and aurantiacus and Asteracanthion glacialis
on fibrin stained with different dyes and never saw them enter
into the radial sacs. He concludes: ,,dasz die sogenannten
Radialanhange des Asteridenmagens reine Ausfiihrungsgange des
Leberdriisen sind und dasz in sie am normalen Tier kein Speise-
brei gelangt.”” In connection with my remarks in the introduction,
I do not consider this as a very serious objection to my results.
Just as in the snails the food substances here enter into the
liver to be resorbed there.

One peculiarity, however, of the snail's liver is absent here,
Biedermann and Moritz describe were definite and active
movements of the separate pouches of the Helix liver, causing
currents which move the food through the whole organ. They
compare the liver of an animal in full digestion to a kind of
boiling jelly, bubbling here and there. This does not occur in
starfishes as far as I could observe. Their radial sacs are com-
pletely motionless during the period of food ingestion, probably
the cilia do most of the work here (compare also the chapter on the
histology where we mentioned that muscle fibres are either
absent or only occasionally present in these organs).

Nearly all natural food in starfishes seems to be digested
very completely. The anus is almost never used and is very narrow.
Shells if taken up are removed by the mouth. This also might
furnish evidence in favor of a very complete resorption into
and digestion in the radial sacs. In the other groups of Echino-

73

derms we always find very large faeces and a very incomplete
digestion — see chapter 9 and 10.

Note: 1. Griffiths 48) has considered the function of the
radial sacs to be partly of pancreatic, partly of excretory nature.
The first author who supposed that they have the function of
kidneys, was Johannes Miiller 91). Griffiths demonstrated
— see the chapter on excretion —, that uric acid is present in them.
After injection of acid fuchsin, indigo-carmine and methyl-green
Cuénot found the epithelial cells of the pyloric coeca of A.
rubens full of these substances; this surely proves that they
must play some rdéle in excretion. The same thing was observed
in urchins; the cells of the last part of the gut (second curvature
and rectum) also appeared to contain these substances. Jordan
63) in his paper on the excretory function of the ,,liver’” of
Astacus fluviatilis, points out that one must distinguish between
the true function of an organ, an accessory function and a merely
incidental activity- between: ,,Hauptfunktion, Nebenfunktion
and Nebenerscheinung (i.e. zufallige Reaktion auf zufallige Be-
dingungen)”. As a matter of fact, there is reason enough to think
of an excretory function on the part of these invertebrate ,,livers’’.
Kovalevski 75) finds indigo-carmine, injected into Squilla, in
their liver, de Saint-~Hilaire 111) finds peptones there in
crawfishes, so does Bruntz 12) with other substances. Darboux
29) finds uric acid and urates in the coeca of Aphrodite and
describes ,,cellules excrétrices’’ in these organs. For Arachnoidea
it has been established with an almost absolute certainty by
Berlese 5), that their ,,liver’’ has excretory function. He demon-
strated uric acid in these livers. Plateau, Bertkau and others
also found guanin in them.

Mc. Munn 93) comes to similar conclusions for Molluscs
and decapod Crustacea. Jordan himself finds injected methy-
len-blue back in the liver of Astacus. When we remember that all
these ,,livers’” are nothing much more than simple appendages of
the mid-gut, serving for the extension of its surface and
that the Malphigian tubes, the organs of excretion in arthropods,
are also appendages of the mid-gut, except as far as their
ontogenis is concerned, we must believe that the mid-gut plays
a role of some kind in excretion. Jordan believes that this
excretory function, at least in Astacus, is a ,,Nebenerscheinung”’,
not a true function — just as in the case of the excretion of
small quantities of urea, anisoil, alizarin etc. by the mammary
glands —.

Delage 31) is opposed to their excretory function. as far
as the physiological injections are concerned, he calls it phar-
macology, Cuénot 24) explains the excretion of dyes as due
to the desire of the organism to get rid of the excess of water,
in which the colored substance has been dissolved. As far as

a

74

the excretion of iron is concerned, Jordan is certainly right;
the presence of uric acid however, a very important excretion
product in lower animals, in the radial sacs of Asterias, in
the coeca of Aphrodite etc. makes me believe, that this ques-
tion is not yet completely settled, the more because Roaf 109)
found creatinine to be present in many of these organs. This
fact seems to me to need further confirmation, considering the
fact that this substance only rarely occurs in lower animals;
if it appears to be true, however, it is another indication in
the same direction.

2. The function of the so-called siphon in the urchins has
never been investigated and is still completely unknown. The
assumption of Perrier 99), that it might have respiratory
function, does not seem very probable to me, because at least
in the species I have studied, one never observes any active
movements in them') and because other organs of respiration
are known in Echinoidea.

A priori it might be thought of as an organ of resorption.
This is not probable however for two reasons: 1. its histology
is more primitive than that of the main-gut — see the
chapter on the histology —, 2. in sections of guts of animals
which had been fed on ammonium carminate and other sub-
stances I always found these substances present in large quan-
tities in the main-gut.

Thirdly, one might assume that it is a kind of hydrostatic
apparatus for the transport of the food in the gut. The food
is in our sea urchins transported in round masses, included ina
mucous membrane — food-vacuoles as observed already by
Roaf and Scott; see p. 33 —. They fill the whole gut and
owing to the incompressibility of fluids, it becomes necessary
to have a device of some kind to permit their forward move-
ment, the siphon might serve for this purpose.

22. THE RESORPTION FROM THE
PERIVISCERAL FLUID.

When we take for granted that the perivisceral fluid has the
function of ,,blood”, of distributor of food substances, it seems
very strange that we never find these substance in it. In the
chapter dealing with this liquid we have seen that it contains
almost no organic substances, except for a small quantity of
uric acid and some corpuscles. Ordinarily no monoses, no amino-
acids nor peptones nor proteins are present.

In the chapter on resorption in Echinoidea and Holothurians
we have learned that after the artificial ingestion of pure food

1) V. Henry 53) however saw rhythmical contractions.

75

substances into the digestive tract these products of hydrolytic
dissociation can as a rule be demonstrated very easily. This
suggests a rapid withdrawal from the blood by the tissues and
it seemed to me worthwhile to investigate this problem a little
more closely.

We know that in nature the Echinoderms obtain their food
at very irregular intervals and I have often been impressed by
the fact that almost all marine animals are, so to say, ina state
of constant starvation.

If this is true it must be of the utmost importance for the
animal to secure whatever it gets and whenever it get sit, in the
most rapid and effective way possible. Seen from this standpoint,
this rapid withdrawal of substances is without any doubt a
property which is highly purposeful in the struggle for life.

There is one very essential feature by which these bloods
are entirely different from mammalian blood. They are in no
respect a store of anything, they are only a transmitting medium.
Nothing is stored in dissolved form; the protein crystalloids,
described by List 81) and the fats contained in the interior
of the corpuscles, are not yet present in dissolved form, but
in formed elements.

These bloods are not the carefully balanced system which
the blood of vertebrates represents; they are just dissolving and
transporting media. The question whether they are absoluty
stagnant as has frequently been supposed, has been discussed
on p. 39.

To study this question of the withdrawal of food constituents
from the perivisceral fluid and to get some information about
the food-consumption by the tissues, I made the following set
of experiments on starfishes. Various quantities of either glucose
or glycine, being the most easily available monose and amino-
acid, were injected by means of a syringe into the coelomic
cavity. They had been dissolved in sea water and were distributed
as equally as possible over the whole system. To avoid the
objection that part of the injected fluid might have escaped
through the wound made by the injection, I first made the
following control experiments. Several starfishes into which a
large quantity of glucose had been injected, were put into a
finger-bowl in a minimal quantity of seawater. After an hour
or so this liquid was tested with Fehling and always gave
a completely negative result, even if Benedict's qualitative
reagent was used. The same tests in the case of glycocoll
gave a negative ninhydrin.

Possibly this retention of injected fluids is due to a contraction
of the muscle tissue which is found everywhere under the peritoneal
epithelium, very much like when in intraveneous injection in
mammals no blood escapes. It may still have another explanation.
Cuénot 23) and 27), in his paper on the biological importance

76

of the clotting of the urchins blood, describes the formation
of a clot in animals from which he removed a very small part
of their shell. After two hours he found the hole covered with
a crust just as a wound in mammals is covered up. All kinds
of corpuscles were seen in these clots, the cells do not any
longer form amiboid pseudopodia, but are gradually transformed
into connective tissue (,,semblent déja en voie de transformation
conjonctive’). The same thing takes place in many other
species — the original experiments were conducted on Echinus
acutus. Lmk. —. It does not take place if the wound is too
large. Then the rims of the wound are closed, but the animal
dies, before it has been able to repair the damage.

The biological importance of such protective clotting is very
evident, especially in the case of the Spatangidae, whose very
thin-walled alimentary tube seems to be especially liable to be
hurt by the sharp particles which are frequently taken in
with the natural food. Something of this nature may take place
in the case of injections of fluid into the coelomic cavity.

In order to avoid all objections however, I injected the liquid
from very near the tip of the arm with a fairly long needle.
‘Close to the proximal side of the opening, I tied off the arm
with a cord. In that way it was not possible for any fluid to
escape. About one fifth of the total quantity was injected into
each arm and with some force so that one might suppose that
it was fairly well equally distributed through the whole perivis-
ceral liquid.

At definite intervals a sample of one c.c. was drawn from
the coelomic fluid, with a syringe. In these samples the quantity
of sugar was determined by means of Folin and Wu's micro-
sugar-method for blood filtrates. In this method the liquid to
be tested is boiled with an alkaline copper solution for 6
minutes. Phosphomolybdic-phosphotungstic acid is added to
dissolve the cuprous oxide. This is oxidised and the molybdic
reagent reduced with the production of a blue color, which is
then compared with a standard. This standard was in the present
case made up in sea water.

The corpuscles were first removed by means of a hand cen-
trifuge; after that they were washed once more with about
one c.c. of seawater. When sodium carbonate was added in
order to produce the blue color, a slight precipitate came down.
This precipitate was removed by means of the centrifuge, before
the colometric reading was made.

In that way the results represented in table 11 were obtained
in a series of glucose experiments. The quantities injected were
75, 150, 350 and 700 mgr., which covers a wide range. On
the average — 6 animals were used — a starfish appeared to
contain 22 c.c. of perivisceral fluid. On this assumption and
given the quantity of liquid in which the sugar had been dis-

77

solved — 5 c.c. —, it was possible to figure out the quantities
of sugar present in one c.c. of coelomic fluid at any desired
moment. Such figures are represented in table 11; in the first
column the time which has elapsed since the injection, is indicated,
in the second the quantities of glucose remaining at that time
in one c.c. Diagrams have been plotted which illustrate these
relations still more clearly (Fig. 7).

“~~ 100. *

Fig. 7.

Resorption of food materials from the perivisceral fluid.
For explanation see text.
glucose.
—s—s—: glycine.
suntees corpuscle experiment.

A. 700 mgr.; B. 350 mgr.; C. 150 mgr.; D. 75 mgr.

Similar experiments have been made with glycocoll. The
same quantities were added and the whole experimental pro-
cedure was the same. The quantity of amino-acids present
was estimated by means of Sérensen’s formol-titration method.

78
Table 11.

Resorption of glucose from starfish coelom').

75 mgr. injected. 150 mgr. injected. 350 mgr, 700 mgr.
e R(emaining). : T. R.Q.
Tiime). Q(uantity). T. R. Q. T. R.Q. Q
0 2.88 0 5.77 0 13.46 0 | 26.92
16 0.71 23 3.00 54 4.88 67 5.83
43 0.29 65 1.08 106 3.01 lil 6.67
106 | 0.048 107 0.31 - — 170 1.21

This method depends on the amino-acids having their basic
character destroyed by formaldehyde and thus bringing about
greater ionisation. A neutralised formalin solution is added in
order to bind the NH, groups, the COOH group is then titrated
in the usual way. A correction was made for the titrable
alcalinity of the sea water. The results of these experiments
are represented in table 12 and fig. 7.

Table 12.

Resorption of glycine from starfish perivisceral fluid?),

= — =

75 mgr. injected. 150 mgr. injected, 350 mgr. 700 mgr.
Time). cua T. RG. |e (Re | a tree
0 2.88 0 5.77 0 | 13.46 | 0 26.92
41 0.74 38 1.48 41 2.09 35 4.88
a - 60 0.19 57 0.74 66 3.67
= - ~ ~ - — 184 1.86

This rapid withdrawal of materials from the ,,blood” is really
very striking. The most essential feature of vertebrate blood
is lacking, for we do not have a balanced and storing system here,
but everything that comes in, leaves the transmitting medium
just as rapidly as possible. Everything coming in, is consumed
with surprising rapidity, the waste products also disappear
rapidly and there are no ,,threshold substances’. This con-
sumption is not only due to the tissues, but also, and in large
part, the corpuscles seem to take away the food substances.
I could make sure of this by the following experiment. The

1) The time-figures indicate minutes, the quantities are given in milligrams.
*) Figures as in table 11.

79!

total quantity of perivisceral fluid of a starfish was secured. This
can easily be done by cutting off the tips of the arms and
allowing the ,,blood” to drip out. A cut in another arm facili-
tates this dripping out. 23 c.c. were secured. To this 5 c.c. of sea
water were added in which 350 mgr. of glucose had been
dissolved. The liquid was now put into a small Erlenmeyer
flask and shaken constantly by means of a hot-air automatic
shaker, in order to prevent the clotting of the corpuscles and
to mix the liquid thoroughly. From time to time a sample of
one c.c. was removed, as in the experiments mentioned above.
The corpuscles were centrifuged of and the sugar determination
made, as described above.

The results of this experiment are given in table 13 and
plotted in fig.7 (E).

Table 13.

Consumption of glucose (350 mgr.) by the corpuscles of
a starfish.

T(ime). Remaining) Q(uantity).
0 12.5
82 8.56
130 5.6
180 4.3

These experiments have given us an opportunity, exceptional
from the general physiological standpoint, to study the disap-
pearance of food substances from the ,,blood” into the tissues.
One fact is very striking: that the consumption is not, or at
least not primarily, dependent on the quantity which is offered.
In the 700 mgr. experiments we have after the lapse of a certain
time just as little left as when 75 mgr., less than one ninth
of the amount, is injected. There is no proportionality between
quantity offered and consumption, the law of mass action can
therefore not be applied to the present case. The needs of
the tissue seem to be the factor of primary importance.

They explain at the same time why the blood picture of the
invertebrates is so very inconstant,as I have already pointed
out in the chapter on the perivisceral fluid, when mentioning
the experiments of Dr. Morgulis. The concentration of the
products of hydrolytic cleavage of food substances and of waste
constituents depends largely or wholly on the feeding condition
of the moment. Constantly starving and having almost no reser-
ves — as we will see in another chapter —, these animals
live from day to day and have to fight for existence.

80

23. EXCRETION IN ECHINODERMS. FUNCTION OF
THE RECTAL COECA OF THE ASTEROIDEA.

About excretion in Echinoderms we are, as far as my know-
ledge of the literature goes, only very poorly informed. One
thing however is absolutely clear: the representatives of this
group do not posses any definite kidney for the elimination of
nitrogenous waste. These kidneys, present in most other divisions
of the animal kingdom, are, as comparative morphology teaches
us, specialised parts of the coelomic epithelium; in many cases
this appears from embryology, in other cases part of the coelomic
wall still participates in the excretory activities. Since in the
Echinoderms specialised organs have never been demonstrated,
the whole coelomic lining apparently must have excretory func-
tion. Cells from this peritoneal wall are constantly thrown into
the surrounding fluid, where they move about. They are called
from the way in which they move, amibocytes and they seem to
have a great importance in excretion. Durham calls them sphae-
ruliferous corpuscles. Substances like indigo-carmine which in the
higher groups, are eliminated by the kidneys are here found to be
absorbed eagerly and to be stored by these ,,nephrocytes ')’’. Later
these cells in starfishes accumulate in the dermal gills, the papulae,
in Holothurians in the wall of the waterlungs, where they move
to the outside — diapedesis — and degenerate. Sometimes
they form large clumps containing the material to be eliminated,

as I myself could observe in Stichopus. Part of them also disap-
pear through the ambulacral feet. Sometimes the papulae when
loaded with these ,,nephrocytes’, are autotomised.

The picture given above was, in its general lines, first
concieved by Durham 34) and 35), also by Chapeaux. Though
nobody has ever tried to demonstrate that this method of elimi-
nation which has beer observed in the case of physiological
injections — of suspended substances —, is the normal one,
it sounds rather probable that some natural waste~products
might be eliminated in this way.

The réle which in the starfishes is played by the papulae, is
played by the water-lungs in the holothurians. These have
been considered to be organs of excretion by a great many
authors. Pourtalés 102), Oken, Huxley, Bartels 4),
Hérouard 55), Schultz 116), Bordas 11) and Danielssen
and Koren 28) all found that migrating cells loaded, either with
artificially injected substances (ink etc.) or with normal granules
left the coelomic cavity through the waterlungs 2), A beautiful
description of this process of elimination has been given by

) This name has been given by de Ribaucourt. C. R. Biol. 19 Janv.
1901.
?) Jourdain 70) also assumes an excretory function for the migrating cells.

81

Schultz 116). Jourdan 72) also saw these cells loaded with
yellowish, refringent granules in the wall of the water lungs.

It must therefore be considered as definitely established
that such elimination takes place, the more because Hérouard
found definite stomata for the outlet of these phagocytes.
Bordas demonstrated uric acid and urates in them by means
of Garrod’s test, Schultz however finds that they do not
give a murexide test. Neither are they guanin, as Carus
supposed '), nor calcium salts, as Jaeger assumed (De Holo-
thuriis, Inaug. Diss. 1833), or carbonates — they do not
dissolve in HCl.

It does not astonish me either, that such elimination takes
place, since these nephrocytes leave the body at almost any
place where there is a thin wall — as we shall see presently ~.
That it is not the only way of elimination, however, is proved
by the fact, that in many Holothurians — the Spatangidae und
Clypeastroidea — the respiratory trees are completely absent.
Our own conception of the process of excretion will be dis-
cussed later on in this chapter.

Perhaps all these phenomena can most easily be explained in
the following way. All ,,Fremdkérper’, all foreign bodies are
grasped by the amibocytes. All amibocytes are, as general
physiology teaches us, positively chemotropic to oxygen. Con-
sequently they will all tend to move towards places where the
oxygen tension is highest. Possibly it is even justified to assume
a certain ,, Umstimmung” of these cells after they have taken
up the excretion products.

Now, it is very remarkable indeed that almost all the organs
mentioned above as supposed organs of excretion are at the
same time organs of respiration. This is true for the papulae
and for the water lungs, as also for the _,,ciliated funnels”
(Wimpertrichter) of the Synaptidae where respiratory trees and
podia are lacking. These little funnel-shaped organs, attached
to the mesenteries in the region near the body-wall, are
supposed to maintain a water-current which aids in respiration
through the skin. This same fact, the high oxygen tension in
the neighborhood of these organs, might account for the accu-
mulation of the amibocytes at that particular place, which other-
wise would seem strange. This hypothesis would also account
for the statement of some authors, among them Chapeaux,
that the phagocytes, not only those loaded with injected sub-
stances, but also young ones — eventually leave the body
through the stone-canal, and madreporite, notwithstanding the
fact that the current in this tube is directed inward — this current
caused by the cilia of the endothelial lining, is supposed to take
care of the internal pressure of the water vascular system —. This

1) He found this substance in the organs of Cuvier which probably is a
mistake (Bordas). :

82

also explains why sometimes the water vascular system has been
considered to play the rdle of an organ of excretion — because
it also contained these amibocytes. — The importance of this
system for respiration can not be doubted, as we shall see in the
chapter on respiration, especially not because hemoglobin,
whenever it occurs in this group, is found in this very system.
I myself could see this once very beautifully in a starfish which
had been fed on ammonium carminate; the whole dissepiment
of the water vascular system appeared on dissection to be colered
brilliantly red even into the very tips of the arms.

The well known paper of Kovalevski 75) on organs of
excretion in lower organisms, has given rise to a whole series
of analogous investigations. This author injected small quantities
of a certain dye into the animals to be studied, and observed
where it went to. That such organs would have the function of
excretion is, however, not necessarily true. On the contrary,
the diversity of the results obtained by such methods with different
dyes, makes this rather improbable. Cuénot 23) distinguished
between ,,néphrocytes 4 carminate” and ,,néphrocytes a indigo”.
These nephrocytes, according to him, desintegrate in the coe-
lomic liquid, are taken up by the phagocytes and eliminated
through the skin-gills, as described above.

One fact must however be mentioned as the result of many
such experiments, i.e. the frequent accumulation of the injected
dyes in the so-called axial organ ') and in the bodies of Tiede-
mann. Injecting a suspension of carmine or Bismarck-brown
into the water vascular system by means of one of the tube-feet,
he found the bodies of Tiedemann stained in the starfishes;
injecting such substances into the coelomic liquid of urchins,
he found them in the axial organ. Leipoldt 79) denies their
excretory function, pointing out that these organs do not have
a glandular epithelium and do not stand in connection with the
perivisceral fluid; furthermore that the current of the stone-canal
moves inward. It is rather remarkable to observe here that again
we have to do with an organ in the neighborhood of the stone-
canal, which should thus be furnished with much oxygen.

The great objection to all such experiments is that they are
,unnatural’”” and that, though they have given valuable infor-
mation in mammalian physiology, they may not have any value
in these groups, because we only have to do with a kind of
vital staining. The same thing also holds true for many experi-
ments on the amibocytes as excretors, especially for the expe-
riments of Metschnikoff who made his original studies
on phagocytosis on this very group. The fact that the phago-

‘) The axial organ or ovoid gland has been supposed to have excretory
function by a great many authors, among which Jourdan, Perrier, Koehler,
Hamann, the Sarasin's etc.

83

cytes eliminate dying mesodermal cells, and that they take up
all kinds of foreign bodies, dyes and bacteria, does not prove
that they are the only, or even the chief, organ of excretion.
Something of this nature is also done by the mammalian
leucocytes, and yet they are not considered to take care of
excretion. This is especially true in cases where foreign bloods
or pathogenic bacteria are injected.
The same thing has already been pointed out for the cases in which
these phagocytes take up food substances. Even if these are digested as in

the cases of injection of starch or olive oil emulsions, this does not prove
that they play a réle in the natural process of digestion.

The possibility that the ,,liver” plays a réle in excretion, has
already been discussed in chapter 21.

Not satisfied with the little evidence known about excretion
in our group, | made some experiments of my own on this
problem which have led me to absolutely different conclusions.

In our study of the perivisceral fluid we have seen that it
is nearly the same as sea water, except for a small quantity
of uric acid which discloses itself when the fluid tested by
means of Folin’s uric acid reagent. This substance is always
present though in varying concentrations and in all three of
our species. This proves that is a regular waste product in their
metabolism.

The next step was to find out in what way it is eliminated.
Faeces of Arbacia were collected. shaked with destilled water
in a test-tube, then the liquid was filtered. In the filtrate Folin’s
uric acid test always gave positive evidence, in a few cases the
color was rather faint, but generally it was strongly positive.

The same material proved to be present in the rectal coeca
of Asterias. It is difficult to isolate these organs as they are
very small and lie in the neighborhood of the anus. The whole
stomach of the starfish is rather fragile, but the rectal part always
sticks to the dorsal tegument. This consists not only of the
rectal coeca, but also of a part of the rectum. The whole mass
was simply taken as such and extracted with cold destilled water.
This extract after having been filtered, showed the same typical
blue coloration.

In looking up the literature on the subject I found a paper
of Griffiths 48) in which the same uric acid is reported to
be present in the five pyloric coeca of Uraster rubens, a fact
which I mentioned in the chapter on the function of these organs.
He examined chemically and microchemically the secretion of
the five stomach pouches and the radial sacs and came to this
conclusion from the following evidence:

1. Treatment with NaOH gives a flaky precipitate. Crystals
of various forms appear to be present. If treated with alcohol, only
rhombic crystals are found. With HNO, they give the murexide test.

84

2. The secretion is evaporated down, treated with absolute
alcohol, then filtered. The residue is taken up in boiling water,
then filtered. Typical uric acid crystals result.

These two’ concordant and independent observations make
the presence of uric acid probable, if not certain. They show
that it may be excreted into different parts of the digestive tract.

Whether or not the uric acid is excreted into the radial sacs and passed
over to the stomach pouches or excreted into the pouches themselves is
not stated by Griffiths. Neither whether these,, livers’ are capable of
destroying uric acid, like many true livers, especially those of the dogfish
(Scyllium catulus); see Scaffidi 114).

The same substance was also found in our Thyone. If the
liquid contents of the rectum, which forms a distinct swelling
in this species, or of the end parts of the gut are collected and
a test is made for uric acid in them, strongly positive evidence
is as a rule obtained. The same thing is frequently true of the
contents of the middle gut. Here also uric acid seems to be
excreted into the intestine. Remarkably enough Co hnheim didnot
obtain a positive murexide-test in the faeces of Holothuria tubulosa.

From all this evidence I have concluded that uric acid plays
a very important rdle in the meta- or catabolism of the Echi-
noderms. It seems to be the solution of the dark problem of
excretion in this group. None of the other waste products of
the animal machinery could be detected. In our chapter on the
perivisceral fluid we mentioned already the absence of ammonia
in detectable quantities. Neither does urea seem to be present.
Griffiths searched for it in vain in the radial sacs of
the starfish which he considered to be organs of excretion. Four
tests: 1. the mercuric nitrate test — no white precipitate, 2. the
Na-hypochlorite test — no N-bubbles, 3. the absence of urea
nitrate after treatment with nitric acid, 4. a negative Nessler,
all gave negative results which makes the presence of this
substance very improbable. Neither are guanin or Ca-phosphate,
the regular products of excretion in Cephalopodes and Lamelli-
branchiates, present here (compare p. 81 on the Holothurians).

The gut, especially the end parts, seems therefore to be
the organ of excretion in Echinoderms and the most important
substance excreted uric acid.

This method of excretion is by no means rare or strange. In
almost all arthropods we find just the same thing and the
Malphigian tubes in insects and other groups, seem to be merely
parts of the mid-gut, specialised for the purpose of excretion.
They are however by no means the only organs with excre-
tory function, for the whole mid-gut may be involved in the
like duty, as stated by von Gorka 46) on p. 329 of his paper
on the physiology of the Malphigian tubes, where he says:
,»Hieraus folgere ich. dasz auch der Mitteldarm an der Aus-
scheidung der Harnsdure teilnehmen kann’.

85

Sitovski 121) in a paper on the biology of the catterpil-
lars of the moth Tineola biselliella, has explained the acidity of
the contents of their end-gut as due to the presence of uric acid.
This does not seem very probable considering the very low
degree of dissociation of this acid, but it seemed to me tobe
desirable to ascertain whether those pa.ts of the gut which
excrete uric acid actually have a lower Py. Therefore I made
some determinations especially of the rectal coeca of the star-
fishes. When we remember that in the cucumbers there is a
very decided alcalinity which increases towards the rectum, we
must agree that here at least the evidence is entirely negative.
But in these rectal coeca some authors have occasionally found
a rather strongly acid reaction. I have made some determi-
nations for the solution of this question in exactly the same
way as described in the chapter on the hydrogen-ion con-
centration. To one single drop of the rectal-coeca-preparation
described above was added a certain amount of indicator and
its color compared with that of a set of standard drops. The follo-
wing values were found in different experiments: 7.2; 7.4 and
7.4. These figures may demonstrate a slightly stronger acidity
than that of the stomach contents, but I do not believe that this
difference has any significance at all. It would be very dangerous
to draw from this conclusions like Sitovski’s especially
since Roaf occasionally found a strongly alkaline reaction in the
liquid squeezed out of the rectal coeca on treatment with NaOH.

The importance of uric acid as a product of excretion in
Echinoderms may be greater than one would at first suppose
I refer here especially to some very interesting obser-
vations of Mangold 82) on auto-intoxication in sea urchins.
The form on which he worked was Arbacia pustulosa a
species rather closely related to our A. punctulata. His obser-
vations hold equally true for our form, and I would have
described these phenomena if he had not done it.

In Arbacia the faeces, greenish or in our case white, round
bodies, are expelled through the cloaca which is situated in
the middle of the periproct. The spines show an avoiding reflex
— first described by von Uexkiill — as soon as one of
these little bodies comes in contact with them. In this way and
by the aid of the trifoliate pedicellariae the bodies are carried
over and dropped down gradually. In nature, of course, the
waves promptly remove them, so that here this complicated system
of remo al is of not much use.

Arbacia is a rather weak form and soon dies in quiet
water. This is doubtless due to a toxic action of the faeces
on the skin. Even in a well aerated aquarium the animals die
because the removing force of the waves is lacking. Irritability

86

and capacity for reaction disappear, the skin shows traces of
decay and the spines fall off one after another.

These phenomena can be prevented by regularly cleaning
the animal with sea-water. After the faeces had worked
for one hour, this recovery, according to Mangold, could
no longer be obtained.

This doubtless proves that a toxic action of some kind is exerted
on the animal by its own faeces. Since faeces of other aminals do
not cause the same phenomena, we can not have to do with
a mechanical lesion, but a substance must act here, ,,deren
physiologische Wirkung auf die lebende Arbacia der Wirkung
von Sauren (! v. d. H.) gleicht’”” (Mangold).

Ueber die Natur dieses Stoffes etwas bestimmtes aus zu
sagen, ist vorerst nicht mdglich, die Frage wird sich aber jeden-
falls erledigen miissen, wenn einmal die Verdauung der Echino-
dermen iiberhaupt die langerwiinschte Bearbeitung gefunden hat.”

It seems probable that these phenomena may be due to
the presence of uric acid. Isolated pieces of shell, with the
spines on them, live much longer than the intact animal, even
when no care is taken to change the water. Spines and pedi-
cellariae keep alive easily for four days on such pieces. The
following observation is interesting in this connection: A
specimen of Arbacia was brought into the laboratory in a
badly injured condition. Almost half of the shell, including the
periproct, was lacking. This piece of animal remained alive
for 5—6 days, while normal animals frequently already begin
to show loss of spines and other symptoms of bad health
in less time. It is ‘a beautiful illustration of the autonomy
of the different parts of the urchins body, of a coordinated
functioning of this ,,republic of reflexes’ without central impulse,
on the other hand it shows that the symptoms observed in the
intact animals are due, if not exclusively, yet chiefly to the
accumulation of faeces on their body.

Something of the same nature has been observed by von Uexkiill, who
saw brittle stars moving around for days after the major part of the disc,
including the stomach, had been removed.

Arbacia seems to be much more liable than any other genus
to injure itself by its own faeces. This might be due to two
of its peculiarities: 1. the spines of this species converge toward
any stimulated point (von Uexkiill) and in that way will
prevent the faeces from falling down, 2. the ectoderm is devoid of
cilia so that the faeces are not swept off by ciliary action. These
forms are largely dependent on the washing produced by the
ripples on the shore.

The substance which von Uexkiill postulates as se-(ex-)-
cretion of the skin of the Echinoderms, may be identical with this
uric acid. The pedicellariae of .the starfishes are known to take
hold of every strange object whatever it may be and to hold

87

to it: they are the fighting members of this ,,republic of reflexes”.
Yet they will never attack any part — tissue, pedicellariae or
spines — of the animal which bears them nor of animals of the
same species. This phenomenon has been called by von Uexkiill
,autodermophily” and he explains it as the result ot a negative
chemotropism towards some substance, which he calls ,,auto-
dermine’’. Uric acid, the toxic substance present here, might be
the autodermine, in fact the substance can be dissolved away
by means of hot water, after which a spine of the animal
itself is attacked just as vigorously as anything else. It may
be however that in this case we have to do with some other
substance as yet unknown; the gemmiform pedicellariae also
contain toxic substances in their ,,poison-glands” (von Uexkiill
129)), the bite of one of them may stop the frog’s heart, kill
the ischiadicus etc. Little animals (marine snails etc.) are killed
at once. Cuénot has come to the conclusion that the toxix
substance was secreted by certain glands of the skin, called
»glandes mériformes’”. Parker 96) could kill young animals,
e.g. cats, by means of extracts of the skin of starfishes ').
Other experiments of Dr. Hiroshi Ohshima, unpublished
as yet, in which touching the skin of Arbacia made their eggs
unfit for fertilisation and, indeed, proved to be lethal for them,
may furnish additional evidence in favor of my view. If one
removes the skin of Arbacia from the shell and tests a watery
extract of it for uric acid, one invariably gets a positive result.
Further experiments will be necessary for a definite decision.

Note: 1. There is one other phenomenon which I want to
mention in connection with this chapter. I have observed it
frequently but have not found it mentioned anywhere in the
literature.

If one takes a starfish from an aquarium and cleans it tho-
roughly with a clean towel, it is as dry as dry can be. Keeping
it for some time in his hand, one can observe that water
begins to exsude through the skin. After a little while one
observes, especially on the aboral side of the arms, long lines of
water-drops. This process goes on until finally drop after drop
falls from the tips of the arms. Meanwhile the arms loose
their rigidity.

Whether or not this process is a normal one, | do not dare
to decide. It might be due to the breakiag of the papulae or
other thin membranes under the internal pressure of the peri-
visceral fluid. It may, however, also be a process which takes
place under normal conditions and then it must be a great
help in excretion, unless the papulae etc. are completely semi-
~~ 4) Notwithstanding its toxicity the skin of the starfishes carries parasitical

Protozoa and a Caprella species, which must in that way havea kind of natural
immunity for their poison.

88

permeable. Further work will be necessary to clear this question.
2. The arguments collected by the Italian investigator Russo
110) to prove that the genital organs in Holothurians would
have an excretory function, have not convinced me. It may
be that one of the accessory functions (see p. 73) of these
organs is to store or possibly even to excrete certain substances,
but my solution seems to me to be the more probable one.
In sea-urchins Giard 45) also observed crystals of calcium
phosphate in deutoplasmatic bodies in the genital organs.

24. RESPIRATION.
a. General observations.

It has repeatedly been suggested that the perivisceral fluid
might play the rdle of a carrier of oxygen, of an internal
respiratory medium. But this is only one of the numerous
guesses which have been made with regard to respiration in
Echinoderms, for no experimental work has ever been done
as far as the writer knows, to obtain more definite information
as to the reality of this function.

But first let us see to what organs a respiratory function
has been attributed. In the starfishes we find the so called
skin-gills (papulae), lacking only in one genus, Brisinga, which
are supposed to have respiratory importance. On their réle
in excretion we spoke in the preceding chapter. They are
little blind sacs of the body wall, in which the coelomic fluid,
with its corpuscles, is kept in constant motion by the cilia of
the epithelial cells of the coelom. Peritoneum and epidermis
are here in contact according to K.C. Schneider; Delage
and Hérouard state (p. 48) that occasionally muscle fibres
are found in the wall. Probably the ambulacral feet and
therewith the whole water-vascular system have a certain
importance in respiration, as later on we shall see. The con-
stant movements of these tube feet makes them very suitable
for such function, and in the chapter on excretion we have
mentioned other facts which make this still more probable.
Their walls are easily permeable.

In many sea urchins the peristome is continued into
branched outgrowths, called internal gills or organs of Stewart,
two in number, situated in each interradius. Every tube foot
is moreover connected by two canals with its ampulla, and
cilia cause a current up on one side, down on the other
side of the tube foot, thus allowing O, to be taken up in
that way and CO, to be given off by the perivisceral fluid.
I have already stated that some authors attribute respiratory
function to the accessory intestine of the urchins, but that this
did not seem to me very probable.

In the brittle stars the so called bursa is supposed to be an

89

organ of respiration, in which a constant current is kept up
by means of cilia. In the third part of this chapter we shall
speak of the occurrence of hemoglobin in Ophiactis virens.

In the Holothurians we have very definite organs of respi-
ration, the so called water-lungs') or respiratory trees in which
an alternating in- and outflow of water through the cloaca can
be observed. They have a more or less tree-like appearance,
the stem of the tree being represented by the two main tubes
opening into the cloaca. The finer branches of these gills end
in rounded, thin-walled swellings, termed ampullae. The rhythm
of the process of in- and outflow appears to comform to van
't Hoff’s rule 18). The inflow seems to take place inter-
mittently, the outflow however in one strong current (Peach.
Ann. Mag. Nat. Hist. Vol. XIII. 1881. p. 418; Ayres
(Thyone). Proc. Boston Soc. Nat. Hist. Vol. IV. 1851~—1854;
Sluiter. Natuurk. Tydschr. v. Nederl.-Indie. Bd. XL. Batavia.
1880), They are not the chief organs of excretion, as Hérouard
supposed, but phagocytes loaded with granules (excretion
products) or artificially injected substances, leave the body
through their wall.

Since however as we have pointed out in the preceding chapter,
these ,,znephrocytes” leave the body by almost any route provided
that oxygen is present. this is not a specific function of the
respiratory trees.

One observation certainly is an argument for their respira-
tory function. The animals which J used were kept in a very
shallow aquarium with a standing overflow. Now, occasionally
several specimens could be seen to move their cloaca upward
above the surface of the water and to open it -to the air.
This attitude is frequently kept up for a considerable length
of time and has apparently some importance for respiration.

The same behavior was observed carefully by Winter-
stein 133), who found that it occurred especially in water
which had been polluted ,,durch den Abgang des Exkremente’”’.

‘It is not a pure air-breathing act. The lungs are kept full of
water and this water is even changed every now and then —
the cloaca again being moved under the surface —. The gaseous
exchange between the water and the air is greatly facilitated
by this procedure, however, as analogous cases show (compare
e.g. the larvae of Aeschnidae and the buccal respiration of
fishes. their ,Notatmung’’). In case of lack of oxygen the ani-
mals first contract, then they relax and faint, finally they lose
all tonus and ,,Reaktionsfahigkeit’’.

One thing which certainly proves that they are not the only
organ concerned with respiration, is the fact that they are so

1) The water-lungs have also frequently been supposed to be lymph glands
(e.g. by Hérouard) and organs of excretion (see the chapter on this topic).

90

frequently discharged from the body which as a rule is not
done with important organs. The same thing holds true for
the so called Cuvierian organs, situated in their immediate
neighborhood, the function of which is as yet unknown except
for their importance as organs of defence. For these organs
Mines 85 and 86), has shown that their discharge takes place
simply by an increased internal pressure, not by any intrinsic
action of the tubes themselves.

Winterstein has studied the relative importance of the
water-lungs in the gaseous exchange. He estimated the exchange
in normal conditions and also when the water-lungs are put
out of commission by means of a rubber condom, using
Winkler’s method for the estimation of O, 50°/) of the
total respiratory exchange appears to go through the lungs.
The fore-gut and tentacles play no important réle, as similar
experiments in which the head was covered, show.

Crozier 19) has calculated the amount of water pumped
through by the large Bermudian species, Stichopus moebii.
This was done by determining the amount of water pumped
through in one pumping and figuring out the average number
of pumpings in one day.

He concludes that 20—21 L. is pumped through per day
by a large Stichopus 24.5 c.M. in length. Compared with
the figures for some other animals this is fairly low. In
the sponge Spinosella Parker 97) found, that by a single
finger’ as much as 78 L. a day is pumped through, and for
Ascidia atra Hecht 52) found as much as 173 L.a day. This,
however, is not strange if we remember that in these animals
the watercurrent has the double function of carrying oxygen
and food, whereas in Holothurians only the oxygen is needed.

1. In this big species Crozier found a difference in PH between
coelomic and sea water, (contrary to my findings in the starfish, mentioned
in the chapter on the perivisceral fluid) due to CO, of course. The
PH of sea water is 8.2; that of the perivisceral fluid is 7.6. The water
expelled during spouting has a PH = 7.8; this shows: 1. that actually
respiration takes place through the lungs, 2. that the short stay of the
water in the lung is sufficient for the respiratory exchange.

2. Frequently small fishes are found in the cloaca of large Holo-
thurian species, f.i. Fierasfer in the cloaca of Holothuria.

Summarising these remarks on the organs of respiration in
Echinoderms, we may quote the words of Delage and
Hérouard: ,,L’animai respire par tous les points ou une
membrane mince sépare les liquides de l'économie d'une eau
de mer plus ou moins renouvelée”’.

b. Are there catalases and peroxydases
in the perivisceral fluid.

We must in the first place try to get some information
as to whether the perivisceral fluid may be considered as a medium

91

of respiration, The studies of the last decennia have revealed
the great importance of catalases and peroxidases in respi-
ratory and metabolic processes and the question arises whether
these enzymes can also be demonstrated in forms as low as
the Echinoderms. The reader of course knows that, according
to Schénbein (J.f. prakt. Chem. Bd. 75. 1858 and Bd. 89.

1863), their discoverer, their properties were common to all
enzymes, but that at present we have distinguished them as
separate enzymes.

Perivisceral fluid was secured from Asterias, Arbacia and
Thyone and filtered. On these liquids the four following tests
were made:

1. To a few c.c. of the perivisceral fluid some hydrogen
peroxide was added. The three samples appeared to be fairly
active and even after a few minutes many bubbles of oxygen
were seen. This shows the presence of a catalase acting directly
upon hydrogen peroxide with the formation of oxygen.

For the demonstration of peroxydases, phenolic substances
are used as a substrate together with hydrogen peroxide, at
least when no natural peroxide or oxygenase is present. The
peroxydase produces ,,active’” oxygen -oxygen in statu nascendi-
which oxydises the substrate.

2. A freshly prepared one per cent solution of guiacum in
alcohol was colored blue in contact with the perivisceral fluid
and hydrogen peroxide. It took some time for the color to
develop, but a very dark color resulted finally.

3. A one per cent solution of a-naphthol in equal parts of
water and alcohol was colored dark lavender.

4. A one per cent solution of the same kind of benzidine
became blue. A brown precipitate was formed, but only after
about 16 hours.

These four tests showed convincingly the presence of both
catalases and peroxidases in the three species investigated. The
benzidine test gave the least striking results, it took a rather
long time before the brown color appeared, but the next day
a brown precipitate was always seen. In Thyone all the three
tests gave a very strongly positive result.

“A paper by Portier 101) in which, according to Kobert
73) many valuable data concerning the occurrence of oxidases
may be found, could not be secured. Kobert found catalases
to be present in a great many insects, spiders etc., even in their eggs
and in alcoholic specimens etc., also in living Ascarids, and he
is of opinion that they occur ,,in allen lebenden funktionsfahigen
Zellen”. In the blood of fishes, some worms, Sipunculus, Octopus
and Eledone he also found them present, in Eledone and Octopus
even if the corpuscles were removed. Oxidases also occur in
the blood and organs of a great many invertebrates, as Giard

(C.R. Soc. Biol. T. 48. 1896. p. 483), Pieri and Portier (CR.

92

Acad. Sc. Paris. T. 123. 1896. p. 1314), Abelous and Biarnés
(C.R. Soc. Biol. T. 49. 1897. p.175 and 249) and Portier 101)
have shown. In Kobert’s paper I found an indication, that
the latter author had found them in Echinoderm blood; unfor-
tunately I was not able to consult the original paper.

What their exact function is in all these groups, nobody can
tell. We can not say, whether the catalases serve only as im~-
mune bodies against the highly toxic hydrogen peroxyde —
toxicity due to the fact that it causes air embolisms —, as
Oskar Loew supposed (Catalase. U.S. Deptm. of Agriculture.
1901. Rep. No. 68. After Kobert), or whether the peroxide-
theory of A. Bach (Du réle des peroxydes dans les phéno-
ménes d’oxydation lente. C.R. Acad. Sc. Paris. T. 124. 1897.
p. 251) and of Engler and Wild (Ueber Sauerstoff-activie-
rung. Chem. Ber. Jahrg. 30. 1897. p. 1669. Both after Kobert)
holds true. The latter theory assumes the formation of peroxides
as the primary stage in every combustion of organic substances
in the living organism and their destruction by catalase. It is

certainly not a measure for metabolic activity, according to
Morgulis 88).

Note. 1. The so called melanosis in the blood of catterpillars etc. has also
been supposed to be due to peroxydases. Here we surely have to do with
a tyrosinase according to Biedermann 8).

2, The results of Portier who found oxidases to be present nearly
everywhere have partly been denied by Kobert. For this reason
a repetition of his work does not seem superfluous.

c. Contents of the Polian vesicles of Thyone. Importance
of the water vascular system in respiration.

The writer 60) was struck by the brilliant red color of the Polian
vesicles of Thyone each time that he dissected a specimen.
Curiosity drove him to examine this phenomenon a little more
closely though it did not seem to have any direct bearing on
the problem in hand.

In Thyone briareus these vesicles which form as we know
a part of the water-vascular system, are present in numbers
varying from one to four. Scott 118) has studied their anato-
mical relations a little more closely and just in this particular
species. He finds that usually one is present, often two,
occasionally three and rarely four. The general tendency for
them is to go to the left side, and thus to disturb the bilateral
symmetry. The retractor muscles of course vary with the
number of the Polian vesicles. In size as well as in number
there is a considerable variety.

The Polian vesicles form, as mentioned above, a part of the
water vascular system and therefore they are on the one hand in
connection with the ambulacral feet, which are found all over
the outside of the body. At the inside of the muscular body

93

wall these podia are represented by their ampullae and remark-
ably enough, all these little ampullae can be seen on dissection
to contain the same colored material. On the other hand this
system communicates freely with the coelomic fluid. The stone-
canal stiffened by carbonate of lime, which in other Echino-
derms and in larval Holothurians communicates freely with
the sea water, forms here the so called ,,internal madreporites”,
of which there may be one or even five, as in Holothuria
tubulosa. Furthermore the water vascular system stands in
connection with the tentacles, the tree-like feelers, which by
their active movements may assist greatly in respiration.

The Polian vesicles are generally supposed to be the regu-
lators of the internal pressure of the water-vascular system.
Their wall contains muscle fibres and by a slight contraction
of these fibres the tentacles may be extended very easily.

It seems however that it also plays a réle in respiration.
The red material namely appears to be hemoglobin. The color
is due to the contents; if one cuts a little hole in the wall of
the vesicle, the color ,,flows away’. It can be centrifuged off
and a microscopical examination of the fluid reveals that the
color is bound to corpuscles.

These corpuscles look under the microscope very much like
mammalian blood-corpuscles. They do not exhibit any active
movements as far as could be observed, have the same yello-
wish color that is characteristic for the erythrocytes and appear
to be perfectly spherical. A nucleus is in most cases clearly
visible, the protoplasm has a granular structure and a cell mem-
brane gives them a perfectly sharp outline.

Hemoglobin is known to occur in many invertebrates, Though
it was originally thought to be characteristic for the verte-
brates, it has since been found in almost all groups of the
animal kingdom. Even in animals as low in the scale of phylo-
genesis as the worms, we find it in many forms, either in the
coelomic liquid (common earthworm) or in corpuscles (Glycera,
Capitella, Phoronis, etc.). In molluscs and arthropods it is gene-
rally found in solution. It is chiefly found in mud-dwellers or,
to put it more generaly, in animals, living in a medium where
oxygen is scarce. The larva of Chironomus is a classical example.

Our knowledge of the occurrence of respiratory pigments
in Echinoderms is very incomplete. In one case, in the brittle
star, Ophiactis virens, hemoglobin has been found by Foet-
tinger 39). Here it is also present in the tubes of the water
vascular system and in the Polian vesicles, two of these being
present in each interradius here. It is bound to spheroidal
corpuscles, which are sometimes flattened and eventually may have
the form of discs. By means of different chemical reactions, Foe t-
tinger demonstrates that we have to do here with hemoglobin
The same pigment perhaps is also found in a species of Ophiolepis.

94

The presence of these corpuscles in the water-vascular system
in both these species, clearly demonstrates the respiratory im-
portance of the latter. Probably this is due to the fact that
it connects all the tube-feet which surely have respiratory
importance.

Other statements with regard to respiratory pigments in Echinoderms,
e.g. the assumption of such a function for the so-called echinochrome (Mc.
Munn. 1885. 92)) in Echinus and Strongylocentrotus, are to be accepted
with some reservation, Winterstein e.g. could demonstrate, that a solution
of echinochrome did not take up any more oxygen than the same amount
of sea water.

The red pigment in Thyone seems to be hemoglobin. The
following tests have led the author to this conclusion:

1. A small quantity of the liquid was dried on a slide, heated
a few times under cover-glass, until bubbles appeared, then
cooled. Blue black prismatic crystals could be seen which though
not absolutely congruent with the figures of the text-books of
biochemistry, were doubtless hemin crystals.

2. To a solution of one or two drops of 1 °/, benzidine in
a large quantity of hydrogen peroxide a few drops of the
blood’ were added. A dark blue color developped at once
and a foaming to way up on top of the test tube could be
seen (peroxidase action).

3. Laked and centrifuged ,,blood’’ showed the typical ab-
sorption spectrum of oxy-hemoglobin—one broad band in con-
centrated solution, two on dilution, one of which can easily be
identified with the D-sodium line.

4. On reduction with Stokes’ reagent the single band of
hemoglobin could be seen. After continued vigorous shaking,
the double band of oxyhemoglobin could be produced again.

5. A small quantity of the material gave a positive prussian-
blue test for iron.

6. Material treated with KCN and kept in the 37° incu-
bator for some time, became orange-yellow and showed the
absorption band in the green which characterises cyanhemo-
globin.

From this evidence, | have concluded that hemoglobin is
present in these corpuscles. This fact is very interesting in
connection with the fact that Thyone is a mud-dweller and
thus lives in a medium, in which oxygen is scarce. Though
there is still a diversity of opinions as to whether oxygen
acts as a store of oxygen or not, it is beyond all doubt that
the presence of a respiratory pigment like hemoglobin is a
great help in the struggle for oxygen.

In how far the Polian vesicles act as ,,movers” of the cor-
puscles, as a kind of very primitive heart in that way, or as
formers — these organs have frequently been called ,,lymph-
glands” (Cuénot) —, could not be investigated. The same cor-

95

puscles occur however not only in the water vascular system,
but also in the wall of the water-lungs e.g. In a preparation
of the tip of one of these organs between cover-glass and slide,
cells were seen which seemed to be identical with the cells
in the Polian vesicles.

Thus they might also occur in the ,,blood-system”; and
so in all the three fluid systems, viz. the water vascular system,
the perivisceral fluid and the blood system.

25. RESERVE SUBSTANCES IN ECHINODERMS.

It has frequently been assumed in the previous chapters that
the Echinoderms live from day to day, so to say, resorbing
with surprising rapidity everything that comes into their peri-
visceral fluid and constantly starving and hunting for food.
For this reason it seemed desirable to make sure whether they
are perhaps in the possession of many reserve substances available
under such circumstances.

As far as the fatty reserves are concerned, I made some
experiments in order to ascertain whether fats do occur at all
in the starfish and, if so, in what quantities. | made these expe-
riments for a double pucpose, in the first place for the reason
mentioned above, in the second place however in order to eluci-
date the question of the digestion of fats, discussed from another
angle in a special chapter. The presence of fats in certain animals
of course, as stated before, does not necessarily prove the pre-
sence in them of sieatolytic enzymes, these may be derived from
other sources, from carbohydrates or even from proteins. But
nevertheless it makes their presence more probable.

In order to determine the quantity of fats present in one
starfish, I extracted two specimens with a mixture of alcohol-
ether. The arms of one specimen and its disc suspended in a
filter-paper cone, were extracted in a soxhlet till the extraction
fluid was watery clear, after this another specimen was treated
in the same way.

The liquid thus obtained appeared to contain a large quantity
of pigment. Not all the pigment was dissolved away from the
animal though, a small quantity seemed to be deposited in some
way in the calcarous skeleton. In to get rid of the pigment,
the liquid was boiled for some time with charcoal, then filtered.
The charcoal was again extracted with cold ether, this ether
was added to the rest of the extract; the treatment was then
repeated, again with a fresh quantity of charcoal.

By repeating this procedure numerous times I succeeded in
getting rid of all the pigment. Whether or not I lost any fat,
I do not dare to decide, but it does not seem to me probable.

The liquid was now gradually evaporated in a large watch-
glass over a water-bath. The residue which did not seem to

96

contain much pigment, was dried in an exsiccator and weighed.

The quantity of fat present in two large, freshly caught spe-
cimens, proved to be 103 mgr. But 51.5 mgr. of fat per starfish
seems to be an exceedingly small quantity, especially when
we take into consideration that part of it may have been
pigment.

In Holothurians also Piitter 105) found a very small quantity
of fats. According to his figures 0,014°/) of the wet weight
of Cucumaria grubei consists of fats and lecithins. This means
that in an average specimen of this fairly large species 203
mgr. of these substances are present. This figure is also very
low and might represent only the lecithins of the nervous
system and some pigment. It need not be mentioned that in
our figures the lecithins, of course, are also included.

A second group of reserve substances are the carbohydrates,
generally represented in the animal kingdom by glycogen. This
substance present in almost all invertebrate groups, is wanting
in starfishes. To make sure of this I treated several sections of
the radial sacs of these animals, made in the usual way — al-
cohols, paraffin ~ with J in KJ, after the paraffin had been
dissolved away with xylol, the xylol replaced with alcohol
and the alcohol with water. Whereas in many other invertebrate
groups, as f. i. in snails ~ see Biedermann and Moritz9)~,
the ,,liver” contains an abundance of glycogen, so that every
section immediately shows the typical glycogen reaction, nothing
of the kind occurs here, the reagent failing to give any indi-
cation of the presence of glycogen. Krukenberg also sear-
ched in vain for glycogen in the starfishes ').

A similar test was made on the guts of Thyone fixed in
several different ways. Here every section gave a strongly
positive test. This explains at the same time the nature of
the ,,insoluble carbohydrates” found by Piitter 105) in his
Holothurians. Not less than 15.8°/, of the dry weight of his
Cucumaria is insoluble carbohydrate.

“A third class of reserve substances are the proteins. We find
protein crystalloids in many invertebrates of different groups,
from the flat worms (Saint-Hilaire 113)) up to the highest
and in many cases they have been considered as reserves.
These protein crystalloids are also found in Echinoderms. They
occur in the amibocytes (Wanderzellen) of the sea-urchins, where
they were discovered by Cuénot 23). In the same cells we find
numerous grains of pigment. These pigments are produced, accor-

1) Stone 125) also failed to find glycogen in the starfish radial sac; she
extracted them by means of Pfliiger’s method and did not obtain any evidence
of its presence.

97

ding to the general opinion by accumulation or transmutation of
waste-products. The origin and fate of these protein-crystalloids
has been studied in detail by List 81), They are very regular
crystals, generally hexaeders or rhomboeders, which show the
typical reactions with J in KJ, with eosin, Millon’s reagent
and picric acid. They are found in the nuclei of the corpuscles,
also in those of the cells of the middle-gut, as in many other
cases (see ')), and occur especially in the ,,amibocytes incolores”
of Cuénot. In the middle-gut of Tenebrio they are found not
only in the nucleus, but also in special inclusions of the cell-body,
reminding one of the aleuron-grains in plants and even free in the
cell protoplasm. More than one crystal is never found in our
urchins, they are preceded by small, round bodies (protein vacuo-
les?) which later on unite to form larger ones. Whether or not
these crystalloids actually are reserves, is not clear from this
research; it is the opinion of the author that ,nach den am
lebenden und conservierten Materiale gemachten Beobachtungen,
es sehr wahrscheinlich ist, dasz die Krystalloide sich schliesslich
in Pigmentkérner umwandeln”’.

All the evidence collected in this chapter shows clearly that
large amounts of reserves are not present in our group. Lack of
time has prevented me from investigating this point more in detail.

26. GENERAL METABOLISM.

Though I do not have to offer any experiments of my own
in this chapter, it has been added because as I said in the
introduction, this paper aims to give not only original work,

‘but also a review of the literature on the subject. Moreover
some critical remarks must be made on one of the most para-
doxical modern theories on nutrition, on Piitter’s theory of
the nutrition of marine animals.

Two papers in which an attempt is made to study the
metabolism of the Echinoderms have come to my attention,
one by Cohnheim 17) and one by Piitter 105), Both con-
cern themselves with dendrochirote Holothurians, Cohnheim
works on Holothuria tubulosa, Piitter on Cucumaria grubei.

Cohnheim’s paper is of a very preliminary nature, in
fact, it does not contain much more than a few experiments
on the CO, cutput. His experimental procedure is of the sim-
plest kind; he puts some animals in a stoppered bottle having
two outlets. O,, purified by NaOH, is bubbled through the
apparatus. The outcoming air is led through baryta and the
amount of CO, produced estimated by titration.

The output per K.G. hour appears to be somewhere in the
neighborhood of 0.147 gr. In the case of Ophioderma longi-

1) See eg. Joh. Frenzel. Berl. Ent. Ztg. Bd. 26. 1882. p. 267—361.
C. Rengel. Zs. wiss. Zool. Bd. 28. 1896. p. 1—60.
P. Mangazzini, Mitt. Zool. Stat. Ncapel. Bd. 9. 1889. p. 1—60.

7

98

cauda a somewhat larger amount is found : 0.228 gr. The relatively
low CO, output in the Holothurians is doubtless due to their
lack of extensive and rapid movements. In the experiments the
animals did not move at all and even in their natural environ-
ment they are entremely sluggish, Cohnheim also warns
against comparing the metabolism, measured by the CO, output,
of representatives of different groups of the animal kingdom.
It may be justified to compare two mammals e. g., but it is
absolutely impossible to compare an animal with a calcareous
or chitinous skeleton with a jelly fish which has only 0.24 °/,
dry weight or a Holothurian the major part of whose weight
is formed by the perivisceral fluid.

The paper by Piitter is doubtlessly more ,,griindlich”, but
I regret to say that in my opinion it contains some very
serious errors, which I will discuss briefly. I believe that it is
justifiable to give serious attention to the work of this author
because his conceptions on the nutrition of sea animals are so ex-
cedingly strange that they are either fundamentally wrong or
will revolutionise all our conceptions on nutrition.

Serious criticism has also been made against his methods and
the ,,superstructure”’ built by him on a very weak experimental
basis. While his whole theory tries to emphasise the importance
of dissolved C- and N-compounds as a source of food for
most marine animals, in all his papers we only seldom find
estimates of the amounts of such substances present. And the
few estimates he has given contain a very serious methodical
error as the work of Moore 87) and others has shown.

He digests sea water with sulfuric acid for the determination
of the dissolved carbon. The hydrochloric acid which is set
free in heating sea water with sulfuric acid, is passed through a
wash-bottle containing lead acetate for the purpose of absorbing
it. This is the serious error; the acid is not only bound and
kept back by the lead acetate, but also sets free acetic acid
which after combustion is interpreted as ,,dissolved organic carbon”
of the sea water. In his O, determinations an error of the same
kind is made in which the chlorides present in the sea water
are not taken into account.

But for the present we shall take the methods of Piitter as
methodically justified and shall confine ourselves to a criticism
of his theoretical considerations.

The paper as I have said, deals with Cucumaria grubei, a
dendrochirote Holothurian, rather closely related in that way
to our Thyone. Piitter first determines the general composition
of the animal by means of a simple, general analytical procedure.
The water content is 78,8°/, of the total weight. 5°/, of the
dry weight is extractive materials (inorganic salts etc.), 17.1 °/g
soluble carbohydrates, 5.6 °/, fats, 1.2 °/, lecithins, 15.8 °/, insolu-
ble carbohydrates and 55.2 °/, proteins.

99

The same determinations are made with animals that have been
starving for six months. All dry components have dimished,
except for the ,,insoluble carbohydrates’; the ratio of C:N
which was 1:5.1 at the beginning, appears to be 1:5.6, so
that the nitrogenous constituents have diminished more than
the carbon compounds.

The loss of weight is determined for every month and in
combination with the above mentioned data he finds that the
animal loses on the average 0.0155 mgr. C and 0.00327 mgr. N
in an hour.

The author then determines the oxygen consumption. This
appears to be dependent on three factors: light (!), temperature
and physiological condition. The temperature relation is quite
clear; not clear however is the influence of light and the fact
that animals which have been starved for three month consume
about twice as much oxygen as before starvation.

Carbon is ex-(se-)creted in four different ways:

1. As CO,, partly dissolving in the sea water, partly disap-
pearing in the air.

2. As a volatile hydrocarbon, probably CH, (methene).

3. As formed material, e.g. as mucus and cells.

4. As complex watersoluble carbon-compounds,

These four factors are all studied quantitatively. CO, and the
hydrocarbon are very regular, the two other factors very irre-
gular. The respiratory quotient is very high in the beginning
(about 3.8), later on it is about 0.8.

The total C-production is now determined and the relative
importance of the three groups. The fresh animals secrete 1.64
times as much C. The nitrogenous excretion is also studied.
The production of nitrites and nitrates is quite regular, but the
KjeidahlN shows no regularity whatsoever. Average values
are used. The irregularity may partly be due to the excreted
cells which come very irregularly.

Since in the combustible excreted gases more H is present
than agrees with the formula CH,, it is evident, that free H,
is also excreted in small amounts.

The quantity of ,,volatile acids” and ,,volatile alkalies” excreted
is also determined.

From all these data the author tries to get an impression
of the ,,total metabolism”. Here he enters into the field of
speculation.

His ,,point de départ’’ is the total N-metabolism, figured
from the N-excretion. On the assumption that this represents
the protein consumption. he finds the corresponding C-produc-
tion. The remaining C is now divided up in several ways, an
ingenious and clever system, based on various assumptions and

facts.
Let us first consider the fundamental figure of the whole

100

system and this in connection with other statements made by
the same author a little further on.

The author postulates that nine tenths of the C which has
been produced by the animals is derived from the soluble car-
bonaceous constituents of the sea water which have been used
as food. This also seems to be the case for the nitrogen, but such
substances account for only 60°/, of the total N-metabolism
however, 40°/, comes from the proteins of the body.

Now here we are: at the very basis of his theoretical con-
siderations the N-excretion is supposed to be the product of
the protein consumption. On this basis figures on the C-meta-
bolism are secured which finally after many detours lead to
the above mentioned conclusions. This is apparently a circulus
vitiosus, an escape from which does not seem to be very easy.

Confining ourselves for the present to the N-metabolism, we
find another discrepancy. From the fact that in the metabolism
experiments the ratio of C:N always remained within the
range of 1:27 and 1:30, whereas in the figures on the com-
position (see above) this ratio is between 1:5 and 1:5.6,
»folgt schon ohne weiteres dasz Stickstoff erspart wird, indem
stickstoffhaltige Abbauprodukte mit Hilfe aufgenommener Kohlen-
stoffverbindingen wieder zu Kérperstoffen umgearbeitet werden.”

If this is true — I do not feel capable of forming a definite
opinion on this subject — we see another fundamental mistake
in Piitter’s logic. In that case more C than the quantity
corresponding with a certain amount of N according to the
empirical protein formula, must be produced together with that
amount of N.

Here are two errors in the fundamental: figure. If actually
nitrogenous water-soluble substances are taken up from the sea
water in order to play a réle in the N-metabolism, they may
have any composition, but that of proteins. The amount of
CO, resulting from the decomposition and combustion of these
substances may have any desired ratio to that of the Kjeldahl-N
and not that of the proteins. At the same time more CO, than
one would expect is obtained by means of the N-saving system,
postulated by Piitter.

From this evidence we see that the conclusion: ,,Es bleiben
also 141.2 mgr. Kohlenstoff iibrig, die nicht aus Eiweisz stammen’”’,
is excedingly questionable and that the speculations based on this
assumption, are to be taken with the greatest reservation. It is
of no particular use to go into details about them, it may only
be mentioned that the assumption of a ,,Methangarung’” and
a ,,Buttersaiiregarung’” of sugar as a source for the observed
CH, and H, is a rather arbitrary one and that their analogies
in mammalian physiology are at their best very hypothetical,
as I mentioned in the foot-note of p. 50.

Piitter’s deductions are also weakened by the following con-

101

siderations. There is still another thing to be thought of, sugges-
ted by a paper of Bohn 10). This eminent French biologist
shows that starfishes can produce O, in the daylight. The
enormous quantity of pigments, present in Echinoderms (von
Firth 138), p.518) might have one among them with respi-
ratory importance. The same thing had previously (C.R. Acad.
Sc. Paris. 19 Oct. 1908) been demonstrated by the same author
for certain actinians (A. equina, Sagartia erythrochila), even when
they do not have any symbiotic algae. It seems, according to him,
to bea general property of lower animals. In oxygen-poor water
the starfishes suffer in the dark, even as far as to autotomise
their arms. In the same water in diffuse daylight there is pro-
duced in five hours as much as 1.4 mgr. of O, in excess of the
quantity consumed by respiration; the animals are perfectly
normal. In supersaturated sea water much more O, is consumed
in the dark than in the light. Several controls have been made
so as to exclude the action of chlorophyll-carrying micro-
organisms, of an incidental introduction of O, and of a sick
condition of the animals used, but the same result invariably
is obtained. Starfishes need much oxygen and suffer in water
which does not contain more than 3 mgr. of oxygen, but they
endure large quantities of potassium cyanide, a property which
Bohn, Drzewina and others always found in animals with
anoxybiotic respiration.

Unexpected as this result may seem, it is made highly probable
by the few, but critical and sufficiently controled experiments
given. In the beginning of this chapter I mentioned that
Piitter finds in his Holothurians a big difference in CO, pro-
duction in the light and the dark. This phenomenon has its
minimum in the freshly caught animals, as we can see from
the table 14. The figures in this table indicate the oxygen
consumption.

Table 14. From Piitter.

In dark. In light. Ratio.
1st month. 31 31 1:1
24 month. 61 38 1:1.6
34 month. 60 23 1:2.6

It is, as Piitter says, hard to imagine that the metabolic
processes can be influenced to such an extent by light; ana-
logies from other parts of the animal kingdom fail, unless we
assume, that also by these Holothurians O, is set free in the
light in some way.

The possibility of the presence of symbiotic algae does not
seem to be very great. The only thing that is left, is the active

102

production of O, in some way by the cucumbers themselves.
Whether this is a phenomenon of the metabolism of the species
or whether respiratory pigments of the nature of chlorophyll and
numerous others simply decompose CQO,, is a question into
which we can not and do not have to enter. I could not
perform any experiments on this question myself, because
Piitter’s work only came to my knowledge after my experimental
work had been completed. The latter assumption however has
its peculiar charms, especially when we remember that analo-
gous facts have been established for the starfishes.

Piitter’s calculations are based on CO, production, CO,
is one of the most important sources of C in his calculation
of the ,,Gesammtkohlenstoffausscheidung” and the total carbon
metabolism. On these figures his whole system of analysis of
the ,,total metabolism” of Cucumaria is based. If this second
corner-stone of his building appears to be weakened, if in fact
the O,-production in the light is important enough to influence
the respiratory quotient in a measurable degree — and the
above mentioned evidence seems to make this probable ~—, the
whole system falls down and the conclusions are no longer
logical postulates. This is also true of his assumption of water-
soluble C- en N-compounds in the sea water as a source of
food. The work of Moore and his coworkers has shown this
very clearly. In the beginning of this chapter I had occasion
to call attention to one of Piitter’s serious methodical mistakes.

But determinations even with these’ wrong methods are very
scarce in the work of Piitter. Several facts are taken for
granted from the work of other authors. One of the most
striking examples is his assumption of the presence of a fatty
acid, dissolved in the sea water. Here he quotes Natterer who
in 1892 succeeded in destilling a ganz geringe Menge” of a
substance, resembling palmitin or stearin by its smell when
heated, from 200 L. of sea water. Piitter quotes this, but
does not try to repeat the experiment.

From destillation experiments P iitter concludes that 36 mgr.
of such acids are present in one Liter of sea water. But he
does not try to study the nature of this relatively enormous
quantity of acids more in detail.

It is true that many circumstances make estimations of this
kind particulary hard, chiefly the presence of a large quantity
of chlorides and CO, which are normal constituents of the
sea water. Henze (Pfliiger’s Arch. f. d. ges. Physiol. Bd.
CXXIII. 1908. p. 487), however, using appropriate methods —
taking a tube of metallic antimony and a glowing tube of lead
chromate and copper oxide to prevent hydrochloric acid or
other Cl-compounds from reaching the CO, apparatus, con-
cludes that the amount of organic material present, falls within
the limits of error.

103

The same results have been obtained by Moore c.s. with
another method. Knowing the scarcety of Piitter’s own expe-
riments, we may conclude that there are many reasons to be
doubtful with regard to his hypothesis, and that for the Holo-
thurians we certainly have many reasons of doubt of various
nature.

1 do not flatter myself that these few remarks disprove as
general an hypothesis as the one of Piitter. But I may have
succeeded in pointing out some weak spots in his building.

Addendum after the paper had partly been printed:

Prof. Dr. Withrow Morse who had the kindness to look
over the manuscript called my attention to the fact that the
enzyme which I studied in the starfishes shows more similarity
to erepsin than to trypsin. In fact, the former enzyme digests
native proteins’, albumins and globulins to amino-acids. Trypsin,
according to the modern conception, works only on lower
peptides, proteoses and peptones, prepared by the gastric pepsin.

As far as the control experiment on p. 20 is concerned, we
must remember the Merck's ,pure trypsin” is made from
extracts of the pancreas and activated by enterokinase from
the gut. The latter is mixed with intestinal erepsin.

The Py-range of really pure trypsin is very limited. Cf. the
recent papers of Mc. Clendon and Dernby in the J. Biol.
Chem. and the very recent work of Northrup, just published
in the J. gen. Physiol. and the J. Biol. Chem.

16,

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p.

py Py PP sy

PRINCIPAL ERRORS IN THIS PAPER:

1. line 20. we will

must he:

several places between p. 1 and p. 48.

1. line 20. from where .
1: line 22. till now .

2. line 8. f, i.

places between p. 1 and > 48.

3. line 7. came
6. line 14.
6. line 28.
11. line 3
13. line 2.
13. line 27.
13. line 36.
18. line 26.
28. line 26.

reminding weakly .
they can close. ,
from below. fishers
rifs .

Has a Thyone.
pro.

which. . . . ,

alive

must be:
must be:

must be:

must be:
must be:
must be:
must be:
must be:
must be:
must be:
must be:

must be:

we shall. This error occurs on

whence.
until now.

e.g. This error occurs on several

have come.

reminding one slightly.
they close.

fishermen.

reefs.

If a Thyone has.

per.

whom.

live.

Fig. 3.

Fig. 5.

Resorption of iron in the radial sacs of Asterias.

2
Resorption of ammonium carminate in the radial sacs of Asterias.

STELLINGEN

I

Men mag met Broman aannemen, dat het organon vomero-
nasale Jacobsoni het voor het landleven gemodificeerde
»Wassergeruchsorgan” der Anamniota is en slechts werkzaam
kan zijn, als de reukstoffen door een waterig vloeibaar medium
het zintuigepitheel ter plaatse bereiken.

II

De opvatting van Austin H. Clark, dat de Echinodermen
aberrante Arthropoden zijn, is onjuist.

III

Bij de resorptie van de staart der kikkerlarven speelt autolyse
een primaire rol.

IV

De natuurlijke immuniteit van het konijn voor atropine is
niet te danken aan de eigenschap van het bloedserum van
deze dieren om dit alkaloid te binden en daardoor physiologisch
onwerkzaam te maken.

Vv

Er zijn geen bewijzen, dat ideeénvorming voorkomt bij
sub-anthropoide dieren; door gewoontevorming kan wel een
»Komplexqualitat” in ,,Teilqualitate’ worden ontleed.

VI

De boleten behooren niet tot de Polyporeeén, maar zijn
Agaricaceeén.

VII

De komplex-theorie van Renner geeft geen voldoende ver-
klaring voor de resultaten van het Oenothera-onderzoek.

Vill

Vele planten-gallen vertoonen structuren, die duiden op
»fremddienliche Zweckmassigkeit (Becher)’’.

IX

De reconstructie van den schedel van Eoanthropus dawsonii
door Smith Woodward is onjuist.

x \

De mammoeth was niet voldoende aangepast aan de kou
en is onder den invloed van het ruwe klimaat van den ijstijd
gedegenereerd en tenslotte uitgestorven.

XI

De zgn. wet-Limburg maakte als ,,nood-wet’’ een eind aan
onrechtvaardige verhoudingen. Te betreuren is het echter, dat
zij bewerkt heeft, dat een zij het dan ook elementaire klassieke
opleiding niet langer een voorvereischte is voor universitaire
examens en het is gewenscht, dat ten spoedigste een herziening
plaats vinde.

XII

De vergelijkende physiologie dient ten spoedigste te worden
ingevoerd als officieel leervak aan onze universiteiten.

4