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RHYTHMICAL PULSATION
SCYPHOMEDUSA:

BY
ALFRED G. MAYER

Director of Department of Marine Biology of the
Carnegie Institution of Washington,
Tortugas, Florida

WASHINGTON, D. C.
Published by the Carnegie Institution of Washington

1906

IN

CARNEGIE INSTITUTION OF WASHINGTON

PUBLICATION NO. 47

FROM THE PRESS OF
THE WILKENS-SHEIRY PRINTING CO
WASHINGTON, vu. u.

RHYTHMICAL PULSATION IN ANIMALS.

1. PULSATION OF JELLYFISHES, ARMS OF LEPAS, HEART OF
SALPA AND OF LOGGERHEAD TURTLE.

I. CONCLUSIONS NEW TO SCIENCE.

1. If we cut off the marginal sense-organs of the scyphomedusa
Casstopea, the disk * becomes paralyzed and does not pulsate in sea-
water. ‘The disk will pulsate in sea-water, however, if we make either
a single ring or a series of concentric broken-ring-like cuts through
the muscular tissue of the sub-umbrella. Then upon momentarily
stimulating the disk in any manner, it suddenly springs into rapid,
rhythmical pulsation so regular and sustained as to recall the move-
ment of’clockwork.

Pulsation “will notstart unless the disk be momentarily stimulated,
as by a mechanical -or electrical shock or by a single touch with a
crystal of K,SO,, but once started it continues indefinitely in normal
sea-water without further external stimufation.

The waves.of pulsation all arise from the stimulated point, and the
labyrinth ofsub-umbrella tissue around this center must form a closed
circuit. Itisnotnecessary that the cuts through the sub-umbrella tissue
of the disk be concentric circles, for any shape will pulsate which
allows contraction waves to travel through tissue forming a closed cir-
cuit from the stimulated center and back to this center. When each
wave returns to the center it is reinforced and again sent out through
the circuit; and thus the center sustains the pulsation.

Note.—It is a pleasure to express my gratitude to those who have aided me in the
prosecution of this research. To Prof. H. S. Jennings for his kindness in sending to
me lists of the coefficient 7 for the making of, isotonic solutions; to Dr. Leon J.
Cole and Dr. Charles Zeleny for important suggestions and criticisms; to Mr. Daven-
port Hooker for collecting Gonzonemus and Dactylometra, and to Prof. H. F. Perkins
for aid in collecting Casszofea at Tortugas ; to Professors Ulrich Dahlgren and Edward
L. Mark for instruction and aid.

*In this paper the term ‘‘disk’’ will be used to designate Medusa from which the
‘marginal sense-organs have been excised ; while the term ‘‘ Medusa’’ will designate the
normal perfect animal.

J

2 PULSATION OF JELLYFISHES.

The pulsating labyrinth may be simplified after the rhythmic move-
ment has started, by cutting parts of it away, or cuts may be made in
such manner as to increase its complexity. Any cut which breaks
the circuit, however, stops the wave of pulsation, and continuous
movement can not again be started.

The rate of pulsation of the disk is fully twice as fast as that of the
normal perfect Medusa. This rate remains constant in the pulsating
disk, and when pulsation ceases the movement stops zustantly, never
gradually. The rate of pulsation in disks deprived of marginal sense-
organs depends not upon the area of the tissue forming the circuit,
but only upon the length of the circuit. Short circuits pulsate more
rapidly than do long ones. In this respect it differs from the con-
trol normally exercised by the marginal sense-organs; for small pieces
of tissue with a marginal sense-organ attached pulsate slower than
large ones. Moreover, when a sense-organ is present, tissue of any
shape will pulsate even if its shape does not form a closed circuit.

The disks of Aurelia and Dactylometra, if cut as described above,
will pulsate as does the disk of Cassiopea,

These experiments show that the rhythmical pulsation in Medusze
must arise from a definite center or centers, but this center may be
established at any point in the muscular layer of the sub-umbrella.
Once established it remains at a fixed point, while the disk continues
to pulsate. Sustained pulsation zz disks occurs only in tissue forming
a complete circuit, and depends upon an electric transmission of
energy, and the pulsation is self-sustaining (z.¢e., sustained by internal
stimuli) once it be started by an external, momentary stimulus.*

2. If normal perfect Medusz be lifted out of water and then
thrown back, the rate and amplitude of their pulsation suddenly
increases. Pulsating disks react in a similar manner, but in their case
the amplitude only increases, the rate remaining practically constant.
The presence of marginal sense-organs is therefore not necessary for
the display of ‘‘excitement.”’

3. The stimulus which causes pulsation is transmitted by the
diffuse nervous or epithelial elements of the sub-umbrella. Newly
regenerated sub-umbrella tissue, which lacks muscular elements and
can not itself contract, will still serve as a bridge to transmit the
stimulus which causes contraction in muscular tissue attached to but

* Professor W. T. Porter (1897) found that any part of the ventricle of the mamma-
lian heart (heart of the dog) will beat for hours if supplied with defibrinated blood
through its nutrient artery. Isolated portions of the heart of the hag-fish continue to
beat rhythmically for hours even in the absence of nutrition. (See A. J. Carlson, 1905,
Amer. Journ. Physiol., p. 220.)

CONCLUSIONS NEW TO SCIENCE. 3

beyond the bridge. In this connection, Carlson has demonstrated
that the stimulus which causes the pulsation of the heart of Limulus
is nervous in nature.

4. The paralyzed disk of Cassiopea is stimulated into temporary
pulsation by all salts of potassium, sodium, lithium, barium, iodine,
bromine, platinum, weak acids (hydrogen), ammonia, and glycerin.
Magnesium, calcium, strontium, urea, and dextrose do not stimulate
the disk, and produce no contraction.

5. The sodium chloride of the sea-water is the chief stimulant to
pulsation in Casstopea, while magnesium is the chief restrainer of pul-
sation, and counteracts the influence of the sodium chloride. Thus
Casstopea will pulsate in a pure 54n NaCl solution for more than half
an hour, but usually comes to rest in less than two minutes in a solu-
tion containing the amounts and proportions of NaCl and magnesium
found in sea-water.

I find also that the heart of Salpa democratica, the branchial arms of
Lepas, and the heart of the embryo loggerhead turtle pulsate actively
in solutions containing only NaCl, K, and Ca, magnesium being absent.
Magnesium zzzdcts pulsation in all of these cases, as it does also in
Casstopea.

The general réle of NaCl, K, and Ca in all of the above cases is to
combine to form a powerful stimulant producing an abnormally
energetic pulsation, which, however, can not continue indefinitely ; and
magnestum is necessary to control and reduce this stimulus so that
the pulsating organ is merely upon the threshold of stimulation.

A Ringer’s solution is an optimum combination of NaCl, K, and
Ca, and is only a stimulant, not an inorganic food, as has been com-
monly assumed. The organism must in time become exhausted under
the influence of this stimulant unless a certain proportion of magne-
sium be present to restrain its action. Indeed, Ringer’s solution prob-
ably acts by withdrawing magnesium ions by osmosis, and replacing
them by a stimulant composed of salts of Na, K, and Ca. Mag-
nesium is therefore a most important element in controlling and
sustaining pulsation. If magnesium be precipitated in the pulsating
Cassiopea, the NaCl, K, and Ca immediately produce a violent pulsa-
tion which soon passes into sustained tetanus, and all movement ceases
in cramp-like contraction.*

* Loeb, J. 1906; Journ. Biological Chemistry, vol. 1, p. 331; finds that in the hydro-
medusa Polyorchis the mouth and tentacles are permanently contracted in any solu-
tion which lacks magnesium ; and that magnesium serves to relax the muscles of the
bell, thus counteracting the tetanus caused by other constituents of the sea-water and
guaranteeing the relaxation after a systole.

4 PULSATION OF JELLYFISHES.

The calcium of the sea-water assists the NaCl to resist the retarding
effects of magnesium. Thus Casszopea will pulsate from half an hour
to an hour in a solution containing the amounts and proportions of
NaCl, magnesium, and calcium found in sea-water, but usually ceases
to pulsate in less than two minutes in a solution containing only the
NaCl and magnestum,

Unlike calcium, potassium does not assist the NaCl to overcome the
stupefying influence of the magnesium.* ‘Thus Casstopea ceases to
pulsate almost as quickly in a solution containing NaCl, magnesium,
and potassium of sea-water as it does in a solution containing only
the NaCl and magnesium.

The potassium of sea-water serves, however, to stimulate pulsation
in connection with both calcium and NaCl. Thus Cassiopea pulsates
only from 20 to 120 minutes and at about a normal rate in NaCl
+ K,SO,, or in NaCl+ KCl, whereas it pulsates for more than three
hours at fully twice its normal rate in NaCl + K,SO, + CaSQ,, or
NaCl + KCl + CaCl,.

We see, then, that the NaCl, K, and Ca of the sea-water unite in
stimulating pulsation and in resisting the stupefying effect of the
Mg. All four salts conjointly produce, in sea-water, an indifferent, or
balanced, fluid which neither stimulates nor stupefies the disk of Cas-
stopea, and permits a recurring internal stimulus to produce rhythmic
movement.

6. Cassiopea does not pulsate when its marginal sense-organs are
removed, simply because the sea-water does not stimulate it. Ifstimu-
lated in sea-water, in any manner, itreadily pulsates. This is also true
of Gontonemus, and Loeb’s statement that both the K and Ca of sea-
water inhibit pulsation is not supported; for the center of Gontonemus
will pulsate actively, though temporarily, in sea-water whenever it is
touched by a crystal of any potassium salt, or otherwise stimulated.

On the other hand, the disks of Auvelia and Dactylometra begin to
pulsate in sea-water in a few minutes, as soon as they recover from
the shock of the operation resulting in the loss of their marginal sense-
organs. Unlike Casstopea and Gonionemus, both Aurelia and Dactylo-
metra are weakly stimulated by the sea-water as a whole and pulsate
almost immediately after the removal of their margins.

*The general anesthetic effect of magnesium has been well known since the re-
searches of Tullberg, 1892; Archiv. Zool. Exper. et Gen., Tome x, p. 11.

+ As a matter of fact, the disk of Gonzonemus is often seen to give isolated pulsa-
tions, at irregular intervals, in sea-water without apparent external stimulation. (See
Yerkes, 1902.)

CONCLUSIONS NEW TO SCIENCE. 5

The disk of Cassiopea usually pulsates spontaneously in an irregular
manner, immediately after the removal of its marginal sense-organs,
if it be placed in a solution containing NaCl, NaCl+KCl, NaCl+
CaCl,, or NaCl + KCl + CaCl, in the amounts and proportions found
in sea-water; but it will not pulsate in any solution which contains
magnestum.

7. The central disk of Casstopea, if set into pulsation, will pulsate
longer than an hour ina solution resembling sea-water but lacking
calcium, whereas the normal perfect Medusa, or parts of the margin
containing sense-organs, cease to pulsate in this solution in less than
six minutes. The marginal sense-organs can not send forth stimuli
producing contractions unless they be constantly supplied with calcium
from the sea-water, whereas the sub-umbrella tissue of the disk itself
is relatively independent of the calcium of the sea-water.

On the other hand, both the disk and the perfect Medusa will pul-
sate in sea-water saturated with CaSQ,.

8. The normal Cassiopea Medusa will pulsate fully three times as
long in a solution of Na,SO, containing the same amount of sodium
as is found in sea-water as it will in a solution of Na,SO, isotonic
with sea-water. This indicates that the amount and proportion of
sodium in the sea-water is more important to pulsation than is its
osmotic property.

9. The contractions of the heart of the loggerhead turtle are con-
ducted and maintained exclusively by the thin peripheral muscular
part of the wall of the heart, the thick cavernated tissue of the heart
being passive. Moreover, the outer muscular part of the heart’s wall
is a better electrical conductor than is the cavernated tissue. A sim-
ilar condition is seen in Casszopea, where the thin sub-umbrella tissue
of the disk is the only part which conducts and maintains the stim-
ulus for pulsation, and is a better electrical conductor than is the thick
gelatinous substance of the disk.

10. The chief results of the paper are the discovery of a new method
of restoring pulsation in paralyzed Meduse, and also that magnesium
plays a most important réle in restraining, controlling, and prolonging
pulsation in animal organisms.

In Cassiopea the ectodermal, epithelial, or diffuse nervous elements
of the sub-umbrella transmit the stimulus which produces rhythmical
contraction.

Rhythmical pulsation can be maintained only when a stimulus and
an inhibitor counteract one another, and cause the organism to be
upon the threshold of stimulation; thus permitting weak internal
stimuli to promote periodic contractions.

6 PULSATION OF JELLYFISHES.

MINOR CONCLUSIONS.

There are certain minor conclusions, mainly confirmations or am-
plifications of the excellent work of Romanes upon Scyphomeduse.

In Cassiopea the sub-umbrella and mouth-arms are the only parts
which respond to mechanical or chemical stimuli. The ex-umbrella is
wholly insensitive.

There is no essential difference in kind between the physiological
action of the sense-organs, in pulsation, and that of any other part of
the sub-umbrella.

Casstopea will live for more than a month in absolute darkness.
Its plant cells then degenerate, but the Medusa does not suffer; hence
its vitality is not dependent upon the commensal plant cells within
its tissues.

Starved Medusz will shrink to about one-sixteenth their initial
volume and still survive. They will live in brackish water contain-
ing 75 per cent fresh water better than they will if we maintain the
amounts and proportions of calcium and potassium, merely reducing
the amounts of NaCl and magnesium of the sea-water.

The fluids of the gastro-vascular space and of the body of the
Medusa are only slightly alkaline, while the sea-water at Tortugas
is decidedly alkaline.

The sense-organs tend to send out contraction stimuli at various
rates, but the fastest working sense-organ controls the Medusa.

Excitement of the disk forces the sense-organs to maintain a higher
tate of pulsation than they are capable of maintaining if cut off,
and it is evident, from other experiments, that the disk reacts recipro-
cally upon the sense-organs, stimulating them into activity.

Small pieces of the disk, enervated by sense-organs, pulsate slower
than large ones.

Small young Meduse pulsate faster than large o/d ones.

The sub-umbrella surface of the disk exercises a reflex control over
both sense-organs and mouth-arms.

Repeated stimulation of any one part of the disk finally tires the
stimulated place so that it ceases to respond. Other parts of the disk
still respond as readily as did the tired place in the first instance.

Having stated the principal conclusions, we will now proceed to
give a detailed account of the experiments upon Cassiopea, Lepas,
Salpa, and the loggerhead turtle.

MAYER PLATE 1

Aboral views of Cassiopea xamachana Bigelow. From life. Natural size.

Above, rare, small variety. This bears a close superficial resemblance to the common Cassiopea ndrosia of the Fiji Islands.
(See Agassiz and Mayer, 1899, Bull. Mus. Comp. Zool. at Harvard Coll., vol. 32, p. 175, pl. 14.)

Below, the common variety.

NORMAL MOVEMENTS IN SEA-WATER. 7

II. PULSATION OF CASSIOPEA IN SEA-WATER.
INTRODUCTION—-NORMAL MOVEMENTS.

The rhizostomous Scyphomedusa Casstopea xamachana (plates 1,
11), is very abundant during spring and summer in the salt-water moat
of Fort Jefferson, at Tortugas, Florida. It was described by Bigelow
(1892, 1900) from a salt-water lagoon in Jamaica, and also under the
name of Cass7opea frondosa by Fewkes (1883), who found it at the
Tortugas.

The Medusz are usually found gathered in clusters upon the weedy
bottom of the moat in water about four feet deep. They lie with the
aboral side of the disk pressed downward upon the bottom, and with
the 8 mouth-arms, with their numerous suctorial mouths, spread out
above. A sucker-like concavity on the aboral side of the disk allows
the Medusa to adhere with considerable strength to the bottom or sides
of an aquarium, and the tenacity of its hold is still further enhanced
by the rhythmical movement of the disk, which beats with considerable
regularity, thus tending to hold the bell firmly against its fastening,
and also to drive a current of water out over the mouth-arms.

If moved from its normal position and placed in the water with its
disk uppermost and arms downward, the rhythmical beating of the
disk causes it to swim upward, but if the water be of considerable
depth it soon topples over and thus swims downward to the bottom
or reaches the side of the aquarium. If, however, itshould reach the
surface, the concavity at the center of the aboral side of the disk often
serves to permit the surface tension to hold the Medusa upon the sur-
face, where it may float for a long time, pulsating normally with the
concavity relatively dry, although lower than the general surface of
the water.

The Medusa pulsates with a regular rhythmical movement, pauses or
irregularities in the rhythm being exceptional. Occasionally, how-
ever, its rate suddenly increases, with or without apparent cause, and
the pulsation may become so active as to cause the Medusa to break
away from its anchorageand glide over the bottom. A regular unex-
cited movement is, however, often maintained for hours at a time, and
in general this rate of pulsation is faster in small than in large Medu-
see, as will appear from table 1, on page 8.

The relatively rapid rate of small Medusz is probably due to their
being young and possessed of more vitality than are the large, old
animals; for not only do small Medusz regenerate lost parts more
readily, but we also find that specimens which have become reduced
in size through starvation pulsate at a slower rate than young and

8 PULSATION OF JELLYFISHES.

well-fed Medusze of the same size. Thus one Cassiofea was starved
for three months, and the diameter of its disk shrank from 78 to 21
millimeters, while at the same time its rate of pulsation declined from
about 40 to16 per minute. It is also interesting to observe that if
we cut off the margins of the disks of Medusz of various sizes, the
severed rims of the small Medusz pulsate at a more rapid rate than
do those of the large Medusz, although in both cases this rate is
slower than that of the uninjured Medusa.

TaBLE 1.—felation between the rates of pulsation and the diameters of the
disks in Meduse of Cassiopea xamachana.

Diameter of | No, of pulsa- Diameter of | No. of pulsa- Diameter of | No. of pulsa-

Medusa in tions per Medusa in tions per Medusa in tions per

millimeters. minute. millimeters. minute. millimeters, minute.
13 78 28 39-55 62 29
15 82-86 28.5 16-23 63 35
16 Q4-III 30 53-63 82, 40-50
18 68 31 55-61 84 28-39
20 36 32 58-62 90 16-28
20.5 45-65 36 42 102 20-21
aa 60 42 51-54 107 23-24
23 44-71 46 45-46 118 7
23.5 40 47 43-36 124 12-16
26 43-52 50 27 136 9-12
27 41-56 57 36-37 153 7

EXCITEMENT.

As we have said, the pulsating Medusz occasionally exhibit a sud-
den increase in their rateand amplitude of pulsation without apparent
cause. This can, however, be invariably brought about as a response
to any stimulus, such as a water current, a mechanical shock, or the
introduction of some irritating chemical into the water. When lifted
wholly or partially out of water, and replaced, the Medusz pulsate at
about twice their normal rate for two or three minutes, and the ampli-
tude of their pulsations is also increased. Even small fragments of
the disk containing a marginal sense-organ will usually display this
excitement, although the duration of the period of excitement is shorter
for small than for large pieces, and their rate of pulsation slower.

However, the presence of marginal sense-organs is not necessary for
this ‘‘excitement,’’ for, as we shall soon show, we have succeeded in
causing disks deprived of marginal sense-organs to pulsate constantly
and regularly in sea-water; and if such disks be pinched or lifted out
of water or otherwise disturbed the amplitude of their pulsations
becomes suddenly increased, while the va/e remains practically con-
stant. In normal uninjured Medusz both rate and amplitude increase,

NORMAL MOVEMENTS IN SEA-WATER. 9

but as we shall see, disks without sense-organs pulsate at the maxi-
mum rate at which their tissue is capable of transmitting the wave
of pulsation, and they can therefore exhibit ‘‘excitement’’ only by
an increase in amplitude.

It is worthy of note that if the forceps used to stimulate the Medusa
be made to seize upon only a small area of tissue, the Medusa will not
respond, but on bringing a larger area between the forceps the response
is sudden and violent. In this connection it will be recalled that
Romanes showed that the bell of Savsza, when deprived of its margin,
will respond to mechanical shocks by pulsations, each stimulus usually
giving rise to one or two pulsations, and this is also true of the par-
alyzed disk of Casstopea. We must conclude that the presence of
marginal sense-organs is not necessary for the display of that sudden
increase in activity which we have called ‘‘excitement,’’ and that this
response may come from many or all parts of the undifferentiated tissue
of the sub-umbrella.*

A

A;

AS

KY
. ° ‘
B
B f

Fig. 1. Fig. 2.

Romanes showed that in 4urelia annular cuts separating the margin
from the center of the disk caused the rhythm to become slower, and he
was led to suspect (1885, p. 163) that a stimulus of an afferent character
emanates from all parts of the sensory surfaces of the sub-umbrella
to the marginal sense-organs, although of this he had no direct proof.
I think we can prove that this is the case in Casstopea, for if we cut
off all but one marginal sense-organ, and then make cuts through
the sub-umbrella tissue (fig. 1) radiating outward from the sense-organ
and therefore not interfering with any stimulus which may travel by the
shortest path from any point in the disk to the sense-organ, the final
rate of pulsation, after the excitement due to the operation has sub-

*It is interesting to observe that Bancroft and Esterly (1903) find that while con-
tractions normally originate from the ganglionated ends of the heart of Ccona, they
may originate from any other region.

10 PULSATION OF JELLYFISHES.

sided, will remain the same as it was before the radiating cuts were
made. Moreover, its excited rate, due to being lifted out of water and
dropped back, remains the same as it was before the cuts were made.
On the other hand, cuts designed to successively reduce the area of the
sub-umbrella tissue enervated by a sense-organ (such as are shown in
fig. 2, A and B) usually cause the normal rate of pulsation to decline.
The excited rates, however, are less influenced by reduction of area,
small pieces sometimes pulsating almost as rapidly as large ones, but
the duration of the excitement displayed by small pieces is much
reduced. For example, in the a series of figure 2—

Normal rate | Excited rate
Area, per minute, per minute.
Bigevanss vetiewa 280 17 32
54 12 40
I 6 II

In the B series the relative areas and rates were as follows:

Ate; | Meee’ | er niinaee”
B jgsseciac antes 271 27 50
Baeeesesceeene 153 14-20 50
Bgeesseeeeeves 5 17 48
Biaeeauanaastacs 2 14 19
Bigwersrvyannss I 14-20 35

The above results are quite similar to those of Romanes upon Aurelia,
and are opposed to the conclusion of Eimer that severed portions of the
disk pulsate at rates approximately proportionate to their respective
areas.

It is interesting to observe that if we stimulate a Medusa into pro-
longed and active pulsation at an ‘‘excited’’ rate and then cut out the
marginal sense-organs, each sense-organ, together with the piece of
tissue attached to it, instantly subsides into a s/ow rate of pulsation,
never faster than the average unexcited rate of the entire Medusa.
Moreover, these pieces with sense-organs attached can not immediately
be stimulated into a display of excitement, although after an interval
of time they will readily respond and exhibit an excited rate com-
mensurable with that of the perfect Medusa. As we have seen, the
display of ‘‘excitement”’ is a function of the undifferentiated tissue of
the sub-umbrella, and it appears that the excited rate of the Medusa
may be maintained by the influence of the general sub-umbrella tissue

INTERDEPENDENCE OF SUB-UMBRELLA AND SENSE-ORGANS. II

upon the sense-organs even after the sense-organs have become too
exhausted to themselves maintain an ‘‘excited’’ rate. Moreover, if
we stimulate the sub-umbrella surface by touching it repeatedly with a
crystal of K,SO, the disk responds by active contractions and forces
the sense-organs to respond at the same rate. Then after the stimu-
lus is withdrawn the sense-organs are found to have been exhausted
by the contractions of the disk and can not again resume pulsation
until after a long interval of rest.

Direct evidence showing that the sub-umbrella may exert a control-
ling influence on all parts of the sensory tissues of the Medusa is also
afforded by the following experiment: If we cut off the basal plate
with the 8 mouth-arms, the mouth-arms remain normally expanded
in sea-water. If now we place the mouth-arms in a solution which
resembles sea-water, but lacks potassium, the arms contract into a
close bunch, and will zot again expand as long as they remain in the
solution. If, however, we place a perfect Medusa in the solution it
exhibits periods of active pulsation alternating with periods of rest.
Immediately after it comes to rest its mouth-arms contract into a
close bunch, but they always expand again as soon as the Medusa
resumes pulsation. It will be remembered that Romanes showed
that removal of the margin of the bell in Sarsza caused the manu-
brium to elongate and lose its muscular tonus. He also found that
in Sarsia stimulation of the sub-umbrella caused the manubrium to
contract, and that the manubrium of Zzavopsis indicans would apply
its mouth to any stimulated part of the sub-umbrella, provided the
stimulus could travel radially inward from the stimulated spot to the
manubrium. Otherwise the manubrium executed ill-directed or
wandering movements.

We will soon show that any difference between the physiological
action of the marginal sense-organs and that of the general sensory
tissue of the sub-umbrella is one of degree, not of kind.

CONTROL OVER PULSATION EXERCISED BY THE MARGINAL
SENSE-ORGANS.

Romanes found that the potency of the marginal sense-organ attached
to a segment of the disk has more to do with its rate of pulsation than
has the size of the segment; nevertheless small segments usually pul-
sate slower than large ones.

In Cassiopea xamachana there are 13 to 23 marginal sense-organs,
and I find that the average rate of the perfect Medusa is apt to be the
same as the rate of its most rapidly working sense-organ. As Romanes
saw in Aurelia, the sense-organs tend to initiate stimuli at various

I2 PULSATION OF JELLYFISHES.

rates, but the fastest controls all the others and forces them to beat
in unison with it. To test this, I took a Casstofea having 19 margi-
nal sense-organs and a normal unexcited rate of 12 to 16 pulsations
per minute. I then made 19 radial cuts
midway between the 19 sense-organs,
so as to divide the disk into 19 practi-
cally equal sectors, each enervated by a
single sense-organ. These radial cuts
through the sub-umbrella completely
separated the sectors one from another
in so far as the transmission of nervous
impulses were concerned (fig. 3). Un-
der these conditions one of the sectors
pulsated 18 times per minute; 2 pul-
sated 17 times; 2 pulsated 16 times; 1 Fig. 3.

pulsated 15 times; 3 pulsated 9 times; 1 pulsated 8 times; 4 pulsated
7 times; 2 pulsated 6 times ; 2 pulsated 5 times, and 1 failed to pulsate.

The sense-organs gradually change their rates, so that at the end of
an hour or two the fastest may sink to second or third place, etc.
Quite often one or more of the sense-organs either failed to send out
pulsations or did so at very infrequent intervals. These sense-
organs appeared normal, however, and if stimulated by being thrown
into sea-water containing 1 per cent excess of K,So, they initiated
pulsations at a rapid rate.

As Romanes and Eimer showed, if we cut off all but one of the mar-
ginal sense-organs this one will maintain a rhythmical pulsation of
the disk, whereas if this last sense-organ be removed the disk at once
becomes more or less paralyzed. The disks of Aurelia or of Dactylo-
metra, however, begin to pulsate irregularly a few minutes after the
loss of the last marginal sense-organ, but Casszopea remains practi-
cally paralyzed for about 24 hours after the operation, rarely executing
a pulsation unless stimulated. On the following day, however, it
occasionally pulsates without apparent stimulation, and three days after
the operation the disk rarely remains for a minute without pulsating.
The pulsations are, however, isolated, single, and separated by irregular
intervals of time, until the marginal sense-organs begin to regenerate.

Romanes showed that in Hydromeduse the least discernible rem-
nant of the bell-margin if left intact will maintain the rhythmical
movement of the bell, but that in Scyphomedusz the marginal sense-
organs are the only parts of the rim which normally control the
thythmical pulsation. I find thatif one cuts off the tip of the last
remaining sense-organ of Cassiopea, thus removing the otoliths and

CONTROL OF SENSE-ORGANS OVER PULSATION. 13

pigment spot but leaving the stalk of the sense-organ intact, the disk
is instantly paralyzed. Also, when the marginal sense-organ regen-
erates, regular pulsation is resumed as soon as the pigment spot and
a few small otoliths begin to appear. For example, figure 4 shows
the appearance of the normal sense-organ, and figure 5 the condition
of a regenerating sense-organ that has become capable of control-
ling the rhythm of the disk. Immediately after death the pigment of
the sense-organ dissolves out into the sea-water; on the other hand
it appears remarkably stable in the living animal, and is not faded by
the most intense sunlight, nor changed by one month’s confinement
in absolute darkness.

Fig. 4. Fig. 5.
Fig. 4.—Enlarged views of a sense-organ of a mature Medusa of Cassiopea. A, aboral

view; B, side view; C, oral view.

Fig. 5.—Enlarged oral view of a regenerating sense-organ, showing the beginning of the
formation of pigment spot and otoliths. A wide strip of new tissue (dotted) separates
the sense-organ from the old muscular layer of the sub-umbrella.

If a sense-organ be cut out with the merest remnant of sub-umbrella
tissue left attached to it, examination under the microscope shows
that this tissue continues to pulsate rhythmically, and it is apparent
that the area of the sub-umbrella tissue attached to a sense-organ may
be reduced to a practical zero without any more marked effect than a
not very pronounced slowing of its rate of pulsation. On the other
hand, if we remove all but one of the sense-organs and then place the
disk in sea-water charged with CO,, keeping the sense-organ itself
out of the fluid, the disk becomes paralyzed and can not be enervated
into contraction by the sense-organ. In some of these experiments
the sense-organ was also paralyzed, although it had not been in the
CO, solution. In others the sense-organ continued to send contrac-
tions out over the adjacent tissue, but these could not extend over the
parts of the sub-umbrella which were bathed by the CO,.

14 PULSATION OF JELLYFISHES.

All experiments serve to demonstrate that the nervous relation-
ship between the sense-organs and the general sub-umbrella tissue is
reciprocal, as has been clearly shown by Romanes, who found that if
we cut a strip of tissue from the disk of Aurelia, leaving a sense-organ
at one end, and then gently stroke the end remote from the sense-organ
with a camel’s hair brush, the marginal sense-organ at the other end
of the strip will be stimulated into sending a contraction wave back
over the strip (Romanes, 1885, pp. 74-77). This discharge is therefore
of a reflex nature. Nagel (1894) supports the idea that the marginal
sense-organs are reflex centers, while von Uexkiill (1901), upon evi-
dence which to me appears insufficient, concludes that the marginal
sense-organs in Rhizostoma pulmo are merely centers for the reception
of mechanical stimuli, and that each pulsation of the bell causes the
sense-organs to swing to and fro, and this stimulation calls forth a
new pulsation.

We will show later that any point in the sub-umbrella surface may
be made to start and maintain impulses which will set the whole disk
into sustained and perfectly regular rhythmical pulsation. There is,
therefore, no difference of kind between the nervous activities of the
marginal sense-organs and those of any other parts of the sensory
surface of the sub-umbrella.

As to the function of the otocysts in Hydromedusze, Murbach (1903)
showed that in Gonionemus they have no static function, for if they be
removed the normal movements of the Medusa will be resumed before
they are regenerated. Murbach’s conclusion that the seat of the static
function is ‘‘muscular sensation in the velum’’ requires confirmation.
Injury of so important a swimming organ as the velum may readily
cause irregularities in movements by abnormally deflecting the water
currents passing through the opening of the velum at each contrac-
tion. Moreover, Yerkes (1902), in his study of the sensory reactions
of Gonionemus, found that the velum is unaffected by stimuli of any
sort.

Romanes (1885) found that the ocelli of Savsta and Tiaropsis are
sensitive to light, and Yerkes (1902) demonstrated that the tentacles
of Gonionemus are very sensitive to chemical, mechanical, and photic
stimuli.

The rates at which waves of contraction travel over the disk in
Casstopea range from 150 to 1200 mm. per second, each individual
displaying a characteristic and constant rate. Apparently there is no
relationship between the size of the Medusa and the rate of trans-
mission of waves over its sub-umbrella tissue. These rates were

FUNCTIONS OF THE MARGINAI, SENSE-ORGANS. I5

determined by cutting spiral strips reaching from the margin inward,
in the manner of Romanes. It was observed that when the spiral was
made 5 mm. or less in width only powerful stimuli would travel from
one end of the strip to the other, and if under these conditions a single
sense-organ was left at the outer end of the strip, waves of contraction
which started from this sense-organ might or might not reach the cen-
tral part of the disk. If, however, the end containing this sense-organ
were touched with a crystal of K,SO,, or any other potassium salt, a
powerful wave of contraction immediately ensued and always traveled
completely through the spiral. But if the inner end of the spiral were
touched with the crystal of potassium salt, not only did the wave not
always reach the sense-organ, but it traveled only three-quarters as
fast as did the waves from the sense-organ. When the sense-organ
was cut off, however, the waves traveled at the same rate from ezther
end of the spiral strip, and this rate was the slower of the two men-
tioned above. Evidently the sense-organ reinforced the stimulus
given by the potassium salt.

In this connection Romanes showed that in Aurelia strong stimuli
may initiate waves that may travel over the disk at twice the rate of
weak ones.

Peripheral parts of the disk transmit stimuli at a faster rate than do
parts near the center of the disk. This was shown by Romanes to be
the case in Aurelia. Altogether the outer parts of the sub-umbrella
are more sensitive than the inner.

As Romanes showed, there must be an appreciable interval of rest
between two successive responses to stimuli, and rhythmical waves
can not follow one after another faster than a certain frequency.
Waves traveling in opposite directions through the same strip of tissue
meet and reinforce, but do not pass each other, for a stimulus can not
produce a contraction over the tissue that has been in contraction only
the instant before.

The sensory field of the Medusa is confined to the sub-umbrella
and the mouth-arms. The ex-umbrella surface exhibits no reactions
to stimuli, and indeed the epithelium of the ex-umbrella may be killed
by such penetrating reagents as Gilson’s fluid, and, provided the poi-
sonous liquid does not reach the sub-umbrella, the rhythmical move-
ment will not be altered in rate. Even near the margin of the disk,
close to the sub-umbrella surface, the ex-umbrella is inert to stimuli
of all sorts. The action of the sucker-like concavity at the aboral
center of the ex-umbrella is entirely passive, and a Medusa deprived of
all marginal sense-organs will still ‘‘cling’’ to the bottom or side of
the aquarium, although paralyzed and motionless.

16 PULSATION OF JELLYFISHES.

VITALITY, ETC.

The fluids of the central stomach of Cassiopea are practically neu-
tral to litmus test, whereas the sea-water at Tortugas is decidedly alka-
line. For example, litmus paper tinged pink by HCl is changed to
blue in the sea-water in from 9 to 12 minutes, whereas a portion of
the same litmus paper thrust into the central stomach cavity of
Cassiopea will not become blue until it has remained in the stomach
for 6 to g hours. ‘The whole surface and all of the tissues of the
Medusa are almost neutral and much less alkaline than is the sea-water.
The stomach cavity may be filled with sea-water charged with CO,:
or we may place crystals of K,SO, within it, and yet little or no effect
will be produced upon the movements of the Medusa, although, as we
shall see, these substances produce a profound effect if applied to the
sub-umbrella surface. Remarkably little CO,is given off by the
Medusz in metabolism. A large Medusa was confined for 12 hours
in a small quantity of sea-water tinged pink by rosolic acid, and the
decoloration of the fluid was barely perceptible.

Cassiopea pulsates regularly and at its usual daylight rate through-
out the night, and even red light has no apparent effect upon its rate
of movement. If long confined in absolute darkness, however, the
rate of pulsation becomes slower, and the plant cells within the tissues
of the Medusa become shriveled and greatly reduced in number, so
that the Medusa becomes pale blue in color and translucent. Only
the filaments of the mouth-arms retain their greenish color. (Pl. 1,
fig. B.) The whole color of the Medusa becomes lighter and more
uniform than the normal, as will be seen upon comparing figures A
and B of plate 11. Two Medusz of Casstopea xamachana were main-
tained in absolute darkness and without food for one month. When
first placed in the dark their diameters were 82 and 42 mm., and their
rates of pulsation 40 to 50 and 51 to 54 per minute, respectively. At
the end of one month the large Medusa had shrunken so as to be but
58 mm. and the small one 25 mm. in diameter, and their rates of pul-
sation 23 and 17 per minute, respectively. On their being removed
to the diffused daylight of the laboratory, the color remained un-
changed for three weeks, but the diameters of the Medusze continued
to decrease; finally, however, the plant cells in the mid-region of the
sub-umbrella and ex-umbrella became dark brown and densely crowded,
so that these parts of the Medusz were dull brown in color. After
being in the light for one month the large Medusa was only 29 mm.
in diameter, and its rate of pulsation was less than 1 per minute.

On the other hand, when the Medusa is maintained without food in
the light it becomes dark brown in color (pl. 11, fig. ¢), as will be
seen upon comparing its photograph with that of a normally colored

PLATE Il

A. Oral view of a normal, recently captured specimen of Cassiopea xamachana.

B. A specimen which has been maintained for one month in absolute darkness, showing its
pale coloration. The plant cells are much reduced in number.

C. A specimen which has been starved for one month in the light, showing its very dark
brown color,

EFFECTS OF STARVATION, AND OF DECREASED SALINITY. 17

Medusa. The greenish color of the oral filaments disappears, and the
plant cells become shriveled and densely crowded. A Medusa starved
in light is more active and shrinks more rapidly than does one starved
in darkness, and thus it appears that metabolism proceeds more rap-
idly in light than in darkness. For example, a Medusa starved in
diffused daylight had a diameter at the beginning of the experiment
of 78 mm. At the end of 2 months its diameter was 37 mm., and at
the end of 3 months, 21 mm., being still vigorous and pulsating at the
rate of 16 per minute.

These starved Medusz exhibited certain phenomena of degenera-
tion. The mouth-arms became reduced to mere stumps, most of the
mouths closed over, and the oral tentacles and filaments were absorbed
or cast off, so that the oral surfaces of the mouth-arms became quite
smooth and rounded. The marginal lappets of the disk became
blunted, and the dull-white peripheral ring of the ex-umbrella was
much reduced in width. Only immature eggs were found in the
gonads of starving Medusz. It appears remarkable that the first
parts to degenerate are the mouths and mouth-arms, although these
are the most important to the organism if in danger of starvation.
The marginal sense-organs remained normal in size and appearance.

Cassiopea xamachana lives in salt-water lagoons having but limited
communication with the sea, and it is therefore not surprising that it
will survive considerable alterations of salinity. Fresh water (rain-
water) is quickly fatal to the Medusee, for they shrivel rapidly; all
pulsations cease, and even if removed to salt water after less than five
minutes’ exposure to the fresh, recovery is very slow. On the other
hand, if every night and morning we decrease the salt and increase
the fresh water 5 per cent, the Medusz can be brought into a mixture
of 25 per cent sea-water plus 75 per cent fresh water, and still sur-
vive. Their rates of pulsation become successively slower as the salt
water is reduced.

Thus, two Meduse in pure sea-water had rates of pulsation of 20 and
60, respectively ; in 60 per cent sea-water plus 4o percent fresh water,
18 and 18, respectively; in 50 per cent sea-water plus 50 per cent fresh
water, 14 and 18, respectively; in 4o per cent sea-water plus 60 per
cent fresh water, 8 and 4, respectively; in 35 per cent sea-water plus
65 per cent fresh water, 7 and 2, respectively; in 30 per cent sea-water
plus 70 per cent fresh water, 3 and 2, respectively; in 25 per cent sea-
water plus 75 per cent fresh water, 3.

The small Medusa ceased to pulsate in 75 per cent fresh plus 25 per
cent sea-water, and its sub-umbrella surface became insensitive to the
most powerful stimuli, such as a touch of a crystal of KCl or K S0,;
yet when transferred to 50 per cent fresh plus 50 per cent sea water it

18 PULSATION OF JELLYFISHES.

recovered and pulsated at the rate of 11 perminute. The large Medusa,
which pulsated only 3 times per minute in 25 per cent sea-water plus
75 per cent fresh water, revived quickly and pulsated 18 times per
minute in 50 per cent salt plus 50 per cent fresh water.

If instead of mixing the sea-water with distilled water, we employ
a solution of fresh water containing the amounts of potassium and
calcium found in the sea-water, the Medusz do not survive as well
as they would in ordinary brackish water, and their rates of pul-
sation are much slower, as will appear from the following: Three
Medusz in pure sea-water had rates of pulsation of about 60 per
minute ; the same Meduse in 55 per cent sea-water plus 45 per cent
rain-water containing the same amounts of potassium and calcium
as sea-water, pulsated 8 to 14 times per minute; in 45 per cent sea-
water plus 55 per cent rain-water containing the same amounts of
potassium and calcium as sea-water, they pulsated 2 to 9 times per
minute; in 40 per cent sea-water plus 60 per cent rain-water containing
the same amounts of potassium and calcium as sea-water, they pul-
sated 1 to 6 times per minute; in 35 per cent sea-water plus 65 per cent
rain-water containing the same amounts of potassium and calcium as
sea-water, they pulsated 0 to 2 times per minute; in 25 per cent sea-
water plus 75 per cent rain-water containing the same amounts of
potassium and calcium as sea-water, two dead, one pulsated about
once every 10 minutes.

Evidently a uniform reduction of the magnesium, sodium, potassium,
and calcium is less injurious than a reduction of the sodium chloride
and magnesium alone. As Ringer, Loeb, and others have shown,
a balance in the proportions of the constituents of the sea-water is more
important than the presence of any single salt.

As might be expected in Medusee living in shallow lagoons, where
evaporation is great, Cassiopea will withstand a considerable concen-
tration of the salt water; however, Medusz in 100 cc. sea-water plus
1 gram NaCi will survive for 12 hours, but their pulsation becomes
irregular, although on the average of about normal rate. The mouth-
arms, however, are strongly contracted, and the Medusa exhibits
alternate periods of rest and activity in its rhythmical movements.

Casstopea will pulsate normally in sea-water saturated with CaSQ,.

As will be apparent from the above, Casstopea xamachana is one of
the hardiest of Scyphomedusz. It survives for months in aquaria
with but ordinary care, and exhibits wonderful recuperative powers
from the effects of poisons. If subjected to constant shaking, as ina
floating live-car, it does not thrive as well as in stationary aquaria
where the water is not so pure.

NATURE OF THE PULSATION-STIMULUS., 19

THE NERVOUS OR EPITHELIAL NATURE OF THE STIMULUS WHICH
PRODUCES CONTRACTIONS.

If the sub-umbrella be injured by scraping parts of it away, as in
figure 54, I, or if the margin be cut off as in figure 54, III, the removed
parts are soon partially regenerated, as shown in the dotted areas, but
this newly regenerated tissue is at first epithelial in character, and
lacks muscular elements. It therefore can not contract, yet if it be
touched with a crystal of K,SO,, or otherwise stimulated, it trans-
mits the stimulus across itself to the adjacent muscular tissue of the
sub-umbrella, which contracts vigorously, although the newly regener-
ated tissue which conducted the impulse does not itself contract. This

Fig. 5A.—Newly regenerated sub-umbrella tissue which lacks muscular elements,
and can not itself contract, can still transmit the stimulus to pulsate to normal
tissue adjacent to it. In fig. 5A, II, the stimulus crosses areas A and B, which

do not contract, while C, D, and S contract in the order named. In fig. 5A, IV,
the bridge of newly regenerated tissue does not itself contract, but serves nevertheless to transmit the
stimulus causing contraction in E. and F.

can best be demonstrated by making the newly regenerated tissue
serve as a bridge connecting two pieces of uninjured sub-umbrella
tissue, as is shown in figure 54, I, or 5A, Iv. Then, upon touching
figure 5a, 11, at S with a crystal of K,SO, or other stimulant, a con-
traction wave passes from S through B-D-A-C; but Band A, being
newly regenerated tissues without muscular elements, do not contract,
although the stimulus which produces contraction passes across them.
Similarly in figures 54, 1v, if E, which is normal sub-umbrella tissue,
be caused to contract, every contraction is followed by F, although the
bridge of newly regenerated tissue which connects them does not con-
tract. These experiments tend to show that the stimulus which causes
pulsation is transmitted by the epithelial or nervous elements to the
muscular elements, and not primarily by the muscular elements them-
selves. I have examined many specimens of newly regenerated tissue

20 PULSATION OF JELLYFISHES.

which did not itself contract, and yet transmitted the impulse which
produced contraction in muscular tissue attached to it, and there
appear to be no muscular elements in the newly regenerated tissue,
although these often develop later. In these examinations I made
use of intra vitem methylene blue, Retterer’s method, Flemming’s
fluid followed by Ehrlick’s acid hematoxylin, corrosive sublimate
followed by aqueous carmine stain, and Hermann’s fluid, but in no
case could I find muscular elements in sections of the newly regener-
ated tissue which appeared to be a simple columnar epithelium, under-
laid by a thin nervous net-work (see fig. 36). The muscle fibrillze
of the sub-umbrella are striate, and are easily demonstrated by any of
the above methods.*

oe

gl

Figs. A-C.—Cross-sections of the sub-umbrella of Cassiopea,
Fig. D.—Surface view of newly regenerated sub-umbrella
tissue. ect, ectodermal epithelium. G, gelatinous substance of

the disk. M, muscle fibers. S, basal membrane.

Figure A is a cross-section of the normal uninjured sub-umbrella of
Casstopea, cut across the trend of the circular muscle fibers ; while fig-
ure B is a cross-section through regenerated sub-umbrella epithelium
which has grown over an area from which all cellular elements had
been cut away about 40 hours before. This newly regenerated tissue
can not itself contract for, as yet, it lacks muscular elements; but it
will nevertheless transmit the stimulus which produces contraction in

*Hesse (1895) finds that the nerve fibers in the sub-umbrella of Rhzzostoma pulmo
extend in all directions, but are mainly grouped in clusters extending from sense-
organ to sense-organ. Bethe (1903) finds that in Rhzzostoma and Cotylorhiza the
epithelium of the sub-umbrella is connected with the deep-lying muscles by means of
an intermediate plexus of nerve fibers.

THE PULSATION-STIMULUS IS NON-MUSCULAR. aI

muscular tissue adjacent toit. There area few spindle-shaped (gang-
lion?) cells upon the basal membrane at the base of the regenerated
epithelium, and occasionally one sees a large rounded cell in the
gelatinous substance below the basal membrane. Occasionally these
rounded cells have one or more delicate processes which extend into
the gelatinous substance.

Figure C is a somewhat slanting section through regenerating sub-
umbrella tissue about 4 days old, which is beginning to regenerate the
muscle fibers and can now contract feebly. The muscle fibers appear
as elongate processes of deep-lying epithelial cells, and extend par-
allel one with another over the basal membrane, trending circumfer-
entially around the sub-umbrella.

Figure D is a surface view of newly regenerated sub-umbrella epi-
thelium which transmits the pulsation-stimulus, but can not yet con-
tract, as it still lacks muscular elements.

It is well known that Carlson has demonstrated the nervous nature
of the stimulus which produces pulsation in the heart of Limulus.
Indeed, I believe that all of the facts brought to light by Gaskell in
his attempt to prove the muscular nature of the transmission of the
stimulus of pulsation in the vertebrate heart will apply equally well if
we assume that the impulse is transmitted by diffuse nervous elements.
In the heart of the loggerhead turtle I find that the stimulus causing
pulsation is transmitted entirely through the thin outer muscular part
of the wall of the heart, and the thick cavernated inner part of the
heart’s wall may be cut away without affecting the pulsation. Also,
the stimulus to pulsate is not transmitted through this cavernated
tissue to the muscular tissue.

Fig. 5B.—Showing that the sub-um-
brella tissue is a better electrical
conductor than is the gelatinous
substance of the bell. The cur-
rent travels around through the
long way, rather than across the
shallow scratches which insulate

the area B.

The sub-umbrella tissue of Casszopea is a good conductor of electri-
city, while the gelatinous substance of the Medusa is a poor conductor.
Thus in fig. 5 B, if we insulate an annulus by the shallowest possible
scratch through the sub-umbrella, and then isolate a small sector,
B, by shallow radial cuts; on touching the large sector 4 at 1 and 2

ae PULSATION OF JELLYFISHES.

with the electrodes the contraction travels all the distance around 4 ;
but the sector B does not contract. The path of least electrical resist-
ance is evidently through the long strip of sub-umbrella tissue, while
the short path across the cuts interposes a greater resistance.

PULSATION WITHOUT MARGINAL SENSE-ORGANS.

Romanes, Eimer, von Uexkiill, and others, have shown that in Scy-
phomedusz the marginal sense-organs are centers which discharge the
stimuli producing the rhythmical movements of the disk; and that
if we remove these sense-organs, a more or less complete paralysis of
the disk occurs. In some forms, such as Aurelia and Dactylometra,
this paralysis lasts but a few minutes, and then more or less zvvegular
contractions commence. In Rhizostoma pulmo, according to Hargitt,
the paralysis is much more pronounced than in Aurelia. In Cassiopea
xamachana the paralysis is practically complete for at least 24 hours,
the disk responding only to definite stimuli, and very rarely giving
a contraction without evident cause. On the second day after the
operation the disk is much more sensitive to stimuli of all sorts
and gives occasional isolated contractions without apparent stimu-
lation, and at the end of a week the disk can rarely be observed for a
minute without one’s seeing it give a number of quick, isolated contrac-
tions. Regular rhythmical pulsation never sets in, however, unless
the marginal sense-organs be regenerated.

Hitherto, disks without sense-organs have always been maintained
in sustained pulsation by constant artificial stimulation, or by being
placed in more or less injurious stimulating solutions. It will be
recalled that Romanes obtained regular pulsation in the disks of
Aurelia by passing through them a constant, or faradaic, current of
electricity of minimal strength. He thus demonstrated that rhythmi-
cal movements might result from a constant stimulus, and he showed
that one contraction could not follow another until the sub-umbrella
tissue had recovered from the exhaustion caused by the previous
contraction ; then, and then only, can the tissue respond to the ever-
present stimulus. Romanes concluded, therefore, that the ganglia of
the marginal sense-organs may exert a constant stimulus, and yet give
tise to periodic contractions. Romanes also found that the paralyzed
bell of Sarsta could be set into a ‘‘flurried shivering’’ pulsation for
one hour by a solution of 10 to 20 drops of acetic acid in 1000 cc. of
sea-water, and that it would also respond by rhythmic contractions
to a solution of 5 per cent glycerin in sea-water.

In 1900 Loeb found that the paralyzed disk of Gonionemus will
pulsate rhythmically for an hour in a solution of 5¢n NaCl or 54n

PULSATION WITHOUT MARGINAL SENSE-ORGANS. 23

NaBr, but that a small amount of calcium or potassium added to the
Na solution will prevent the disk from pulsating. Loeb concluded
that the calcium and potassium ions of the sea-water prevented the
center of the bell of Gonionemus from pulsating. This is untrue for
Casstopea, for not only will the disk when deprived of sense-organs
pulsate regularly for more than an hour in an artificial sea-water
without calcium, but will also pulsate indefinitely in natural sea-
water, and will contract rhythmically in solutions containing NaCl +
KCl, or NaCl + CaCl,, or NaCl + KCl + CaCl, in amounts and propor-
tions found in sea-water. All solutions containing magnesium tend
to prevent pulsation in the disk of Casszopea.

Asa result of his work upon
the skeletal muscles in 1899 Loeb
concludes that rhythmical con-
tractions occur only in solutions Cs wy
of electrolytes, z. ¢., in com-
pounds capable of ionization,
and that in solutions of non-con-
ductors such as glycerin these
thythmical contractions are im-
possible. However, Romanes
found that glycerin caused rhyth-
mical pulsation in Sarsza.

Greene (1898) and Howell (1901,

p. 189) found that strips of heart

muscle, after having ceased to

pulsate in NaCl, will again pul-

sate if immersed in a pure solu-

tion of cane sugar or dextrose

isotonic with the NaCl solution, Fig. 6.—A disk of Cassiopea pressed by a con-
and I find that the heart of centric series of block-tin rings so as to insulate
Salpa will pulsate normally for circuits of tissue. A disk so pressed may be
more than half-an-hour in dex- caused to pulsate continuously.

trose, isotonic with sea-water (see table 6). Thus automatic beats
may occur in a solution entirely free from electrolytes, but, as How-
ell shows, these beats are probably dependent upon the presence of
electrolytes in the tissue itself.

When we come to consider the effect of ions, etc., upon Casszopea, it
will appear that one must be cautious of drawing general conclusions,
even from the most evident effects upon any one animal. Thus I find
that chemicals which produce certain perfectly definite and invariable
responses upon Casszopea act differently upon Aurelia, Dactylometra,

24 PULSATION OF JELLYFISHES.

Gonionemus, Lepas, Salpa, and the loggerhead turtle. If there be
marked differences between the reactions of closely related Scypho-
medusz, one may expect even greater disparity between those of ver-
tebrates as compared with invertebrates.

Romanes, Loeb, von Uexkiill, Hargitt, and others have caused disks
to pulsate temporarily by subjecting them to the influence of NaCl
solutions, etc., but in all cases more or less toxic effects resulted from
the experiments and the sensibility of the sub-umbrella tissues
became impaired or destroyed, so that further stimulation soon became
impossible, We will now describe a method by which the disk of
Casstopea when deprived of marginal sense-organs may be made to
pulsate indefinitely in sea-water with the production of effects no more
injurious than those of fatigue. This may be most readily accom-

SN

Fig. 7,—Oral view of Cassio-
pea xamachana. Four of
the mouth-arms are cut off,

v )
\¥
NY

>)
rs

NY
ay

WIA LER
WE
Or SARE and the muscle layer of the
Cys y sub-umbrella in the upper

right-hand quadrant removed
to show the underlying vas-
cular system. ab, Mouth-
arm plate; ma, mouth-arm;
ml, muscular system of the
“sab sub-umbrella; us, vascular ca-
nals of the sub-umbrella.

plished by cutting off all marginal sense-organs, and then making a
series of concentric, discontinuous, ring-like cuts through the muscu-
lar tissue of the sub-umbrella, as is shown in figures 8 to 19.* Then
upon stimulating the disk in any manner it instantly springs into rapid
rhythmic pulsation, so regular and ceaseless as to remind one of the
movement of clockwork. The cuts must be so made as to permit
a free passage of contraction waves through sub-umbrella tissue form-
ing a closed circuit. The simplest circuit is, of course, a single ring

* A glance at figure 7 will show that the muscular area of the sub-umbrella is a wide
annulus with the mouth-arm disk and stomach in the center. In figures 8 to 33 we
have represented the disk as a circle, the small concentric circle at the center being
the mouth-arm disk, while the wide annulus is the sub-umbrella.

PULSATION WITHOUT MARGINAL SENSE-ORGANS. 25

(annulus) of sub-umbrella tissue; and such a ring can readily be set
into sustained pulsation.

It is not necessary, however, that cuts be made through the sub-
umbrella tissue ; for mere pressure prevents the transmission of con-
traction waves across the pressed region, and we may form circuits by
pressing lightly upon the sub-umbrella with a concentric series of
metallic rings, as is shown in figure 6. Then upon stimulating the
disk in any manner it pulsates rhythmically.

Disks which have been cut, or pressed, as described above do not
pulsate until they have been momentarily stimulated at some definite

Figs. 8-19a,—Shapes cut from disks without marginal sense-organs. These will pulsate
continuously in sea-water.

point by a touch of some potassium or sodium salt, a mechanical or
electrical shock, or by suddenly cutting off the last remaining sense-
organ immediately after it has sent out its contraction wave.

A contraction wave travels outward from the stimulated place
through the circuit of sub-umbrella tissue, and when it returns to the
point whence it started it is immediately reinforced, and again sent

26 PULSATION OF JELLYFISHES.

through the circuit. Thus there is normally but one contraction wave
which proceeds from its center, travels through the labyrinth of sub-
umbrella tissue, and returns to the center whence it came, only to be
again augmented and sent forth.

It is thus the function of the center to reinforce and maintain the
contraction wave. This is well shown in a long circuit such as is
shown in figure 30, I-11; where on account of the great length of
the circuit the course of the wave may readily be followed by the eye.
The outer annuli of the sub-umbrella tissue are more sensitive, and
conduct contraction waves * better than do the inner parts of the disk;

Figs. 20, 21, 22, 23, 25, 27a, disks cut so that they can not be set into continuous pulsation.
Figs. 21a, 23a, 24, 26 can be set into sustained pulsation in sea-water.

and if we touch the disk at 4, figure 30, 1, the greater part of the con-
traction wave takes the short path of /east resistance into the interior of
the labyrinth, as is shown by the full arrow, and only a very weak
wave goes in the direction of the dotted arrow. The strong contraction

* The sub-umbrella tissue is a good conductor of electricity, but the gelatinous sub-
stance of the Medusa is a poor conductor.

PULSATION WITHOUT MARGINAL SENSE-ORGANS. 27

wave then proceeds as is shown by the sequence of arrows and num-
bers until it finally returns with lowered amplitude to the center,
where it is instantly restimulated and again sent through the circuit
with its energy restored. The same conditions apply to figures 31,
I and Ir.

When in regular pulsation we always find that the waves of contrac-
tion start from a definite place. The position of this center tends to
bear a certain relation to the geometrical figure formed by the cuts.
It is marked S in figs. 8 to 19a, and the arrows show the observed
courses of the wave of pulsation. Usually the center of pulsation lies
near the periphery of the disk at a place where the tissue is widest
and least interfered with by cuts, and it also tends to lie upon the axis
of bilaterality of the labyrinth of tissue.

If we stimulate the disk by dropping it upon a glass plate, etc.,
the waves of pulsation start from the point S; and this is the place
where we must touch the disk if we wish to stimulate it into sus-
tained pulsation. Wherever we touch the disk with a crystal of
K,SO,, waves of contraction immediately start out from the touched
point, but it is usually impossible to establish a permanent center of

A B

Fig. 28. Fig. 29.

pulsation at any point other than one upon the geometrical axis of
the figure. Centers at other places either cease to initiate pulsations
when the effect of the initial stimulus dies out, or the center
quickly shifts to the geometrical axis. Sometimes, however, when
a disk is stimulated by a severe mechanical shock, two or more per-
manent centers of pulsation appear and waves of contraction start
out from each independently and interfere where the opposing waves
meet one another. Such conditions are shown in figures 14 and 17.

It will be observed that with the exception of the very elongate
spiral (fig. 14) all of the labyrinths formed by the cuts are closed circuits,
the tissue being merely a more or less complicated circuit, with the
center of pulsation at the geometrical center of the figure. A/ter the
disk has begun to pulsate we may cut away portions of the laby-
rinth, and the part containing the center will still pulsate, provided it
remains a closed circuit. Thus the crescent (figure 18a) is cut out
from figure 18 and the ring (figure 19a) is made from figure 19,

28 PULSATION OF JELLYFISHES.

by cutting them out after the more complicated circuits had been set
into pulsation. Instead of simplifying the pulsating labyrinth, we
may increase its complexity, but as long as the waves proceeding from
the center can find a single uninterrupted circuit, the figure pulsates.
Thus, a disk cut asin figure 28, A, is set into pulsation and then all of
the inner rings are cut so as to be converted into ‘‘cut-off” paths as
in figure 28, B; but the disk continues to pulsate until we cut across
the outermost ring, when it stops instantly. Every one of the forms
shown in figures 8 to 19a can be thus stopped by even the smallest
cut which breaks the last circuit, although they continue to pulsate
despite any cutting which does not sever the circuit. Thus, figure 16
stops at once if we cut across one of the narrow places between the
rays of the star.

The center of pulsation usually establishes itself in a large uncut
area, but once it be established we may greatly cut down this area
and not interfere with the center. Thus, the ring shown in figure 19a
may be thinned by cutting at S, but the center remains undisturbed.

Fig. 30.—Very elongate circuits showing that the peripheral parts are better conductors of pul-
sation than are the inner parts of he sub-umbrella. These circuits can be caused to pulsate
continuously.

Sustained pulsation without marginal sense-organs can be main-
tained only in tissue forming a closed circuit. These circuits may be
complex and constricted at intervals to mere thread-like connectives,
as in figures 31, A-c, where every annulus is crossed by radial cuts ; or
they may be very simple, as in figure 31, D. The circuits may either
cross or trend with the muscle fibers.*

On two occasions disks were set into sustained pulsation when only
the marginal sense-organs were cut away; uo other cuts having been

*The statement in my preliminary paper in the Carnegie Institution Year Book for
1905 that the circuits must trend with the muscle fibers is erroneous.

PULSATION WITHOUT MARGINAL SENSE-ORGANS. 29

made. This can rarely be accomplished, however, for the returning
wave must usually be focused back upon the center in order to be
sustained ; and in a wide annulus it is dissipated and returns with
too little force to call forth the latent ability of the center to restimu-
late the wave. Similarly figures 20, 21, 22, 23, and 25 represent forms
which dissipate and confuse the contraction wave, setting up ‘‘ eddy
currents’? which weaken the wave and prevent its returning definitely

A
Cc D
Fig. 31.—A, B, and C, disks having every annulus crossed by radial cuts, but which may

be set into sustained pulsation. D, a simple circuit which may be set into sustained
pulsation.

and forcefully to the center. Hence these figures can not be set into
sustained pulsation. If, however, we cut partial rings, as in figures
21a and 23a, or convert figure 21 into a shape such as is shown in
figure 24, we find no difficulty in setting them into sustained pulsa-
tion. In all of these cases the figures oblige the contraction wave to
return definitely and forcefully to the center. I could not obtain sus-
tained pulsation in a disk cut out of the side of a Medusa as in figures

30 PULSATION OF JELLYFISHES.

27, 27a. This, I believe, is due to the fact that the contraction
wave returns so quickly to the center that an insufficient time elapses
before the center is again called upon to restimulate the wave. As
Romanes showed, an appreciable interval of time must elapse before
tissue which has been in contraction can again contract.

Very elongate, many-whorled spirals, such as one sees in figure 14,
are the only forms zo/ closed circuits that we have succeeded in set-
ting into constant pulsation. This occurs only when two or more
centers arise simultaneously in the spiral, as in S, S’, and S”, figure 14.
These centers mutually sustain one another, the contraction wave from
one being restimulated and reflected back from the other. If one
attempts to convert a series of partial rings (fig. 32, A) into a spiral
by successive cuts, as shown in the dotted lines, 1-5, (fig. 32, B) the
tissue ceases to pulsate as soon as the final cut (5) is made which
breaks the last circuit.

A B
Fig. 32.— Showing that sub-umbrella tissue can not maintain itself in pul-
sation unless it has the shape of a closed circuit. If cuts be made as
shown in the dotted lines in the order 1, 2, 3, 4, 5, the tissue ceases to
pulsate as soon as cut number 5 breaks the last complete circuit.

It must be borne in mind that cuts through the sub-umbrella tissue
heal over in the course of a day or two and will then transmit pulsa-
tion more or less imperfectly across the healed lines, and then a spiral
will pulsate, for it is, physiologically speaking, only a series of concen-
tric rings of readily conducting tissue with numerous more or less
imperfect points of conduction between the annuli. Similarly a disk
having complede circular cuts through the muscular tissue of the sub-
umbrella, such as is shown in figure 26, can not be made to pulsate
continuously as a whole until two or three days after the operation,
although each annulus may be made to pulsate independently. After
several days of healing the cuts will allow a more or less imperfect
conduction of impulses across from one ring to another, and the con-

PULSATION WITHOUT MARGINAL SENSE-ORGANS. 31

traction waves will be unimpeded circumferentially, but more or less
hindered radially. That this is the true explanation of the matter is
proven by the fact that the disk shown in figure 12, wherein the
circumferential cuts are numerous and the spaces between are as wide
as the cuts are long, will pulsate continuously.

Mere mutilation of a disk without sense-organs will not cause it to
become capable of continuous pulsation. Thus the disk shown in
figure 20, having about 800 punctures made through its sub-umbrella
tissue, can not be set into a sustained rhythm.

Although I had several hundred paralyzed disks of Casszopea capa-
ble of being set into pulsation by a stimulus, such as a momentary
touch of acrystal of K,SQO,, only one of these started into pulsation
of its ‘‘own accord.’’ Ordinarily they might remain for days in the
aquaria awaiting the momentary stimulus which alone could call forth
their latent power of rhythmical pulsation.

If disks without marginal sense-organs be set into rhythmical pul-
sation they move with machine-like regularity, without pauses, and
without any of the irregularities shown by normal Medusz with sense-
organs intact, Their rates of pulsation are not only practically uni-
form, but they are much faster than are those of the uninjured normal
Medusze from which the disks were prepared, as will be shown by the
following table:

TABLE 2.—Rate at which normal Meduse of Cassiopea pulsated and the rates of
pulsation of their disks when the sense-organs were excised and circumferen-
tial cuts were made in the sub-umbrella.

ee of . Figure
pulsation Oo Rate of pulsation of disk showing the
Kee ama Sathout sense-organs. Senne
operation. the disk.
25-30 77-88 8
32 63-78 9
40 183 bee
51 85 13
Outermost centerS .. 117
15-20 { Mid-region center S’. rox I4
Innermost center S’’. 78
Outermost center. ..80-82
47 { Inner center ...... 66-68 t 17

When disks without sense-organs are set into pulsation we may re-
duce the area of pulsating tissue by cutting parts of it away, but the
rate of pulsation will remain constant, provided we do not alter the
length of the circuit through which the wave must pass. If, however,
we make cuts in such manner as to increase the length of the circuit

32 PULSATION OF JELLYFISHES.

the rate of pulsation becomes slower. For example, twenty disks
were cut as shown in figure 33, A, and after they had been set into pul-
sation they were cut across as shown in figure 33, B. This cut made
the circuit twice as long as it was formerly, and obliged the contrac-
tion wave to travel double the distance in order to traverse the circuit.

©) ©)
| © O

Fig. 33.—Showing how cuts may be made so as to increase the length of
the pulsating circuit, thereby decreasing its rate of pulsation.
We might then expect the pulsation to be reduced to one-half its former
rate, but as a matter of fact the wave traveled on an average 1.16
times as fast in the long as it did in the short circuit, so that the cut
reduced the rate to but 58 per cent of its former value.

Similarly, if we set disks cut as shown in figure 33, Cc, into pulsation,
and then make two cuts as shown in figure 33, D, making the circuit
almost three times as long as it was before, the rate becomes about
0.4 of its former value, not 0.33 as we would expect. I believe that
the faster rate of the contraction wave in the long circuit is due to the
longer rest which the tissue enjoys, thus allowing it the more com-
pletely to recover and regain its sensibility to the stimulus which
calls forth the contraction. Romanes showed that strong contraction

SENSE-ORGANS AND PULSATION. 33

waves travel faster than weak ones, and that strong stimuli repeated
at short intervals soon tired the tissue, so that it failed to respond.

The rate of pulsation of disks is greater than their most excited
tate when the sense-organs are intact; in other words, the disk itself
can maintain pulsation at a faster rate than can the marginal sense-
organs. The rate of pulsation in the disks deprived of sense-organs
depends simply upon the time required for the waves to traverse the
circuit and restimulate the center. The wave travels faster through
peripheral than through the inner annuli of the disk. When pulsating
disks are suddenly seized, moved, or otherwise stimulated, the ampli-
tude of their rhythmical movement suddenly increases, but the vate
remains practically the same, and thus the presence of the marginal
sense-organs is not necessary for the display of excitement. The disks
of small Medusz pulsate at a faster rate than do those of large ones,
other things being equal.

These pulsating disks may continue to give regular rhythmical con-
tractions in sea-water for 140 hours or more, but at the end of that
time, if they have been deprived of their mouth-arms and central
stomach, they become exhausted, and the amplitude of their pulsa-
tion decreases, although the vaze remains practically constant. Sud-
denly the center fails to restimulate the returning wave, all movement
ceases, and the disk can not be re-stimulated until after a period of
rest. Indeed, the tissue appears much exhausted and responds feebly
even to the strongest stimuli, such as K,SO,, KCl, etc. Complete
recovery takes place, however, in normal sea-water, so that disks may
be maintained in condition to pulsate for weeks.

While in sea-water it is almost impossible to set a Medusa, with mar-
ginal sense-organs intact, into any form of pulsation other than that
controlled by the sense-organs. If, however, we cut partial rings in
the sub-umbrella of a Casstopea, leaving the sense-organs and margin
intact, and then place the Medusa in a solution resembling sea-water
but lacking calcium,* all pulsations will cease in from 2 to 6 minutes.
Then, after the Medusa has remained motionless in the solution for
one hour, if we touch the disk for an instant with a crystal of K,SO,
it immediately springs into a rapid rhythmical pulsation at a much
faster rate than that previously maintained by the sense-organs. This
pulsation, indeed, exhibits all of the features shown by disks with-
out sense-organs, and therefore we see that the absence of calcium has

*965 HeO + 26.74 NaCl + 3.75 MgCle + 1.64 MgSOu + 0.85 KeSO,-+ 0.07 MgBr,
or Van 't Hoff’s solution consisting of 100 NaCl+ 2.2 KC1+ 7.8 MgCle + 3.8
MgS0O,, all of 56n concentration.

34 PULSATION OF JELLYFISHES.

caused a paralysis of the marginal sense-organs, but not of ‘the sub-
umbrella tissue of the dzsk.

This we can prove directly, for disks without sense-organs, once
they be set into pulsation, will continue to pulsate for over three hours
in a solution resembling sea-water but lacking calcium. The amplitude
of their pulsations, however, decreases steadily, but may be 7vestored
by adding calcium to the solution. It is evident that the central parts
of the sub-umbrella of Casstopea may pulsate both in normal sea-water,
and for a long time in sea-water deprived of calcium, whereas the mar-
ginal sense-organs are quickly paralyzed by a deficiency of calcium
in the sea-water. On the other hand, perfect Medusz and disks
deprived of sense-organs will pulsate in sea-water at 82° F. containing
CaSO, + CaCO; to saturation, the only effect being a slight slowing
of the rate of pulsation in the case of the perfect Medusze. Hence the
marginal sense-organs require calcium* to perform their function,
whereas the general tissue of the sub-umbrella is relatively unaffected
by the presence or absence of calcium. This is, however, a relative
matter, for while the lack of calcium produces less effect upon the disk
than upon the sense-organs, nevertheless the disk itself will jizally
cease to pulsate in the absence of calcium. It is interesting to observe
that while the disk is almost unaffected by a wide range in the amount
of calcium in the sea-water, it is very quickly affected by a change in
the amount of the potassium. Such disks cease to pulsate in a few
minutes either in a solution resembling sea-water but /acking potassium
ot in a solution of 4% gram K,SO, in 100 c.c. of natural sea-water.
Indeed, the center of the disk is fully as sensitive to changes in the
amount of potassium in the water as is the entire Medusa.

Under normal conditions pulsation is controlled by the marginal
sense-organs, the rate being that of the fastest working sense-organ.
The general sub-umbrella surface has considerable influence in sus-
taining the sense-organs, for if we reduce the area of the sub-umbrella
enervated by the sense-organs the rate declines. Normally the pul-
sation is controlled by the sense-organs, not by centers of pulsation in
the undifferentiated sub-umbrella tissue. Among thousands of nor-
mal Meduse I observed only two individuals in which a center in the
sub-umbrella controlled the pulsation. These two were pulsating
slowly when I lifted them out of water and threw them forcibly back.
They instantly began to pulsate in the rapid, uniform, clockwork-like
manner characteristic of pulsation maintained by a center in the sub-
umbrella, their rates being fully four times as great as the normal. I
then cut off their marginal sense-organs, and the disks still continued

“The chief réle of calcium is to counteract the anesthetic effects of magnesium.

SENSE-ORGANS AND PULSATION. 35

to pulsate without alteration in their rates. They both ceased in-
stantly as soon as a radial cut was completed from center to margin,
thus breaking the circuit of the waves of contraction.

We have seen that a center of pulsation in the undifferentiated sub-
umbrella tissue sends out its stimulus only when the contraction wave
teturns to it through the circuit, and that therefore the rate must be
constant, for it depends only upon the length of the circuit and the
rapidity of the wave; and no pulsation can be maintained by a center
in the sub-umbrella tissue unless the contraction wave can pass through
a circuit and finally travel back to restimulate the center.

The marginal sense-organs behave differently. They send forth the
stimulus, which produces contraction, at a slow, irregular rate, and
they are not restimulated into immediate action by a returning wave,
and can maintain tissue in pulsation even if its shape is not that of a
closed circuit. They function only when calcium is present in solu-
tion in the sea-water, and if lifted out of water and dried with blotting
paper they cease in a few minutes to initiate pulsations; but if then
they be moistened with distilled water containing the amount of cal-
cium found in sea-water, they recommence pulsation. Indeed, the
sense-organs behave as if a slow chemical change takes place within
them, the result being a contraction-stimulus; and this state of con-
traction in turn reducing the built-up compounds to their original
condition. Calcium has the peculiar power to offset the stupefying
influence of the magnesium of the sea-water, but calcium is of primary
importance only when magnesium is present. If magnesium be absent
the presence of calcium is relatively unimportant in the pulsation of
Cassiopea. Indeed, the Medusa pulsates longer and faster in a solution
containing the amounts and proportions of NaCl + KCL, found in sea-
water than it does in NaCl + CaCl).

Before closing the account of these experiments upon disks it should
be stated that the disks of Aurelia flavidula and Dactylometra quin-
quecirra may also be set into sustained and regular rhythm by cutting
partial rings, as has been described in the case of Cassiopea. These
Scyphomedusz, however, soon recover to some extent from the loss
of their marginal sense-organs, and the chief difference between their
usual behavior after the loss of the margin and their behavior when
cut by partial rings and then set into pulsation is that in the latter
case the pulsation is of machine-like regularity and without pauses,
whereas under normal conditions itis irregular. Dactylometra is more
favorable for these experiments than Aurelia, for Aurelia is extremely
sensitive to mechanical shocks and to chemical stimuli. It is of
interest to observe that the rate at which the tissues of the disk of

36 PULSATION OF JELLYFISHES.

Dactylometra maintain these pulsations is only a little higher than that
maintained by its marginal sense-organs. For example, a Dactylo-
metra which pulsated 39 times per minute when intact pulsated 46
times per minute with perfect regularity when all sense-organs were
removed and partial rings were cut in its sub-umbrella.

It will be recalled that Romanes briefly mentions a specimen of the
hydromedusa Staurophora laciniata, in which there were three centers
of spontaneous contractions after the bell margin was removed. I have
not succeeded in causing the disk of Gonionemus to pulsate contin-
uously by cutting partial rings in its sub-umbrella after the margin
had been removed. There were, however, but a few small specimens
at my disposal. As Yerkes found, the central disk of Gonionemus,
when deprived of its margin, often gives isolated contractions without
external stimulation.

III. REACTIONS OF CASSIOPEA TO CHEMICAL STIMULI.
CHEMICAL STIMULATION OF PARALYZED DISKS.

As we have seen, the loss of the marginal sense-organs paralyzes
the disk of Casszopea, but it still reacts strongly by contractions if the
surface of its sub-umbrella be touched by certain substances, while
others have no effect upon it.

Strong solutions or crystals of the following produce contractions:
KA1(SO,)2, KBr, KCN, K,CO;, KCl, KC1O;, K,CrO,, K,Cr.O;,
K,Fe,C},N1;.6H,O, KI, KMnO,, KNO;, KOH, KHSO,, K,SO,,
K,S,0,; also Na,CO;, NaHCO;, NaCl, NaClO;, Na,HPO,12H.0O,
NaNO;, NaOH, NaSO37H,O, Na,SO,10H,O, and sodium oxalate;
also LiCl, BaCl,2H,O, BaSO,, Ba(OH),, NH,OH, glycerin, dextrose,
CuSO,, Fe,Cl,, PtCl,, and iodine, etc. Contractions are also produced
by very weak solutions of the following acids: Acetic, chromic, oxalic,
sulphuric, hydrochloric, picric, nitric, and formic. This effect is doubt-
less due to hydrogen, the only element common to all of these acids.

The following substances produce no contractions, even when the
crystals themselves, or their saturated solutions, are applied to the
surface of the sub-umbrella: MgBr, MgCl,, MgCO;, MgSO,; also
CaCO;, CaCl,, CaO, CaSO,, and SrCO;, SrCl,6H,O, SrSO,, HgCl,,
FeSO,7H,0, CH,N,0.

Summarizing the above, we see that allsalts of potassium, sodium,
lithium, barium, and platinum produce contractions, as do also weak
solutions of acids, glycerin, dextrose, ammonia, and iodine. By far
the strongest contractions are produced by potassium salts, while
sodium salts produce much weaker effects. Nevertheless the NaCl
of sea-water is a more powerful stimulant than the potassium (K,SO,

REACTION TO CHEMICALS. ay

or KCl), owing to its far greater amount. The salts of calcium, mag-
nesium, and strontium do not stimulate the disk and fail to produce
contractions, even when in saturated solutions.

Combinations of Mg or Ca with Na or K may or may not give con-
tractions, for the Mg always, and Ca in some cases,* tends to inhibit
pulsation. Thus a series of contractions are produced by 5K,So,.-
Na,SO,, Na,SO,.8K,SO,, MgCl,.2KC1.6H,O, K,Mg(SO,)., Na,Mg-
(SO,).4H,0, K,Ca(SO,).2H,0.MgSO,, MgCl,K,SO,6H,O, and Mg-
Cl,.NaCl.2H,O; the first named giving powerful and the last weak
contractions. On the other hand, Ca,.K,Mg(SO,). and CaCl,.2MgCl,.-
12H,0 give no contractions. The salts act in accordance with their
mass-effects. It is interesting that solutions of the ashes of the
Medusa will not produce contractions, although Merunowicz (1875)
found that an aqueous solution of the ashes of the blood will stimu-
late the vertebrate heart into action.

Loeb (1905) states that Ba, Li, Na, Rb, Cs, F, Cl, Br, and I are capa-
ble of bringing about contractions in skeletal muscles; whereas K,
Mg, Ca, Sr, Mn, and Co give rise to no contractions or inhibit them.

It is evident that the stimulating effects of the electrolytes are gen-
erally due to their cations rather than to their anions, but contrac-
tions may also be produced by substances which can not be ionized,
such as glycerin and dextrose, and weak contractions are sometimes
produced by CaBr,, the effect being due to the bromine. It will be
recalled that Greene (1899) and Howell (1901) also found that heart
muscle will pulsate in pure solutions of cane sugar and dextrose,
and I find that the heart of Sa/pa and the branchial arms of Lepas
will also pulsate in dextrose or glycerin. In his former papers Loeb
maintained that rhythmic pulsation was impossible in non-ionizable
solutions, but his views appear to have changed upon this point.

EFFECTS OF CALCIUM IN RESTORING PULSATION.

We have the well-known experiment of Howell (1898) and others
showing that when heart muscle has ceased to beat in Ringer’s solu-
tion it may be made to beat again for a short time by adding any cal-
cium salt. This is also true for Casszopea, for the Medusa will pulsate
for a short time in any solution containing Na and K in amounts found
in sea-water, and then after all pulsations have ceased they can be
revived by adding calcium. This is illustrated in the following list of
trials (table 3), wherein if the sodium chloride was replaced by any

* Taken alone calcium inhibits or fails to stimulate pulsation, but in combination
with sodium chloride and potassium, as in NaCl + KCl + CaCls, it becomes a power-
ful stimulant.

38 PULSATION OF JELLYFISHES.

other salt this was made isotonic with the NaCl of sea-water. The
potassium was so introduced as always to give the same amount of the
element (K) as is found in sea-water.

TaBLE 3.—How calcium revives rhythmical pulsation in Cassiopea after all
movement has ceased in solutions contatuing Na or Li, isotonic with the NaCl
of sea-water, and potassium in the same amount as is found in sea-water.

ak scone ¥ eduse Were restored to Poona s by
n from sea- A the addition of any of the
water and placed They ceased to pulsate in— following calcium salts, tried
in— separately.
NaCl+ KCl......... About 120 JOIQUUES cess renenenuncaavereenss CaCle, CaSOg.
NaCl + KeSOq....... 20 to 30 minutes, Very rapid pulsation | CaSOq, CaCOs, CaClz,or CaH2Og.
at first, followed by periods of rest and
activity.
NaCl+-EKe2C0g....... 12to18 minutes. Pulsation not so rapid | Very active pulsation restor-
as in NaCl + KeSO4 7 ed by CaSO4,CaCle,or CaH2Og.
NaCl+KCl03....... 4 to 10 minutes, Pulsation not very | CaSQq, CaClz, or CaH2Oe.
rapid at first, Slower than in NaCl +
KeCO3 |
NagCO3+ KeCOs....] 3 to a minutes. Pulsation slow and | CaCQs revived weakly.
weak.
NaNO3+ KNO3..... Less thani minute. Pulsation rapid at | CaCle or CaH2O0g. Some of the
first. Medusz did not revive pul-
. sation,
P(e es ol ot Meee 1to6minutes. Pulsated slowly at first. | CaClo. All three Medusz
revived weakly.

Table 3 shows that Cassopea pulsates longer and more rapidly in
a solution of NaCl+KCl than in any other solution named in the
above table. Also, sodium and potassium zzévates are more injurious
than a solution in which the sodium is replaced by an isotonic amount
of lithium. Evidently the axons as well as the cations of the salts
have a decided influence upon the rhythmical movement. This is
also shown by the fact that Meduse pulsate longer and with greater
regularity of movement in NaCl + K,SO, + CaSO, + CaCO; than
they do if we omit the CaCO, and replace it by an equivalent amount
of CaSO,. It will be recalled that Rogers (1905, p. 249) found that
the addition of small amounts of Na,CO; or NaOH to solutions
have a beneficial effect in maintaining the rhythm of the crab’s heart,
and he attributes this effect to the neutralization of small amounts
of free acid in the solutions. Ammonia, KOH, or NaOH in small
amounts have, however, little effect upon the rhythm of Cassiopea,
but if the sea-water be rendered almost neutral by HCl (it is nor-
mally decidedly alkaline at Tortugas) the pulsations of the Medusz
lose energy, and finally the rate declines, and movements, although
regular, are feeble and slow, Thus the rates of three Medusze declined
in six hours from 37-50 to 13-17 per minute, due to the effect of a
minute quantity of HCl in the sea-water, causing it to become almost
neutral, but still alkaline to litmus test. It seems improbable, how-

EFFECTS OF SODIUM AND CALCIUM. 39

ever, that the addition of CaCO;, which improves the regularity of
pulsation of Medusze in NaCl + K,SO,, has only the effect of neutral-
izing acids. Distilled water and the purest obtainable salts were used
in making solutions and there is no reason to suppose that there were
any more free acids in the solutions than in the natural sea-water itself.

Physiologists have generally assumed (see Howell, Text-Book of
Physiology, p 502) that the chief réle of sodium chloride in pulsa-
tion is to maintain the osmotic pressure of the solution. I find, how-
ever, that Cassiopea pulsates more than 24 minutes in a solution of
Na,SO, containing the same amount and proportion of Na as is found
in sea-water; whereas it will not pulsate more than 14 minutes in a
solution of Na,SO, isotonic with sea-water. This would lead one to
believe that the sodium of the sea-water exerts a specific action, and
that the salts have a specific chemical effect independent of their
osmotic action. Indeed, the various salts of sodium behave very
differently ; for example, Casszopea pulsates less than 1 minute in
Na,CO;, 11 to 12 minutes in NaClO;, and more than half an hour in
NaCl, or NaNO; isotonic with sea-water.

When pulsations have ceased in 96 c.c. H,O + 2.7 grams NaCl +
0.085 gram K,SO, they may be revived temporarily by Na,CO;, more
NaCl, KCl, K,CO;, or weak acids. These cause only a few irregular
contractions, however, and are quite different in their effects from
the long, steady revival of pulsation upon the addition of calcium.
Potassium is, however, capable of reviving temporary pulsation in
any solution which lacks magnesium, but if magnesium be present it
can not usually revive pulsation. It is interesting to observe that
after Medusz have ceased to pulsate in the NaCl+K,.SO, and have
been revived by potassium, they will not again pulsate upon the addi-
tion of calcium to the solution. On the other hand, if pulsations have
ceased and have been revived by adding more sodium, they can be
revived a second time by adding calcium. Potassium in excess at
first stimulates the disk powerfully, but soon it poisons the tissues and
inhibits the sensibility, while calcium is not a stimulant, but is neces-
sary for pulsation in connection with sodium and potassium. The
chief réle of calcium is, however, to counteract the inhibiting effect
of the magnesium.

‘This is shown by the fact that if we were to place Casstofea in nor-
mal sea-water, and then add sufficient sodium oxalate to precipitate
the calcium, pulsation ceases in less than five minutes, but is quickly
restored if we place the Medusa in NaCl + KCl + sodium oxalate, or
in NaCl +KCl. Pulsation is not restored, however, if we place the
Medusa in NaCl + magnesium. These experiments prove that the

40 PULSATION OF JELLYFISHES.

pulsation is inhibited by the magnesium of the sea-water, not merely by
the loss of calcium; for pulsation may be restored in solutions which
lack calcium. They also show that when calcium is present the
magnesium does not inhibit pulsation.

EFFECTS OF MAGNESIUM UPON PULSATION.

The magnesium salts in sea-water retard pulsation in Casszopea, and
reduce its rate, amplitude, and energy. Cassiopea pulsates at about
twice its normal rate in a solution resembling sea-water but lacking
magnesium, but if we add the magnesium to this solution the Medusa
immediately pulsates at normal rates. Also, an excess of magnesium
added to sea-water causes the rate and energy of pulsation to decline,
although Medusz will tolerate 1.6 grams MgCl, in 100 c.c. sea-water,
and will pulsate slowly for half an hour without the least apparent
injury, their normal rate being regained in a few minutes after they
are returned to pure sea-water. Magnesium acts only as a restrainer,
never stimulating the disk of Casstopea. When the disk, deprived of
marginal sense-organs, is placed in a solution of MgCl, or MgSO,
isotonic with sea-water it does not pulsate. Indeed, the rate of pulsa-
tion of normal Medusz in natural sea-water becomes successively
slower as we add more and more magnesium.

The réle of magnesium is, however, an essential one in pulsation,
for it counteracts the strongly stimulating action of the combination
of NaCl, K, and Ca which occurs in Ringer’s solutions, or in sea-water.
For example, if we place Casszopea in a solution of NaCl+KCl+CaCl,
in amounts and proportions found in sea-water * the Medusa is highly
stimulated and pulsates at fully twice its normal rate. If now we pre-
cipitate the magnesium in its tissues in any manner, the stimulating
effect of the sodium, potassium, and calcium is unchecked, and after a
short period of violent pulsation the Medusa passes into a strong sus-
tained tetanus and remains motionless, with its bell highly contracted.

I find also that sustained pulsation is impossible in the heart of
Salpa or the branchial arms of Zepas unless magnesium be present,
and that in these cases also NaCl+KCl+CaCl, is a powerful stimu-
lant, producing rapid but not permanently sustained pulsation, but
normal sustained pulsation is attained on the addition of magnesium.
It appears, therefore, that a Ringer’s solution is not an inorganic food
for the pulsating organ, as has been commonly assumed by physiol-

*100 NaCl + 2.2 KCl + 3 CaCl, all of 36n concentration, as in Van 't Hoff’s solu-
tion.

+The magnesium may be precipitated by a small amount of Ba(OH)z, KOH, NaOH,
or sodium phosphate + ammonia + ammonium chloride, etc.

ANESTHETIC EFFECTS OF MAGNESIUM. 41

ogists, but is only a stimulant which in the end produces injurious
effects by the withdrawal of magnesium through osmosis. It can not
sustain permanent pulsation unless a certain proportion of magnesium
be present to preserve a balance.

It is interesting to see that Meltzer and Auer (1905-’06) find that
magnesium affects the nervous system in such manner as to produce
in mammals a deep anesthesia, with relaxation of all the voluntary
muscles. It is inhibitory, never stimulating in its effects, but it does
not interfere with the trigeminal reflex inhibition of respiration. Also,
Carlson (1906) finds that magnesium and calcium depress the gan-
glionic rhythm of the heart of Zzmulus without primary stimulation.
Indeed, the anesthetic effects of magnesium salts upon aquatic animals
have been known since Tullberg’s researches in 1892.

Macallum (1903) finds that there is about 10 per cent less magnesium
in the bodies of Cyanea and Aurelia than in sea-water. Rogers (1905),
however, found that the optimum solution for the continuance of
rhythmic movement of the crab’s heart contains fully as much magne-
sium as the sea-water.

Loeb (1906) finds that in Polyorchis the NaCl + KCl + CaCl, of sea-
water produce sustained contraction without pulsation, and that
magnesium is necessary in order to overcome the tetanus and permit
of rhythmical pulsation. Also, this effect of magnesium can be inhib-
ited by the addition of an equivalent amount of calcium or potassium.
Also, Romanes (1885) found that the vigor of the swimming move-
ments of Savsza is impaired in a pure NaCl solution of the same
strength as that of the sodium chloride in sea-water, but that this vigor
of movement is somewhat restored by adding MgSO, to the same amount
found in sea-water. In the case of Cassiofea all movement would
cease in less than six minutes in NaCl+ MgSO, in amounts found in
sea-water ; whereas irregular pulsation continues for half an hour in
NaCl alone, although after that the Medusze would show periods of
quiescence alternating with periods of pulsation. I find also that
I per cent magnesium added to sea-water slowly lowers the rate of
the rhythmical movement of the arms of Zepas. It seems probable,
therefore, that magnesium, while always inhibitory, plays a somewhat
different rdle in the efficiency of its control over rhythmical movement
in various animals.

EFFECTS OF POTASSIUM UPON PULSATION.

Potassium in small amounts temporarily stimulates and then retards
pulsation. Unlike magnesium or calcium in excess, it is quite poi-
sonous. All potassium salts, with the exception of those consisting

42 PULSATION OF JELLYFISHES.

of combinations of potassium with magnesium and calcium, are pow-
erful stimulants to the disk of Casszopea, causing strong but temporary
contractions. Repeated touches of a crystal of K,SO, to any one spot
on the sub-umbrella of Casszopea soon renders the place insensitive to
further stimulation of any sort. For example, a single spot upon a
disk, deprived of sense-organs, was touched 17 times, in rapid succes-
sion, with a crystal of K,SO, and each time a contraction resulted.
The next 2 touches, however, gave no contractions; then followed 2
touches with contractions, 7 without contractions, 1 with, and finally
11 without contractions, etc.

If normal Cassiopea with sense-organs intact be placed in sea-water
+0.125 to 1.55 per cent K,SO,, KC1O;, KCl, or K,CO; they immedi-
ately pulsate at an abnormally high rate, but the movement soon
loses force, and the disk comes to rest expanded with the mouth-arms
strongly contracted. Medusz in 0.125 per cent excess of K,SO, will
pulsate quickly at first and then more and more slowly, so that at
the end of 13 hours their rates are only about half the normal rate in
sea-water. On the other hand, Medusz in sea-water+1.55 per cent
K,SO, will pulsate with great activity for a few moments, but will
cease all movement in less than 4 minutes. Also, a solution of K,SO,
isotonic with the NaCl of sea-water az once reduces the rate of pulsa-
tion of normal Medusze and quickly brings them to rest without an
initial display of excitement. It appears that a small excess of potas-
sium acts as a temporary stimulus, whereas a large excess at once
inhibits pulsation. It is possible that the initial stimulation is due
to the physiological reaction of the tissues against the injurious
effects of the potassium. ‘Temporary activity is commonly called
forth in animals by sudden injurious stimuli. In this connection it
is interesting to see that Carlson (1906) finds that potassium is a
primary stimulant for the heart of Lzmulus, but its action is quickly
followed by depression.

An excess of I per cent potassium in the sea-water quickly lowers
the rate of movement of the arms of Lefas, causes tetanus-like con-
traction, and may be fatal in 10 minutes.

The effect of potassium upon the disk without marginal sense-
organs is, however, different from its effect upon the normal, perfect
Cassiopea, for disks without sense-organs are actively stimulated into
pulsation for a short time in all excess of potassium from sea-water +
0.25 per cent K,SO, to a pure solution of K,SO,, or KOl, isotonic
with the NaCl of sea-water. Perfect Medusz, however, show no
increase in rate of pulsation in isotonic K,SO,, but steadily decline.
It seems probable, therefore, that a strong excess of potassium impairs

EFFECTS OF POTASSIUM. 43

the activity of the marginal sense-organs sooner than it affects the
disk itself. The disk without sense-organs will, however, cease to pul-
sate in a solution resembling sea-water but lacking potassium quite
as quickly as will the perfect Medusa. It wouldseem, therefore, that
the sense-organs and the sensory surface of the sub-umbrella are
equally intolerant of a lack of potassium in the sea-water. This is
interesting in view of the fact that the disk without sense-organs is
relatively indifferent to calcium, or magnesium, and will pulsate either
in sea-water saturated with CaSO,, in normal sea-water, or for more
than an hour in a solution resembling sea-water but without calcium.
The Medusa with sense-organs intact, however, ceases to pulsate in
a solution containing all of the elements of sea-water excepting cal-
cium in less than six minutes, but will pulsate in sea-water satu-
rated with CaSO,. It is evident that the accurate balance between the
proportions of calcium, potassium, and sodium insisted upon by Loeb
as being zecessary for the continuance of pulsation need not be main-
tained and yet pulsation may continue. As Howell has pointed out,
marine animals are attuned to the sea-water in which they live, and
any change in its constituents must be expected to affect them more
or less adversely. Loeb’s theory of the influence of ions upon pulsa-
tion, although of fundamental value, unfortunately neglects, in some
measure, to consider the effects of the salts as a whole. As we shall
soon see, however, Cassiopea will pulsate for at least 30 minutes in a
pure 5gn NaCl solution, whereas it is paralyzed in less than a minute
in an isotonic solution of Na,CO3;. Indeed, the various potassium
salts stimulate in different degrees. KI, K,SO,, and KCl are powerful
stimulants, whereas KMnO,, KAI(SO,)., and potassium metabisulphite
produce weak contractions.

Matthews (1905) concludes that valence, as such, either of the anion
or cation, is of secondary or no importance in determining either the
toxic or antitoxic action of the salt.

Loeb (1900) concluded that the potassium and calcium ions of sea-
water prevent the center of the bell of Gonzonemus from pulsating
rhythmically. His experiment, however, does not prove this point,
for he found that the center of the bell of Gonzonemus would pulsate
in 44n NaCl, but not in sea-water; and thus he concluded that the K
and Ca of sea-water inhibited pulsation,* but he neglected to consider

*While this paper was in press Loeb (1906: Journ. Biol. Chemistry, vol. 1, p. 431)
concludes that magmestum and calcium inhibit the center of Gonionemus. In so far
as the effect of magnesium is concerned his view now accords with the researches of
Tullberg (1892), Meltzer and Auer (1905-06), and Mayer (1906) that magnesium is
anesthetic or inhibitory.

44 PULSATION OF JELLYFISHES.

the effects of magnesium. I find, indeed, that the center of the bell
of Gonionemus does occasionally pulsate spontaneously in sea-water,
and always pulsates actively whenever one touches it with a crystal of
KCl or K,SO,. It is not stimulated by the sea-water, but the inhib-
itory effect of the sea-water is probably due to magnesium, not to
potassium or calcium. The center of Gonionemus is strongly stimu-
lated by Na salts, and the reason it pulsates in $n NaCl is that magne-
sium, as well as calcium and potassium, is withdrawn from the tissues
by osmosis by the pure NaCl solution, thus giving a preponderating
influence to the Na, which acts as a stimulant. Indeed, Loeb himself
found that the center of Gonionemus pulsates slowly in 96 c.c. 54n NaCl
+2c.c. 44n KCl +2 c.c. 1°/sn CaCl. I also find that Gonionemus
pulsates slowly but wzthout pauses in a solution resembling sea-water*
but lacking magnesium salts. The characteristic pauses which occur
periodically in the normal pulsation of Gonionemus are thus due to
magnesium. Magnesium fails to stimulate the center of Gonione-
mus, and, indeed, if the center be touched with MgSO, or MgCl, it
deadens the part touched, so that it responds weakly or not at all to
such powerful stimuli as the touch of a crystal of NaCl or K,SOQ,.
The disk of Casstofea deprived of sense-organs behaves exactly as does
Gonionemus, for it does not pulsate spontaneously in sea-water but
does so in 54n NaCl, or in any solution containing NaCl + K or Ca,
but lacking magnesium. If, however, we stimulate it with KCl or
K,SO, it gives some active pulsations in sea-water; or better still, if
we cut partial rings in its sub-umbrella and then stimulate it mechan-
ically by a shock, it pulsates indefinitely in sea-water.

It is significant that the disks of Aurelia and Dactylometra, when
deprived of marginal sense-organs, still pulsate irregularly in sea-
water; and the disks of both of these Scyphomedusz sometimes
respond by weak contractions to MgSO, and MgCl,.+ They therefore
pulsate in sea-water as soon as they recover from the shock-effects
tesulting from loss of their marginal sense-organs, because their
disks are stimulated by everything (Na, K, Mg) in the sea-water,
except the calcium, which, taken singly, exerts only a slight inhibitory
action. In the case of Cassiopea, Gonionemus, and Polyorchis the sea-
water is a balanced fluid. Na stimulates while Mg inhibits pulsation.
Ca in connection with Na and K is necessary to, and stimulates,
pulsation.

* 96 c.c. HeO + 2.7 grams NaCl + 0.124 CaSO, + 0.01 CaCO3 + 0.085 KeSOu.

+ These reactions are so irregular and the Medusz so extremely sensitive to mechan-
ical effects that I am in doubt concerning the validity of this statement. It may be
that the occasional response is due to some chemical shock-effect.

EFFECTS OF CALCIUM. 45

The disk of Casstopea does not pulsate in sea-water, because the
sea-water as a whole does not stimulate it. Disks of Aurelia and
Dactylometra behave in sea-water as if they were weakly stimulated.

Howell (1901, pp. 200, 204) concludes as a result of his own work
and a review of the labors of others that potassium acts somewhat as
an inhibitory agent upon the rhythmical pulsation of the heart muscle
of the ventricle of the terrapin, for it lengthens the period of diastole,
causing the rate to become slower,* but at the same time the heart
muscle pulsates longer when potassium is present than it does when
only sodium and calcium are present. A small excess of potassium in
physiological doses is not toxic in its effects, yet it inhibits the pulsa-
tion of the heart muscle; but the muscle will beat again in solutions
containing less potassium or more calcium. Other physiologists con-
clude that small amounts of potassium stimulate ‘‘the vertebrate
heart.’’ (See Carlson, 1906, p. 397.)

It is interesting to observe that Macallum (1903) finds that the
bodies of Cyanea and Aurelia contain considerably more potassium
than does sea-water. He found the various elements to exist in the
following proportions :

oa Na. | Ca. K. | Mg.
Sea-water........ Ioo | 3.84 | 3.66 | 11.99
Cyanea arctica ...| 100 | 3.86 | 7.67 | 11.31
Aurelia flavidula..| roo | 4.13 | 5.18 | 11.43

GENERAL INFLUENCE OF CALCIUM UPON PULSATION.

Calcium is essential for pulsation on account of its power to coun-
teract the inhibiting influence of magnesium. Its importance in con-
nection with sodium and potassium in maintaining pulsation has been
known since Ringer’s important experiments in 1883.

If we place perfect Medusze of Casstopea, with marginal sense-
organs intact, in a solution resembling sea-water but merely lacking
calcium,t the Medusz pulsate more and more weakly, and all move-
ment ceases in less than 6 minutes. The Medusz are not poisoned,
however, for if, after remaining motionless for fully an hour we add
calcium to the solution, or restore the Medusz to sea-water, pulsation
is resumed almost at once, beginning feebly at first but rapidly regain-
ing its normal vigor in a few minutes.

*J find that in the embryo loggerhead turtle, 14 days old, the heart pulsates faster
in NaCl-+ KCI than it does in pure NaCl.

+96 c.c. CH,0 + 2.7 grams NaCl-+ 03.7 gram MgClz + 0.16 gram MgSO, + 0.085
gram KsSOx, or 100 NaCl+ 2.2 KCl + 7.8 MgCl, + 3.8 MgSoa, all of $n concentra-
tion.

46 PULSATION OF JELLYFISHES.

The Medusz are, however, inhibited from pulsating by the presence
of magnesium, not by the mere aésence of calcium ; for if magnesium
be absent, calcium may also be absent and the Medusz will pulsate
fully two hours.

A large excess of calcium lowers the rate of pulsation of Cassiopea,
after a momentary increase. The inhibitory effect of calcium is, how-
ever, far less marked than that of magnesium, or than the final toxic
effect of potassium. For example, if we add CaSO, + CaCO; to sea-
water at 82° F., to saturation, normal perfect Medusz of Casstopea
pulsate at about two-thirds their normal rate after being in this solu-
tion 12% hours. One gram of CaCl, in roo c.c. sea-water also slightly
reduces the rate of pulsation without injurious effects, recovery being
almost immediate in normal sea-water. Perfect Cassiopea with sense-
organs intact when placed in a pure solution of CaCl, isotonic with
the NaCl of sea-water ceases to pulsate in 10 seconds, and can not be
restored to pulsation by being placed in NaCl + K,SO, in amounts
found in sea-water. A strong solution of K,SO, in NaCl, however,
revives them into active pulsation. Evidently their sensibility to
stimuli is impaired but not destroyed.

Calcium salts never stimulate the disk of Cassiopea into pulsation,
even when placed upon it in concentrated solutions.

We see that calcium, while not of itself a stimulant, is 2ecessary to
pulsation and is a stimulant iz connection with sodium and potassium.
An excess of calcium tends to retard pulsation, but even a saturated
solution of CaSO, in sea-water exerts no appreciable toxic influence.
It is far more important to pulsation than potassium ; for Casséopea
will pulsate for more than an hour with irregular periods of rest and
activity in the absence of potassium, but in the absence of calcium
pulsation ceases in less than 6 minutes. This importance is due solely
to the remarkable ability which calcium has to counteract the inhibit-
ing effect of magnesium.

EFFECTS OF SODIUM UPON PULSATION.

All of the sodium salts are weak stimulants to the disk of Cassiopea
deprived of its marginal sense-organs, producing not very powerful
contractions. The sodium salts, however, vary considerably in their
stimulating power, NaCl or NaOH giving strong and Na,CO; or
Na,SO, weak contractions.

The disk of Casstopea deprived of marginal sense-organs pulsates
for about 20 minutes in a pure 34n NaCl solution, and alsoin NaCl +
K,SO, or NaCl+K,50,+CaSO, or NaCl+CaSO,.*

* The proportions of Na, Ca, and K were such as are found in sea-water.

EFFECTS OF SODIUM. 47

It will not pulsate, however, in NaCl+ MgSO, or MgCl, or both,
and it is evident that the magnesium salts contained in sea-water
counteract the stimulating effect of the sodium. Disks that have
ceased to pulsate in 5n NaCl will revive a few pulsations if supplied
with calcium, or with a strong excess of potassium, or both, but no
revival results when magnesium is added to the NaCl solution.
Indeed, it may be said of the sea-water that the chief stimulant,
owing to its large amount, is sodium chloride, and the chief inhibitor
of pulsation is the magnesium. As is well known, however, pure
sodium chloride solutions can not sustain pulsation, for in all known
cases of rhythmical movement from that of Medusz to that of the
vertebrate heart, calcium and potassium must be associated with the
sodium, and I find that magnesium must also be present to vestrain the
highly stimulating influence of the combination of sodium, calcium,
and potassium. Indeed, in order to pulsate rhythmically an organ
must be in that delicately balanced state known to physiologists as
being upon the threshold of stimulation. When in this condition a
constantly accumulating internal stimulus, which is reduced at each
contraction, will maintain rhythmical pulsation.

Normal Medusz of Casszopea with marginal sense-organs intact will
pulsate fora short time with abnormal rapidity in a pure 4gn NaCl
solution, but their rate quickly declines so as to become abnormally
slow, and in about 10 minutes they begin to pulsate only at intervals
with longer and longer periods of rest between periods of pulsation.
Practically all movement ceases at the end of about 30 minutes. Little
or no toxic effect is produced, however, for recovery is almost instan-
taneous in sea-water, and pulsation can be revived, even after several
hours, by the addition of any calcium salt to the NaCl solution.

Pulsation of normal Casstopea ceases in 1 to 6 minutes in a solution
containing the amounts of NaCl and MgSO, + MgCl, found in sea-
water, but it can sometimes be revived temporarily by adding potas-
sium, or always by the amount of calcium found in sea-water.

Normal Meduse of Cassiopea are but little affected by an excess of
NaCl in the sea-water, and will pulsate for more than 18 hours in sea-
water+1 percentexcessof NaCl. Their pulsation, however, becomes
somewhat irregular, although of practically normal average rate, but
the mouth-arms are strongly and abnormally contracted. Recovery in
sea-water is, however, very rapid and no apparent toxic effects are
produced. A Medusa in sea-water+1.55 per cent excess of NaCl
pulsates with abnormal rapidity for half an hour, and although shriv-
eled, recovers quickly on being replaced in normal sea-water.

48 PULSATION OF JELLYFISHES.

When we proportionately reduce the sodium chloride and mag-
nesium, but at the same time maintain the amounts of calcium and
potassium of the sea-water, the rate of pulsation and general energy
of the Meduseze steadily decline. This was done by diluting sea-water
with distilled water containing the amounts of calcium and potassium
found in sea-water, as is described on page 18. If we simply dilute
the sea-water with distilled water the rate of pulsation does not decline
so tapidly, and the injurious effects are not so pronounced.

These experiments show that a relative excess of Ca and K retards
pulsation, even when the actual amounts of Ca and K are such as are
found in sea-water.

Cassiopea will pulsate longer in LiCl+K,SO,+ CaSO, than in a
solution wherein the NaCl is replaced by Na,CO,;. In these solutions
the LiCl and Na,CO; were isotonic with the NaCl of sea-water, while
the amounts of K and Ca were the same as are found in sea-water.
The Medusz ceased pulsating in about 6 minutes in the LiCl solution,
but it seems somewhat remarkable, in illustrating the effects of salts
as a whole, that LiCl should replace the NaCl with less injury than
Na,COs.

We have seen that NaCl in excess or in pure solutions has very little
toxic effect upon Cassiopea. ‘This appears remarkable, for its marked
toxic effects have been made known by Loeb, Lingle, Cushing, and
others upon a number of animals, and I find that pure solutions of
NaCl have a very rapidly injurious effect upon the movement of the
branchial arms of Lepas. We must remember, however, that Cassiopea
normally lives in semi-stagnant salt-water lagoons where considerable
range in density must take place through evaporation and rainfall.
It is also one of the most hardy of marine animals and will survive
without serious effects several minutes’ immersion in sea-water con-
taining such poisons as 0.1 per cent KCN.

It will be recalled that Macallum (1903) found that while the amount
of NaCl in brackish estuaries might change greatly with the condition
of the tide, the amount of NaClin the bodies of the Aurelia and Cyanea
remained practically constant. It is therefore possible that Casszopea
may resist osmosis of NaCl to some extent and thus avoid its possibly
toxic influences.

We conclude that NaCl is a stimulant and is counteracted in this
respect by the magnesium of sea-water so as to produce a balanced
solution. It can not maintain pulsation except in connection with
calcium and potassium, in combination with which it forms a powerful
stimulant which produces a rapid but only temporary pulsation, mag-
nesium being necessary to reduce and sustain its action.

INTERACTION OF SALTS. 49

ARTIFICIAL SHA-WATER AND THE EFFECTS OF THE SALTS OF SEA-
WATER, AS A WHOLE, UPON PULSATION.

In the experiments upon Cassiopea the solutions containing some or
all of the chief constituents of sea-water were made up in accordance
with the formula given by Dittmar (1884)*, and also according to
Van ’t Hoff’s formula (100 NaCl+2.2 KC1+7.8 MgCl, +3.8 MgSO, +
3 CaCl,, all of 54n concentration.

Medusz pulsate normally in an artificial sea-water made according
to Van ’t Hoff’s formula, but pulsation is somewhat irregular in a
sea-water made according to Dittmar’s formula. Table 4 shows the
results of experiments with Dittmar’s formula, and table 5 gives the
results obtained by using Van ’t Hoff’s formula.

Tables 4 and 5 show the effects upon Cassiopea of various solutions
containing one or more of the constitueuts of sea-water. It will be
apparent that magnesium is the chief restrainer of pulsation, and that
it prevents the spontaneous contraction of disks deprived of mar-
ginal sense-organs and retards pulsation in perfect Meduse. When
magnesium is present the absence of calcium quickly stops pulsation,
but when magnesium is absent we may have calcium also absent and
the Medusze will pulsate for a considerable time. It is apparent,
therefore, that calcium assists the NaCl to counteract the retarding
influence of magnesium. This is also shown by the fact that Medusze
pulsate for a long time in Na + Mg + Ca, whereas all movement ceases
very soon in Na+ Mg.

Potassium, however, does not assist the NaCl to resist the stupefy-
ing influence of magnesium, for Medusz cease to pulsate almost as soon
in Na+Mg+Kas they doin Na+Mg. Potassium serves mainly to
stimulate movement in connection with both calcium and sodium ; thus
Na+K and Na+Ca give temporary pulsations at about normal rate;
whereas Na + Ca + K gives strong pulsations at fully twice the nor-
mal rate, but these can not be sustained indefinitely unless magnesium
be present to counteract the too powerful stimulating effects of the
Na+Ca+kK. A Ringer’s solution is only a powerful stimulant, and
can not sustain pulsation indefinitely unless tempered by magnesium,
Potassium has little power to revive pulsation, whereas calcium pos-
sesses this power to a marked degree; thus, when pulsations have
ceased in NaCl they can always be revived by calcium, but at best
only a very few isolated contractions can be revived by potassium in
the amount and proportion found in sea-water.

*Reports of voyage of H. M. S. Challenger, Chemistry, vol. 1, p. 204.

PULSATION OF JELLYFISHES.

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51

TABLE SHOWING EFFECTS OF SALTS.

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PULSATION OF JELLYFISHES.

52

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EFFECTS OF VAN "I HOFF’S SEA-WATER.

53

TABLE 5.—Lffects upon the rhythmical pulsation of Cassiopea, exerted by solutions
containing Na, Ca, K, and Mg in amounts and proportions found in sea-
water according to Van ’t Hoff’s formula, wherein sea-water is supposed to
contain roo NaCl+ 2.2 KC1+ 7.8 MgC + 3.8 MgSOs+ 3CaCh, all of San

concentration.

Composition of the solution.

Normal Medusez taken from
sea-water and placed in the
solution pulsate as fol-
lows :

Disks made by cutting off
the marginal sense-organs
of Cassiopea behave as
follows :

NaCl ..sceeee covey Cie meaciie)

NaCl4CaClg...... ..eeee wate orate

NaCl+ KCI ...eecee cess eee is

NaCl+ KCl-+ CaClg.........-

NaCl + MgCly...... pcainapReateey
NaCl + MgS0O4.......... seen Te

NaCl-+ MgCle+ MgSO .......

NaCl + MgCle+ MgSO4+ KCl,

NaCl + MgCle + MgSO« +
CaCle.

NaCl + MgCle+ MgS0O4+KCl
+CaClg.

Pulsation is abnormally
rapid at first, but in from
7 to 10 minutes periods of
rest appear and these in-
crease in duration and
frequency while the pe-
tiods of active pulsation
decrease. All movement
dies out before the end of
45 minutes.

At first the rate is about
normal. Periods of rest
begin to appear after be-
ing about 15 minutes in
the solution. These peri-
ods of rest increase in dur-
ation and frequency so
that all movenient ceases
before the end of 1% hours.

Pulsation is abnormally rap-
id at first, but at the end
of about half an hour
pauses set in, and these
periods of rest gradually
increase in length and fre-
quency. All movement
ceases after the Medusa
has pulsated somewhat
more than 2 hours.

Pulsation is maintained
without pauses at fully
twice the normal rate for
more than 2hours; then
periods of rest are apt to
commence. The Medusa
pulsates over 4 hours.

Pulsation declines steadily
and ceases before the end
of 6 minutes.

Pulsation declines at once
and all movement ceases
before end of 1o minutes.

Pulsation declines rapidly
and usually ceases before
the end of 30 seconds.
Some Meduse may pulsate
longer than 5 but less than
6 minutes. It is restored
if we add CaCle, but not
usually by KCl,

Pulsation steadily declines
and ceases before the end
of 6 minutes. It is effectu-
ally restored by adding
CaCle. @

Rate normal at first but de-
clines slowly with pauses,
so that all movement
ceases before the end of 40
minutes. Pulsation is re-
stored by adding KCl.

Pulsation is normal as in
natural sea-water,.

Newly cut disks give a few
contractions, then subside
into quiescence. Disks
two or more days old usu-
ally pulsate slowly and ir-
regularly for hours,

Disks behave very much as
they do in NaCl, but are
somewhat more actively
stimulated.

Disks behave as they do in
NaCl+CaClg, but are more
strongly stimulated,

Disks are powerfully stimu-
lated, and even newly
made disks may com-
mence spontaneous, irreg-
lar pulsation which may
continue for several hours,

No pulsation.

No pulsation.

No pulsation,

No pulsation occurs.

No pulsation.

Newly made disks do not
pulsate. Disks 2 or more
days old pulsate irregular-
y.

54 PULSATION IN BARNACLE, TUNICATE, AND TURTLE.

IV. PULSATION OF THE BRANCHIAL ARMS OF LEPAS, THE HEART
OF SALPA, AND THE HEART OF THE LOGGERHEAD TURTLE.

The Medusz are the most primitive of the metazoans which display
rhythmical pulsation, and therefore a study of the laws which control
their movement is important, for it is practically certain that pulsation
began to attain physiological importance in primitive marine animals,
and that the vertebrate heart developed in creatures living in salt
water. In the most primitive forms the body pulsates as a whole, but
finally pulsation is assumed by or restricted to special organs. It is
therefore interesting to consider various sorts of pulsating organs in
order to see whether some fundamental conditions may not apply to
all of them.

Accordingly studies were made of the pulsation of the heart of the
solitary asexual form of Salpa democratica, the rhythmical move-
ment of the branchial arms of Lefas, and the pulsation of the heart of
the embryo loggerhead turtle, 7halassochelys caretta, and these varied
sorts of pulsation were compared with that of the jellyfish Casszopea.

The results are presented in condensed form in table 6 (p. 60) which
shows the number of minutes that pulsation endures in various solu-
tions consisting of one or all of the ingredients NaCl, KCl, CaCl,,
MgSO,, and MgCl,. In the experiments upon Casszopea, Lepas, and
Salpa Van t’ Hoff’s sea-water solution was employed. This con-
sists of 100 NaCl + 2.2 KC1+7.8 MgCl, + 3.8 MgSO, + 3CaCl,, all of
54n concentration. In experiments upon the heart of the logger-
head turtle the proportions of the above-named salts were changed so
as to beo.7 per cent NaCl + 0.03 per cent KCl + 0.1 per cent MgCl, +
0.025 per cent CaCl,. The various animals were placed in solutions
containing one or all of these salts in the amounts and proportions
stated above. Where + follows a number it means that pulsation occa-
sionally lasts a few more minutes than is here recorded, and on the
other hand, — following a number means that the pulsation does not
usually last as long as is recorded.

An inspection of table 6 (p. 60) will show that pulsation in all of
theselforms (jellyfish, barnacle, tunicate, and reptile) is most powerfully
stimulated by solutions composed of sodium chloride, potassium, and
calcium, and that all are depressed by magnesium. Nevertheless
sustained pulsation can only take place in a solution containing
sodium, potassium, calcium, and magnesium, the last-named element
being necessary to ‘‘tone down”’ and restrain the strong stimulation
caused by the first three, thus giving a slower but indefinitely sus-

ained pulsation. This important réle of magnesium has hitherto

BARNACLE, TUNICATE, AND TURTLE. 55

been unsuspected, and we see that Ringer’s solutions, which consist of
combinations of sodium, potassium, and calcium chlorides, are only
stimulants, and must be partially inhibited and restrained by mag-
nesium in order that they may sustain pulsation indefinitely.

In simple marine animals such as Medusz, barnacles, and Sa/pa the
optimum solution for pulsation is the sea-water itself, but in the higher
terrestrial forms the proportions and amounts of the ingredients of the
optimum solution have changed, although still composed of sodium
chloride, potassium, calcium, and magnestum. In Cassiopea, Lepas,
and Salpa it is the special réle of calcium to assist the sodium chloride
to overcome the anesthetic effect of magnesium, whereas potassium
practically lacks this power.

A further inspection of table 6 shows that there are considerable
differences in the effects of various elements upon different animals.
For example, pulsation is sustained fairly well in Casszopea, the heart
of Salpa democratica, and the loggerhead turtle embryo by a pure
NaCl solution, but this quickly stops the movement of the branchial
arms of Lepas. Also, the addition of KCl to NaCl greatly improves
the solution in its ability to sustain the pulsation of Casszopea, whereas
it has but little beneficial effect in the case of the arms of Lefas. Cal-
cium, on the other hand, has but little power to sustain pulsation in
connection with NaCl in Casszopea, but in the case ofthe arms of Lepas
it is very efficient. In Cassiopea pulsation ceases almost instantly in
such non-ionizable solutions as urea, dextrose, and glycerin, but the
heart of Salpa democratica will pulsate for a considerable time in these
solutions, and the heart of the embryo loggerhead turtle pulsates as
long in dextrose as it does in NaCl. These differences in the effects
of the several salts upon pulsation in different animals areso consider-
able that we must be cautious of drawing general conclusions from
the behavior of any one animal and applying them to related forms.
For example, Cassiopea can not pulsate for 6 minutes in a solution
resembling sea-water but simply lacking calcium, whereas another
Scyphomedusa, Linerges mercurius, will pulsate for 45 minutes in the
same solution. Both Linerges and Casstopea are, however, restored to
normal pulsation by the addition of calcium, and the difference in
their behavior is one of degree, not of kind. The papers of physiol-
ogists abound in general conclusions concerning the action of ‘‘the
vertebrate heart’’ when only the heart of the terrapin or the dog has
been studied, and undoubtedly these sweeping conclusions are often
misleading. For example, when the loggerhead turtle embryo is 11 to
14 days old its heart ceases to pulsate in less than 22 minutes in the

56 HEART OF LOGGERHEAD TURTLE.

albumen of its own egg, but when it is 41 days old it pulsates from 3

to 7 hours in the albumen of its egg, which then sustains it better than
cana Ringer’s solution, or any solution I could devise.

The albu-
men contains Na, K, Ca, and Mg.

70

60

/
\
50 SS

5
w

wo
Va

w
&
S

PULSATIONS PER MINUTE

tw
=
Len = =
a
zg
to
~~
Does
@

1
{
‘i \P i x .
\\ \ N \
\ \ \—-
\

\
\ \ Nv IT? :
\ \ \ \ \
\ \ N39 \ \ \
10 4 1 Sa t
\ rN : MN \ \ '
“a \ 1 NY \ '
ra \ is. \ 1
\l \ \ \ NV \ !
‘A ‘ \ \\ \ \
i! \ \ ! \ \ 1
i! & \ l AN \ \
(eo) 10 20 30 40 50 60
MINUTES

Fig. 34.—Showing the decline in rate and also the length of time that pulsation
endured in the hearts of 10 loggerhead turtle embryos (11 to 14 days old)
placed in 0.7 per cent NaCl--0.1 per cent MgCle. The heavy dark line shows
the average condition, and the fine full lines show the behavior of individual

embryos. The dotted lines cover periods from the last observation to the time
when the heart ceased to beat.

The ‘‘allor none ’’ principle in pulsation does not apply to the pul-
sation of the heart of the embryo loggerhead turtle, for the ventricle
ceases first, then after a long time the auricles cease to pulsate, but
the sinus still pulsates. Normally, as is well known, the heart-beat
originates in the sinus; then after an interval the auricles respond,
and finally the ventricle contracts. After the heart which has been
removed from the body has ceased to pulsate, however, we may stim-

HEART OF TURTLE. Ley

ulate the ventricle by an induction current, and after the current has
been removed the heart may pulsate for several minutes in a reverse
manner, each contraction originating at the stimulated place in the ven-
tricle, then after a pause the auricles, and finally the sinus contracting.

60

Pa em

a
is}

PULSATIONS PER MINUTE
ry
is}

Ny
is}

oO 40 20 30 40 50 60
MINUTES

Fig. 35.—The full heavy line shows the average rate and duration of pulsation of
the hearts of 10 loggerhead turtle embryos (11 to 14 days old) in 0.7 per cent
NaCl. The dotted line shows the same things for the hearts of 10 embryos in
0.7 per cent NaCl+-0.1 per cent MgCle. It appears that the NaCl+-MgCle does
not affect the rate, but nevertheless it stops the heart sooner than does the pure
NaCl solution.

The heart of the loggerhead turtle often revives temporarily im-
mediately before it ceases to pulsate in solutions. This is seen in
figures 34 and 36, which show the decrease in the rates of pulsation of
the hearts of 20 loggerhead turtle embryos 11 to 14 days old. Ten of
these (fig. 36) were placed in 0.7 per cent NaCl, and 10 others
whose pulsation is shown in figure 34 were placed in 0.7 per cent
NaCl+o.1 per cent MgCl,. The MgCl, has no effect upon the rate,
but it stops the heart sooner than does the pure NaCl. (See fig. 35.)

After the heart of the loggerhead turtle has ceased to pulsate in
NaCl it may be revived temporarily by CaCl,. KCl will also revive

58 PULSATION OF TURTLE’S HEART.

it, but not so powerfully, and even distilled water or MgCl, will often
give rise to a few final, weak pulsations. In other words, the heart
responds to any osmotic change, be it beneficial or injurious. It is
worthy of note, however, that if the heart ceases to beat in NaCl +
MgCl, it is usually impossible to revive it, even by CaCl,.

The heart of the loggerhead turtle embryo, 14 days old, pulsates
more rapidly, and usually longer, in 0.7 per cent NaCl+ 0.03 per
cent KCl than it does in 0.7 per cent NaCl. Thus the addition of a
small amount of KCl acts as a stimulus. Physiologists are in dispute
concerning the action of potassium upon the ‘‘vertebrate heart,’’ the
general opinion being that potassium depresses the heart. The litera-
ture of this subject is reviewed by Carlson (1906, Amer. Journ. Phys-
iology, vol. 16, p. 397). Much of the discrepancy in results arises
from the sweeping conclusions which physiologists have drawn in
applying to all vertebrates the results achieved from experiments
upon a few forms. Moreover, in some papers experiments are con-
ducted upon each salt separately, and the assumption is made that
the effect of a mixture of these salts is merely the summation of their
individual effects. Nothing could be moreerroneous. For example,
calcium alone never stimulates, but even inhibits pulsation in Cassz-
opea, but in connection with sodium and potassium chlorides it forms a
most powerful stimulant.

In closing we will state that the heart of the embryo loggerhead tur-
tle behaves quite differently from that of the animal after hatching, but
we will leave the discussion of this and other points to a future paper,
wherein we hope to treat of the general effects of different salts upon
the hearts of various vertebrates and invertebrates.

In conclusion it may be said that rhythmical pulsation can be sus-
tained only when an external stimulant is counteracted by an inhibi-
tor, so that the pulsating organism is in a state bordering upon the
threshold of stimulation. This allows the weakest internal stimuli to
produce periodic contractions. Each contraction either produces a
chemical change which periodically reduces the internal stimulus, or
the tissue can not again respond to the ever-present, constant stimulus
until after a period of rest.

PULSATION OF TURTLE’S HEART. 59

PULSATIONS PER MINUTE

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Fig. 36.—Showing the decline in rate and also the length of time that pulsation lasted in the
hearts of 10 loggerhead turtle embryos (I1 to 14 days old) placed in 0.7 per cent NaCl. The
heavy line shows the average condition, and the fine unbroken lines show behavior of individual
embryos numbered from | to 10. The dotted lines cover periods from the last observation to
the time when the heart ceased to beat.

DURATION OF PULSATION IN SOLUTION.

60

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PAPERS CITED. 61

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