ARTIFICIAL

PARTHENOGENESIS
BY MARTIN

[F~*om

the

PhysioZogicnZ

H.

Labordory

IN

NEREIS.

FISCHER.

of the

Uhiversity

of CnZzjhzia.lJ

Loeb and I 1 published a note in this journal in

L ASTwhichyearweDr.stated
that it is possible to cause the unfertilized
eggs of Nereis limbata to develop into swimming larvae by keeping
them for some time in sea-water, the concentration of which has been
raised by the addition of potassium chloride, and then returning them
to ordinary sea-water. We expressed the opinion that we were probably dealing with osmotic effects, and not with the specific effects of
K-ions, since we obtained swimming larvae only from those mixtures
of sea-water and potassium chloride in which the osmotic pressure
had been considerably raised. We based our conclusions upon a
series of experiments on the eggs of a single female, but did not
publish them, as we were not able to obtain any more mature females
upon which to corroborate our findings. This year we were more
fortunate, however, and succeeded both in repeating our experiments
of last year, and of obtaining further proof of the correctness of our
conclusions.
All the precautions necessary to prevent the infection of the eggs
with sperm, which have been so often described by Loeb,2 were followed in these experiments.
Each female Nereis, as soon as caught,
was kept in a separate dish of sea-water sterilized by heating to
80C. After swimming about in this for some time, the worms were
washed in several changes of sea-water, then in fresh water, and
finally were opened in sterile sea-water with sterilized instruments.
I shall give first of all a detailed description of the series of experiments which formed the subject of our first communication.
1 FISCHER,
NEILSON
3 LOEB,

M. H.: This journal. , 1902, vii, p. 313; LOEY,, FISCHER,

Archiv fiir die gesammte Physiologic, Igor, lxxxvii, p. 594.
J . : This journal, IC)OI, iv, p, 424.
100

and

A rtijciad

Parthenogenesis

in Nereis.

101

Experimerrt Jfl~ly 20, 1901. - I 2.30 A. M. The eggs of two females were
gently shaken together and distributed
into the following
mixtures of
potassium chloride and sterile sea-water :1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

95

C.C. sea-water

924 C.C. sea-water

90

C.C. sea-water

87; C.C. sea-water

85
S2i
80
774

C.C. sea-water
C.C. sea-water
C.C. sea-water
C.C. sea-water
75 C.C. sea-water
729 C.C. sea-water
70 C.C. sea-water
Sterile
sea-water

+ 5
+ 7+
+ 10
+ 129
+ 15
+ 174
+ 20
+ 22;
+ 25
+ 274
+ 30
(colltrol).

C.C.
C.C.
C.C.
C.C.
C.C.
C.C.
C.C.
C.C.
C.C.
C.C.
C.C.

KC1
KC1
KC1
KC1
KC1
KC1
KC1
KC1
KC1
KC1
KC1

24
2+
24
23
2;
24
2;
2;
2;
2$
24

m.
m.
m.
m.
m.
m.
nt.
m.
~2.
m.
m.

The eggs were taken out of the solutions and transferred to sterile seawater at intervals of thirty, forty-five, and sixty minutes.
These will be
designated as Lots I, II, and III.
The first evidences of segmentation were found in the eggs exposed for
thirty minutes to the mixture of 80 cc. sea-water + 20 C.C. KC1 29 SW.
One hour and twenty minutes after returning the eggs to sea-water several
were found in the two-cell stage. Ten minutes later an egg was found in
the four-cell stage. After two hours, with rare exceptions, all the dishes
contained segmenting eggs. In some of the dishes the eggs did not pass
through the two and four-celled
stages, but. broke up immediately
into
eight and twelve cells. Up to five hours after the beginning
of the
experiment,
it was difficult to decide which salt solution would give the
most favorable results.
After ten and a half hours (10.55 A. M.), . however,
it was clearly noticeable that the optimum sea-water-salt solution mixture
lay between 77+ C.C. sea-water + 22+ C.C. KC1 2&m and 85 C.C. sea-water
+ 15 C.C. KC1 28 iv,?. All these solutions contained
eggs in the thirtytwo to sixty-four cell stages.
The first swimming larvae were found in the afternoon, fourteen hours
after the return of the eggs to sea-water, in Lot III of the mixture of 82+C.C. sea-water + I 74 C.C. KC1 2+ m. Lots II and I showed the same condition of affairs. In one field of the latter every egg was in the blastula
stage, and many were beginning to swim.
At four oclock, Lot I of the mixture of 80 C.C. sea-water + 20 C.C.
KC1 a&v2 showed even better results than the foregoing, the dish teeming
with swimming larvae. In Lots II and III of the same mixture many less
were found.
Lots II and III of 77$ C.C. sea-water + 22+ C.C. KC1 2+2
showed some swimming
larvae. The solutions
containing
a greater
amount of KC1 than this showed many segmented eggs but no swimming

102

larvae, In solutions less concentrated

than 85 C.C. sea-water + 15 C.C.
A few
KC1 2-i m, up to this time, no swimming larvae had developed.
hours later occasional swimming larvae were found in these solutions also.
At 1.00 A. M., twenty-four hours after the return of the eggs to sea-water,
with an occasional exception, every egg from Lot I of the mixture of 80
C.C. sea-water + 20 C.C. KC1 z$ m was swimming.
In Lot I of 824 c.c.
sea-water + I 7+ C.C. KC1 z& m many were swimming.
In the rest of the
dishes all were dead.
The control eggs left in the sterile sea-water contrasted sharply with
Not a
those which had been in the sea-water-salt solution mixtures.
single egg segmented throughout
the night.
Eighteen
hours after the
beginning
of the experiment
a few were found in the two to four cell
stage. Six hours later, when the other dishes were teeming with swimming larvae, the control eggs were granular and surrounded by a granular
debris.
The dead eggs floated in the water, and were held together by a
gelatinous material.
Not a single swimming larva developed.
Many of the swimming larvae lived through the next day. One was
removed to fresh sea-water in a separate dish. This embryo lived fiftyseven hours.
Exfehnent
It was necessary, fi .rst of all, to
14 $4) 6, 1902. - II.05 P.M.
repeat the experim ent of last year. I d .istributed the eggs of a freshly
caught Nereis into the following
solutions :
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

25 C.C. KC1 2$ m + 75 C.C. sea-water.

22; C.C. KC1 2; m + 776 C.C. sea-water.
20 C.C. KC1 2i m + 80 C.C. sea-water.
174 C.C. KC1 2% nc + 82% C.C. sea-water.
15 C.C. KC1 24 m + 85 C.C. sea-water.
123 C.C. KC1 2; 772 + 87$ C.C. sea-water.
10 C.C. KC1 24 1~2+ 90 cc. sea-water.
74 C.C. KC1 23 m + 92Q C.C. sea-water.
5 CC. KC1 26 m + 95 C.C. sea-water.
2+ C.C. KC1 24 m + 97+ C.C. sea-water.
Sterile sea-water
(control).

I removed one lot of eggs at I 2.00

midnight, a second at I 2.25 A. M.
These will be designated as Lots I and II.
A few eggs
At 3.00 A. M. July 7, every egg in the control was intact.
were removed from Lot I of Solution 4 for microscopic
study.
These
were somewhat shrunken and opaque, had lost their nuclei, and no longer
showed the peripheral arrangement of the oil globules which is so characistic of the unfertilized
egg. At 4.30 A. M. I found the same condition of
affairs in Lot II of Solution 5. The eggs were fissured and broken up
into irregular
masses (cells ?). Only occasionally
were these arranged
regularly as in the normal cleavage of the fertilized egg.

At 3.00 A. M. conditions
were much the same, except that cleavage
had gone further.
At r0.,30 I noticed that many of the lines of cleavage
had become obscure or had disappeared
entirely, so that the eggs were
again spherical.
The control eggs were still intact.
At I 1.30 A. M.,
however, a few were found in which the peripheral
arrangement
of the
oil globules was lost. The eggs from Solutions 7 and 8 were nearly all
intact.
A few were somewhat shrunken, irregular in outline, and had
lost the peripheral
arrangement
of their oil globules.
At 9.30 P. M. conditions were as follows : Not a single swimming larva
was found in the control.
Some of the eggs had undergone
a granular
A few had lost the peripheral
degeneration
and were going to pieces.
arrangement
of the oil globules ; the remainder were intact, and looked
as though they had just been removed from the ovaries.
Both lots of
Solutions I and z yielded no swimming larvae. The eggs were irregular
and contracted, with thick, clear membranes.
Many were dying, and no
Several
traces of segmentation could be found in Lot II of Solution I.
swimming
larvae were obtained in both lots removed from Solution 3.
The optimum
salt-solution
sea-water mixture was Solution
4. This
yielded many swimming larvae, as did also Solution 5. A few swimming
larvae were found in Lot II of Solutions 6 and 7. One swimming larva
was found in Lot II of Solution 8, and one in Lot II of Solution g. An
occasional egg, segmented into two or four cells, was found in Solution IO.
At noon, July g, rgoz, larvae were still swimming in the dishes containing the eggs from Solutions 3, 4, and 5. The control eggs still showed
the peripheral arrangement of the oil globules, or else were broken up into
I kept the control
a granular debris.
In the other dishes, all were dead.
eggs for another day, but no swimming larvae developed.

The experiment
seemed to leave no doubt as to the correctness of
the findings of last year. The unfertilized eggs of Nereis, if left undisturbed in sea-water, will not develop into swimming larvae. After
several hours, however, they may show the changes which precede
cleavage, or may actually divide into two or four cells. If the eggs
are, however, kept for an hour in a mixture of 17&- C.C. KC1 z& ITZ+
824 C.C. sea-water, and are then removed to ordinary sea-water, they
will develop into swimming larvae.
Experiment
114 Jz~2y 8, 1902 - 2. IO A.M.
I wished once more to assure
myself of the correctness of the results obtained in the last experiment ;
I wished also to discover whether the KC1 acted only through its osmotic
effects. The following solutions were therefore prepared :

1.
2.
3.
4.
5.
6.
7.

8.
9.
10.
11.
12.

25 C.C. KC1 2; m + 75
22+ C.C. KC1 2; m + 77;
20 C.C. KC1 2+ m + 80
17~~c.c.
KC1 2+ m + 821
15 C.C. KC1 2& m + 85
124 C.C. KC1 24 m + 82Q
10 C.C. KC1 24 1.2 + 90
7$ C.C. KC1 24 m + 92$
5 C.C. KC1 24 nt + 95
2; c.c. KC1 Zg m + 974
17+ CL NaCl 21 m + 82;
Sterile
sea-water
(control).

C.C. sea-water.
C.C. sea-water.
c.c. sea-water.
C.C. sea-water.
C.C. sea-water.
C.C. sea-water.
C.C. sea-water.
C.C. sea-water.
cc. sea-water.
c.c. sea-water.
C.C. sea-water.

Lot I was returned to sea-water after 45 minutes ; Lot II, after 75, and
Lot III, after 105 minutes.
To avoid repetition, I will only state in which solutions swimming larvae
were obtained, leaving a description of the histological findings until later.
At 4.00 P. M. the first ciliated larva were discovered in Lot II of Solution 4. At T I .30 P. M., when it was certain that all the larvae which would
develop were swimming, conditions were as follows :
The majority of the eggs in the control were somewhat shrunken and
opaque, and many had lost the peripheral arrangement of the oil globules,
and were segtnented into two and four cells. None were swimming.
All three lots of the NaCl-sea-water
mixture were full of swimming
larvae. Many swimming larvae developed in Lots I and II of Solutions 4
and 5. Several ciliated larvae were also found in Lot II of Solutions 3,
6, and 7. The eggs from Solutions I and z were shrunken and granular.
In soluThe capsules were swollen and the eggs were going to pieces.
tions 8, g, and TO, the majority of eggs had segmented into two and four
cells, but none were swimming.
At noon on the following day, no swimming larvae had developed in the
control; a large number of the eggs were dying.
Lots II and III of
Solutions 4, 5, and 6 still contained
many swimming larvae. The eggs
which had been in the sodium-chloride
solution were still swimming
An occasional ciliated larva was found in Solution 7. In the
beautifully.
other dishes everything was dead.
The experiment again showed that when the unfertilized eggs of Nereis
are left for a certain time in sea-water, the concentration
of which has been
increased to a definite degree by the addition of either KC1 or NaCl, they
will develop into swimming larvae when returned to ordinary sea-water.
In the experiments described
IV,
Jzlo
IO,
1902. - 2.15 A. M.
thus far, only electrolytes have been used to increase the concentration
of
the sea-water.
If parthenogenesis
in Nereis is due simply to the ab-

Exjeriment

straction of water from the egg, we should be able to bring about the
development of the unfertilized egg, not only by mixtures of sea-water and
electrolytes, but also by mixtures of sea-water and non-electrolytes.
TO
test this point, the following solutions were prepared :
1.
2.
3.
4.
5.

50 C.C.
40 C.C.
30 cc.
10 C.C.
Sterile

cane-sugar
cane-sugar
cane-sugar
cane-sugar
sea-water

2 972+ 50 C.C. sea-water.

2 m + 60 C.C. sea-water.
2 m + 70 C.C. sea-water.
2 m + 90 C.C. sea-water,
(control).

The first lot of eggs was removed from the solutions after go minutes, the
second, after I IO minutes,
At ~2.30 P. M., all the eggs which had been in Solutions I and 2 were
dead.
I3oth lots of eggs from Solution 3 were full of swimming larvae.
In the remaining
dishes, every egg was intact.
A mixture of 30 C.C. cane-sugar z m + 70 C.C. sea-water has about the
same osmotic pressure as a mixture of 15 c.c. KC1 2* m + 85 c.c. seawater. It seems? therefore, as though the essential factor in bringing about
artificial parthenogenesis
in Nereis is an abstraction of water from the egg,
and that it does not matter decidedly whether the concentration
of the
sea-water used for this purpose is raised by the addition of electrolytes or
by the addition of non-electroIytes.

The above experiments

were repeated several times, but alwavs
with the same results. The eggs of Nereis are not naturally parthknogenetic, but cleave and develop to the swimming stage if immersed
for from one-half to one and one-half hours in sea-water, the concentration of which has been raised a definite amount, and then returned
to ordinary sea-water. It does not matter whether electrolytes or
non-electrolytes are used for this purpose, - sodium chloride, potassium chloride, cane-sugar may be used,- though sodium chloride
usually yields the largest number of swimming larvae, while sugar
yields the least.
A series of experiments had been begun in which I attempted to
obtain swimming larva by altering the ion concentration of the seawater without altering its osmotic pressure, when the material gave
out, and put an end to further work. Thus far the experiments in
this direction have yielded only negative results; but they will be
continued next year.

106

Mart232

Ii? Fischer.

DIFFERENCES BETWEEN THE NORMALLY FERTILIZED, PARTHENOGENETIC AND UNFERTILIZED

EGGS OF NEREIS LJMBATA.

THE

At ordinary
summer
temperature,
the fertilized
egg of Nereis
limbata throws out its polar bodies about one hour after fertilization,
and cleaves for the first time some fifteen or twenty minutes later.
The second cleavage occurs about one hour and forty-five
minutes
after fertilization.
Cleavage then proceeds in a regular manner, and
the blastula stage is reached in seven hours; the eggs swim in about
eleven hours, varying somewhat with the temperature.
When the unfertilized
eggs are brought to the swimming
stage
through a temporary
residence in sea-water,
the osmotic concentration of which has been increased a definite amount, no special changes,
save a slight shrinkage
of the protoplasm,
a disappearance
of the
nuclear membrane, and a slight increase in opacity, are noted while
the eggs remain in the concentrated
sea-water.
An hour after being
returned
to ordinary
sea-water,
the egg protoplasm
becomes more

1. - A. Unsegmented
egg of Nereis
limbata immediately
after its return
to
ordinary
sea-water after immersion
for seventy-five
minutes
in a mixture
of 176 C.C.
2+ m NaCl + 824 C.C. sea-water.
The eggs were returned
to sea-water
at 3.25 A.M.
Experiment
3. B. Same egg, one hour later.
The increase in the opacity
of the
protoplasm
is not indicated
in the drawing.
C. Same egg, 4.55 A. M. I). Same egg,

FIGURE

5.10

A.M.

opaque and its outline somewhat

irregular.
The characteristic
peripheral arrangement
of the oil globules also disappears, and they
become clumped irregularly
in the central portions of the egg (Fig. I,
W . During the second hour after their return to ordinary sea-water,
many of the eggs cleave into two cells, after which cleavage into four,
eight, and sixteen cells may occur in a more or less regular manner
(Fig. I, C, 0).
Much oftener, however, no change whatever
takes
place in the two hours following the return of the eggs into normal
sea-water, and then the eggs suddenly fissure irregularly,
and break

up at once into a number of spherules (cells ?) (Fig. 2, B, C). Cleavage may then continue until such pictures as are shown in Figures 3,
A, B, C, D, E, are obtained which were made when the first evidences
.jr.

.p

.+b
.*
2.. ..x
.<.
C
:t

*-...

* . . . p..
-.

-.-.*..
. -

..y.:.

<-!-

.a,,
.&
-.
*

;.

,: .2. ,i

0
-

r- 54
32

:
.
-8 .Y
( **a
.;

CB
, .:
.\- *
.y..

-*.i:;

. .%

.,*
?
i

*.
* . ...

I*-

,..a.

FIGURE
2. -A.
Control
egg from Experiment
3. B, C. Eggs which had been in a solution of 90 C.C. sea-water + 10 C.C. KC1 2Qm for seventy-five
minutes, and were then
returned
to sea-water.
The eggs were removed
from the solution
at 3.25 A. M. The
drawings
were made at 6.00 A.M.

of ciliary movement were noticeable among the eggs.

This stage is
reached in about fourteen hours, though the majority of the eggs do
not swim until six or ten hours later.
The swimming
stage is therefore reached at a much later time than when the eggs are fertilized

FIGURE 3. -Parthenogenetic
larvae from Experiment
2. The drawings
were made fourSome
teen hours after the return of the eggs from Solution 5 to ordinary
sea-water.
of the eggs were just beginning
to swim.

by a spermatozoon.
At other times, cleavage occurs in a much more
regular manner (Fig. 4, A, C), and the swimming
larva have an
appearance which cannot be distinguished
from that of trochophores

produced through fertilization

with sperm.
It often occurs that lines
of cleavage disappear, and cells coalesce, as Loeb found to be the
case in Chaetopterus.z
The unfertilized
eggs of Nereis, when left in ordinary sea-water,

4. - Parthenogenetic
swimming,
but the cilia

FIGURE

larvae from Solution

11 of Experiment
are not indicated
in the drawings.

3.

The eggs were

5. - Unfertilized
control eggs of Nereis, showing the various changes they suffer
when left in ordinary
sea-water.
A, B. Control
eggs twenty-eight
hours after their
removal from the ovaries.
The eggs cleaved, and then underwent
a granular
degeneration.
C. Control
egg from the same dish, practically
intact.
D, E, F. Control
eggs
in which cleavage occurred.
G. A control egg in a state of granular
disintegration.

FIGURE

1 LOEI:,

J. : This

journal,

Igor,

iv, p. 423.

usually show no change during the first eight or twelve hours after
their removal from the ovaries.
Often they are absolutely unaltered
after remaining
in sea-water
for thirty-six
hours (Fig. 5, C), when
they become opaque and granular and suddenly
break up into a
granular debris (Fig. 5, G).
At other times the unfertilized
eggs
become slightly opaque after lyin, u in sea-water for several hours, lose
the peripheral arrangement
of the oil globules, and go through one or
two cleavages (Fig. 5, D, E).
They then become granular and go
to pieces (Fig. 5, A, H).
Some of the eggs may even divide into
a dozen cells (Fig. 5, F).
Never, however, do the unfertilized eggs
develop into swimming
larvae. It can often be seen that while one
dish of control eggs will show no evidences of cleavage whatsoever,
another dish of the same eggs will show a large number in the two or
four cell stage.
Mere mechanical
agitation is certainly not responsible for this change, for I have often found that while none of the
eggs which had been transferred
from one dish to another sho,wed
any signs of development,
those which had been kept absolutely undisturbed showed a large number of eggs in the two or four cell stage.
In looking over the notes of my experiments,
I find that such evidences of development occurred most often in dishes containing large
numbers of eggs.
It is possible, therefore, that lack of oxygen or
the formation
of carbon dioxide lie at the basis of the process, - a
question which will be studied next summer.