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ARTIFICIAL PARTHENOGENESIS IN NEREIS.

BY MARTIN H. FISCHER.

[F~* om the PhysioZogicnZ Labordory of the Uhiversity of CnZzjhzia.lJ

L

AST year Dr. Loeb and I 1 published a note in this journal in

which we 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 prob-

ably 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,

conclusions.

and of obtaining further proof of the correctness of our

All the precautions necessary to prevent the infection of the eggs

with sperm, which have been so often described by Loeb,2 were fol-

lowed in these experiments. Each female Nereis, as soon as caught,

was kept in a separate dish of sea-water sterilized by heating to

80°C.

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 experi-

ments which formed the subject of our first communication.

1 FISCHER, M. H.: This journal.

,

1902, vii, p. 313; LOEY,, FISCHER, and

NEILSON :

Archiv fiir die gesammte Physiologic, Igor, lxxxvii, p. 594.

3 LOEB, J . : This journal, IC)OI, iv, p, 424.

100

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102

larvae, In solutions less concentrated than 85 C.C. sea-water +

15

C.C.

KC1 2-i m, up to this time, no swimming larvae had developed.

A few

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

those which had been in the sea-water-salt solution mixtures.

Not a

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 swim-

ming 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 fifty-

seven hours.

Exfehnent

14 4) 6,

1902. - II.05

P.M.

It was necessary, fi rst of all, to

repeat the experim ent of last year. I d .istributed the eggs of a freshly

caught Nereis into the following solutions :

1. 25 C.C. KC1 2 m

+ 75 C.C. sea-water.

2. 22;

C.C.

KC1 2; m + 776 C.C. sea-water.

3. 20

C.C.

KC1 2i

m + 8 0 C.C. sea-water.

4. 174

C.C.

KC1 2% nc + 82% C.C. sea-water.

5.

15

C.C.

KC1 24 m + 85 C.C. sea-water.

6. 123 C.C. KC1 2;

772

+ 87 C.C. sea-water.

7. 10 C.C. KC1 24 1~2+ 90 cc. sea-wa ter.

8. 74

C.C.

KC1 23 m + 92Q C.C. sea-water.

9. 5

CC.

KC1 26 m + 95 C.C. sea-water.

10. 2+ C.C. KC1 24 m +

97+

C.C. sea-water.

11. 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.

At 3.00

A. M.

July 7, every egg in the control was intact.

A few eggs

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 charac-

istic 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.

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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

degeneration and were going to pieces.

A few had lost the peripheral

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

traces of segmentation could be found in Lot II of Solution

I.

Several

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 contain-

ing the eggs from Solutions 3, 4, and 5.

The control eggs stil l showed

the peripheral arrangement of the oil globules, or else were broken up into

a granular debris. In the other dishes, all were dead.

I kept the control

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 un-

disturbed 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 :

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1. 25 C.C. KC1 2; m +

75

C.C. sea-water.

2. 22+ C.C. KC1 2; m + 77; C.C. sea-water.

3. 20 C.C.

KC1 2+ m + 80 c.c. sea-water.

4.

17~~c.c. KC1 2+ m + 821 C.C. sea-water.

5.

15 C.C. KC1 2& m + 85 C.C. sea-water.

6.

124 C.C. KC1 24 m + 82Q C.C. sea-water.

7. 10 C.C. KC1 24 1.2 + 90 C.C. sea-water.

8. 7 C.C. KC1 24 m + 92 C.C. sea-water.

9.

5 C.C. KC1 24 nt + 95 cc. sea-water.

10. 2; c.c. KC1 Zg m + 974 c.c. sea-water.

11.

17+

CL NaCl 21 m + 82; C.C. sea-water.

12. Sterile sea-water (contro l).

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 Solu-

tion 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.

The capsules were swollen and the eggs were going to pieces.

In solu-

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

beautifully.

An occasional ciliated larva was found in Solution 7. In the

other dishes everything was dead.

The experiment again showed that when the unfertilized eggs of Nereis

are lef t 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.

Exjeriment IV, Jzlo IO,

1902.

- 2.15 A. M.

In the experiments described

thus far, only electrolytes have been used to increase the concentration of

the sea-water. I f parthenogenesis in Nereis is due simply to the ab-

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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. 50 C.C. cane-sugar

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

2. 40 C.C. cane-sugar

2 m + 60 C.C. sea-water.

3. 30 cc. cane-sugar 2 m +

70

C.C. sea-water.

4. 10 C.C. cane-sugar 2 m + 90 C.C. sea-water,

5. Sterile sea-water (control).

The fi rst 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 . sea-

water. 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 parthk-

nogenetic, but cleave and develop to the swimming stage if immersed

for from one-half to one and one-half hours in sea-water, the concen-

tration 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, potas-

sium 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 sea-

water 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.

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106 Mart232 Ii? Fischer.

THE

DIFFERENCES BETWEEN THE NORMALLY FERTILIZED, PARTHE-

NOGENETIC AND UNFERTILIZED EGGS OF NEREIS LJMBATA.

At ordinary summer temperature, the fertilized egg of Nereis

limbata throws out its polar bodies about one hour after fert ilization,

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 concentra-

tion 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

A

B C

D

FIGURE 1.

- A. Unsegm ented egg of Nereis limbata im mediate ly after its return to

ordinary sea-water after imm ersion for seventy-five minute s 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,

5.10 A.M.

opaque and its outline somewhat irregular. The characteristic

peripheral arrangement of the oi l 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

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up at once into a number of spherules (cells ?) (Fig. 2, B, C).

Cleav-

age 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

. j r . - . - . * . .

* . . . . . . -

.p - . - . ‘ . y . : . - .

i

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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,

A

6 C

FIGURE

4. - Parthenog enetic larvae from S olution 11 of Experiment 3. The eggs were

swimming, but the cilia are not indicated in the drawings.

FIGURE 5. - Unfertilized control eggs of Nereis, sho wing the various chan ges 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 degener-

ation. 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 disintegra tion.

1 LOEI:,

J. : Th is jou rnal, Igor, iv, p. 423.

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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 cel l stage. Mere mechanical agitation is certainly not respon-

sible 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 un-

disturbed 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 evi-

dences 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.