the effect of alkaloids on the early development of toxopneustes variegatus

The Effect of Alkaloids on the Early Development of Toxopneustes VariegatusAuthor(s): Sergius MorgulisSource: Proceedings of the American Academy of Arts and Sciences, Vol. 44, No. 5 (Nov.,1908), pp. 133-146Published by: American Academy of Arts & SciencesStable URL: http://www.jstor.org/stable/20022408 .

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CONTRIBUTIONS FROM THE BERMUDA BIOLOGICAL STATION FOR RESEARCH. ?No. 14.

THE EFFECT OF ALKALOIDS ON THE EARLY DEVEL OPMENT OF TOXOPNEUSTES VARIEGATUS.*

By Sergius Morgulis.

Presented by E. L. Mark. Received October 1, 1908.

It was found by Mathews (:01) that upon adding small quantities of pilocarpine hydrochloride to the sea-water the process of develop

ment could be hastened and abnormally large embryos produced, while the addition of atropine sulphate resulted in a slowing of the

developmental process and in the production of dwarf embryos. The

effect, according to this author, is especially well marked on the devel

oping eggs of the star-fish, Asterias Forbesii; Torald Soliman (:04), whose work upon the influence of atropine and pilocarpine on the

development of star-fish and sea-urchin eggs was done apparently under Mathews' direction, maintains (p. 355) that "the effects [i. e.

acceleration or retardation] of the poisons were very similar on both Arbacia and Asterias."

A size above the normal is rather an unusual condition, and it therefore seemed highly desirable to find out in what relation the

overgrown lavae stand to the normal ones, especially from a cytolog ical point of view, as such knowledge might contribute something to the understanding of the general problem of growth. The work the results of which are given in the present paper was undertaken origi nally for the purpose of studying the cellular nature of the larvae, both those larger and those smaller than the normal ones, as well as to test the influence of other alkaloids upon the rate of growth and the size of developing embryos.

Although my experiments have not yielded the anticipated results, a brief statement of the work may not be without interest. The ex

1 Contributions from the Bermuda Biological Station for Research, No. 14.



134 PROCEEDINGS OF THE AMERICAN ACADEMY.

periments were all made on eggs of Toxopneustes variegatus.2 Obvi

ously the first point to be determined was the size relations of the larvae developing in various alkaloid solutions. As the outcome has shown no marked influence in the direction of either an increase or a decrease in size, it is clear that no basis for a cytological study pre sented itself.

This work was done at the Bermuda Biological Station during the

past summer, and it gives me pleasure to express my thankfulness to Dr. E. L. Mark, the Director of the Station, for the many courtesies extended me while there.

Experiments with Atropine Sulphate and Pilocarpine Hydrochloride.

Bearing in mind the fact that the developing echinoderm eggs are

very sensitive to changes in their environment, and are more or less

easily affected by external conditions, a few special precautions were taken in carrying out the experiments. The eggs to be fertilized were

kept in finger bowls containing sea-water about an inch deep, and were mixed with very small quantities of the spermatic fluid. In this

way there was eliminated a possible disturbing factor due to the dis

integration of superfluous sperms. The eggs were allowed to settle to the bottom of the dish and then transferred to finger bowls each

containing 300 c.c. of the solution to be tried. To insure also an

equal distribution of eggs among the several dishes, thus avoiding another possible source of error, the same number of drops of fluid

containing the fertilized eggs was added, by means of a pipette, to each dish. It goes without saying that each experiment had its own

control, and that the eggs of the same individual were used in both

experiment and control.

The eggs were examined at intervals, and outline camera drawings

were made of the developing larvae. If any differences in the larvae of a set were observed, drawings were prepared of each type sepa rately. Besides, the drawing of each larva was compared with a few other larvae, so that every drawing

was representative of a number

of larvae. These drawings served later for reference, and also for a comparison of the sizes attained by embryos in different solutions.

Measurements, wherever such are given,

were made on the draw

2 Toxopneustes variegatus is found in great abundance in Bermuda, and its

eggs may be easily obtained, according to the writer's observation, from about the middle of May till the middle of August, June and July being the most favorable period.



MORGULIS. ? DEVELOPMENT OF TOXOPNEUSTES VARIEGATUS. 135

ings, the numbers indicating the full length of the drawing in milli

meters, and though not giving the actual size of larvae, offer a basis for comparison of the larvae with one another.

Solutions of atropine and pilocarpine of a very weak concentration

(about 1 : 60000) exert no influence whatever upon the developing eggs, neither during the cleavage stages nor later when the larval

stage is reached. But with the increase of concentration of those solutions their effects become pronounced, the necessary strength, however, being different for the two reagents. Definite results may be obtained with atropine by adding i c.c. of a 0.5 per cent aqueous solution to 100 c.c. of sea-water (1 : 40000), while pilocarpine in the same concentration does not produce any noticeable influence. In

no case, except when the concentration of the atropine or

pilocarpine was strong enough to injure the eggs, has there been any influence

produced upon the developing eggs during segmentation; the effect was shown only in stages involving the transformation of the gastrula to a pluteus and in those following it.

The larvae developing in atropine solutions of the strength indi cated are invariably smaller than the normal ones. The pilocarpin ized larvae, when they develop in sea-water to which there has been added from 1 c.c. to 2 c.c. of a 0.5 per cent aqueous solution of pilo carpine per each 100 c.c. of sea-water (1 :10000 or 20000), are also smaller than normal ones; but in weaker solutions, those containing from 0.5 c.c. to 1 c.c. of the 0.5 per cent pilocarpine solution to every 100 c.c. of sea-water, the larvae may be either quite normal, so far as

size is concerned, or they may vary from the normal, being either

slightly larger or slightly smaller. The following two Tables (I, II), presenting the notes of two experiments started at the same time, but with eggs of different animals, well illustrate this point.

From these tables it will be seen that cleavage is not in the least affected by any of the three different strengths of atropine and pilo carpine used. But the influence became apparent on the next day,

when the surface of the water of control dishes was teaming with plutei, while in atropine the young were still in the gastrula stage, or just begin ning to change to plutei, and very few were

swimming at the surface.

It will also be seen that in one case the pilocarpinized embryos are

slightly larger than the normal ones, while in the other set of experi ments they are smaller by just as much. In addition to the fact that the differences in the size-relations of the embryos are quite insignifi cant, the fact that those differences are not of a constant nature indi cates that they are chance variations rather than the result of the action of pilocarpine.




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Since my results did not agree with those obtained from the similar

investigations of my predecessors, and since they are derived from

entirely different species, I repeated the experiments with atropine and pilocarpine a great many times, but always with the same result.

Although a small reduction in the size of embryos did occur, there was no increase of size nor acceleration of the development under the influence of pilocarpine.

The suggestion has been made that atropine and pilocarpine respec tively inhibit and accelerate the oxidizing processes going on in the cells, thus causing either a decrease or an increase in the size of the embryos. It might he expected, therefore, that a mixture of appropriate quan tities of atropine and pilocarpine would neutralize each other's action. In none of my own trials have I succeeded in neutralizing their effects, but, as had been already observed by Soliman, the depressive action

predominates, and the embryos show a greater tendency to die out in the mixture than in either atropine or pilocarpine alone. In all of

my experiments the larvae developing in the mixture of atropine and

pilocarpine were intermediate in size as compared with those develop ing in either of those solutions alone. This will be seen from Table III.

Experiments with Morphine Sulphate.

Eggs were placed in sea-water with various quantities of a 0.5 per cent aqueous solution of morphine sulphate soon ^fter they had been fertilized. In none of the experiments,

save those where the concen

tration proved directly injurious, has there been an influence exerted

upon the developing eggs during the segmentation stages, the effect

becoming apparent after the first day only. \In sea-water with but

fo to s\j of 1 c.c. of the standard morphine s?lutiojn (0.5 per cent) to each 100 ccihe rate of development as well as the size of the larvae remained absolutely normal, but in concentration of J to J c.c. of the

morphine solution to 100 c.c. of sea-water the size of the developing embryos suffers a slight, though noticeable reduction. The segmen tation, however, is perfectly normal. With stronger solutions the effect becomes more

pronounced, of course; and when 1 e.c. of the

morphine solution is added to 100 c.c. of sea-water the effect is no

longer limited to the size of the plutei, but is seen also in a general slowing of the developmental process. In solutions two and three times that strength cleavage is very much retarded and is quite ab normal. ^ In the following Table (IV) are given the records pertaining to the concluding experiment with various; strengths of the mor

phine solution.




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morgulis. ?

development of toxopneustes variegatus. 141

Experiments with Cocaine Hydrochloride.

I have not performed as many experiments with cocaine as with some of the other alkaloids, but the effect of cocaine upon the size of larvae can be inferred even from the rather insufficient data at my

disposal. As was also observed in the experiments with other reagents, the influence of cocaine is not revealed during the first day, segmenta tion going on normally. Even in sea-water containing 2 c.c. of the standard cocaine solution (0.5 per cent aqueous solution) to each 100 c.c.

gastrulae may appear at the same time as in the control, and

they are in all essentials normal. In sea-water with i to 1 c.c. of the

cocaine solution per 100 c.c. the plutei are invariably from J to } smaller than the normal ones. But in weaker concentrations (J to

? c.c. of a 0.5 per cent solution of cocaine to 100 c.c. of sea-water),

though the size of the plutei may be slightly reduced, there is consid erable variation in size between the plutei of different lots of eggs. It may be assumed, however, that the limit of toxicity of the cocaine is probably J to ^ c.c. of a 0.5 per cent solution to 100 c.c. of sea-water.

Experiments with Strychnine.

The sulphate of strychnine was used in a 0.5 per cent aqueous solution. As in all foregoing experiments no effect has been observed

upon segmenting eggs in sea-water to which from ^ c.c. to 1 c.c. of

the strychnine solution was added. The bl?stula stage is reached at the same time in all the several concentrations. But from this stage on the influence of the poison becomes quite pronounced in the stronger solutions, where fewer larvae come to the surface, and where also the

process of gastrulation lags behind that of the control. The limit of

toxicity of strychnine differs for eggs of different animals, but -fe c.c. of the standard solution (0.5 per cent) diluted in 100 c.c. of sea-water is invariably ineffective. The plutei developed in various strychnine solutions (TV c.c. to 1 c.c.) are smaller than normal ones; the differ

ences, however, are not constant, being greater or smaller in different

sets of eggs, as was also the case in experiments with all other reagents.

Table V contains the records of one of the experiments. From this table it can be seen that as the strychnine solution reaches

an effective concentration, it also causes a reduction of the size of

the larvae, although the early stages in the development are not in the least modified.




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MORGULIS.?DEVELOPMENT OF TOXOPNEUSTES VARIEGATUS. 143

Experiments with Digitalin and Quinine.

As in the previously described experiments, five tenths per cent

aqueous solutions of digitalin and of quinine sulphate have been used as standard solutions, and of these, various quantities of each were

added to the sea-water. Each of these substances proved to be more

toxic than the other alkaloids with which I experimented. The addi tion of from ^ c.c. to 1 c.c. of the digitalin solution to 100 c.c. of sea-water was sufficient to retard cleavage, and to

produce various ab

normalities in the segmentation process. (The eggs would divide into two unequal portions, from which other cells are budded off quite irregularly, so that after a time the whole egg is broken up into a mass of small and large fragments, of either round, oval, or triangular shape.) In sea-water with half that amount of digitalin (? c.c. to

J c.c.) cleavage is also retarded, but no abnormalities are to be ob served. In none of these dilutions of digitalin, however, can develop

ment proceed very far, rarely beyond the gastrula stage. But in still more dilute concentrations of digitalin, as when only one or two drops of the standard solution is added to 100 c.c. of the sea-water, the eggs

develop more or less normally, differing with different lots of eggs, and reach the pluteus stage. But the plutei are as a rule smaller than those developed in pure sea-water. In Table VI are combined the data from two separate experiments to illustrate the above statement.

Quinine is likewise very injurious to the developing eggs in concen

trations ranging from 1 c.c. to 2 c.c. of the standard solution in 100 c.c. of sea-water. The effect is shown in a retardation of the segmenta tion process, which is greater the stronger the solution. But when

much smaller quantities of the quinine solution (J c.c. to | c.c.) are diluted in 100 c.c. of sea-water, the segmentation of the eggs is normal and unchecked. In none of the solutions do the eggs develop very far, but the stage reached in the various solutions is inversely propor tional to the concentration; while in a concentration 1 : 80000 (J c.c. to 100 c.c. of sea-water) the eggs may develop up to the gastrula stage, they probably never go beyond the 8-cell stage in a concentration 1 :10000. Table VII reproduces the record of one of the experiments.

Unfortunately, lack of time did not permit me to complete the

experiments with quinine and to determine the limit of toxicity of this alkaloid and its effect upon the size of the developing larvae. It does not seem to me improbable, however, that, as in the previous experiments, the size of embryos would have been reduced in quinine solutions in which their development was possible.




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Summary.

From the facts obtained in the foregoing experiments it may be

inferred that alkaloids, such as atropine, pilocarpine, morphine, digi talin, strychnine

or quinine, when present in sea-water in very small

quantities, produce no influence upon the developing eggs of Toxop neustes variegatus, and become effective only with the approach to a

certain concentration, which is different for the different alkaloids and

also for different lots of eggs. The length of time from the moment

the eggs are subjected to the influence of these alkaloids till the effect

becomes noticeable differs, t>f course, for the various alkaloids, but as

a rule the stronger the solution the earlier in the developmental process does its effect become pronounced. In the weaker solutions the effect

is seen only in later stages, the earlier stages (segmentation) remaining unaffected. The increasing influence is not due to a gradual concen

tration of the originally weak solution through evaporation of the

water, as was determined by measuring the volume of water in dishes

before and after the experiment, but seems rather to be the result of

accumulated effects due to a prolonged action of the poison. In solu

tions which were effective and yet not sufficiently strong to check

noticeably the development of the eggs in the earlier stages, the larvae as a rule were smaller than the normal ones.

Pilocarpine does not hasten the development of eggs of Toxopneustes variegatus, and larvae developing in pilocarpine solutions are either

of the normal size or else they are smaller than the normal ones, depend

ing upon the strength of the solution and the lot of eggs. Pilocarpine and atropine mixed in various proportions

do not, in my experience,

neutralize each other's action, but the depressing effect predominates.

Papers Cited.

Matthews, A. P.

:01. Action of Pilocarpine and Atropine on Embryos. Amer. Jour.

Physiol, Vol. 6, pp. 207-215.

Soliman, T.

:04. Simultaneous Action of Pilocarpine and Atropine. Amer.

Jour. Physiol, Vol. 10, pp. 352 361.



the effect of alkaloids on the early development of toxopneustes variegatus

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