THE ACTION OF ISOTONIC SALT SOLUTIONSON THE UNFERTILISED EGGS OF THALASSEMA

NEPTUNI

BY A. D. HOBSON, M.A.,

Lecturer in Experimental Zoology, University of Edinburgh.

{Received 12th April 1928.)

(With Four Text-figures.)

THE action of isotonic solutions of single salts in causing artificial parthenogenesiswas first described by R. S. Lillie (1910, 1911 «, 1911 b). He found that while thesalts of sodium and potassium caused development, those of calcium and magnesiumwere ineffective. Addition of calcium or magnesium inhibited the action of sodiumor potassium. In a later paper (1916) Lillie showed that activation of the eggy

whether by fertilisation or by artificial methods, is accompanied by an increase inpermeability of the surface membrane of the egg to water.

It seemed desirable to repeat and extend these observations especially as, inLillie's experiments, the hydrogen ion concentration was uncontrolled. The eggs ofThalassema neptuni were found to provide favourable material for work on this andallied subjects. The animals are readily obtainable at Plymouth where this work wascarried out and they have an unusually long breeding season, extending from themiddle of July until the beginning of October.

Compared with other marine forms the number of eggs laid by Thalassemaneptuni is small. A large individual probably contains not more than about 40,000eggs, and small specimens are considerably less prolific. The ripe eggs or sperma-tozoa are contained in the four nephridia which have powerful muscular walls. Thenephridia can usually be seen through the semi-transparent wall of the animal andthe difference between the dead white of the sperm and the pale orange colour ofthe eggs enables the sex of the animal to be distinguished in most cases without thenecessity of opening it.

The technique of handling the animals for experiment is as follows. Theselected individuals are thoroughly washed in fresh water to destroy any sperm thatmay adhere to the surface. They are then pinned down and the body wall is slit.The contents of the body cavity are quickly washed out with fresh water and thenephridia taken out and put into sea-water. The muscular walls of the nephridiacontract rhythmically and drive out the eggs or sperm.

The eggs are very tightly packed in the nephridia and, as the result of the pres-sure to which they are subjected, they are much distorted. Usually the eggs do not

BJEB'Vli 5

66 A. D. HOBSON

become spherical if allowed to remain unfertilised in sea-water; if they do so at allit is only after some hours. If, however, fertilisation takes place, the spherical formis acquired in less than a minute. The eggs of Thalassema undergo this change almostas rapidly when treated with an activating agent, such as isotonic calcium chloride,as when fertilised. Lefevre (1907) has recorded a similar change occurring in theeggs of Thalassema mellita as the result of natural or artificial activation.

The eggs of Thalassema neptuni appear to be similar to those of Thalassemamellita described by Griffin (1899) and by Lefevre (1907). They are about 100 //. indiameter after fertilisation. The nucleus of the unfertilised egg is large and is usuallysomewhat eccentrically placed. The cytoplasm is fairly transparent and uniformlygranular; there are no large yolk droplets. The egg is surrounded by a thick vitellinemembrane.

Maturation does not normally begin until the egg is fertilised. A sample of eggswhich has been standing, unfertilised, in sea-water for so long as eight hours mayshow a few in which maturation has begun, but these have rarely been observed toform more than 5 per cent.

The changes undergone by the egg after fertilisation may be briefly described.The first sign that fertilisation has occurred is that the egg becomes spherical. Thisprocess takes less than one minute, and by the time it is completed the vitellinemembrane can be seen to be separating from the surface of the egg as the fertilisationmembrane. The elevation of the fertilisation membrane to its final position takesseveral minutes to complete. During the early stages of separation of the fertilisa-tion membrane a completely transparent gelatinous substance appears on the out-side, carrying away with it the excess sperm which can now be seen as a ringsituated at some distance from the surface of the egg. In this respect the behaviourof the egg of Thalassema is similar to that of Nereis according to the description ofF. R. Lillie (1919) and other workers. Soon after fertilisation the nuclear membranebegins to disappear; then the two polar bodies are formed. Normally a definitivefemale pronucleus is not formed and maturation is directly succeeded by the pro-cesses leading to the first cleavage. Eggs which have been activated by experimentalagents, however, often proceed no further than maturation. The female pronucleusis formed and the egg undergoes no further change until cytolysis occurs.

The experimental procedure consisted of treating the eggs for various times withisotonic solutions of single salts or with series of mixtures of two salts. Kahlbaum'spurest salts were employed. The chlorides of lithium, sodium, and potassium weremade up in o-6 M. solution and those of calcium and magnesium in 0*4 M. solution.These concentrations were assumed to be approximately isotonic with sea-water.The hydrogen ion concentration was kept, as nearly as possible, constant at aboutpH 8-2 which is equivalent to that of sea-water at Plymouth. The sodium, lithium,and potassium chloride solutions were buffered by the addition of one drop ofsaturated sodium bicarbonate solution to 200 c.c. All the solutions were thoroughlyaerated and the final adjustment of pH was made by the addition of carbonate orsaturated Ca(OH)2 solution. In the solutions where the calcium content is high,adequate buffering is impossible. In such cases the variation in/>H was prevented

Isotonic Salt Solutions on Eggs of Thalassema neptuni 67as far as possible by filling the experimental vessel up to the top and covering with aglass plate so that practically no air was in contact with the solution. In this way avariation of̂ >H of more than 0-3 was avoided and in many experiments the alterationwas less.

The solutions with which the eggs were to be treated were contained in glassvessels with flat bottoms and vertical sides. Into each of these was fitted a smallsieve formed by a ring of celluloid over which was fastened a piece of fine boltingsilk with a rubber band. These sieves were found to be very convenient for handlinga large series of egg samples which require to be exposed to a long set of mixtures ofsalts for the same length of time. With care the time error can be reduced to a fewseconds. The sieves are taken to pieces and washed thoroughly after each experi-ment. Before being used for the first time all the size was removed from the boltingsilk by repeated washing. Careful control experiments were made which showedthat the eggs were not in any way influenced by the technique employed or by thematerials of which the sieves were composed. As soon as the correct time of exposureof the eggs is completed the sieve is lifted out, the fluid drains off quickly and theeggs are washed off the sieve in a finger bowl containing sea-water. As soon as theeggs settle at the bottom, the supernatant fluid is poured off and the bowl is filledwith fresh sea-water. All the sea-water used in these experiments was "outsidewater," that is to say, water which was collected in carboys from the open sea, out-side the breakwater. This had always, when used, been in the laboratory for two orthree days, and consequently contained no living spermatozoa. In all experimentscontrols were kept of unfertilised eggs in sea-water.

In each experiment the eggs of only one individual were used. In nearly allexperiments the eggs were examined alive but in a few cases the eggs were preservedin 5 per cent, formalin. Counts were made in almost every case of 300-1000 eggs,depending on the number available.

The temperature was not specially controlled. In different experiments it variedfrom 130 C. to 18° C. but in any one experiment the variation was less than i° C.

It should be noticed that in the following pages for the sake of convenience theterm "activation" when not otherwise qualified is used to designate the conditionof those eggs which have begun maturation but which have not undergone cleavage.Maturation is assumed to begin, for purposes of description, with the completedisappearance of the membrane surrounding the oocyte nucleus and not with theearlier stages of this process. Under the heading "activation" are included also alleggs which have completed maturation and developed asters but which, because ofthe abnormal form or number of these structures, have failed to divide.

In the experiments to be described the eggs were only allowed to reach theearlier cleavage stages although ciliated larvae can readily be obtained as the resultof artificial activation by the agents employed. When quantitative observations arebeing made on the effects of various agents in initiating cell division it is importantthat the estimates should be based on the examination of as many eggs as possible.The counts are made much more easily during the earlier stages of developmentwhen the blastomeres are large. At this stage it is also easier to distinguish true

5-3

68 A. D. HOBSON

cleavage from the cases of fragmentation which are liable to occur in all experimentson artificial parthenogenesis.

THE ACTION OF ISOTONIC SOLUTIONS OF SINGLE SALTS.

The salts whose action on the eggs of Thalassema has been investigated are thechlorides of sodium, lithium, potassium, and calcium. The effect of the first threeof these salts on the unfertilised eggs is similar. When the eggs are exposed to thesolutions of sodium, lithium, or potassium chloride at the pH of sea-water forperiods up to 60 min. no visible effect is produced, either in the solution or afterremoval to sea-water. The eggs preserve the distorted shape which they possessedbefore the treatment, and there is practically no maturation. Strikingly different isthe effect of calcium chloride. Shortly after immersion in this solution all the eggshave become spherical and soon maturation begins in a varying number and con-tinues whether the eggs remain in the solution permanently or are transferred to

7 0 -

Minutes

Fig. 1. Action of isotonic calcium chloride. ©—® Cleavage. A—A Total activation. Controlin sea-water: 1*2 per cent, maturation; no cleavage. Date 1. x. 27. Temperature 14-5° C.

sea-water. A certain number of the eggs removed to sea-water begin cleavage sometime afterwards. The time factor is of prime importance in all these experiments indetermining the proportion of maturing eggs and especially the percentage ofcleavage. Cleavage may also occur among those eggs left permanently in the calciumchloride solution but the percentage is small and the dividing eggs do not usuallyproceed beyond the 2-cell stage. Table I illustrates the result of a typical experimentand Fig. 1 shows graphically the effect of the calcium chloride.

It is somewhat unexpected to encounter an instance such as the one here de-scribed in which calcium is an effective agent in inducing development while thealkali-metals, under the conditions of these experiments have no such action. It istrue that some other cells, such as the eggs of Echinus esculentus, are remarkably littleaffected by pure, isotonic solutions of sodium or potassium chlorides, but calcium

Isotonic Salt Solutions on Eggs of Thalassema neptuni 69

and magnesium chlorides have also little action under similar conditions. On theeggs of Thalassema, however, calcium has a very marked action although it isgenerally considered to be a substance whose effect is to decrease permeability towater and salts. The action of calcium chloride is not over-emphasised by theexperiment quoted. In many cases the proportion of activation induced exceeds90 per cent.

Table I.

Time ofex-

posuremins.

369

12IS3045

Calcium chloridepH8-z

Cleavage

0//o

0-41*28-i5\5O'S

18-4ii'4

Standarderror

0-42O'590-941'290-232-321'23

Totalactivation

/o

2'64-1

42*356-8I5'952-245'O

Standarderror

i*o61-071-712'8lI-I83-001-41

Sodium chloride£H8-2

Cleavage0//o

0000000

6 6 6 6 6 6 6

Totalactiva-tion %

o-5O'Oi-6O'OO'OO'OO'O

Lithium chloridepH8-2

Cleavage%

0000000

0000000

Totalactiva-tion %

O'Oo*60-4o-5O'l0-40-3

Potassium chloridepHS-2

Cleavage0//o

0000000

0000000

Totalactiva-tion %

0-3O'OO'OO'OO'OO'O0-3

Control in sea-water: i#2 per cent, maturation; no cleavage.Date 1. x. 27. Temperature 14-5° C.

An experiment may be quoted which shows that pure isotonic potassium chlorideis not entirely devoid of action on the eggs. The material used in this experiment wasunusual in that it formed the only exception encountered to the general rule that thegreat majority of the eggs of this animal do not mature when left in sea-water. Inthe control batch of eggs left in sea-water just 50 per cent, underwent maturationbut developed no further. That the eggs were otherwise normal was shown by thefact that 100 per cent, perfect larvae were obtained from another lot which werefertilised. It is possible that under natural conditions if the eggs are depositednormally by the ripe female they are capable, like those of other Annelids, of be-ginning maturation in the sea-water before fertilisation. If this is true it furnishesan explanation for the exceptional behaviour of an otherwise normal batch of eggs,which may have been on the point of being extruded by the female. The eggs whichwere used for the experiment were immersed in isotonic KCl^H 8-i. At intervalsbatches were removed. Some of each batch were placed in sea-water free from spermand some were placed in another dish with sea-water and inseminated immediately.The results are shown graphically on Fig. 2. It will be seen that the potassiumchloride had an immediate inhibitory effect on both the spontaneous maturation andon the capacity for fertilisation of the eggs. Two minutes* exposure caused the pro-portion of maturation to fall from 50 per cent, to 14 per cent., a value which remainednearly constant even after 60 min. treatment with KC1. It may be presumed thatthis value represents the percentage of maturation already attained at the beginningof the experiment. The graph representing fertilisation capacity shows a sudden fall

A. D. HOBSON

from ioo per cent, to about 80 per cent, after 2 min. exposure to the KCl. Thisvalue is maintained until after 8 min. exposure when the graph descends again to avalue of 2-5 per cent, after 60 min. exposure.

The reaction of the eggs to treatment with isotonic calcium chloride is in everyrespect different from that described for the alkali-metal chlorides. The eggs becomespherical almost at once and the fertilisation membrane begins to separate in a fewminutes. After about half an hour the nuclear membrane begins to disappear.Polar bodies are formed by many eggs and probably by all which are capable ofundergoing cleavage. The time at which cleavage takes place is very variable but it

100*-

90

30

Minutes

Fig. 2. Action of isotonic potassium chloride. A—A Total activation of unfertilised eggs.W—V Cleavage of eggs inseminated after treatment with potassium chloride. A few of theunfertilised eggs divided. The highest proportion was o-8 per cent, for 8 min. exposure.Control in sea-water: 50 per cent, maturation; no cleavage. Date 9. vii. 27. Temperaturei6-5° C

does not usually begin until two or three hours after activation. The period beforecleavage is characterised by a considerable degree of pseudo-amoeboid activity onthe part of the eggs such as has been described by various authors (e.g. F. R. Lillie,1906) for the eggs of other animals. The cytological details of the events culminatingin cleavage have yet to be worked out by means of preserved material. A few seriesof sections have been cut of eggs activated by means of calcium chloride and fixedin corrosive-acetic. These indicate that the period of pseudo-amoeboid activitybegins when the nuclear wall is breaking down, preceding somewhat the develop-ment of asters, but the movements of the cytoplasm continue until shortly beforecleavage. Excessive treatment with calcium chloride frequently inhibits cleavagethrough the development of too many asters (cf. Herlant, 1918). Another type of

Isotonic Salt Solutions on Eggs of Thalassema neptuni 71abnormality due to over-treatment is the formation of a large monaster which com-pletely fills the uncleaved egg or the blastomeres of one which has cleaved. Fre-quently uncleaved eggs are found to contain several nuclei in a resting conditionsimilar to those described by Lefevre (1907) in the artificially activated eggs ofThalassema mellita which this author suggests arise as the result of multipolarmitoses. Differentiation without cleavage has not hitherto been observed. In thisrespect and in the various types of abnormal behaviour of artificially activated eggsThalassema neptuni shows a remarkably close resemblance to Thalassema mellita asdescribed by Lefevre.

A remarkable and characteristic feature of these experiments is well shown byTable I and Fig. 1. When the eggs are treated with the activating solution forvarious periods of time it is found that there are two optimal times of exposure.As will be shown later, this is true not only when the activating solution is composedof pure calcium chloride but also when a mixture of two salts is used. The firstmaximum usually occurs at about 9 min. exposure and the second at about 30 min.or more. Discussion of this matter is reserved for a later part of this paper but it maybe pointed out here that the first optimal time for cleavage as a rule slightly precedesthat for activation. The second optimal time appears to be the same for both cleavageand activation but this may be due to the longer intervals in this part of the experi-ment.

Calcium chloride does not inhibit rapidly the capacity for fertilisation of theeggs.

Table II shows the results of an experiment made to investigate this point. Theprocedure was similar to that already described for the experiment demonstratingthe inhibitory action of potassium chloride.

Table II.

Time ofexposureto CaCl2£H8- 3mins.

369

12

15304560

Unfertilised eggs

Cleavage

/o

O'O2-8O'O0-3O'O

14-624-932-8

Standarderror

0-72

0*29

2-482'536 1 6

Total activation

0//o

O'O

42'356-855'552-845-663-858-7

Standarderror

2-142-482-632-663'5°2'8l

6-58

Eggs inseminated afterreturn to sea-water

Cleavage

0//o

IOO'O

97*491*358-046-052-564-866*3

Standarderror

o-77i*333-932-763*593*44470

Control in sea-water: no maturation or cleavage.Date 27. ix. 27. Temperature 14-0° C.

Inspection of the figures in the above table shows that the percentage of cleavagein the eggs inseminated after the treatment with calcium chloride exceeded that ofthe uninseminated eggs. Hence a considerable proportion of the eggs which wouldnot otherwise undergo cleavage are capable of being fertilised and dividing. In

72 A. D. HOBSON

other words, the period during which fertilisation is possible does not end whenmaturation begins.

THE ACTION OF ISOTONIC SOLUTIONS CONTAINING TWO SALTS.

If the activating solutions contain a mixture of two salts the results are morecomplex than those described above. The nature of the salts and their relative con-centrations are of great importance in determining the proportion of activated eggs.In general a higher percentage of cleavage is obtained as the result of treatment withthe most favourable binary mixtures than with calcium chloride alone. The cleavageis also, as a rule, more normal and the embryos are consequently more healthy.The morphology of development appears, however, to be closely similar although

60 -

Ca only ' ° 1 5 6 ' ° 6 2 5 *25 i'O 4#0

*-«• "r",y . 0 3 1 2 5 , 1 2 5 #5 2.o 8 . 0 3 2 . 0

Ratio K/CaFig. 3. Action of isotonic mixtures of potassium and calcium chlorides. ©—© Cleavage.

A—A Total activation. Six minutes' exposure. Control in sea-water: 4*7 per cent, maturation;no cleavage. Date 29. viii. 27. Temperature 17-1° C.

this has yet to be confirmed in detail. Polar bodies are certainly formed in mostcases. The various types of abnormalities obtained resemble those found as theresult of treatment with pure calcium chloride.

The activating power of mixtures of calcium chloride with the chloride of analkali-metal shows a remarkable variation corresponding with the relative concen-trations of the two constituents. Table III, and Fig. 3, illustrate the results obtainedby submitting eggs for six minutes to the action of a series of mixtures of potassiumand calcium chlorides. For the sake of completeness the values for pure calciumchloride and pure potassium chloride are given in the table, and are inserted inthe graph joined by broken lines although, strictly, the scale used forbids their

Isotonic Salt Solutions on Eggs^ of Thalassema neptuni 73inclusion. The results obtained in different experiments vary considerably but certaingeneral features can be distinguished. With series of potassium-calcium mixturesten experiments have been made. In two of these little or no development took place.In one other development occurred as the result of treatment only with mixtures inwhich the K/Ca ratio lay between 0*5 and 32. The remaining seven experimentsgave results similar to those illustrated.

Table III.

Ratio K/Ca

0-01560-031250-06250-1250-25o-5i-o2-O4'O8-o

16-032-064-0Pure KClPure CaCl2Control insea-water

pU

8-158-158-io8-258-208-208-208-io8-208-258-208-208-158-208-io

8-2O

Cleavage

%

P6-2

23-719-824-42O-8

2-610-717-114-5o-o0-7o-o0-3

19-0o-o

Standarderror

1-321-252-282-272*372*230-85i-662-231-87

0-41

0-303'4i

Total activation

0/./o

34'O41-245'753*447'93967-5

19*426-518-52-3473*i4*3

49-3

4*7

Standarderror

2-802-552*672-842-762-691-412-132'6l2-070-761-030-971-134*351*19

Date 29. viii. 27. Temperature 17*1° C.

Pure calcium chloride is effective in inducing cleavage but its action is greatlyinhibited by the addition of a small amount of potassium. The graph then rises to amaximum and falls sharply when the K/Ca ratio reaches the value of i-o. As theK/Ca ratio rises still further the proportion of cleavage again reaches a maximumbefore decreasing again to a low value as the mixture approximates more and moreclosely to pure potassium chloride.

The relative ineffectiveness of mixtures in which the concentrations of calciumand of potassium ions is approximately equal is characteristic. This point is wellmarked in the experiment quoted. That the fall in the middle of the curve issignificant can be seen by comparing the value for cleavage as the result of treatmentwith the mixture in which K/Ca = i-o with the two adjacent figures. In one case(K/Ca = 2*0) the difference is over four times the standard error of difference; inthe other (K/Ca = 0-5) the difference is over seven times the standard error ofdifference. The position of this dip between the two principal maxima of the curveis not constant but it remains approximately central.

The graph showing the total activation is in most respects similar to that justdescribed for cleavage. Two points may however be mentioned. There is a tendency,especially for that part of the series in which the calcium concentration is high, forthe maxima of the cleavage graph to fail to coincide with the maxima of activation.

74 A. D. HOBSON

This may perhaps be compared with the somewhat similar phenomenon describedfor eggs treated with pure calcium chloride, although in one case the varying factoris time of exposure and in the other the proportion of potassium. The second pointto be noticed is that high concentrations of calcium generally cause more activationin proportion to the cleavage induced than high concentrations of potassium.Scott (1906) concluded that in Amphitrite calcium tends to stimulate nucleardivision and inhibit cleavage while potassium tends to promote cleavage.

Few experiments of this type have as yet been made with sodium and lithium.Those which have been performed indicate that sodium and lithium are less effectivethan potassium in promoting activation in the presence of small concentrations ofcalcium. The graph thus tends to show a single maximum in the region where thealkali-metal/Ca ratio is low. Lithium seems to resemble potassium more closelythan does sodium.

MinutesFig. 4, Action of isotonic lithium-calcium mixture (Li/Ca = 0-125). ®—® Cleavage. A—A Total

activation. Control in sea-water: no cleavage or maturation. Date 22. ix. 27. Temperature14-5° C.

The time factor is of great importance in determining the proportion of eggsactivated by exposure to mixtures of salts. The time curve has a form closelyresembling that already described for pure calcium chloride. This can be seen byinspection of Table IV and Fig. 4. Table IV gives the results of an experimentundertaken to compare the effects of potassium, lithium and sodium-calciummixtures in which the alkali-metal/Ca ratio was high.

Table IV also illustrates the fact mentioned above that sodium and lithium arenot effective activating agents when their concentration is high compared with thatof calcium. In this example however the difference is more striking than is oftenthe case. Fig. 4 shows the time curve obtained in another experiment in which alithium-calcium mixture (Li/Ca = 0-125) w a s u s e d - The similarity between thiscurve and the curve shown in Fig. 1 is apparent, the only difference being in detail.

Isotonic Salt Solutions on Eggs of Thalassema neptuni 75

DISCUSSION.

Although a very large number of methods have been described for causingartificial parthenogenesis and in many cases solutions of various electrolytes havebeen employed, yet the action of isotonic salt solutions has received little attention.Hypertonic salt solutions have long been known to induce development of the un-fertilised eggs of a considerable variety of animals.

Table IV.

Time ofex-

posureinins.

369

1315

456o

Ratio K/Ca=i6pH 8-15

Cleavage

o//o

o-oo-3

15*530-5

4-5

13-8

Standarderror

0-161-331-350-690-651-480-50

Total activation

0//o

O-20-7

37.3

86-889-998-3

Standarderror

0-170-351-501-531-441-381-390-51

Ratio Li/Ca =16pH 8-15

Cleavage0//o

o-oo-oo-oo-oo-oo-oo-io-o

Totalactivation

/o

o-io-oo-io-i

3-Oo-io-6

Ratio Na/Ca =16pH 8-15

Cleavage/O

00000000

00000000

Totalactivation

/o

0-3o-iO-3O*3

O-30-3o-o

Control in sea-water: no maturation or cleavage.Date 3. x. 37. Temperature 15*0° C.

The papers dealing with this method are too numerous and well known to bedealt with here. A comparatively small number of observations has been made onthe action of salt solutions in the absence of any considerable changes in osmoticpressure. Most of these experiments, however, consisted in subjecting eggs to theaction of sea-water to which had been added varying quantities of salt solutions.It is evident from the experiments described in the earlier part of this paper that therelation between the eggs and the surrounding medium may be complex even inbinary salt solutions. It is, therefore, evident that the results will be very difficultto interpret if the experiment consists of the addition of salts to sea-water which isalready a complex and delicately balanced mixture. Slight alterations in the com-position of these salt-solution-sea-water mixtures may be expected to show greatdifferences in effect and when the additional variable presented by the eggs ofvarious species of animals is considered it is not difficult to see that marked qualita-tive differences may appear. It might be expected that the observed differences in theeffect of a certain salt on the eggs of various species of animals would be quantita-tive rather than qualitative. Apparently qualitative differences may, however, beessentially quantitative or may be indirectly the result of quantitative alterations.The investigation of the activating effect of salt solutions on unfertilised eggs must,therefore, be based on a thorough examination of the action of single salts in isotonicsolution and then of binary mixtures of these salts before the more complex con-ditions in mixtures of three or more salts can be understood. The work of R. S.Lillie (1910, 1911a, 1911 b) is, to the writer's knowledge, the only systematic

76 A. D. HOBSON

attempt which has been made to examine the effects of isotonic salt solutions incausing artificial parthenogenesis. He, using the eggs of Arbacia punctulata andAsterias forbesii, found that isotonic solutions of sodium and potassium salts wouldinitiate development. Lithium chloride behaved like sodium chloride. Pure mag-nesium and calcium chlorides would not cause development and inhibited the actionof sodium and potassium salts when mixed with them. Pure strontium chloride alsofailed to cause parthenogenesis. Lillie's interpretation of these results was thatsodium and potassium salts caused increased permeability while the opposite effectwas produced by the alkali-earth salts. This view was supported by the diffusion ofpigment from the eggs of Arbacia treated with pure sodium and potassium salts andthe prevention of this process by calcium and magnesium salts. His experimentsare, however, open to the objections that the hydrogen ion concentration was un-controlled and that he employed only a limited number of mixtures of sodium orpotassium with calcium or magnesium. The present writer has, moreover, shownthat calcium chloride alone may be very effective in causing artificial parthenogenesisof the eggs of Asterias rubens (1927).

The eggs of Annelids seem to be more susceptible to the action of salt solutionsthan those of Echinoderms. Mead (1896) was the first to show that the eggs ofAmphitrite could be induced to form polar bodies by the action of sea-water towhich a small quantity of potassium chloride had been added. Loeb (1901) foundthat potassium had a specific action on the eggs of Chaetopterus; whereas other saltswould cause development when added to sea-water if thereby the mixture was madehypertonic, potassium chloride was effective in the absence of any increase in thetotal salt concentration. Potassium chloride added to sea-water, usually in hyper-tonic solution, has also been employed as a means of causing artificial partheno-genesis by various other investigators such as Treadwell (1902) working withPodarke, Fischer (1903) with Nereis, Bullot (1904) with Ophelia, F. R. Lillie (1906)and Allyn (1913) with Chaetopterus and Scott (1906) with Amphitrite. Experi-ments have less frequently been made with calcium salts but it is noteworthy thatFischer (1902) and Scott (1906) both found that addition of normal (i.e. approxi-mately isotonic) calcium nitrate to sea-water would cause development of the un-fertilised eggs of Amphitrite. Lefevre (1907) did not obtain satisfactory results bythe action of salt solutions on the eggs of Thalassema mellita.

The work of Horstadius (1923) is also of interest. He found that the eggs ofPomatoceros normally begin maturation when they are extruded into the sea-wateralthough the process is not completed unless fertilisation takes place. Maturationis inhibited in calcium-free sea-water and is promoted in the presence of excesscalcium. The effect of potassium is opposed to that of calcium. The percentage ofmaturing eggs varies inversely as the concentration of potassium.

The account given above of the previous work on the action of salts on un-fertilised eggs does not pretend to be exhaustive but merely indicates the mainresults obtained by investigators in the past. It is evident from their results thatunder certain conditions both potassium and calcium salts can activate unfertilisedeggs, or it might be wiser to say that the addition of potassium or of calcium salts in

Isotonic Salt Solutions on Eggs of Thalassema neptuni 77

certain amounts to sea-water may produce a mixture capable of inducing artificialparthenogenesis. Activation may depend in certain cases on the absolute concen-tration of a particular ion or in other cases on the relative concentrations of two ormore ions to each other. Unfortunately the evidence provided by previous accounts,with a few exceptions, is too limited and unsystematic for any definite conclusionsto be drawn.

Cleavage in Thalassema neptuni is directly continuous with maturation. Thedifference between a stimulus causing maturation only and one which causes cleavageseems to be quantitative. A similar conclusion has been reached by Allyn (1913) forChaetopteras. Within the limits of the experiments described in the present paperthe eggs could not be activated in the absence of calcium. This supports the con-clusion of Horstadius (1923) that calcium favours and is necessary for maturation.The presence of certain concentrations of an alkali metal increases the proportionof both maturation and cleavage, showing that calcium is not the only factor con-cerned. The discovery of two markedly separate optimum potassium-calciummixtures suggests however that the change resulting in activation may be accom-plished in different ways. If, as R. S. Lillie considers, the essential change involvedin the activation of an egg is an increase in permeability to water and salts, it isevident that this is a more complex problem than has hitherto been supposed. Itwould not be difficult to construct a hypothesis whereby could be explained theaction of excess calcium in increasing the permeability to water of the surfacemembrane of the egg but it would be unprofitable to do so until more data areavailable.

The rapid attainment of a spherical shape by the eggs of Thalassema neptuniwhen they are fertilised is of interest as it is probably an optical demonstration of anincrease in the permeability to water of the surface membrane of the egg. Thischange occurs almost as rapidly after artificial activation as after fertilisation. It isconceivable, of course, that a true explanation is to be found in some other changein the physical properties of the thick vitelline membrane. Nevertheless, in view ofR. S. Lillie's (1916) experiments in which an increased permeability to water wasshown to succeed natural or artificial activation in the egg of Arbacia, it seems highlyprobable that the interpretation suggested above is correct.

It is noteworthy that cytolysis does not take place rapidly as the result of treat-ment with isotonic solutions of sodium or potassium chlorides. Only after severalhours is any considerable amount of cytolysis to be seen even if the eggs are allowedto remain in the solutions. This seems to show that the permeability of the eggmembrane to salts is not markedly increased by this treatment.

The relation between the time of exposure of the eggs to the salt solution and thedegree of activation induced presents some points of interest. An important factoris the number of accessory asters formed. Eggs treated for long with calcium chloridealways develop several accessory asters and Herlant (1918) has shown that this con-dition leads to suppression of cleavage. It is significant that the optimum time ofexposure tends to precede that for activation, in view of the similar relationshipfound by Herlant between cleavage and "polycentrie" in Paracentrotus.

78 A. D. HOBSON

I wish to thank the Royal Society and the British Association for the use oftables at the Marine Laboratory, Plymouth. I am indebted to the staff of theLaboratory for their unfailing courtesy and help. I am especially grateful to Pro-fessor J. H. Ashworth, F.R.S., and to Dr E. J. Allen, F.R.S., for their helpful interestin the work.

SUMMARY.

1. The eggs of Thalassema neptuni are stored in the nephridia and, as the resultof their compression by the muscular walls of these organs, are distorted in shape.This distorted shape is maintained until maturation begins. Activation, whethernormal or artificial, is accompanied by the adoption of a spherical shape by the egg.This change probably indicates an increase in the permeability of the egg membraneto water and forms a useful index of activation.

2. Artificial parthenogenesis of the eggs of Thalassema neptuni can be inducedby means of isotonic salt solutions.

3. At the hydrogen ion concentration of sea-water the chlorides of sodium,lithium, and potassium are incapable of causing development of the unfertilisedeggs, but calcium chloride is an efficient activating agent.

4. Mixtures of calcium chloride with the chloride of an alkali-metal in certainproportions cause parthenogenesis.

5. For series of potassium-calcium mixtures two maxima for activation areobtained, one where the calcium concentration is high and one where it is low.Sodium and lithium in presence of low concentrations of calcium are much lesseffective than potassium.

6. For all the activating solutions tested two optimal times of exposure arefound at about 6-9 min. and 30 min.

REFERENCES.ALLYN, H. M. (1913). Biol. Bull. 24, 21.BULLOT, G. (1904). Arch. Entwm. 18, 161.FISCHER, M. H. (1902). Amer.Journ. Physiol. 7, 301.

(*9°3)- Ibid. 9, 100.GRIFFIN, B. B. (1899). Journ. Morph. 15, 583.HERLANT, M. (1918). Arch. d. Zool. Exp. et Gen. 57, 511.HOBSON, A. D. (1927). Proc. Roy. Soc. Edin. 47, 94.HORSTADIUS, S. (1923). Arch. Entwm. 98, 1.LEFEVRE, G. (1907). Journ. Exp. Zool. 4, 91.LILLIE, F. R. (1906). Journ. Exp. Zool. 3, 153.

(1919). Problems of Fertilization. Univ. Chicago Press.LILLIE, R. S. (1910). Amer.Journ. Physiol. 26, 106.

(1911 a). Ibid. 27, 289.(1911 b). Journ. Morph. 22, 695.(1916). Amer.Journ. Physiol. 40, 249.

LOEB, J. (1901). Amer.Journ. Physiol. 4, 423.MEAD, A. D. (1896-1897). Woods Hole Biol. Lects.SCOTT, J. W. (1906). Journ. Exp. Zool. 3, 49.TREADWELL, A. L. (1902). Biol. Bull. 3, 235.