experimental distortion of development in amphibian tadpoles

Experimental Distortion of Development in Amphibian TadpolesAuthor(s): Dorothy E. SladdenSource: Proceedings of the Royal Society of London. Series B, Containing Papers of aBiological Character, Vol. 106, No. 744 (May 3, 1930), pp. 318-325Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/81403 .

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318

591I 392. I 597 8 R aa

Experimental Distortion of Development in Anyhibian Tadpoles.

By DOROTHY E. SLAPPEN, Zoology Research Department, ifmperial College

of Science.

(Oommunicated by E, W, MacBride, F.R.S.-Received March 18, 1930.)

[PLArTP.q 25-27.1

Introduction.

In 1908, Tornier published a paper (1) on the probable causes of the formation

of the abnormal " fancy " races of goldfish. All these races originated in

China where the wild ancestor (Carausius auratus) still abounds in the streams,

It had been suapposed that these races were produced by a secret process known

only to the Chinese breeders. The fish during winter were kept crowded in

earthenware pots, on shelves in dark and insanitary huts; in summer they were

transferred to small and filthy tanks overgrown with weeds. In these tanks

they spawned and mueh of the spawn perished; amongst the fraction whichJ

s urvived, however, all sorts of abnormalities were found. By selecting the

mlost striking of these, the breeders secured the parents of their " fanecy "

breeds, which showed in every succeeding generation a stro:ng tendency to

revert to the normal; only by the most rigid selection was anything like a

pure '" race obtained. Tornier drew the conclusion that the abnormalities were due to the effects

of lack of oxygen in very early stages of development. This lack induced

wlhat he called "' plasma-weakness " in different parts of the formative are(a

of t,he very young embryo. In consequence of these localised areas of weakness

th-e protoplasmic part of the egg was liable to mechanical distortion, indulced

by abnormal pressutre from the yolk; the latter absorbed water and swelled,

thus crushing and destroying the protoplasmic structures from which the

future organs of the adult are formed. These abnormalities once produced were, according to Tornier, hereditary,

biut were not transmitted to the next generation as a " factor " or " gene " for

a speciefi abinormality (e.g., a double tail or a bulldog face) but as a degree of

plasma-weakness, which in every generation produced mechanically the same

morphological results. If, however, the race was replaced into a normal

etivi rornment the weakness tended to disappear after a certain numi ber of

generatious.



Amphibian Tadipoles 819

Tornier attempted to test these conclusions by performinig experinie'tts with the eggs of a large newt, the Axolotl (Siredon piscifornis). HIe sulbj ectcd

the eggs to darkness, which prevented the planits in the water froni producinlg

oxygen, and then placed them for 48 hours in a 5 to 10 per cent. solution of sugar, which he assumed would act as an absorbenit of ainy oxygeni present in. solutioni. In this way he obtained embryos which exhibited all the phenomen1a of " plasma-weakness. " The axis of the emnbryos in question had been shortened and in some cases bent inwards in a concave curve, the head in consequence bent upwards and the mouth cavity anid gill-slits enlarged. He succeeded iti

rearing a few of the embryos to an advanced larval stage. The larva3 had

swollen abdomens, like those of the " Ianchu," a race of fanicy goldfish, When these abnormal specimens were dissected. it was seen that the abdoien was swollen with water, and that the liver had consequeu-tly been impeded in its growth and reduced to less than a quarter of its normal size.

By applying his mrethods to frogs and toads Tornier was able to get further results. In these later cases he used '25 per cent. solution of sugar for a shlorter time. The yolk plug which. niormally fills the blastopore became enormously enlarged and prevented or greatly impeded the growth of the tail.

Berndt (3), who was attracted by Tornier's views, experimented with the "'fancy " breeds of goldfish aild endeavoured to show that the marks of their races were not due to general weakness, as Tornier supposed, but to a specific weakness separately inheritable. Berndt confirms Tornier's idea that life in aquaria leads to rapid degenerationi of stock, buit he does not agree with the latter's tlheory of " yolk pressure," as he found the swelling of the body-cavity might only appear in late stages, from which yolk was absent, the young forms showing no signs of it, aind the goldfish with swollen abdomens (characteristic of certain breeds) appear. to owe the swelling to enlarged ovaries and fat masses. and niot to fluid.

At Prof. MNfacBride's suggestion I repeated- rTornier's experiments in 1928, usinlg the eggs of the commonio frog (Ra,,na [emtporaria) and subjecting themi., at the end o-f segmlenLtatioii, to a 1.0 per ceit. solution of sugar -for 4 hours. At the timef of hatching only a few of the larvw were (abnormal, the principal type of abnormality being the swolleii body-cavity. Without exceptioii these larvL- died slhortly after hatching, leaving onily those which appeared perlfectly niormal. These proceeded to de-velop until jUst before the hind limbs were due to appear (abouti 8 weeks after ha-tchinig), when in a few cases a bend appeared at the base of the tail. These abnormalities were followed by others appearinig at intervals during the Inext four mnoniths, showing the same type )of deformity.



320 D. E. Sladden.

During the followinig year, 1929, experiments were againi carried out with the eggs of Ranta temporaria, this time subjecting them to reduced air pressure. 'I'his experiment gave the same results as that of the previous year, the majority of abnormals appearing as before at a fairly advaniced stage of development. In this case, however, a larger percentage of tadpoles were affected in their advaniced stages. Anotlher batch of eggs was treated with 25 per cent. sugar solution this year, for the same leingtlh of time---namely 4 hours. Also the experimenit of the previous year, using 10 per cent. sugar solutioni, was repeated.

The table givein below shows the percentage of abnormalities obtained ill 1929, from the three experimenits described above, at time of hatchilng anld 5 months later. In the latter case, the percentage is of the total inumnber of living Iarvee, anid niotlani additioinal percentage to that taken at tinme of ha tchling. As already stated, the early abnormals died during the first month of larval life.

Table.- Tercentage of Abniormalities, 1929.

RIteduced 10 25 air Costrol. per cent. Conitrol. pe celt (Jontrol. pressure. ~sugar jsugar pressure. solution. solution.

Per cent. Per centt. Per cent. Per cPiit. Pew cent. Per cent. At time of hatching .... 0 8 3-5 3-2 |e2 1P10 O-0 7

5 nionthe later ............ 9 0.0 6 0.0 2 0.0

Technique.

In 1928 several artificial fertilisations were carried out with the eggs of Rana temporaria previous to treatmenit with sugar, but the best results were obtained froml a pair of frogs in which the eggs were directly fertilisedt. In this case the larval deatlh-rate was conisiderably redluced, anid therefore these egg.s wvere used for the following experiment.

The spawn, 24L hours after fertilisationi, was divided into two, one half beinig subjected to a 10 per cent. solution of cane sugar in tap-water for 4 houLrs, and the other lhalf kept; under nor;mnal conditions to act as conitrol. Tlhe sugar solutioni was stirred at frequeint initervals during the time it conitainied the spawn, to maintain an equal concenitration. After 4 hours the spawn was rinsed in cleani water and tranisferred to running water until the larve hatched. 'rhe niewly hatched larvme were transferred to aquaria containing " still " tap- water and the necessary conlditionis for their development during larval life. ARl l' inetamiorphoses; tlhe youlng frogs were kept in vivatria.



Amphilnb,au Tad _poles. 321

T'he following year (1929) directly fertilised spawn was again useJd, Three. sets of experiments were carried out. In the case of the first set, the spawin, 24 liours after fertilisationi, at the same stage of development as in the previous

year, niamely, at the end of segmenitation, was kept in distilled water, in an atmosphere which was maintained at 300 mm. (of mercury) below the atmio- spheric pressure at the time, for 4 hours by means of a water-vacuum pump. T 'le eggs were then transferred to " still " tap-water aind allowed to develop under the same normal conditions as the control.

The experiment with 10 per cent. sugar solutioin of the previous yea] was repeated, and in additioni a 25 per ceint. sugar solution was used on atnotlher batclh of eggs. The technique of the secoind aind third sets of experinmenits was the same as that of the previous year. In all three cases some of the eggs were used as controls.

How far treatment with sugar solutionis, or exposure to an atmosphere of red;uced pressure, actually influences the oxygeni consumption of the eggs----

as claimed by Tornier-remains to be determined. For immediate purposes, lhowever, it is more important to kvow precisely the results of such treatment thami the precise nature of their underlying causes.

Results.

Wheni the eggs treated with 10 per cent. sugar solution hatched, the greater perceintage of larva) were niornmal. Some died immediately onl hatchiing, but of the survivors a few slhowed marked abnormalities of a definite type, namely, the swollein body-cavity described by Tornier (1 and 2) and Berndt (3) (Plate 25, fig. 2). In a few cases this was accompanied by a bend at the base of the tail (Plate 25, fig. 1). When these larvaw with the swollen abdomens were see- tioned, it was found that the yolk in the gut had expanded----possibly due to the absorption of water as Tornier affirmed---rupturing the walls of the gut and extruding into the body-cavity (Plate 27, fig. 9). In some cases the body- cavity was also swollein the liver and pronephros being considerably reduced. WVith few exceptions these larva) died shortly after hatching, anid those which survived did so only until their supplies of reserve niutriment were exhausted,

wheni, beinig uniable to feed (due possibly to a defect of the alimenitary canal, or, owing to the distention of the body-cavity in cases in wlihl it occurs, it was not possible to bring the mouth in contact with the food), they also died.

Witlhout exceptiomi the newly-hatched abnornmal larva) were seen to lie oni the floor of their taink instead of attachinig themselves to submerged objects in the usual way by mnsctlis of the adhesive organs or suckers. This is due to



322 D. E. Sladden.

the ffact that these organs are conisiderably reduced int size in anl abnornal larva (PJlate 27, fig. 11) as compared with those of a normal one of the same age (Plate 27, fig. 12). In addition to this fact, the adhesive cells are either eintirely absent or, if presenit, not sufhiciently well developed to penetrate the epidernlis and so reach the exterior. The adhesive organs are therefore rendered functionless.

After the above-menitioned abnormal larv died, there remained only those which to all external appearances were perfectly normal at the time of hatchin g. These conitinued to develop in the usual way, over a period of 8 weeks, -until a fairly advaineed stage of development was reached, namely, that at which the hind limbs appear. At this time one or two of the larvae developed a slight tendenicy to swinm in a circle, and on beinig examined more closely the, cause was found to be a bend at the base of the tail, simi'lar to that observed in the younger stages, but to a lesser degree. Such abniormalities as these continued to make their appearance during the next 4 months. The bend in some cases flexed the tail to the right and in others to the left.

As time went on and more individuals appeared showing the same defect, it became clear that the longer the period between the time of hatching and the first external evideince of the bend, the greater was the defect (Plate 25, figs. 3 and 4). Thus the frogs shown in figs. 6 and 8 (Plate 26) with extreme abnormalities were 5 months old before any trace of the defects became apparent in the tadpole.

When one of these later larv-e was sectioned (Plate 27, fig. 13) it was founld that on the inner or " concave ' surface of the bend, the muscle bunldles forming the myotomes were considerably shortened and irregularly arranged, while on the other surface the myotomic formation was perfectly regular and the muscle bundles of normal length. The younger stagre in which the bend was evident at the time of hatching showed a similar internal defect in the miuscle bundles in the region of the bend (Plate 27, ftg. 10).

Towards the end of larval life, in the case of the later stages described in, a previous paragraph, the sacral region of the tadpole commenlced to show sigIus of distortion in relation. to the bend in the tail. This gave rise in a newly metamorphosed frog to a dorsolateral projection or " hump " on the opposite side of the body to the bend. In cases in which the tadpole's tail was flexed to the right, the distortion was on the left side of the body of the metamorphosed frog.

The extent to which this distortion was carried varied, as already stated, in accordance withi the age of the larva. When a period of 5 months or over



Amphibian Tacdpoles. 323

elapsed between the time of hatching and the first appearance of the bend, the distortion was accompanied by a twistiug of the hind limb or pelvie girdle away from the projection (Plate 26, fig. 6). This had the effect of displacing the hind limbs, the left one becoming almost central and the other in contact with the right fore-limb, and vice-versa. This defect prevented the young frog from feeding.

In one case (Plate 26, fig. 8) the twisting of the pelvic girdle was sufficient to displace the left limb, preceded by the anus, to th.e larva's right side; the right limrb was suppressed altogether, an effect which may be due to compression in growth at an early and critical period of development. The projection, which

in this case was towards the larva's left side, was flattenied at its apex and in the young frog was often. brought inito contact with the ground, when in a resting position, to act as a support. This frog was unable to feed itself owing to the same reason given. above. Its movements were even more affected, as the only means of progression. was by a series of shortened leaps (always in an oblique direction) which greatly exhausted it, and often caused loss of balance. As in. the previous case, death followed closely on metamorphosis.

A section (Plate 27, fi.g. 14) transverse to the body and through the sacrnal region of the frog (see Plate 26, fig. 7) appears to have three distinct notochords intercepted by cartilage, but in a reconstruction of the same larva this w(as clearly due to the manner in which the notochord had become distorted. It was seeni to be prolonged up into the projection in the sacral region and then passed back again to the median line of the body, thus becoming almost bent on itself.

Twenty-six weeks after fertilisation a new type of deformity made its appearance among the few apparently normal larva in which metamorphoses were not completed. This took the form of a defect in the articulatior of the femur with the tibio-fibula, resulting in a stiffening of the afected limb (Plate 2 6, :fig. 5). In every case the hind limbs only were affected. This larva died 4 days later. Three other abnormalities of the same type followed, with either right or left limbs affected, and one with both. These four metamorphosed successfully.

Owing to the scarcity of the material it was decided not to sacrifice any of the young frogs in order to examine the internal structure of the affected limb, but this it is hoped will be done at a later date.

The above described abnorinalities are those obtained froTm the experiment, wit}l 10 per cent. sugar solution in 1928. Those resulting froma the experiments in the following year showed exactly the same types of abnormality, t.;ie only



324 D. E. Sladden.

difference beinig the percentage of larvm affected according to thIe nature of the experiment, as shown by the table on. p. 320. The percenttage of abnormalities at the time of hatching, as seenl. by the table, was highest in the case of the larva3 treated with 25 per cent. sugar solut-ion and lowest in the case of the larvae subjected to reduced air pressure. Five montlhs later, however, this condition was reversed..

N-o cases of stiffened. limbs Tave been obtained. thiis year (1929) by any of thle methoods employed.

Summary.

Eggs of Rana temporaria were exposed., at the end of segmentation, 24- hours after fertilisation, to a 10 per cent,t solution of stgcar in tap-water for 4- h.ours;

they were then transferred to normal aerated water. Thie res iltinlg larvam e:.hibited narked strutctural abnormalities, althouLgh these might not be obvious for a prolonged period, e.g., 3 to 4 montlhs after fertilisation. These ablnor- malities have been described as (a) distention of body-cavity ; (b) rupture of Cu.t and extrusion of yolk; (c) flexure of tail.; (d) distortionl of sacral region

(e) non-appearance of limb.

In. conclusion, I would like to express my deep gratitude to Prof. E. W. MIacBride not only for the great enco-urga,ement andl. assistance wich he lhas

given me while I have been. wor-king l1nuder his supervision, but also for htis

original suggestion, th.at this line of research could. be profitabl-y followed. 1 is improb.able that this work would have reached a successful colusIsio-11n withlout l.elp which Mr. H:. R. Hewer, MI.Se., has so freely given mi,e.

REFERENCES.

(I) Tornier, G., "Vorlauffiges iiber das Entstehen de:r a oldIlsebassen" 1Berlin S'il z.

1Ber. Qes. Natf. Freunde,' pp. 40-45 (1908).

(2) Tornier, (G., '0 b'her experimentelles Hlervorrufen nind Natuirentsftehen voni. Mopsk Pfen,

(Cyelopen sind anderen Vorgebtirtliehen -Kopfxterbildtinienll b)ei WiT; ri)erbtie-f enl"'

'Berlin Sitz. Bere. Ges. Natf. Freunde,' pp. 298-315 (1908).

(3) Ilerndt, W., " Vererburngs-stndien an Goldfischrassen." ' Zs. Indukt. Abstamngslehrv,

Berlin,' vol. 36, pp. 161-349, Plate 5 (1925).

DESCRIPTION OF PLATES.

The illustrations on Plates 25 and 26 are of tadpoles and. frogs f rom eggs treate(l with 1 0

per cent. sugar solution in 1928. The bodly-lengtb. is tha,%t of a straight line <Idrawn fri-om the

tip of tie -tose to the base of the tail. Age talken from timie of hatcbhing un-til fixed,



Slaciclcen. RVoy. Soc. Prsoc., B, vol. l106, Pl. 250.

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3 X

(Facintg p. :324.)



Slacdden. Roy. Soc. Proc., B, vol. 106, PI. 26.

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Slaciclen. Roy. Soc. Proc., B, vol. 106, PI. 27.

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Amphibian Tadpoles. 325

PLATE 25.

FiG. 1.-A tadpole showing flexure in tail owing to deformation of myotomes. Age 5 weeks and 2 days. Length of body, 3 mm.

FIG. 2.-A tadpole showing distention of body-cavity, X. Length of body, 8 mm. FIG. 3.-Limbless tadpole, age 20 weeks, showing flexure in tail. Length of body, 10 mm. FiG. 4.-Tadpole with four limbs present. Note the stiffening in the left hind limb and

flexure in tail. Body length, 11 mm. Age, 20 weeks and 2 days.

PLATE 26.

Fia. 5.-Metamorphosed frog showing stiffness in right hind limb, and slight distortion of sacral region. Age, 11 months. Length of body, 17 mm.

FIG. 6.-Metamorphosed frog. Length of body, 10 mm. Note distortion of sacral region and displacement of hind limbs. Age, 26 weeks.

FIG. 7.-Metamorphosed frog showing distortion of sacral region. Length of body, 9 - 5 mm. Age, 26 weeks.

FIG. 8.-Metamorphosed frog. Note twisting of sacral region and absence of right hind limb. Length of body, 10 mm. Age, 22 weeks.

PLATE 27.

FiG. 9.-Microphotograph. Trans. sect. of tadpole shown in Plate 25, fig. 2. Note the extrusion of the yolk into the body-cavity. x 43. n.G., nerve cord; n.ch., notochord; w.g., wall of gut; ex.y., extruded yolk.

FIG. 10.-Microphotograph. Trans. sect. through the sacral region of a 10-mm. tadpole showing a defect in the myotomes. x 32. n.c., nerve cord; n.ch., notochord;

g., gut; my., defect in myotomes. FIG. 11.--Microphotograph. Vert. sect. through sucker of an abnormal tadpole (cf. fig.

12). Note absence of adhesive tissue on surface of suckers. x 90. FIG. 12.-Microphotograph. Vert. sect. through suckers of a normal tadpole (cf. fig. 11).

x 90. ad.t., adhesive tissue. FIG. 13.-Microphotograph. Frontal sect. of a tadpole showing deformiation of tail

myotomes. x 24. n.c., nerve cord; n.ch., notochord; my., defect in myotomes. FIG. 14.-Microphotograph. Trans. sect. of frog shown in Plate 26, fig. 7. Note the noto-

chord sectioned in three places owing to distortion of the sacral region. x 28 n.c., nerve cord; n.xh., notochord.

VOL. CVI.-B. 2 A



experimental distortion of development in amphibian tadpoles

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