STUDIES ON AMPHIBIAN METAMORPHOSIS

XVI. THE DEVELOPMENT OF FORELIMB OPERCULARPERFORATIONS IN RAN A TEMPORARIA AND

BUFO BUFO

BY O. M. HELFF

Department of Biology, University College, New York University

(Received 10 July 1938)

(With Five Plates, containing Figs. 1-50)

INTRODUCTION

THE forelimbs of anurans are derived from small, hemispherical, bud-like rudi-ments situated at the posterior borders of the peribranchial cavities of the larvae.Histologically, they consist of compact masses of mesenchyme covered with one ortwo layers of flattened epidermal cells. The buds can be distinguished before thegrowth of the operculum occurs and hence prior to the formation of the peri-branchial cavity. In fact, Ekman (1922) has shown that the premature destruction offorelimb buds in no way inhibits the later development of normal peribranchialcavities. The operculum bounding the gill cavities arises as an integumentary fold oneither side of the head during late embryonic (early larval) development. Thesefolds, by a process of posterior migration and growth, gradually enclose the gillsand limb bud of their respective sides and eventually fuse with the body wallbehind and below the gill region. Thus the peribranchial cavities are entirelyenclosed with the exception of the spiracular openings which may be present on theleft side only as in the case of Bufo, Hyla, Rand and Pelodytes, or located ventral andmedial as in Alytes, Bombinator and Discoglossus. The position of the spiracularopenings would appear to be dependent on the respective growth rates of the leftand right opercular folds which determine the ultimate location at which the twofolds meet and fuse. Where the spiracle is on the left side, a narrow transversecanal connects the right with the left peribranchial cavity. A peculiar conditionexists in the African anuran Xenopus (Dactylethra), where spiracles are formed onboth right and left sides. The opercula, however, enclose only the gills, since theforelimbs arise from buds located externally as in the case of Urodeles or the hind-limbs of Amphibia in general.

During metamorphosis, the gills undergo progressive degeneration while thelimb buds differentiate and grow rapidly in size. The continued growth of thelimbs in time usually results in a distinct bulging of the opercular skin, especially

Studies on Amphibian Metamorphosis 97

where the elbow is exerting pressure. It is in this location that perforations eventu-ally form through which the forelimbs are released. Where the spiracle is on theleft side, the emergence of the corresponding limb is primarily through this opening,although considerable enlargement of this natural orifice frequently takes place. Inthose forms having a median-ventral spiracle, perforations are developed on bothright and left sides where the histolysis of the operculum and the emergence of thelimbs involve essentially the same processes. Prior to the actual appearance of aperforation, the opercular integument usually presents a more or less orderlysequence of macroscopic changes, although this is subject to some variation betweendifferent species. These include a lessening in pigmentation so that the area con-cerned in time appears yellowish or nearly white in colour. Frequently a typicaltranslucency is produced, and in later stages the integument may become quite thinand transparent. In certain forms the area to perforate is circumscribed by a thinwhite line before the enclosed integument has undergone any pronounced macro-scopic change. This is particularly true of R. catesbeiana and R. clamitans. The site offirst appearance of the perforation is usually where the elbow of the forelimb isexerting considerable pressure against the operculum. The initial perforationrapidly enlarges and the forelimb emerges. Usually, however, the perforation con-tinues to enlarge, especially anteriorly. The steps entailed in the enlargement of aperforation and the ultimate union of the adjacent opercular integument with theskin of the forelimb have previously been described in considerable detail (Helff,1926) and need not be discussed here."

The writer (1926) has shown that the formation of opercular forelimb perfora-tions is the result of a definite and orderly sequence of histological events whichtakes place in the integumentary areas concerned. These may be briefly summarizedas follows: The fibrils of the stratum compactum layer are first thrown into morepronounced convolutions than they ordinarily assume. The region appears com-pressed as evidenced by the increase in numbers of nuclei as compared with otherregions. This condition is shortly followed by a dissociation of the fibrils and aninvasion of blood cells, chiefly of the lymphocyte type. Following this, the stratumspongiosum undergoes dissociation and lymphocyte invasion. The resulting histo-lysis in time brings about the total destruction and removal of both stratum com-pactum and stratum spongiosum. Near the close of this process the subcutaneousconnective tissue and the internal epithelial lining also degenerate and are removed.The outer epidermis, which up to this time has remained peculiarly unaffected, nowbegin to histolyse, the lowermost layer of cells being broken down first. The histo-lysis progresses towards the cuticular side until finally only the cuticle remains. Atthis stage the forelimb usually ruptures the thin single-layered cuticle or, in theabsence of limb pressure, further histolysis soon removes the cuticular layer and soinitiates the perforation.

The first experimental work dealing with the causation of forelimb perforationswas that of Braus (1906) on Bombinator larvae. Braus extirpated the minuteundifferentiated limb buds from larvae 17-19 mm. in total length. The mortality'rate was so high that only three individuals survived subsequent metamorphosis.

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Of these, two developed definite opercular perforations while a thinning of the in-tegument was noted on the third. Autopsy showed that no regeneration of the fore-limb had occurred. A few years later, Braus (1909) obtained similar results with R.esculenta although slow limb regeneration was found to occur in this species. Theforelimbs so regenerated, however, never acquired a size sufficient to maintaincontact with the operculum. Schulze (1924) repeated the experiments of Braus onR. esculenta and obtained similar results.

The writer (1926) removed the forelimbs of fully grown tadpoles of threeAmerican species: R. pipiens, R. sylvatica and R. clamitans. The extirpated limbsvaried from 2 to 10 mm. in length depending on the species, but had never attainedthe size at which pressure is exerted against the operculum. In the first two of thesespecies perforations subsequently developed during metamorphosis, while thinningoccurred in many of the larvae of R. clamitans. These results confirmed those ofBraus and others in that presence of the forelimb is not essential to the formationof opercular perforations. The writer then investigated the effect of homoplastictransplantation of the limb bud beneath the integument of the back. Twenty-fivecases were obtained in which growth and differentiation took place during larvalinvolution. Of these, however, there were only three instances of perforationformation in the overlying back integument. Histological sections through theseperforations gave no evidence, however, that their occurrence had been due toprevious histolysis of the various integumentary layers as is characteristic of oper-cular skin during perforation formation. In fact, similar perforation of integumentwas obtained by the implantation of glass beads beneath the skin of the side, aresult attributed to simple "pressure atrophy". That the perforating area of theoperculum is not peculiarly specific to undergo histolysis during larval involutionwas determined by the reciprocal transplantation of opercular skin with that of theback and side. Opercular grafts in foreign locations failed to histolyse duringmetamorphosis, while back- and side-skin transplants grafted to the opercularregion underwent degeneration and perforation formation. It was finally found thatif atrophying gill tissue was transplanted beneath integument, the latter underwentrapid histolysis typical in all respects to that described as preceding the appearanceof normal opercular perforations. The writer concluded, therefore, that the atro-phying gills of the anuran are primarily concerned in initiating the histolysis ofopercular integument during metamorphosis, although the forelimb may have asecondary effect through pressure in hastening the actual appearance of a perfora-tion.

Weber (1931) has disagreed with the writer, as regards the importance of thegills, on the basis of results obtained following the removal of limb rudiments inBombinator pachypus and R. temporaria. Weber prevented the growth and differenti-ation of forelimbs by destroying the structure while still in the bud stage. Toaccomplish this a heated needle was first employed, but better results were obtainedby the use of an electro-cautery. During subsequent metamorphosis of larvae sotreated, a variety of results were secured. These included individuals developingpractically normal opercular perforations and those exhibiting no external macro-

Studies on Amphibian Metamorphosis 99

scopic changes or histological signs of integumentary thinning. According to thisauthor, the degree of opercular histolysis and perforation formation was directlycorrelated with the presence or absence of cutaneous glands within the peribranchialcavity. Weber states that where the cauterization had been complete, no remnantsof the integumentary coverings of the limb and shoulder girdle developed, resultingin the complete absence of cutaneous glands. In such individuals the operculumfailed to histolyse and perforate in spite of the fact that the gills underwent normalatrophy. Conversely, the presence of even a few microscopic glands, following in-complete cauterization, was sufficient to bring about thinning of the operculum andeven perforation formation in some cases. Thus, to Weber the normal causation offorelimb perforations is due to the activity of large cutaneous glands located in theintegument covering the limb and part of the shoulder girdle.

More recently, Blacher et al. (1934) and Liosner & Woronzowa (1935), workingwith R. temporaria and R. ridibunda, claim that perforation of the operculum is dueto a definite specificity of the integument. Their conclusions are based on the resultsof reciprocal skin transplantation between the perforation region of the operculumand the integument of the back and tail. Contrary to the results of the writer, theyobtained perforations during metamorphosis in opercular transplants previouslymade to the back and tail, while integumentary grafts from the latter regions refusedto perforate when placed in the normal perforation area. They furthermore statethat opercular perforations occurred following complete removal of the limb, andhence in those cases where no cutaneous glands persisted or developed as shown bysubsequent histological examination. These last results are therefore in directcontradiction to those of Weber on the same species (R. temporaria).

The histolysis of anuran operculum during forelimb perforation formation has,therefore, been accredited to the action of autolysing gill tissue, to cutaneous glandsecretions, and to self-degeneration of the operculum by the writer, Weber, andBlacher et al. respectively. Two possibilities present themselves to account for thevariance in results obtained and conclusions drawn by the above workers. The factthat different species were utilized might account for the discrepancies recorded, inthat the causative factors involved might not always be the same in the variousAnura studied. Secondly, it seemed quite possible to the writer that more than onefactor might be involved in any one species for the production of the perforation.The opportunity, therefore, to work on European species (R. temporaria and B.bufo) was welcomed by the writer, and the present paper contains the results ofexperiments performed in connexion with a leave of absence spent in the Depart-ment of Zoology at the University of Cambridge, England.

MATERIALS AND METHODS

A plentiful supply of R. temporaria and B. bufo tadpoles were obtained duringthe latter hah0 of June. The frog larvae were collected near Whittlesford and the toadlarvae in the vicinity of Cambridge. Since tadpoles collected so late in the seasonwould have all begun metamorphosis in from 1 to 2 weeks, it was necessary to

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inhibit the onset of involution in order to prolong adequately the time available foroperative work. This was accomplished by keeping the larvae in a cold-temperatureroom in large glass aquaria constantly aerated with compressed air. The stocks werefurthermore fed regularly with Spirogyra and the water changed once a week.Using these precautions, a sufficient number of larvae were maintained throughoutJuly and August for operative work.

For any one series of operations, the larvae were carefully selected as regardssize, absence of metamorphic signs, and general healthiness. In general, a 2%solution of ether was employed for anaesthesia. It may be stated here that thewriter has found ether to be definitely superior to chloretone, especially where aquick, safe anaesthesia is desired. There is little danger of overdosage if the larvaeare removed from the anaesthetic as soon as they cease to respond to tactile stimula-tion. Part of the effectiveness of ether is no doubt due to the coldness of the solutionwhich in itself tends to stupefy the larvae, while the non-lethality may be accredited,at least in part, to the rapid volatilization of the anaesthetic when the anaesthetizedanimal is placed in air for operative purposes. Chloretone solutions, if used inconcentrations weak enough to require 10-15 min. for anaesthesia, are recommendedin cases where a long operation is entailed. The immersion of the animal in freshwater every 2 or 3 min. during the first 10 or 15 min. following the operation is oftenvery helpful in counteracting the undesired accumulative action of the anaestheticwhich frequently proves to be lethal. Although operations involving skin transplan-tations required previous anaesthesia, the extirpation of forelimbs was usuallyperformed by simply holding the larvae between two pieces of damp cloth, theoperation being made through a suitable hole in the upper cloth.

Considerable difficulty was at first experienced in obtaining recovery, especiallyin the case of skin transplantation and regardless of the care taken to induceanaesthesia. Parenthetically, it may be stated here that both R. temporaria and B.bufo are decidedly more delicate and difficult to work with as compared with thelarger more resistant American species such as R. pipiens, R. palustris, R. clamitansand R. catesbeiana. It was finally found that the high mortality experienced was duenot to the operation performed but to the loss of lymph and blood during andimmediately following the operation. A method was consequently devised wherebyrapid congealing of these vital fluids was brought about by the application of astream of compressed air. The air is applied to the wound areas by means of a6 in. length of glass tubing, one end of which is drawn out and slightly curved. Theother end connects with a length of rubber tubing leading to a needle valve controlfor the compressed air supply. An arrangement whereby the operation of theneedle valve can be controlled with the foot is of considerable convenience in thatboth hands of the operator are thus free to work with the animal. In addition to themaking of otherwise lethal operations possible, the use of compressed air has thedecided advantage of greatly reducing and in some instances of nearly eliminatingwound areas. Thus, where reciprocal skin transplantations have been made and thetendency of the transplant to contract has resulted in a wide exposed wound areaaround it, it is possible gently to stretch the transplant with forceps and bring its

Studies on Amphibian Metamorphosis 101

edges in contact with that of the surrounding host integument. The stream of air isthen applied resulting in a congealing of the lymph and a drying of the integumen-tary edges. The adherence of the two integuments is also partially due to the peculiarquality of the skin to become sticky or gummy as it dries. This method is especiallyrecommended in cases where a slit has been made in the integument for the purposeof extirpating or operating on an internal organ. Usually, in such cases, the edges ofthe slit remain apart, due to the normal tension of the surrounding integument, anda wound area requiring from 3 to 10 days or more to heal is presented. By theuse of air the edges of the slit, when drawn together, can be sealed and frequentlyremain sealed even after the animal has been immersed in water. Since the twointegumentary edges soon fuse together, the total time required for healing of thewound is thus greatly shortened. It is perhaps unnecessary to point out that thismay be a deciding factor in the successful completion of certain experiments.

All operated larvae were placed in individual aquaria at room temperature andfed Spirogyra. Under these conditions natural metamorphosis ensued within 1-2weeks afterwards. The animals were eventually fixed in Bouin's (aqueous) fixativefor later histological study or preserved in 70 % alcohol for photographic purposes.Serial sections were made of representative cases and stained with Heidenhain'siron-hematoxylin and eosin.

TRANSPLANTATION OF THE INTEGUMENT

(1) Technique of skin transplantation

The technique involved in making skin transplantations may be describedbriefly as follows: The anaesthetized animal is placed on a damp pad of cloth anda square or rectangular incision made with iridectomy scissors around the areaof integument to be removed. It is best first to puncture the skin carefully with asurgical needle in order that one blade of the scissors can be inserted. This isespecially necessary when skin from.the side is to be removed, since rupturing of thethin underlying peritoneum results in extrusion of the internal organs and con-sequent death. The outlined area of skin is now removed by gently maintainingtraction with forceps while the connecting septa are cut with a small sharpspearheaded scalpel. Previous to this procedure, the region on the host to which thetransplant is to be grafted has been denuded of its integument. The transplant isnow fitted to this wound area and its edges prevented from curling under by runningthe point of a spearhead scalpel around and under the periphery of the graft. Theedges of the transplant are now attached to the surrounding host integument by theuse of compressed air, as previously described.

The grafts were taken from three regions, viz. (1) opercvlar grafts from the rightside to include the integumentary area which normally perforates during meta-morphosis; (2) side grafts from the right side just anterior to the hind leg; and (3)back grafts from the median region of the back just posterior to the eyes. Theseregions also indicate the locations of the host wound areas to which transplantationwas made. All transplantations were made with R. temporaria larvae.

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(2) Homoplastic transplantations

Series HB consisted of the transplantation of opercular grafts to the back regionof other larvae. Twenty-seven larvae were operated on of which twenty survived thestage of metamorphosis characterized by emergence of both forelimbs. The animalswere killed for fixation or preserving within a few days following this period atwhich time many had progressed to the stage where complete union of forelimbintegument with that surrounding the perforation had been established. Thus, thetransplanted opercular grafts were allowed adequate time to undergo histolysis andperforation, assuming that the potentialities for such degeneration were inherent andsubject to manifestation under the influence of general metamorphic stimuli.

The macroscopic appearance of the transplants presented considerable variation.In a few cases normal pigmentation, general coloration, and size were maintained.In others, the only apparent external change consisted in a generalized darkening ofthe entire transplant with no appreciable reduction in area (PI. II, fig. 13). Quitefrequently the epidermis of the transplant underwent definite disintegration and inmany cases became loosened and was shed. The twenty transplants were carefullyexamined under a binocular microscope, while still on the living animal, for signs ofperforation formation. In no instance could the smallest perforation be detected,although a cursory examination might at first lead one to suspect one especiallywhere epidermal erosion had been more or less localized.

Representative transplants were later sectioned to determine the histologicalcondition of the various cell layers. In general, the histolysis present was correlatedquite closely with the degree of area reduction, darkening, and general degenerativeappearance that had been observed while on the living animal. Thus, those trans-plants which had simply assumed a darker coloration with no reduction in surfacearea, presented only slight signs of histolysis; usually associated only with thestratum compactum layer. Where area reduction had occurred, sections revealed thedissociation of all layers. The epithelium was usually either hypertrophied ordefinitely breaking down, the stratum spohgiosum reduced in thickness or lackingentirely and the stratum compactum and subcutaneous connective tissue dissociatedand invaded with lymphocytes. In many cases large masses of blood cells hadaccumulated beneath the transplant (PI. IV, fig. 31).

Series HS was similar in all respects to series HB with the exception that thegrafts were transplanted to the right side of the host animals. Fifteen transplanta-tions were made of which eleven survived metamorphosis. The results, in general,were practically the same as described for the HB series, although a smaller per-centage underwent pronounced macroscopic and histological degeneration. In fact,four of the transplants remained normal and healthy in all respects even to thepreservation of the finer details of all external and histological structures (PI. II,fig. 14). In two instances, where only slight histolysis was apparent, it was, interest-ing to note the presence of large cutaneous glands (PI. IV, fig. 37). One case wasunique in that a raised bulbous-like area appeared near the centre of the transplant.The integument in this region was quite thin and evidently considerably histolysed.

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Beneath this area a degenerating, brownish, semi-liquid mass was found, and itseems quite probable that it had been derived from a small piece of gill tissueaccidentally transplanted along with the skin graft. It should be emphasized here,however, that in all other cases where histological degeneration occurred in seriesHB and HS, the histolysis was quite generalized throughout all regions of the trans-plant. There were no indications of a particular area being especially susceptible tohistolysis.

Series HO involved the reverse transplantation of back gratts to the opercularregion. Thirty-five larvae were so transplanted of which twenty-eight lived past thestage characterized by the eruption of the right forelimb. The metamorphosinglarvae were not all fixed or preserved at this time, but at varying intervals afterwardsin order to study the enlargement and ultimate fate of the perforations.

The site of the first appearance of the perforation in relation to the transplantwas subject to considerable variation. In nine instances the perforation was firstseen entirely within the boundaries of the transplant and in three cases occurred atthe exact centre. PI. II, fig. 15, and PI. Ill , fig. 24, represent one of these, althoughat the time the photographs were made the perforation had enlarged to includenearly all of the transplant. In the remaining nineteen cases, however, the perfora-tion first appeared at the edge of the transplant in the region of fusion with the hostintegument. This does not mean that adjacent portions of the transplant and hostintegument had not undergone pronounced histolysis but rather that the fusionregion is more easily ruptured. • It is difficult to orient the transplant on the un-metamorphosed tadpole so that the point of greatest pressure of the limb (elbow)will be directed against the centre of the graft during metamorphosis. Prior to thefirst appearance of the perforation the transplant is bulged out under considerablepressure. If at this time the position of the limb elbow is eccentrically placed inrelation to the centre of the transplant, it usually soon shifts to the fusion regionwhere the tensile strength is least and rupturing consequently takes place. Follow-ing the emergence of the limb at this point, however, the perforation rapidly en-larges to involve varying amounts of the transplant and adjacent host integument(PI. II, figs. 16 and 17, and PI. I l l , fig. 25). PI. II, fig. 22, and PI. Ill , fig. 23, repre-sent a case of premature forelimb emergence at the anterior boundary of the trans-plant, no doubt due to faulty healing of the transplant at this point coupled withabnormal pressure of the limb for this early stage of larval involution. It will benoted that the transplant appears crowded and bunched up, while anterior to thelimb the ruptured fusion area is filled with gill tissue.

Histological sections made of transplants just prior to perforation and throughperforations in various stages of enlargement left no doubt that a process of cellulardegeneration had occurred typical in all respects with normal opercular histolysis.PI. IV, fig. 34, shows a section made through a perforation several days after theemergence of the forelimb through the posterior central region of the transplant.The section shows the histolysed condition of that portion of the transplant formingthe anterior border of the perforation. This region of the perforation is filled withatrophying gill tissue. In PI. IV, fig. 33, the forelimb has emerged between trans-

104 0. M. HELFF

plant and host integument. The transplant appeared wrinkled and crowded asviewed on the living animal, while the section reveals the presence of pronouncedhistolytic degeneration.

Series HSO resembled series HO in all respects except that the transplant wasderived from the side of the donor. The side skin transplants in the opercular regionof the eleven hosts which survived larval involution exhibited, in general, the samereactions towards histolysis and perforation formation as described for series HOand hence need no further description here. PI. IV, fig. 35, represent a sectiontaken through a perforation which involved practically the complete histolysis of thetransplant. Only a remnant of the latter still persists which, in itself, is undergoingrapid dissociation.

(3) Autoplastic transplantations

The results of the homoplastic transplantation of opercular integument to theback and side in those instances where partial degeneration of the transplantoccurred, showed that a generalized histolysis involving the entire graft was typicalof all cases. Since this condition is frequently met with in homoplastic grafts, thedesirability of autoplastic transplantation was suggested; although the presence oftwo wound areas on one larva would obviously impose a more severe strain on theviability of the operated animal as compared with homoplastically transplantedindividuals where only one wound area was involved.

Two series of operations were made as follows: Series S involved the reciprocaltransplantation of opercular and back integument, while series SS entailed the inter-changing of opercular and side skin. The general technique employed here was thesame as described for the homoplastic transplants with the exception that greatercare was necessary to prevent the detachment and consequent loss of the transplantsduring the first hour or so following the operation. This was accomplished by partialsubmersion of the operated animal for a period of 15-20 min. in such a way that thetwo transplanted regions were exposed to the air and the snout maintained beneathwater. Following this, the transplants were moistened with a few drops of waterfrom a medicine dropper and the entire animal submerged by the careful addition ofwater to the bowl or dish in which the tadpole was to be kept. In this manner theaction of surface tension, which frequently tends to pull the delicate transplants offwhen submerging the animal, can usually be prevented.

At the time the operations were made (late in August), only a limited number oflarvae were available in which no signs of metamorphosis could be detected.Fifteen of these were used for series S, of which nine survived metamorphosis.Five of the opercular transplants on the back remained normal and healthy asdetermined by macroscopic observation and subsequent histological examination.In two cases a definite thinning of the transplant occurred which was quite general-ized in one and somewhat localized in the other. The remaining two operculartransplants were of considerable interest in that perforations developed. Althoughone of these was represented only by a minute hole in the centre of a rather largetranslucent thin area, the other developed into a good-sized oval-shaped perforation

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with a regular border resembling quite closely the appearance of a normal opercularforelimb perforation. Sections made through this perforation (PI. IV, fig. 32) gaveevidence of the occurrence of pronounced histolytic changes in those portions of thetransplant surrounding the orifice. Nine reciprocal transplantations of opercularand side skin (series SS) were made, of which but four survived larval involution.Three of the opercular transplants on the side remained normal in appearance whileonly one showed signs of a thinning process which, however, did not develop to theperforation stage. All back-skin grafts of series S and side-skin grafts of series SS,previously transplanted to the opercular region, underwent histolysis and perfora-tion formation during metamorphosis as already described for the homoplastictransplantations. PI. IV, fig. 36, shows a typical example of histolysis in a side skingraft shortly before the appearance of a perforation.

EXTIRPATION OF LIMB AND ADJACENT SHOULDER GIRDLE

(1) Technique of extirpation

The removal of the forelimb and the adjacent portions of the shoulder girdlewith which it articulates was performed in order to determine the effect, if any,on opercular histolysis and perforation formation during larval involution. Thetechnique involved may be described, briefly, as follows: The animal to be operatedon was first placed on a damp pad under a dissecting microscope and covered with apiece of wet cloth in which a small hole had been cut to expose the field of operation.A dorsoventral incision 1-2 mm. in length was next made through the operculumjust dorsal to the location of the limb base. The small forelimb was now located withthe help of a strong direct light and pulled out through the integumentary incisionby means of fine-pointed and slender forceps. By maintaining sufficient tractionwith the forceps, not only the limb but also that portion of the attached shouldergirdle which is covered with a cutaneous patch continuous with the integument ofthe limb was exposed to view. The shoulder girdle was now carefully cut throughjust external to and following the clearly marked boundary of the more or less oval-shaped cutaneous patch. Considerable care must be exercised during this procedurein order not to rupture or cut the nearby pericardium with resultant exposure ofthe heart and probable death of the animal. The cutting was consequently doneusing only the points of iridectomy scissors whose blades had been ground quiteslender. Following the removal of the limb and attached shoulder-girdle portion,the opercular incision was finally closed with the help of compressed air and thelarva immediately immersed in water to await metamorphosis.

(2) Extirpation in R. temporaria

The R. temporaria larvae used in this series (series TRE) were fully grown tad-poles measuring from 29 to 38 mm. in total length and with hindlimbs but 2-5 mm.long. In such individuals the extirpated right forelimbs varied from 1 to 2 | mm.,the average length being 1 £ mm. The integument is pigmented only on the dorsalside (extensor surface) of the limb at this stage of development.

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Sixty-five animals were operated on of which forty-four survived the stage atwhich the forelimbs are normally released through opercular perforations. Intwenty-four instances, perforations developed on the right or operated side. Usually,these were of small size although several cases were observed in which the extentof the perforated area was several times that which would have been necessary forthe emergence of a forelimb. The perforations always developed within a muchlarger, angular-shaped, translucent area which had previously formed. Eleven caseswere recorded in which translucent areas developed without subsequent perforationformation. Sections made through such regions showed the presence of histolysiswhich was typical of normal opercular degeneration during metamorphosis (PI. IV,fig. 38). In the remaining nine cases the region of perforation was clearly indicatedin that all layers of the integument had undergone complete histolysis with theexception of the epidermis which in most instances had been reduced to one or twolayers of cells.

The macroscopic appearance of the perforations which formed was subject toconsiderable variation. The smaller perforations were always oval in shape andpossessed regular edges (PI. I, fig. 1), while the larger perforations were apt to bemore irregular and variable in outline (PI. I, fig. 2, and PI. II, fig. 21). Quitefrequently, atrophying gill tissue was seen filling all or part of the smaller andinvariably the anterior regions of the larger perforations (PI. I, fig. 2, PI. II, fig. 21,and PL IV, fig. 39). In several of the larger perforations the posterior half of theopening was occupied by a regenerated outgrowth from the shoulder girdle which,however, was devoid of any cutaneous covering (PI. I, fig. 2, and PI. II, fig. 21).Certain of the smaller perforations also developed without either shoulder girdle orgill tissue occurring directly beneath (PI. IV, fig. 40), although atrophying gill wasfound to occur just anterior to the opening and underlying the translucent histolys-ing opercular integument of that region. Finally, it is interesting to note that inseveral cases where perforations developed and the animals lived through completeor nearly complete metamorphosis, considerable diminution in the size of theperforation took place. In two instances this healing process resulted in completeclosure of the opening. Similar cases of perforation healing following previous limbextirpation have been reported by the writer (1926) in R. pipiens.

In view of Weber's (1931) statement that perforation formation can only takeplace (in the absence of the forelimb) when cutaneous glands are present in theperibranchial cavity, the desirability of a careful histological examination wasevident. Serial sections were consequently made of the entire anterior halves oftwelve animals in which perforations of various sizes had formed during meta-morphosis. Thus the entire peribranchial cavity could be examined for the presenceof cutaneous fragments containing glandular structures which might have resultedfollowing incomplete removal of the cutaneous patch covering the shoulder girdleat the time limb extirpation was made. A thorough study of the serial sectionsshowed that cutaneous glands persisted in only two cases. In one of these a singlegland was found and in the other a total of three glands located quite close togetherand on the inner wall of the gill cavity. The right peribranchial cavities of ten indi-

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victuals, however, were found to be devoid of even a single cutaneous gland (seePI. IV, figs. 39, 40). Hence Weber's contention (that opercular histolysis andperforation formation can occur only in the presence of cutaneous glands) could notbe confirmed.

(3) Extirpation in B. bufo

The B. bufo tadpoles selected for extirpation of the right forelimb and adjacentregions of the shoulder girdle (series BRE) were mature larvae 20-27 mm. in totallength with hindlimbs varying from 1J to 4^ mm. The forelimbs in such individualswere but 1-3 mm. in length, and as in the case of R. temporaria, the smaller of thesehad not developed to the stage where differentiation of the digits could be detected.The integumentary covering of the forelimb in B. bufo differs from that of R.temporaria, however, in that it is darkly pigmented on all surfaces although to agreater degree on the dorsal side (extensor surface).

Seventy-five larvae were operated on of which forty-one survived metamorphosis.Of these, thirty-four developed perforations, usually quite large in size, while sevenindividuals showed varying degrees of opercular histolysis. In several of the latter,only a thin partially histolysed epithelium persisted, which under the normal in-fluence of limb pressure would have ruptured to establish perforations.

In twenty-six of the perforations formed, atrophying gill tissue was observedfilling all or part of the opening. The small perforations were usually entirelyoccluded with gill tissue which frequently protruded out through the orifice, whilein the larger perforations the gill tissue was more often limited to the anterior half(PI. I, fig. 10, and PI. II, fig. 19). Several large perforations developed, however,which were entirely filled with red degenerating gill filaments (PI. I, fig. 11, and PI.Ill , fig. 26). As in the case of the R. temporaria series, several examples of healingwere observed during which the perforation area was reduced from 50 to 70 %. Nocases of complete closure of the perforation were observed, however, although it islikely that all would have completely healed had the animals been allowed to livelong enough.

It was consistently observed that following the eruption of the left forelimb, theperforation of that side which at first was little larger than the original spiracularopening, underwent enlargement in an anterior direction. This anterior extensionwas, of course, quite superfluous, and yet it frequently developed to a size evenlarger than the original opening through which the limb had emerged. It wasinvariably filled with red atrophying gill tissue. Following eventual contraction ofthe gills during their later stages of atrophy, this anterior extension of the perfora-tion healed over partially and its edges finally became fused with the integument ofthe limb.

One peculiar case was observed in which a regenerated limb stump fully 3 mm.in length developed. It exerted pronounced pressure on the adjacent opercularwall which was consequently held away from contact with the gills. Although theanimal underwent complete metamorphosis, no perforation formed, which wasremarkable in view of the sharpness of the stump end. The animal was later

108 O. M. HELFF

autopsied and the stump found to be devoid of an integumentary covering. Failureof the operculum to histolyse in this case was probably due to lack of contactbetween it and the atrophying gill tissue.

As in the R. temporaria series-, serial sections were made of representativeanimals to determine the presence or absence of cutaneous glands within the peri-branchial cavity. In none of the gill cavities so sectioned could the slightest trace ofa cutaneous gland be detected (PI. V, figs. 46, 47), and it is reasonable to conclude,therefore, that the original extirpations had been complete. The results emphasizethe conclusion previously reached following a study of the R. temporaria sections inthat the presence of cutaneous glands within the peribranchial cavity is not a pre-requisite for opercular histolysis and perforation formation.

EXTIRPATION OF FORELIMB ONLY

Weber (1931) believes that the histolysis of opercular integument is the directresult of secretions liberated by large cutaneous glands which develop in the skin ofthe limb and adjacent shoulder girdle during larval involution. Contrary to theresults of Weber, the writer has found that opercular histolysis and perforationformation can occur in the complete absence of the cutaneous glands in question.A survey of these latter results (described in the present paper) reveal, however, thatwhile opercular histolysis occurred in all cases of limb and adjacent shoulder-girdle extirpation, actual perforation of the integument was not attained in allinstances. There can be no reasonable doubt, moreover, that had a limb beenpresent in these latter instances, a perforation would have formed. Hence, theprobability remains that the limb and adjacent shoulder girdle, by virtue of thecutaneous glands which they bear in their integuments, may normally exert ahistolytic influence on the operculum. In order to test this conception, other seriesof operations were planned which involved the extirpation of the right forelimbonly, without disturbing the adjacent portion of the shoulder girdle and its integu-mentary covering.

The technique involved in the removal of the forelimb closely followed that aspreviously described in the present paper for limb and shoulder-girdle extirpationand need be only briefly described here. Following the exposure of the appendagethrough a suitable incision made in the opercular wall, the limb was excised at itsbase by a single cut of iridectomy scissors. The wound area so created was thentrimmed of any projecting pieces of tissue, care being exercised not to injure thecutaneous patch on the surrounding shoulder girdle. The opercular incision wasthen closed and the animal allowed to undergo subsequent metamorphosis. Dissec-tions made of several individuals 48 hr. following limb extirpation showed thatintegumentary covering of the shoulder-girdle wound had taken place within thisinterval, although it is doubtful whether all layers of the newly formed skin wererepresented at this early period.

Studies on Amphibian Metamorphosis 109

(1) Results in R. temporaria

Mature tadpoles of the same size and limb development as employed in seriesTRE were used for the limb extirpation experiments (series TE). Sixty-five larvaewere operated on of which fifty lived through subsequent metamorphosis. In thegreat majority of individuals, translucent areas developed in the opercular integu-ment during the early stages of larval involution. As thinning progressed in theseareas and the integument became progressively more transparent, the stump of thelimb was always seen lying directly beneath the thinnest part of the histolysingintegument and usually in contact with it. This stump was either oval or circular inshape, viewed laterally, and was covered with its own integument. In forty-eightinstances perforation of the thin histolysing operculum occurred at some pointoverlying the limb stump. This initial small perforation usually rapidly enlargeduntil the entire lateral surface of the stump was exposed (PL I, fig. 6, and PL II,fig. 20). In several instances slight regeneration had occurred giving the stump aconvex or conical appearance, in which case the structure protruded more or lessthrough the opercular opening (PL I, fig. 7). There were also two individuals inwhich miniature forelimbs had regenerated. The differentiation was complete in allmajor respects even to the formation of the digits, although the limb as a whole wasbut one-third to one-half normal size for this stage of larval metamorphosis. Bothof these limbs emerged through rather small perforations.

Four individuals were observed in which the results differed markedly fromthose as described above. Two of these gave evidence of pronounced opercularhistolysis but failed to form perforations in spite of the late stage of metamorphosisattained. In another instance a much-delayed small perforation finally formed(PL I, fig. 12). The remaining case was of interest in that two perforations de-veloped. The smaller and more ventral of these occurred over the limb stump,while a much larger hole formed just dorsal to and separated from the ventralopening by a narrow band of integument. The anterior part of the dorsal perfora-tion was rilled with atrophying gill tissue.

Following the emergence of the limb stump, further histolysis of the operculumadjacent to the anterior side of the stump usually occurred. This resulted in varyingdegrees of perforation enlargement in this direction and invariably exposed the redatrophying tissue of the gills which frequently tended to protrude slightly throughthe opening. As a result, the healing of the integument of the stump with that of thesurrounding operculum always took place at the posterior, dorsal, and ventralborders first and was frequently much prolonged anteriorly. In fact, healing at thispoint could apparently not take place until continued histolysis of the gills resultedin their contraction and consequent withdrawal from the anterior perforation.

Representative animals were sectioned to obtain serial sections of the perfora-tion region and the inclosed limb stump. In most cases where the stump hademerged through a perforation, numerous large cutaneous glands were observed inthe integumentary covering of the stump (PL V, fig. 49). The granular or poisonglands were usually of much greater size and more numerous as compared with the

no O. M. HELFF

mucous glands. Two instances were recorded in which very few glands could befound (PL V, fig. 50). In both cases, however, the stump integument appeared tobe rather poorly developed.

(2) Results in B. bufo

The animals used for this series (series BE) were of corresponding size andexhibited the same degree of limb development as previously described for seriesBRE larvae. Sixty-five tadpoles were operated on of which forty-four survivedbeyond that stage of metamorphosis at which the forelimb perforations are normallyformed. Forty-one larvae developed perforations on the right or limb-extirpatedside during metamorphosis, while in three cases pronounced histolysis and conse-quent opercular thinning occurred. Since the latter individuals would no doubthave developed actual perforations if the larvae had lived a day or two longer, theyneed not be discussed further here.

The opercular perforations which developed during larval metamorphosisvaried considerably as regards size, shape, and their association with regard toatrophying gill tissue and the stump of the forelimb. Although practically no twoindividuals presented exactly the same type of perforation as regards all of theabove factors, they can be classified roughly into the following five categories:Group 1 was represented by a single case in which a small perforation formedthrough which the limb stump could be seen. The stump was clearly not in contactwith the opercular wall. Group 2 consisted of two individuals in which a largeperforation developed entirely filled with gill tissue. In both cases the Limb stumpcould be seen beneath the thin histolysing opercular wall a short distance ventralto the perforation. Group 3 involved thirteen individuals in which a single perfora-tion formed conforming in shape and approximate size to the lateral surface of thelimb stump and which, in fact, filled the perforation. In several instances the stumpdeveloped into a conical-shaped projection which protruded through the perforation(PL I, fig. 9, and PL II, fig. 18). Group 4 consisted of eleven cases in which a largeperforation developed, the posterior or posterior ventral part being occupied by thelimb stump while the opposite anterior or anterior dorsal region was filled with gilltissue (PL I, fig. 8, and PL III, fig. 30). Fourteen individuals were represented bygroup 5, which was of considerable interest in that two separate perforations de-veloped on the right side, one above the other, and separated in many cases by whatappeared to be normal opercular integument. In nine instances the dorsal perfora-tion was entirely filled with gill tissue while the limb stump completely occupied theopening of the ventral perforation (PL I, fig. 3, and PL III, fig. 27). The upper partof the dorsal perforation was filled with gill tissue in two other cases, while the moreventral regions of the same opening were occupied by part of the limb stump. Theventral perforation in both these individuals was rilled with limb stump (PL I, fig. 4,and PL III, fig. 28). The remaining three cases represented the reverse arrangementin which the entire dorsal perforation and the anterior part of the ventral openingwere filled with gill tissue while the posterior regions of the latter perforation wereoccupied by a portion of the limb stump (PL I, fig. 5, and PL III, fig. 29).

Studies on Amphibian Metamorphosis 111

As in series TE, representative animals were sectioned to obtain serial sectionsof the perforation regions. In general, sections made through perforations or partsof perforations filled with limb stump gave evidence of numerous granular or poisonglands in the integumentary covering of the latter structure (PI. V, fig. 41). Theseglands, however, were disproportionately smaller as compared with those seen inthe R. temporaria series. In several instances where the perforation was filled withboth gill tissue and limb stump, sections of the stump portion revealed the presenceof only a few small glandular structures (PI. V, fig. 42). Sections through gill-filledperforations or those "portions of perforations occupied by gill tissue usually failed todemonstrate glandular structures (PI. V, figs. 44, 43). It was also of interest to notethat where perforations developed in connexion with atrophying gill tissue, thehistolysis was usually very rapid, resulting often in liquefaction of the integumentforming the border of the opening (PI. V, figs. 43, 45).

DISCUSSION

The results of autoplastic transplantation of opercular integument to foreignregions, as described in the present paper, would lead one to suspect that the normalperforation area of the operculum may possess varying degrees of susceptibility tohistolytic factors operating through the circulatory system during larval involution.It will be recalled in this respect that of the thirteen transplantations made, elevenremained normal, two underwent generalized and one a localized thinning process,while in two cases the histolysis gave rise to the formation of actual perforations.Liosner & Woronzowa (1935), working with the same species (R. temporaria),obtained quite similar results in which, although no thinning was observed intwenty-one instances, definite histolysis usually resulting in perforation formationwas observed in fifty-three others. The same authors have obtained a similarvariation in results when working with R. rtdibunda, Bombina bombina, and Pelobatesfuscus larvae, while the writer (1926) was unable to detect any instances of histolysisin opercular grafts of R. clamitans which had previously been transplanted to theback or side. Apparently, therefore, not all species of anurans are the same in thisrespect and in fact the results of Liosner & Woronzowa and the writer indicate thatdifferent individuals of the same species are subject to great variation as regards theabsence or degree of self-degeneration possessed by the opercular integument. It isof course possible, although not very probable, that the failure of certain operculartransplants to histolyse in foreign locations is due rather to the unsuitability of thetransplantation site and not to a hereditary lack of histolytic susceptibility.

The results of homoplastic transplantation of R. temporaria opercular skin to theside or back, as described in the present paper, are difficult to analyse. No instanceof a localized histolysis of one part of a transplant or consequent perforation forma-tion was observed, yet the majority of grafts underwent a generalized process ofpartial degeneration. The fact that all regions of the transplant seemed equallyaffected, would lead one to believe that the degeneration experienced was due moreto individual blood differences between host and donor animals, as is frequently the

i i 2 O. M. HELFF

case in homoplastic transplantation. It could be reasoned from this that the un-favourable environment of the transplant should have operated to induce even morerapid histolysis and consequent perforation formation in that area of the graftwhich was presumably hereditarily specific for perforation formation during larvalinvolution: Since this was not the case, however, some other explanation must besought. It seems more likely, therefore, that the process of generalized degenerationwhich followed homoplastic transplantation might have operated to desensitize thetransplant. This, in turn, may have prevented the specific area of integument con-cerned from reacting with those factors in the blood stream which are normallyresponsible for the induction of opercular histolysis. during metamorphosis.

The results of homoplastic and autoplastic transplantation of back and side skinto the opercular region, as described in the present paper, are significant in thattransplant thinning and subsequent perforation formation occurred in all thirty-nine cases. Apparently, therefore, certain histolytic influences must be operative inthe peribranchial cavity during larval metamorphosis which are capable of inducingcomplete histolysis of opercular skin, whether or not the integument in question iscapable of undergoing self-degeneration under the influence of histolytic factorspresent in the blood stream. Blacher et al. (1934) obtained similar results in twoinstances, working with R. ridibunda, in which the forelimb emerged through thecentre of a back-skin graft previously homoplastically transplanted to the opercularregion. The writer (1926) has furthermore obtained the same results with R.clamitans wherein eighteen instances of perforation formation and four cases ofthinning occurred following the autoplastic transplantation of back and side skin.So far, therefore, all species worked on are similar as regards the ability of histolyticfactors peculiar to the opercular cavity to induce histolysis in suitably transplantedforeign integument.

Whether or not the secretory products of the large cutaneous glands, whichdevelop in the integument of the forelimb and adjacent shoulder girdle, are capableof inducing opercular histolysis, is a question which as yet cannot be answered withany degree of certainty. According to Weber (1931), opercular histolysis and per-foration formation can only occur, following cauterization of the limb bud, whenintegumentary remnants containing cutaneous glands persist in the peribranchialcavity, as in cases of incomplete cauterization. When no glands persist, thenhistolysis and perforation of the operculum is impossible. These results wereobtained not only in R. temporaria but also when working with Bombinator pachypus.Blacher et al. (1934), Liosner & Woronzowa (1935) and the writer (present paper),however, have since shown that opercular histolysis and perforations can occur inR. temporaria in those cases in which subsequent serial sections reveal no traces ofcutaneous glands. As regards Weber's work, it seems quite improbable that one or afew microscopic glands located on the inner wall of the peribranchial cavity andbathed by the continuous flow of water passing through that chamber could producea histolytic effect on a certain definitely located area of the opercular wall with whichthey have no physical contact. Obviously, secretions liberated by such glands mustbe rapidly diluted and so transported from the right to the left gill chambers and

Studies on Amphibian Metamorphosis 113

finally lost as the water passes out through the spiracular opening. How, then, canwe account for those instances in which histolysis and perforation formation failedto occur on the operated side. It can only be suggested here, that in these cases theinitial cauterization process made through the operculum of the young larvae mayhave so injured the potential perforation area physiologically that it was unable toreact during metamorphosis to the histolytic influences of the bloodor atrophying gills.

Although Blacher et al. attribute the histolysis and perforations which theyobtained as due entirely to the self-degenerating potentialities of the integumentaryarea involved, the writer finds it difficult to believe that the large perforations ofvariable sizes encountered in the present work could be the result of this factoralone. The perforations observed were in many instances three or more times aslarge as those normally met with in cases of natural forelimb emergence. Most ofthe perforations, moreover, were filled either in part or wholly with atrophying gilltissue which had been in contact with the operculum of that area during the previoushistolysis of the integument. It must be borne in mind, in this regard, that the fore-limb in exerting pressure tends to produce considerable lateral extension of theoperculum. When the limb is extirpated, the operculum is thereby permitted toassume a closer association with the gills and actual contact is established betweenthe two structures. This may well explain the abnormally large perforations whichhave been observed by many workers in cases of desiccated thyroid or otherwiseartificially induced metamorphosis. The sequence of metamorphic changes isfrequently very abnormal in such cases, and perforation formation may occur beforethe forelimbs have undergone any marked increase in size. The writer has frequentlyobserved perforations so large that union is almost attained between the left andright openings in the mid-ventral line. In such instances the gills have undergoneabnormally rapid histolysis and usually fill the greater part of the perforations, whilethe small forelimbs are unable to establish contact with the opercular wall. Thehistolytic action of the gills is certainly strongly suggested in such cases. The writer(1926) has previously shown that gill tissue is capable of producing pronounced andrapid histolysis in integument beneath which it is transplanted. These results wereobtained in both R. palustris and R. pipiens, while, more recently, Blacher et al.(1934) observed the same effect following the transplantation of R. temporaria gilltissue beneath back integument. Hence, the writer believes that histolysis andperforation formation in R. temporaria and B. bufo, following complete limb extir-pation, is due not only to the self-degenerating potentialities of the perforation areabut also to the histolytic action of the atrophying gill tissue.

The histolysis and perforation of the operculum following previous limb andshoulder-girdle extirpation does not, however, preclude the possibility that thecutaneous glands of these structures may have a definite histolytic function when thelatter are present. During natural metamorphosis of the unoperated animal, thelimb maintains strong contact with the opercular wall and the mechanism is con-sequently present for the direct transference of the glandular secretions to the areaof operculum which exhibits histolysis and perforation formation. Unlike gilltissue, however, the glandular integument of the forelimb and shoulder girdle has

JKB'XVli 8

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not as yet been shown to possess histolytic properties. In fact, such experiments ashave been made with this end in view have given negative results. The writer(1926), for example, was unable to detect any signs of histolysis in R. palustris backintegument under which forelimbs had developed following previous homoplastictransplantation of limb buds. It is only fair to state here, however, that the limbswere always quite small in size and the differentiation of the cutaneous glands mustalso have been correspondingly abnormal compared with those present in theintegument of limbs developing within the opercular cavity. More recently, how-ever, Blacher et al. (1934) transplanted whole forelimbs and parts of limbs beneaththe back integument of R. temporaria larvae, and although the limbs were obtainedfrom larvae undergoing normal opercular histolysis, no signs of thinning or perfora-tion formation were observed in the overlying back integument. It is the experienceof the writer, however, that fully differentiated limbs, homoplastically transplanted,do not usually remain normal, and it is quite probable that glandular secretion stopsin such cases or at least proceeds at a decreased rate. Hence, neither the results ofthe writer nor of the above authors definitely preclude the possibility that activelysecreting glands of the normal forelimb and shoulder-girdle integument may have ahistolytic effect on the operculum when maintained in close contact with the latterstructure for a sufficient period of time.

The results of removing only the forelimb of R. temporaria and B. bufo, withoutdisturbing the adjacent shoulder girdle or its integumentary covering, are of interestin that they further suggest a histolytic action on the part of cutaneous glands andatrophying gill tissue. In R. temporaria the perforations were usually preceded bythe development of a rather large translucent area under which, as thinning pro-gressed, both limb stump and gill tissue could be seen. The actual perforation,however, first appeared over the limb stump. This fact can be explained in severalways. The perforating area may simply have represented the region hereditarilyspecific to undergo self-degeneration during metamorphosis, or the limb stump bymeans of "pressure atrophy" may have hastened perforation in this particulararea of the histolysing operculum. A third possibility exists in that histolytic fluidsderived from the atrophying gills or perhaps even small dissociated pieces of thesestructures may have been held in close contact with the perforating area by means ofthe limb stump. Lastly, and more probably, the cutaneous glands of the limb-stumpintegument may have exercised an additive histolytic action on the particular oper-cular area which first perforated. It is perhaps unnecessary to point out that all ofthese factors may have been functioning simultaneously to hasten histolysis andperforation formation. The results recorded in the B. bufo series are even moresuggestive of the action of more than one histolytic factor. The fact that two separateperforations frequently developed, one of which was filled with limb stump and theother with gill tissue, naturally suggests the importance of both these structures.Unfortunately, as previously stated, we as yet have no definite proof of a histolyticaction on the part of the cutaneous glands., In this respect the writer would like tosuggest an experiment which should serve to throw light on this question. It issuggested that integumentary transplants be taken from the shoulder girdle and the

Studies on Amphibian Metamorphosis 115

limb (at a time when the glands appear to be fully developed and presumably at theheight of their secretory activity) and transplanted beneath the skin of the back orside. The chances of the transplant remaining normal and the cutaneous glandscontinuing to function should be very good, while subsequent histological examina-tion of the overlying host integument would serve to indicate whether or nothistolysis had been induced. Another experiment can be suggested at this timewhich should be an improvement over previous methods of limb and shoulder-girdle extirpation, at least as it concerns the question of cutaneous gland action. Thewriter has found it possible to remove the entire integumentary covering of both theshoulder girdle and the limb without the removal or injuring of these structures inany other respect. The limb and girdle are exposed through a suitable opercularincision and the border of the cutaneous shoulder-girdle patch cut free. This patchof skin together with the entire integumentary covering of the limb can then bepulled off as one can turn a stocking or glove inside out by pulling from the openend. The removal of the integument, and hence all cutaneous gland elements in thismanner, has the decided advantage over limb extirpation methods in that the restof the limb is left intact and can consequently exert its usual pressure against theopercular wall. The normal spatial relationship between gills and operculum wouldalso remain undisturbed. Thus only one of the possible histolytic factors is eliminated,and any change in the resulting opercular histolysis or perforation formation can beattributed to the absence of the forelimb integument and its glandular components.

It is not possible in view of our present state of knowledge to speak with anygreat degree of definiteness as regards the exact causation of the various phases ofnormal opercular histolysis and perforation formation in any one species of anurans.Of the different larvae studied, however, R. temporaria has been worked on morethan any other species, and an attempt to explain normal perforation formation inthis form may perhaps not be out of place at this time. As the writer sees it, thereexists in the perforation region of the right side operculum an area of integumentwhich is more or less hereditarily specific to undergo histolysis during larval in-volution under the influence of inductive factors present in either the blood orlymph or both at this time. This potentiality for self-degeneration may well besubject to considerable variation in intensity between different individuals and mayeven be absent entirely in some. This factor, however, may be capable by itself, insome instances, of producing a perforation sufficiently large enough for the releaseof the forelimb. At approximately the same time this type of histolysis occurs, orperhaps even before, other factors become important. The forelimb has developedto the point where it exerts strong pressure against this general area of the opercularwall, while beneath the limb and also anterior to it the gills are seen to be undergoingrapid degeneration. The limb no doubt accelerates the thinning of the opercularwall through the medium of "pressure atrophy" and also, perhaps, through theaction of secretory products of the cutaneous glands in its integument, the secretionbeing directly transmitted to the operculum wherever the limb is in contact withthat tissue. The atrophying gills also function at this time to release powerfulhistolytic substances. It is doubtful, however, whether any considerable area of

n 6 O. M. HELFF

gill actually comes in contact with the opercular wall, except in certain cases wherelimb development is retarded and the operculum is consequently allowed to sinkdown and so establish contact with the gills. It is possible that the gills can inducehistolysis in the operculum only when in contact with this structure. Under thecombined influences of these various histolytic factors the operculum continues togrow thinner and thinner until finally a minute perforation appears usually locatedjust over the elbow, or the limb may simply rupture the remaining epithelial layersof the opercular wall through pressure. In either event the elbow emerges and issoon followed by the entire limb. The emergence of the limb allows the operculumas a whole to sink down so that certain portions of it are brought into strong contactwith the atrophying gill tissue lying adjacent and anterior to the base of the limb.The perforation now continues to enlarge rapidly anteriorly under the influence ofthe gills, which frequently protrude to some extent through this region of theopercular opening. The final stages involving healing of the periphery of the perfora-tions with the integument of the limb have previously been described by the writer(1926) for other anurans, and since the histological steps involved are probably thesame in all species, there is no need to review them here. It should be pointed out,however, that union of the forelimb integument with the anterior border of theperforation cannot take place until continued atrophy of the gills has withdrawnthese latter structures from the perforation region.

In conclusion, the writer would like to emphasize the point that no general rulecan be established to account for the mechanism of opercular histolysis which willhold true for all anurans. Thus, in R. temporaria, the process is quite probably"triply assured" by the action of three distinct histolytic factors, while pressureatrophy no doubt functions in a supplementary capacity. In other species onlyone or two of these factors may normally be operative in inducing histolysis. Pre-liminary experiments by the writer, for example, indicate that the operculum ofR. catesbeiana larvae is not heriditary specific as regards self-degeneration, whileextirpation of the forelimb always prevents perforation formation. Hence, the gillsin this species apparently cannot in themselves bring about opercular histolysis andperforation formation. Factors associated only with the forelimb are probably theonly ones concerned. Obviously, therefore, great variation exists in the type ofcausal mechanism responsible for opercular histolysis, and an adequate explanationfor the process involved in any one form can be arrived at only when suitable experi-ments are performed on that particular species.

SUMMARY AND CONCLUSIONS

The histolysis of the anuran operculum during forelimb perforation formationhas been accredited to the action of autolysing gill tissue, to cutaneous gland secre-tions, and to self-degeneration of the integument by Helff, Weber, and Blacher et al.respectively. The writer has reinvestigated the problem as follows:

(1) Opercular integument, homoplastically transplanted to the back and side ofR. temporaria larvae, underwent a process of partial degeneration. The histolysiswas not confined to any localized region of the transplant.

Studies on Amphibian Metamorphosis 117

(2) Autoplastic transplantation of opercular integument to the back and sideproduced a variety of results. Normal histological structure was maintained incertain transplants, generalized degeneration was observed in others, while inseveral instances localized histolysis resulting in perforation formation occurred.

(3) Homoplastic and autoplastic transplantation of back and side skin to theopercular region resulted in histolysis and perforation formation in such transplantsduring larval involution.

(4) The right forelimbs (in early stages of development) with attached portionsof the shoulder girdle were extirpated in R. temporaria and B. bufo. Duringsubsequent metamorphosis, normal opercular histolysis followed by perforationformation in many cases was observed. In most instances, serial sections of theperibranchial cavity revealed the absence of cutaneous glands.

(5) Extirpation of the right forelimb only was made in the same two species.Opercular histolysis subsequently occurred in all instances, resulting in perforationformation in the great majority of cases during larval involution. In many of theB. bufo animals two separate perforations developed, one filled with limb stump andthe other with gill tissue.

(6) It is concluded that in R. temporaria a particular area of the operculum mayin some individuals possess self-degenerative potentialities conducive to histolysisand perforation formation during metamorphosis. In both R. temporaria and B.bufo histolytic influences emanating from the atrophying gill tissue and the cutaneousglands of the forelimb are probably also responsible for operfiular histolysis andperforation formation. Limb pressure must be considered a supplemental factor.

(7) The results are discussed in general and attention called to the fact thatopercular histolysis and perforation formation are "doubly assured" in some speciesand possibly even "triply assured" in others. Emphasis is placed on the evidentlywide divergence between species as regards the particular combination of factorsresponsible for opercular histolysis and perforation formation. Apparently, no oneexplanation can serve to account for the phenomenon as it occurs in various speciesof anurans.

REFERENCESBLACHER, L. J., LIOSNER, L. D. & WORONZOWA, M. A. (1934). Bull. int. Acad. Cracovie, B, p. 325.BRAUS, H. (1906). Morph. Jb. 35, 509.

(1909). Morph.Jb. 39, 155.EKMAN, G. (192a). Soc. tci.fetm. Comm. biol. 1, No. 3, p. 1.HBLFF, O. M. (1926). J. exp. Zool. 45, 1.LIOSNER, L. D. & WORONZOWA, M. A. (1935). Bull. int. Acad. Cracovie, B, p. 231.SCHULZE, W. (1924). Arch. mikr. Anat. 101, 338.VAN DER JAGT, E. R. (1929). J. exp. Zool. 54, 225.WEBER, A. (1931). Arch. Anat. micr. 27, 230.

n8 O. M. HELFF

EXPLANATION OF PLATES I-V

PLATE IFig. i. Extirpation of forelimb and shoulder girdle. A small perforation has formed in the oper-culum. (Case RE 54, R. temporaria.) x 3$.Fig. 2. Extirpation of forelimb and shoulder girdle. A large irregularly shaped perforation hasformed, the posterior half of which is filled with a regenerated mass from the shoulder girdle devoidof integument. (Case RE 17, R. temporaria.) x 3.Fig. 3. Extirpation of forelimb. Two perforations have formed in the operculum. The dorsal one isfilled with gill tissue and the ventral with limb stump. (Case E 14, B. bufo.) x 3}.Fig. 4. Extirpation of the forelimb. Two perforations have formed in the operculum. The dorsal oneis filled with limb stump and gill tissue while the ventral shows limb stump only. (Case E 21, B.bufo.) x 4.Fig. 5. Extirpation of the forelimb. Two perforations have formed in the operculum. The dorsal oneis filled with gill tissue while both limb stump and gill tissue occupy the ventral. (Case E 17, B.bufo.) x3f.Fig. 6. Extirpation of the forelimb. A large perforation has formed in the operculum. It is filledcompletely with limb stump possessing its own integument. (Case E 50, R. temporaria.) x 2}.Fig. 7. Extirpation of the forelimb. A large perforation has formed in the operculum throughwhich a regenerated limb stump projects. (Case E 34, R. temporaria.) x 4.Fig. 8. Extirpation of the forelimb. An exceedingly large perforation has formed in the operculum.Gill tissue fills most of the perforation but a portion of the limb stump is seen through the extremeventral part of the opening. (Case E 5, B. bufo.) x 3J.Fig. 9. Extirpation of the forelimb. A perforation has formed in the operculum through whichprojects a conical-shaped regenerated limb stump possessing its own integument. (Case E 23, B.bufo.) X3fFig. 10. Extirpation of forelimb and shoulder girdle. An exceedingly large perforation has formed inthe operculum, the anterior half of which is filled with gill tissue. (Case RE 33, B. bufo.) x 3}.Fig. 11. Extirpation of forelimb and shoulder girdle. A large perforation has formed in the oper-culum entirely filled with gill tissue. (Case RE 16, B. bufo.) x 3!.Fig. 12. Extirpation of the forelimb. A much delayed, very small perforation has formed in theoperculum. (Case E 17, R. temporaria.) x 3J.

PLATE IIFig. 13. Homoplastic transplantation of opercular integument to the back. A generalized darkeningof the transplant has occurred with no reduction in area. There are no signs of localized histolysisorperforation formation. (Case HB 5, R. temporaria.) x 3J.Fig. 14. Homoplastic transplantation of opercular integument to the posterior right side. The trans-plant has remained normal in all respects. There are no signs of localized or generalized histolysis.(Case HS 11, R. temporaria.) x 3^.Fig. 15. Homoplastic transplantation of back integument to the opercular region. An instance inwhich the perforation first occurred in the centre of the transplant. Nearly all of the latter is nowincluded in the perforation area. Gill tissue shows in the anterior part of the opening. (Case HO 11,R. temporaria.) x 3f.Fig. 16. Homoplastic transplantation of back integument to the opercular region. The perforationfirst occurred at the edge of the transplant but has by now enlarged to include most of the graft.(Case HO 7, R. temporaria.) x 3$.Fig. 17. Homoplastic transplantation of back integument to the opercular region. A large perfora-tion has formed resulting in total destruction of the transplant. Atrophying gill tissue protrudesthrough the anterior part of the opening. (Case HO 4, R. temporaria.) x 3J.Fig. 18. Extirpation of the forelimb. An enlargement of the perforation region as shown in Fig. 9.(Case E 23, B. bufo.) x 24.Fig. 19. Extirpation of forelimb and shoulder girdle. An enlargement of the perforation region asshown in Fig. 10. (Case RE 33, B. bufo.) x 24.Fig. 20. Extirpation of the forelimb. An enlargement of the perforation region as shown in Fig. 6.(Case E 50, R. temporaria.) x 24.

Studies on Amphibian Metamorphosis 119Fig. a 1. Extirpation of forelimb and shoulder girdle. An enlargement of the perforation region asshown in Fig. 2. (Case RE 17, R. temporaria.) x 24.Fig. 22. Homoplaatic transplantation of back integument to the opercular region. Prematureemergence of the forelimb at the anterior border of the graft due to faulty healing of the latter. Thetransplant is seen just posterior to the limb and appears wrinkled and crowded. (Case HO 18,R. temporaria.) x 3$.

PLATE III

Fig. 23. Homoplastic transplantation of back integument to the opercular region. An enlargement ofthe perforation region as shown in Fig. 22. (Case HO 18, R. temporaria.) x 15.Fig. 24. Homoplastic transplantation of back integument to the opercular region. An enlargement ofthe perforation area as shown in Fig. 15. (Case HO 11, R. temporaria.) x 15.Fig. 25. Homoplastic transplantation of back integument to the opercular region. An enlargement ofthe perforation area as shown in Fig. 17. (Case HO 4, R. temporaria.) x 15.Fig. 26. Extirpation of forelimb and shoulder girdle. An enlargement of the perforation area asshown in Fig. 11. (Case Re 16, B. bufo.) x 24.Fig. 27. Extirpation of the forelimb. An enlargement of the perforation areas as shown in Fig. 3.(Case E 14, B. bufo.) x 24.Fig. 28. Extirpation of the forelimb. An enlargement of the perforation areas as shown in Fig. 4.(Case E ai, B. bufo.) x 24.Fig. 29. Extirpation of the forelimb. An enlargement of the perforation areas as shown in Fig. 5.(Case E 17, B. bufo.) x 24.Fig. 30. Extirpation of the forelimb. An enlargement of the perforation area as shown in Fig. 8.(Case E 5, B. bufo.) x 24.

PLATE IV

Fig. 31. Homoplastic transplantation of opercular integument to the back. The transplant shows atthe left upper boundary of the section. The epithelium is hypertrophied and the stratum compactumdissociated. Large masses of blood cells appear beneath the transplant. (Case HB 13, R. temporaria.)x4o.Fig. 32. Autoplastic transplantation of opercular integument to the back. The upper boundary ofthe section shows the transplant, in which a large perforation has developed. The integumentaryedges of the perforation are thinned out and give evidence of the histolysis which preceded perfora-tion formation. (Case S 1, R. temporaria.) x 50.Fig. 33. Homoplastic transplantation of back integument to the opercular region. The forelimb,shown at the upper left of the section, has emerged through the fusion region of the host operculumwith the ventral border of the transplant. The transplant, shown at the upper right, appears crowdedand is undergoing pronounced histolysis. (Case HO 13, R. temporaria.) X 50.Fig. 34. Homoplastic transplantation of back integument to the opercular region. The forelimbemerged through a perforation formed in the transplant. The section shows atrophying gill tissuefilling one part of the perforation, while the transplant integument forming the anterior border of theopening (upper right) is undergoing rapid histolysis. (Case HO 16, R. temporaria.) x 80.Fig. 35. Homoplastic transplantation of side integument to the opercular region. A large perforationformed which included the greater part of the transplant. Atrophying gill tissue filled most of theopening. Only a remnant of the posterior part of the transplant remains (upper centre boundary ofsection), which appears in a late stage of histolysis. (Case HSO 2, R. temporaria.) x 40.Fig. 36. Autoplastic transplantation of side integument to the opercular region. The section showsthe histolysed condition of a transplant (upper centre boundary of section) just prior to perforationformation. Note atrophying gill tissue in peribranchial cavity beneath. (Case SS 6, R. temporaria.)X50.Fig. 37. Homoplastic transplantation of opercular integument to the posterior right side. Thetransplant (upper boundary of section) remained quite normal during subsequent larval involution,with only slight signs of histolysis. Note large glands in transplant and blood clot beneath. (CaseHS 2, R. temporaria.) x 80.Fig. 38. Extirpation of forelimb and shoulder girdle. A large translucent area developed in theopercular wall during metamorphosis. The section shows pronounced histolysis in this region. Theepidermis (upper boundary of section) shows definite thinning and dissociation of cells from thelower layers. The degenerating mass beneath, consists chiefly of dissociated stratum compactum withlymphocytic infiltration. (Case RE 26, R. temporaria.) x 70.

120 0 . M. HELFF

Fig. 39. Extirpation of forelimb and shoulder girdle. A large perforation developed during meta-morphosis. The section shows the histolysing borders of the perforation with atrophying gill tissueprotruding through the latter. There are no cutaneous glands in the peribranchial cavity. (CaseRE so, R. temporaria.) x 40.Fig. 40. Extirpation of forelimb and shoulder girdle. A small perforation developed during meta-morphosis. Although gill tissue was found just anterior to the opening, the section reveals nonedirectly beneath the perforation. No cutaneous glands were observed in any part of the peribranchialcavity. (Case RE 6, R. temporaria.) x 40.

PLATE V

Fig. 41. Extirpation of the forelimb. Two perforations developed during metamorphosis. Thesection is cut near the edge of the ventral perforation, at which point only the cuticular layer of theopercular wall persists. The limb stump appears beneath, in whose integument a gland can be seen(right centre of section). (Case E 19, B. bufo.) x 80.Fig. 42. Extirpation of the forelimb. A medium-sized perforation developed filled with limb stumpand gill tissue. The section is through the posterior part of the perforation which is filled with limbstump. (Case E 9, B. bufo.) x 80.Fig.' 43. Same perforation as described in Fig. 42. The section is, however, taken through theanterior gill-filled part of the perforation. No cutaneous glands were observed in that part of theperibranchial cavity lying immediately beneath. (Case E 9, B. bufo.) x 80.Fig. 44. Same case as in Fig. 41. The section is, however, cut through the dorsal gill-filled perfora-tion. There are no traces of cutaneous glands in that portion of the peribranchial cavity lying directlybeneath. (Case E 19, B. bufo.) x 50.Fig. 45. Extirpation of the forelimb. The section is cut through the two perforations whichdeveloped during metamorphosis. Both perforations are filled with atrophying gill tissue, whilecutaneous glands are seen in the integument covering the limb stump beneath the ventral perforation(left Bide of section). Note excessive histolysis (amounting to liquefaction) in integument borderingthe dorsal perforation. (Case E 33, B. bufo.) x 80.Fig. 46. Extirpation of forelimb and shoulder girdle. The section is cut through the anterior, gill-filled half of the large perforation formed during metamorphosis. Note protrusion of atrophying gillsand their close association with the perforation border. No cutaneous glands were seen in any partof the peribranchial cavity. (Case RE 13, B. bufo.) x 50.Fig. 47. Extirpation of forelimb and shoulder girdle. The section is cut through the anterior, gill-filled part of the medium-sized perforation which developed during metamorphosis. The gills repre-sent a somewhat later stage of atrophy as compared with those shown in Fig. 46. There were nocutaneous glands in the peribranchial cavity. (Case RE 20, B. bufo.) x 75.Fig. 48. Extirpation of forelimb and shoulder girdle. A small perforation developed during meta-morphosis. The section is through the opercular wall just anterior to the perforation. Hiatolysis isevidenced by hypertrophy of the epithelial cells and complete dissociation of the stratum compactum.The internal epithelial lining of the operculum is still intact. Note atrophying gill tissue in peri-branchial cavity beneath. (Case RE 54, B. bufo.) x 150.Fig. 49. Extirpation of the forelimb. A perforation filled with limb stump formed during meta-morphosis. The section was made at a late metamorphic stage, by which time the limb stump haspractically healed with the perforation border. Note the three abnormally large cutaneous glands inthe limb-stump integument. (Case E 41, R. temporaria.) x 50.Fig. 50. Extirpation of the forelimb. A perforation filled with limb stump formed during meta-morphosis. The section shows the histolysing integumentary borders of the perforation with the limbstump beneath. The integumentary covering of the latter is poorly developed and only a few smallcutaneous glands were found in it. (Case E 35, R. temporaria.) x 50.

JOURNAL OF EXPERIMENTAL BIOLOGY, XVI, r* PLATE I

HELFF—STUDIES ON AMPHIBIAN METAMORPHOSIS (pp. 96—120).

JOURNAL OF EXPERIMENTAL BIOLOGY, XVI, t. PLATE II

HELFF.—STUDIES ON AMPHIBIAN METAMORPHOSIS (pp. 96—120).

JOURNAL OF EXPERIMENTAL BIOLOGY, XVI, i. PLATE III

HELFF.—STUDIES ON AMPHIBIAN METAMORPHOSIS (pp. 96—120),

JOURNAL OF EXPERIMENTAL BIOLOGY, XVI, i. PLATE IV

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fJELFF—STUDIES ON AMPHIBIAN METAMORPHOSIS (pp. 96—120).

JOURNAL OF EXPERIMENTAL BIOLOGY, XVI, i. PLATE V

42

HELFF.—STUDIES ON AMPHIBIAN METAMORPHOSIS (pp. 96—120).