STUDIES ON AMPHIBIAN METAMORPHOSIS
XVI. THE DEVELOPMENT OF FORELIMB OPERCULAR
PERFORATIONS 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 or two layers of flattened epidermal cells. The buds can be distinguished before the growth 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 of forelimb buds in no way inhibits the later development of normal peribranchial cavities. The operculum bounding the gill cavities arises as an integumentary fold on either side of the head during late embryonic (early larval) development. These folds, by a process of posterior migration and growth, gradually enclose the gills and limb bud of their respective sides and eventually fuse with the body wall behind and below the gill region. Thus the peribranchial cavities are entirely enclosed with the exception of the spiracular openings which may be present on the left side only as in the case of Bufo, Hyla, Rand and Pelodytes, or located ventral and medial as in Alytes, Bombinator and Discoglossus. The position of the spiracular openings would appear to be dependent on the respective growth rates of the left and right opercular folds which determine the ultimate location at which the two folds meet and fuse. Where the spiracle is on the left side, a narrow transverse canal connects the right with the left peribranchial cavity. A peculiar condition exists in the African anuran Xenopus (Dactylethra), where spiracles are formed on both right and left sides. The opercula, however, enclose only the gills, since the forelimbs arise from buds located externally as in the case of Urodeles or the hind-limbs of Amphibia in general.
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 the left side, the emergence of the corresponding limb is primarily through this opening, although considerable enlargement of this natural orifice frequently takes place. In those forms having a median-ventral spiracle, perforations are developed on both right and left sides where the histolysis of the operculum and the emergence of the limbs involve essentially the same processes. Prior to the actual appearance of a perforation, the opercular integument usually presents a more or less orderly sequence of macroscopic changes, although this is subject to some variation between different species. These include a lessening in pigmentation so that the area con-cerned in time appears yellowish or nearly white in colour. Frequently a typical translucency is produced, and in later stages the integument may become quite thin and transparent. In certain forms the area to perforate is circumscribed by a thin white line before the enclosed integument has undergone any pronounced macro-scopic change. This is particularly true of R. catesbeiana and R. clamitans. The site of first appearance of the perforation is usually where the elbow of the forelimb is exerting considerable pressure against the operculum. The initial perforation rapidly enlarges and the forelimb emerges. Usually, however, the perforation con-tinues to enlarge, especially anteriorly. The steps entailed in the enlargement of a perforation and the ultimate union of the adjacent opercular integument with the skin 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 which takes place in the integumentary areas concerned. These may be briefly summarized as follows: The fibrils of the stratum compactum layer are first thrown into more pronounced convolutions than they ordinarily assume. The region appears com-pressed as evidenced by the increase in numbers of nuclei as compared with other regions. This condition is shortly followed by a dissociation of the fibrils and an invasion of blood cells, chiefly of the lymphocyte type. Following this, the stratum spongiosum 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 subcutaneous connective tissue and the internal epithelial lining also degenerate and are removed. The outer epidermis, which up to this time has remained peculiarly unaffected, now begin 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. At this stage the forelimb usually ruptures the thin single-layered cuticle or, in the absence of limb pressure, further histolysis soon removes the cuticular layer and so initiates the perforation.
The first experimental work dealing with the causation of forelimb perforations was that of Braus (1906) on Bombinator larvae. Braus extirpated the minute undifferentiated 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. The forelimbs so regenerated, however, never acquired a size sufficient to maintain contact with the operculum. Schulze (1924) repeated the experiments of Braus on R. esculenta and obtained similar results.
The writer (1926) removed the forelimbs of fully grown tadpoles of three American species: R. pipiens, R. sylvatica and R. clamitans. The extirpated limbs varied from 2 to 10 mm. in length depending on the species, but had never attained the size at which pressure is exerted against the operculum. In the first two of these species perforations subsequently developed during metamorphosis, while thinning occurred in many of the larvae of R. clamitans. These results confirmed those of Braus and others in that presence of the forelimb is not essential to the formation of opercular perforations. The writer then investigated the effect of homoplastic transplantation of the limb bud beneath the integument of the back. Twenty-five cases were obtained in which growth and differentiation took place during larval involution. Of these, however, there were only three instances of perforation formation in the overlying back integument. Histological sections through these perforations gave no evidence, however, that their occurrence had been due to previous histolysis of the various integumentary layers as is characteristic of oper-cular skin during perforation formation. In fact, similar perforation of integument was obtained by the implantation of glass beads beneath the skin of the side, a result attributed to simple "pressure atrophy". That the perforating area of the operculum is not peculiarly specific to undergo histolysis during larval involution was determined by the reciprocal transplantation of opercular skin with that of the back and side. Opercular grafts in foreign locations failed to histolyse during metamorphosis, while back- and side-skin transplants grafted to the opercular region underwent degeneration and perforation formation. It was finally found that if atrophying gill tissue was transplanted beneath integument, the latter underwent rapid histolysis typical in all respects to that described as preceding the appearance of normal opercular perforations. The writer concluded, therefore, that the atro-phying gills of the anuran are primarily concerned in initiating the histolysis of opercular integument during metamorphosis, although the forelimb may have a secondary effect through pressure in hastening the actual appearance of a perfora-tion.
macro-scopic changes or histological signs of integumentary thinning. According to this author, the degree of opercular histolysis and perforation formation was directly correlated with the presence or absence of cutaneous glands within the peribranchial cavity. Weber states that where the cauterization had been complete, no remnants of the integumentary coverings of the limb and shoulder girdle developed, resulting in the complete absence of cutaneous glands. In such individuals the operculum failed to histolyse and perforate in spite of the fact that the gills underwent normal atrophy. Conversely, the presence of even a few microscopic glands, following in-complete cauterization, was sufficient to bring about thinning of the operculum and even perforation formation in some cases. Thus, to Weber the normal causation of forelimb perforations is due to the activity of large cutaneous glands located in the integument covering the limb and part of the shoulder girdle.
More recently, Blacher et al. (1934) and Liosner & Woronzowa (1935), working with R. temporaria and R. ridibunda, claim that perforation of the operculum is due to a definite specificity of the integument. Their conclusions are based on the results of reciprocal skin transplantation between the perforation region of the operculum and the integument of the back and tail. Contrary to the results of the writer, they obtained perforations during metamorphosis in opercular transplants previously made to the back and tail, while integumentary grafts from the latter regions refused to perforate when placed in the normal perforation area. They furthermore state that opercular perforations occurred following complete removal of the limb, and hence in those cases where no cutaneous glands persisted or developed as shown by subsequent histological examination. These last results are therefore in direct contradiction 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 gland secretions, and to self-degeneration of the operculum by the writer, Weber, and Blacher et al. respectively. Two possibilities present themselves to account for the variance in results obtained and conclusions drawn by the above workers. The fact that different species were utilized might account for the discrepancies recorded, in that the causative factors involved might not always be the same in the various Anura studied. Secondly, it seemed quite possible to the writer that more than one factor 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 of experiments 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 during the latter hah0 of June. The frog larvae were collected near Whittlesford and the toad larvae in the vicinity of Cambridge. Since tadpoles collected so late in the season would 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 for operative work. This was accomplished by keeping the larvae in a cold-temperature room in large glass aquaria constantly aerated with compressed air. The stocks were furthermore fed regularly with Spirogyra and the water changed once a week. Using these precautions, a sufficient number of larvae were maintained throughout July and August for operative work.
For any one series of operations, the larvae were carefully selected as regards size, absence of metamorphic signs, and general healthiness. In general, a 2 % solution of ether was employed for anaesthesia. It may be stated here that the writer has found ether to be definitely superior to chloretone, especially where a quick, safe anaesthesia is desired. There is little danger of overdosage if the larvae are 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 solution which 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 anaesthetized animal is placed in air for operative purposes. Chloretone solutions, if used in concentrations weak enough to require 10-15 min. for anaesthesia, are recommended in cases where a long operation is entailed. The immersion of the animal in fresh water every 2 or 3 min. during the first 10 or 15 min. following the operation is often very helpful in counteracting the undesired accumulative action of the anaesthetic which frequently proves to be lethal. Although operations involving skin transplan-tations required previous anaesthesia, the extirpation of forelimbs was usually performed by simply holding the larvae between two pieces of damp cloth, the operation being made through a suitable hole in the upper cloth.
edges in contact with that of the surrounding host integument. The stream of air is then 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 peculiar quality of the skin to become sticky or gummy as it dries. This method is especially recommended in cases where a slit has been made in the integument for the purpose of extirpating or operating on an internal organ. Usually, in such cases, the edges of the slit remain apart, due to the normal tension of the surrounding integument, and a wound area requiring from 3 to 10 days or more to heal is presented. By the use of air the edges of the slit, when drawn together, can be sealed and frequently remain sealed even after the animal has been immersed in water. Since the two integumentary edges soon fuse together, the total time required for healing of the wound is thus greatly shortened. It is perhaps unnecessary to point out that this may be a deciding factor in the successful completion of certain experiments.
All operated larvae were placed in individual aquaria at room temperature and fed Spirogyra. Under these conditions natural metamorphosis ensued within 1-2 weeks afterwards. The animals were eventually fixed in Bouin's (aqueous) fixative for later histological study or preserved in 70 % alcohol for photographic purposes. Serial sections were made of representative cases and stained with Heidenhain's iron-hematoxylin and eosin.
TRANSPLANTATION OF THE INTEGUMENT
(1) Technique of skin transplantation
The technique involved in making skin transplantations may be described briefly as follows: The anaesthetized animal is placed on a damp pad of cloth and a square or rectangular incision made with iridectomy scissors around the area of integument to be removed. It is best first to puncture the skin carefully with a surgical needle in order that one blade of the scissors can be inserted. This is especially necessary when skin from.the side is to be removed, since rupturing of the thin underlying peritoneum results in extrusion of the internal organs and con-sequent death. The outlined area of skin is now removed by gently maintaining traction with forceps while the connecting septa are cut with a small sharp spearheaded scalpel. Previous to this procedure, the region on the host to which the transplant is to be grafted has been denuded of its integument. The transplant is now fitted to this wound area and its edges prevented from curling under by running the point of a spearhead scalpel around and under the periphery of the graft. The edges of the transplant are now attached to the surrounding host integument by the use of compressed air, as previously described.
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(2) Homoplastic transplantations
Series HB consisted of the transplantation of opercular grafts to the back region of other larvae. Twenty-seven larvae were operated on of which twenty survived the stage of metamorphosis characterized by emergence of both forelimbs. The animals were killed for fixation or preserving within a few days following this period at which time many had progressed to the stage where complete union of forelimb integument with that surrounding the perforation had been established. Thus, the transplanted opercular grafts were allowed adequate time to undergo histolysis and perforation, assuming that the potentialities for such degeneration were inherent and subject 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 of the entire transplant with no appreciable reduction in area (PI. II, fig. 13). Quite frequently the epidermis of the transplant underwent definite disintegration and in many cases became loosened and was shed. The twenty transplants were carefully examined under a binocular microscope, while still on the living animal, for signs of perforation formation. In no instance could the smallest perforation be detected, although a cursory examination might at first lead one to suspect one especially where epidermal erosion had been more or less localized.
Representative transplants were later sectioned to determine the histological condition of the various cell layers. In general, the histolysis present was correlated quite closely with the degree of area reduction, darkening, and general degenerative appearance that had been observed while on the living animal. Thus, those trans-plants which had simply assumed a darker coloration with no reduction in surface area, presented only slight signs of histolysis; usually associated only with the stratum compactum layer. Where area reduction had occurred, sections revealed the dissociation of all layers. The epithelium was usually either hypertrophied or definitely breaking down, the stratum spohgiosum reduced in thickness or lacking entirely and the stratum compactum and subcutaneous connective tissue dissociated and invaded with lymphocytes. In many cases large masses of blood cells had accumulated beneath the transplant (PI. IV, fig. 31).
Beneath this area a degenerating, brownish, semi-liquid mass was found, and it seems quite probable that it had been derived from a small piece of gill tissue accidentally transplanted along with the skin graft. It should be emphasized here, however, that in all other cases where histological degeneration occurred in series HB 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 to histolysis.
Series HO involved the reverse transplantation of back gratts to the opercular region. Thirty-five larvae were so transplanted of which twenty-eight lived past the stage characterized by the eruption of the right forelimb. The metamorphosing larvae were not all fixed or preserved at this time, but at varying intervals afterwards in order to study the enlargement and ultimate fate of the perforations.
The site of the first appearance of the perforation in relation to the transplant was subject to considerable variation. In nine instances the perforation was first seen entirely within the boundaries of the transplant and in three cases occurred at the exact centre. PI. II, fig. 15, and PI. Ill, fig. 24, represent one of these, although at the time the photographs were made the perforation had enlarged to include nearly 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 host integument. This does not mean that adjacent portions of the transplant and host integument had not undergone pronounced histolysis but rather that the fusion region 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 the first appearance of the perforation the transplant is bulged out under considerable pressure. If at this time the position of the limb elbow is eccentrically placed in relation to the centre of the transplant, it usually soon shifts to the fusion region where 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. Ill, 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 with abnormal pressure of the limb for this early stage of larval involution. It will be noted that the transplant appears crowded and bunched up, while anterior to the limb the ruptured fusion area is filled with gill tissue.
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plant and host integument. The transplant appeared wrinkled and crowded as viewed on the living animal, while the section reveals the presence of pronounced histolytic degeneration.
Series HSO resembled series HO in all respects except that the transplant was derived from the side of the donor. The side skin transplants in the opercular region of the eleven hosts which survived larval involution exhibited, in general, the same reactions towards histolysis and perforation formation as described for series HO and hence need no further description here. PI. IV, fig. 35, represent a section taken through a perforation which involved practically the complete histolysis of the transplant. Only a remnant of the latter still persists which, in itself, is undergoing rapid dissociation.
(3) Autoplastic transplantations
The results of the homoplastic transplantation of opercular integument to the back and side in those instances where partial degeneration of the transplant occurred, showed that a generalized histolysis involving the entire graft was typical of all cases. Since this condition is frequently met with in homoplastic grafts, the desirability of autoplastic transplantation was suggested; although the presence of two wound areas on one larva would obviously impose a more severe strain on the viability of the operated animal as compared with homoplastically transplanted individuals where only one wound area was involved.
Two series of operations were made as follows: Series S involved the reciprocal transplantation of opercular and back integument, while series SS entailed the inter-changing of opercular and side skin. The general technique employed here was the same as described for the homoplastic transplants with the exception that greater care was necessary to prevent the detachment and consequent loss of the transplants during the first hour or so following the operation. This was accomplished by partial submersion of the operated animal for a period of 15-20 min. in such a way that the two transplanted regions were exposed to the air and the snout maintained beneath water. Following this, the transplants were moistened with a few drops of water from a medicine dropper and the entire animal submerged by the careful addition of water to the bowl or dish in which the tadpole was to be kept. In this manner the action of surface tension, which frequently tends to pull the delicate transplants off when submerging the animal, can usually be prevented.
with a regular border resembling quite closely the appearance of a normal opercular forelimb perforation. Sections made through this perforation (PI. IV, fig. 32) gave evidence of the occurrence of pronounced histolytic changes in those portions of the transplant surrounding the orifice. Nine reciprocal transplantations of opercular and 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 while only one showed signs of a thinning process which, however, did not develop to the perforation 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 formaperfora-tion during metamorphosis as already described for the homoplastic transplantations. PI. IV, fig. 36, shows a typical example of histolysis in a side skin graft 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 girdle with which it articulates was performed in order to determine the effect, if any, on opercular histolysis and perforation formation during larval involution. The technique involved may be described, briefly, as follows: The animal to be operated on was first placed on a damp pad under a dissecting microscope and covered with a piece 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 operculum just dorsal to the location of the limb base. The small forelimb was now located with the help of a strong direct light and pulled out through the integumentary incision by means of fine-pointed and slender forceps. By maintaining sufficient traction with the forceps, not only the limb but also that portion of the attached shoulder girdle which is covered with a cutaneous patch continuous with the integument of the limb was exposed to view. The shoulder girdle was now carefully cut through just external to and following the clearly marked boundary of the more or less oval-shaped cutaneous patch. Considerable care must be exercised during this procedure in order not to rupture or cut the nearby pericardium with resultant exposure of the heart and probable death of the animal. The cutting was consequently done using only the points of iridectomy scissors whose blades had been ground quite slender. Following the removal of the limb and attached shoulder-girdle portion, the opercular incision was finally closed with the help of compressed air and the larva immediately immersed in water to await metamorphosis.
(2) Extirpation in R. temporaria
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Sixty-five animals were operated on of which forty-four survived the stage at which the forelimbs are normally released through opercular perforations. In twenty-four instances, perforations developed on the right or operated side. Usually, these were of small size although several cases were observed in which the extent of the perforated area was several times that which would have been necessary for the emergence of a forelimb. The perforations always developed within a much larger, angular-shaped, translucent area which had previously formed. Eleven cases were recorded in which translucent areas developed without subsequent perforation formation. Sections made through such regions showed the presence of histolysis which was typical of normal opercular degeneration during metamorphosis (PI. IV, fig. 38). In the remaining nine cases the region of perforation was clearly indicated in that all layers of the integument had undergone complete histolysis with the exception of the epidermis which in most instances had been reduced to one or two layers of cells.
The macroscopic appearance of the perforations which formed was subject to considerable variation. The smaller perforations were always oval in shape and possessed regular edges (PI. I, fig. 1), while the larger perforations were apt to be more irregular and variable in outline (PI. I, fig. 2, and PI. II, fig. 21). Quite frequently, atrophying gill tissue was seen filling all or part of the smaller and invariably 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 the opening 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 or gill tissue occurring directly beneath (PI. IV, fig. 40), although atrophying gill was found to occur just anterior to the opening and underlying the translucent histolys-ing opercular integument of that region. Finally, it is interesthistolys-ing to note that in several cases where perforations developed and the animals lived through complete or nearly complete metamorphosis, considerable diminution in the size of the perforation took place. In two instances this healing process resulted in complete closure of the opening. Similar cases of perforation healing following previous limb extirpation have been reported by the writer (1926) in R. pipiens.
indi-victuals, however, were found to be devoid of even a single cutaneous gland (see PI. IV, figs. 39, 40). Hence Weber's contention (that opercular histolysis and perforation formation can occur only in the presence of cutaneous glands) could not be confirmed.
(3) Extirpation in B. bufo
The B. bufo tadpoles selected for extirpation of the right forelimb and adjacent regions of the shoulder girdle (series BRE) were mature larvae 20-27 mm. in total length with hindlimbs varying from 1J to 4^ mm. The forelimbs in such individuals were but 1-3 mm. in length, and as in the case of R. temporaria, the smaller of these had 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 a greater 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 seven individuals 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 observed filling all or part of the opening. The small perforations were usually entirely occluded with gill tissue which frequently protruded out through the orifice, while in 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 healing were observed during which the perforation area was reduced from 50 to 70 %. No cases of complete closure of the perforation were observed, however, although it is likely that all would have completely healed had the animals been allowed to live long enough.
It was consistently observed that following the eruption of the left forelimb, the perforation of that side which at first was little larger than the original spiracular opening, underwent enlargement in an anterior direction. This anterior extension was, of course, quite superfluous, and yet it frequently developed to a size even larger than the original opening through which the limb had emerged. It was invariably filled with red atrophying gill tissue. Following eventual contraction of the 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 of the limb.
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autopsied and the stump found to be devoid of an integumentary covering. Failure of the operculum to histolyse in this case was probably due to lack of contact between it and the atrophying gill tissue.
As in the R. temporaria series-, serial sections were made of representative animals 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 of a 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 emphasize the conclusion previously reached following a study of the R. temporaria sections in that 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 direct result of secretions liberated by large cutaneous glands which develop in the skin of the limb and adjacent shoulder girdle during larval involution. Contrary to the results of Weber, the writer has found that opercular histolysis and perforation formation 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, that while opercular histolysis occurred in all cases of limb and adjacent shoulder-girdle extirpation, actual perforation of the integument was not attained in all instances. There can be no reasonable doubt, moreover, that had a limb been present in these latter instances, a perforation would have formed. Hence, the probability remains that the limb and adjacent shoulder girdle, by virtue of the cutaneous glands which they bear in their integuments, may normally exert a histolytic influence on the operculum. In order to test this conception, other series of operations were planned which involved the extirpation of the right forelimb only, without disturbing the adjacent portion of the shoulder girdle and its integu-mentary covering.
(1) Results in R. temporaria
Mature tadpoles of the same size and limb development as employed in series TRE were used for the limb extirpation experiments (series TE). Sixty-five larvae were operated on of which fifty lived through subsequent metamorphosis. In the great majority of individuals, translucent areas developed in the opercular integu-ment during the early stages of larval involution. As thinning progressed in these areas and the integument became progressively more transparent, the stump of the limb was always seen lying directly beneath the thinnest part of the histolysing integument and usually in contact with it. This stump was either oval or circular in shape, viewed laterally, and was covered with its own integument. In forty-eight instances perforation of the thin histolysing operculum occurred at some point overlying the limb stump. This initial small perforation usually rapidly enlarged until 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 a convex or conical appearance, in which case the structure protruded more or less through the opercular opening (PL I, fig. 7). There were also two individuals in which miniature forelimbs had regenerated. The differentiation was complete in all major respects even to the formation of the digits, although the limb as a whole was but one-third to one-half normal size for this stage of larval metamorphosis. Both of these limbs emerged through rather small perforations.
Four individuals were observed in which the results differed markedly from those as described above. Two of these gave evidence of pronounced opercular histolysis but failed to form perforations in spite of the late stage of metamorphosis attained. 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 ventral opening 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 operculum adjacent to the anterior side of the stump usually occurred. This resulted in varying degrees of perforation enlargement in this direction and invariably exposed the red atrophying tissue of the gills which frequently tended to protrude slightly through the opening. As a result, the healing of the integument of the stump with that of the surrounding operculum always took place at the posterior, dorsal, and ventral borders first and was frequently much prolonged anteriorly. In fact, healing at this point could apparently not take place until continued histolysis of the gills resulted in their contraction and consequent withdrawal from the anterior perforation.
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mucous glands. Two instances were recorded in which very few glands could be found (PL V, fig. 50). In both cases, however, the stump integument appeared to be rather poorly developed.
(2) Results in B. bufo
The animals used for this series (series BE) were of corresponding size and exhibited the same degree of limb development as previously described for series BRE larvae. Sixty-five tadpoles were operated on of which forty-four survived beyond that stage of metamorphosis at which the forelimb perforations are normally formed. Forty-one larvae developed perforations on the right or limb-extirpated side during metamorphosis, while in three cases pronounced histolysis and conse-quent opercular thinning occurred. Since the latter individuals would no doubt have developed actual perforations if the larvae had lived a day or two longer, they need not be discussed further here.
As in series TE, representative animals were sectioned to obtain serial sections of the perforation regions. In general, sections made through perforations or parts of perforations filled with limb stump gave evidence of numerous granular or poison glands in the integumentary covering of the latter structure (PI. V, fig. 41). These glands, however, were disproportionately smaller as compared with those seen in the R. temporaria series. In several instances where the perforation was filled with both gill tissue and limb stump, sections of the stump portion revealed the presence of only a few small glandular structures (PI. V, fig. 42). Sections through gill-filled perforations or those "portions of perforations occupied by gill tissue usually failed to demonstrate glandular structures (PI. V, figs. 44, 43). It was also of interest to note that where perforations developed in connexion with atrophying gill tissue, the histolysis was usually very rapid, resulting often in liquefaction of the integument forming the border of the opening (PI. V, figs. 43, 45).
DISCUSSION
The results of autoplastic transplantation of opercular integument to foreign regions, as described in the present paper, would lead one to suspect that the normal perforation area of the operculum may possess varying degrees of susceptibility to histolytic factors operating through the circulatory system during larval involution. It will be recalled in this respect that of the thirteen transplantations made, eleven remained 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 in twenty-one instances, definite histolysis usually resulting in perforation formation was observed in fifty-three others. The same authors have obtained a similar variation in results when working with R. rtdibunda, Bombina bombina, and Pelobates fuscus larvae, while the writer (1926) was unable to detect any instances of histolysis
in opercular grafts of R. clamitans which had previously been transplanted to the back or side. Apparently, therefore, not all species of anurans are the same in this respect and in fact the results of Liosner & Woronzowa and the writer indicate that different individuals of the same species are subject to great variation as regards the absence or degree of self-degeneration possessed by the opercular integument. It is of course possible, although not very probable, that the failure of certain opercular transplants to histolyse in foreign locations is due rather to the unsuitability of the transplantation site and not to a hereditary lack of histolytic susceptibility.
i i 2 O. M . H E L F F
case in homoplastic transplantation. It could be reasoned from this that the un-favourable environment of the transplant should have operated to induce even more rapid histolysis and consequent perforation formation in that area of the graft which was presumably hereditarily specific for perforation formation during larval involution: Since this was not the case, however, some other explanation must be sought. It seems more likely, therefore, that the process of generalized degeneration which followed homoplastic transplantation might have operated to desensitize the transplant. This, in turn, may have prevented the specific area of integument con-cerned from reacting with those factors in the blood stream which are normally responsible for the induction of opercular histolysis. during metamorphosis.
The results of homoplastic and autoplastic transplantation of back and side skin to the opercular region, as described in the present paper, are significant in that transplant thinning and subsequent perforation formation occurred in all thirty-nine cases. Apparently, therefore, certain histolytic influences must be operative in the peribranchial cavity during larval metamorphosis which are capable of inducing complete histolysis of opercular skin, whether or not the integument in question is capable of undergoing self-degeneration under the influence of histolytic factors present in the blood stream. Blacher et al. (1934) obtained similar results in two instances, working with R. ridibunda, in which the forelimb emerged through the centre of a back-skin graft previously homoplastically transplanted to the opercular region. The writer (1926) has furthermore obtained the same results with R. clamitans wherein eighteen instances of perforation formation and four cases of thinning occurred following the autoplastic transplantation of back and side skin. So far, therefore, all species worked on are similar as regards the ability of histolytic factors peculiar to the opercular cavity to induce histolysis in suitably transplanted foreign integument.
finally lost as the water passes out through the spiracular opening. How, then, can we account for those instances in which histolysis and perforation formation failed to occur on the operated side. It can only be suggested here, that in these cases the initial cauterization process made through the operculum of the young larvae may have so injured the potential perforation area physiologically that it was unable to react during metamorphosis to the histolytic influences of the bloodor atrophying gills. Although Blacher et al. attribute the histolysis and perforations which they obtained as due entirely to the self-degenerating potentialities of the integumentary area involved, the writer finds it difficult to believe that the large perforations of variable sizes encountered in the present work could be the result of this factor alone. The perforations observed were in many instances three or more times as large as those normally met with in cases of natural forelimb emergence. Most of the perforations, moreover, were filled either in part or wholly with atrophying gill tissue which had been in contact with the operculum of that area during the previous histolysis 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 the operculum. When the limb is extirpated, the operculum is thereby permitted to assume a closer association with the gills and actual contact is established between the two structures. This may well explain the abnormally large perforations which have been observed by many workers in cases of desiccated thyroid or otherwise artificially induced metamorphosis. The sequence of metamorphic changes is frequently very abnormal in such cases, and perforation formation may occur before the forelimbs have undergone any marked increase in size. The writer has frequently observed perforations so large that union is almost attained between the left and right openings in the mid-ventral line. In such instances the gills have undergone abnormally rapid histolysis and usually fill the greater part of the perforations, while the small forelimbs are unable to establish contact with the opercular wall. The histolytic 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 and rapid histolysis in integument beneath which it is transplanted. These results were obtained in both R. palustris and R. pipiens, while, more recently, Blacher et al. (1934) observed the same effect following the transplantation of R. temporaria gill tissue beneath back integument. Hence, the writer believes that histolysis and perforation formation in R. temporaria and B. bufo, following complete limb extir-pation, is due not only to the self-degenerating potentialities of the perforation area but also to the histolytic action of the atrophying gill tissue.
The histolysis and perforation of the operculum following previous limb and shoulder-girdle extirpation does not, however, preclude the possibility that the cutaneous glands of these structures may have a definite histolytic function when the latter are present. During natural metamorphosis of the unoperated animal, the limb maintains strong contact with the opercular wall and the mechanism is con-sequently present for the direct transference of the glandular secretions to the area of operculum which exhibits histolysis and perforation formation. Unlike gill tissue, however, the glandular integument of the forelimb and shoulder girdle has
O. M- HELFF
not as yet been shown to possess histolytic properties. In fact, such experiments as have 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 back integument under which forelimbs had developed following previous homoplastic transplantation of limb buds. It is only fair to state here, however, that the limbs were always quite small in size and the differentiation of the cutaneous glands must also have been correspondingly abnormal compared with those present in the integument of limbs developing within the opercular cavity. More recently, how-ever, Blacher et al. (1934) transplanted whole forelimbs and parts of limbs beneath the back integument of R. temporaria larvae, and although the limbs were obtained from larvae undergoing normal opercular histolysis, no signs of thinning or perfora-tion formaperfora-tion were observed in the overlying back integument. It is the experience of the writer, however, that fully differentiated limbs, homoplastically transplanted, do not usually remain normal, and it is quite probable that glandular secretion stops in such cases or at least proceeds at a decreased rate. Hence, neither the results of the writer nor of the above authors definitely preclude the possibility that actively secreting glands of the normal forelimb and shoulder-girdle integument may have a histolytic effect on the operculum when maintained in close contact with the latter structure for a sufficient period of time.
n 6 O. M. HELFF
gill actually comes in contact with the opercular wall, except in certain cases where limb development is retarded and the operculum is consequently allowed to sink down and so establish contact with the gills. It is possible that the gills can induce histolysis in the operculum only when in contact with this structure. Under the combined influences of these various histolytic factors the operculum continues to grow thinner and thinner until finally a minute perforation appears usually located just over the elbow, or the limb may simply rupture the remaining epithelial layers of the opercular wall through pressure. In either event the elbow emerges and is soon followed by the entire limb. The emergence of the limb allows the operculum as a whole to sink down so that certain portions of it are brought into strong contact with 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 of the gills, which frequently protrude to some extent through this region of the opercular 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 the same 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 the perforation cannot take place until continued atrophy of the gills has withdrawn these latter structures from the perforation region.
In conclusion, the writer would like to emphasize the point that no general rule can be established to account for the mechanism of opercular histolysis which will hold true for all anurans. Thus, in R. temporaria, the process is quite probably "triply assured" by the action of three distinct histolytic factors, while pressure atrophy no doubt functions in a supplementary capacity. In other species only one or two of these factors may normally be operative in inducing histolysis. Pre-liminary experiments by the writer, for example, indicate that the operculum of R. catesbeiana larvae is not heriditary specific as regards self-degeneration, while extirpation of the forelimb always prevents perforation formation. Hence, the gills in this species apparently cannot in themselves bring about opercular histolysis and perforation formation. Factors associated only with the forelimb are probably the only ones concerned. Obviously, therefore, great variation exists in the type of causal mechanism responsible for opercular histolysis, and an adequate explanation for 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 formation has 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:
(2) Autoplastic transplantation of opercular integument to the back and side produced a variety of results. Normal histological structure was maintained in certain transplants, generalized degeneration was observed in others, while in several instances localized histolysis resulting in perforation formation occurred.
(3) Homoplastic and autoplastic transplantation of back and side skin to the opercular region resulted in histolysis and perforation formation in such transplants during larval involution.
(4) The right forelimbs (in early stages of development) with attached portions of the shoulder girdle were extirpated in R. temporaria and B. bufo. During subsequent metamorphosis, normal opercular histolysis followed by perforation formation in many cases was observed. In most instances, serial sections of the peribranchial 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 perforation formation in the great majority of cases during larval involution. In many of the B. bufo animals two separate perforations developed, one filled with limb stump and the other with gill tissue.
(6) It is concluded that in R. temporaria a particular area of the operculum may in some individuals possess self-degenerative potentialities conducive to histolysis and perforation formation during metamorphosis. In both R. temporaria and B. bufo histolytic influences emanating from the atrophying gill tissue and the cutaneous glands of the forelimb are probably also responsible for operfiular histolysis and perforation formation. Limb pressure must be considered a supplemental factor. (7) The results are discussed in general and attention called to the fact that opercular histolysis and perforation formation are "doubly assured" in some species and possibly even "triply assured" in others. Emphasis is placed on the evidently wide divergence between species as regards the particular combination of factors responsible for opercular histolysis and perforation formation. Apparently, no one explanation can serve to account for the phenomenon as it occurs in various species of anurans.
REFERENCES
BLACHER, 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.
n 8 O. M. HELFF
EXPLANATION OF PLATES I-V PLATE I
Fig. 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 has formed, the posterior half of which is filled with a regenerated mass from the shoulder girdle devoid of integument. (Case RE 17, R. temporaria.) x 3.
Fig. 3. Extirpation of forelimb. Two perforations have formed in the operculum. The dorsal one is filled 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 one is 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 one is 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 filled completely 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 through which 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 extreme ventral 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 which projects a conical-shaped regenerated limb stump possessing its own integument. (Case E 23, B.
bufo.) X3f
Fig. 10. Extirpation of forelimb and shoulder girdle. An exceedingly large perforation has formed in the 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 the operculum. (Case E 17, R. temporaria.) x 3J.
PLATE II
Fig. 13. Homoplastic transplantation of opercular integument to the back. A generalized darkening of the transplant has occurred with no reduction in area. There are no signs of localized histolysisor perforation 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 in which the perforation first occurred in the centre of the transplant. Nearly all of the latter is now included 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 perforation first 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 destrucperfora-tion of the transplant. Atrophying gill tissue protrudes through 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 as shown in Fig. 10. (Case RE 33, B. bufo.) x 24.
Fig. a 1. Extirpation of forelimb and shoulder girdle. An enlargement of the perforation region as shown in Fig. 2. (Case RE 17, R. temporaria.) x 24.
Fig. 22. Homoplaatic transplantation of back integument to the opercular region. Premature emergence of the forelimb at the anterior border of the graft due to faulty healing of the latter. The transplant 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 of the 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 of the 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 of the 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 as shown 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 at the left upper boundary of the section. The epithelium is hypertrophied and the stratum compactum dissociated. 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 of the section shows the transplant, in which a large perforation has developed. The integumentary edges of the perforation are thinned out and give evidence of the histolysis which preceded perfora-tion formaperfora-tion. (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 operculum with the ventral border of the transplant. The transplant, shown at the upper right, appears crowded and is undergoing pronounced histolysis. (Case HO 13, R. temporaria.) X 50.
Fig. 34. Homoplastic transplantation of back integument to the opercular region. The forelimb emerged through a perforation formed in the transplant. The section shows atrophying gill tissue filling one part of the perforation, while the transplant integument forming the anterior border of the opening (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 perforation formed which included the greater part of the transplant. Atrophying gill tissue filled most of the opening. Only a remnant of the posterior part of the transplant remains (upper centre boundary of section), 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 shows the histolysed condition of a transplant (upper centre boundary of section) just prior to perforation formation. 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. The transplant (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. (Case HS 2, R. temporaria.) x 80.
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 tissue protruding through the latter. There are no cutaneous glands in the peribranchial cavity. (Case RE 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 none directly beneath the perforation. No cutaneous glands were observed in any part of the peribranchial cavity. (Case RE 6, R. temporaria.) x 40.
PLATE V
Fig. 41. Extirpation of the forelimb. Two perforations developed during metamorphosis. The section is cut near the edge of the ventral perforation, at which point only the cuticular layer of the opercular 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 stump and gill tissue. The section is through the posterior part of the perforation which is filled with limb stump. (Case E 9, B. bufo.) x 80.
Fig.' 43. Same perforation as described in Fig. 42. The section is, however, taken through the anterior gill-filled part of the perforation. No cutaneous glands were observed in that part of the peribranchial 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 directly beneath. (Case E 19, B. bufo.) x 50.
Fig. 45. Extirpation of the forelimb. The section is cut through the two perforations which developed during metamorphosis. Both perforations are filled with atrophying gill tissue, while cutaneous 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 bordering the 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 gills and their close association with the perforation border. No cutaneous glands were seen in any part of 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 no cutaneous 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 is evidenced 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 has practically healed with the perforation border. Note the three abnormally large cutaneous glands in the limb-stump integument. (Case E 41, R. temporaria.) x 50.
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