fn Fascicule 2 (Nos 10-24) Juillet 1963










EMILE DOTTRENS Directeur du Museum d’Histoire naturelle de Genève


HERMANN GISIN Conservateur des arthropodes


Conservateur des invertébrés

Ce fascicule renferme les travaux présentés a l’Assemblée

générale de la Société suisse de Zoologie tenue à Genève les 30 et 31 mars 1963.



Ne 1. Jacques DE BEAUMONT. Les Ammophiles paléarctiques du groupe de ‘nasuia. (Hym. Sphecid.). Avec 65 figures dans le texte. .

2. Bernd HORNING. Zur Kenntnis der A nn des Eichhérnch (Sciurus vulgaris) in der Schweiz. Mit 1 Textabbildung .

3. J.L. Perret. Les Gekkonidae du Cameroun, avecla description de deux sous espéces nouvelles. Avec 5 figures dans le texte

4. Hans-Rudolph HAEFELFINGER. Remarques biologiques et sqstematianes au Ir sujet de quelques Tritoniidæ de la Méditerranée ( Moll. Opi Avec 11 figures dans le texte . :

5. Hermann Gisin. Collemboles d'Europe. y. ESS 20 ee Agee ie ns i

6. Georges Dusois. Contribution à l’étude des Trématodes de Chiropteres. Revision du genre Allassogonoporus Olivier 1938 et note additionnelle sur le sous-genre Prosthodendrium Dollfus 1931. Avec 4 figures dans le texte 5

7. G. Mrermop et E. BINDER. Les Types de la Collection Lamarck au Muséum de Genève. Mollusques vivants. V. Avec 34 figures dans le texte . ©

Ne 8. Robert MATTHEY. Polymorphisme chromosomique intraspécifique chez un Mammifére Leggada minutoides Smith Lams ear Avec 15 a La res dans le texte :

9. H. SAINT Girons et E. an te Gee a nes Vipera e re È en. montagne Tonne ¢

. N°10: L. N. RUBEN, Lucké carcinoma implants i in regenerating de regressing | urodele limbs. With 4 figures . 3

41. M. BaLLS, Xenoplastic Dons Bf amphibian lymphoid tino With 5 figures PEA he Are

N°12. J. BovET, Etude, par l’ Analyse du conten ae oies Cote Effraie (Tyto alba), de fluctuations dans les populations de Micromammifères. Avec une tabelle et une figure dans le texte . È

N°13. A. M. Du Bots et F. GRIESSEN, L’activité HORS hei le ich de Tal 4 et de cobaye. Avec une planche Fy Sy ke CR 24

N°14. H. R. HAEFELFINGER, Bedarf die marine aun Mo sicari Kiis- tenzone eines Schutzes? A

Ne15. P. E. Howse, Zur Evolution der Erzeugung von Erschütterungen ae Benachrichtigungsmittel bei Termiten. Mit 3 Textabbildungen

N°16. W. Huser und H. SÄGESSER, Verkeilung und Wölbung der Frontalia beim Reh (Capreolus capreolus). Mit 7 Textabbildungen 3

Prix de l’abonnement :

Suisse Fr. 75.— Union postale Fr. (en francs suisses) |

Les demandes d'abonnement doivent être adressées à la rédactic

REVUE SUISSE DE ZOOLOGITIE Tome 70, n°s 10 à 24. Juillet 1963



Communications publiées ailleurs :

Werden an anderem Orte veröffentlicht:

R. Matthey. Un cas de polymorphisme chromosomique intra- spécifique dans une population du Mammifere Leggada minu- toides Smith (Rodentia, Muridae). Rev. suisse Zool. 70: 173-190.

A. W. Blackler. Stérilité spontanée et provoquée chez Xenopus laevis.

J. Aubert. Observations sur des migrations d’Insectes au col de Bretolet. Bull. Soc. entom. suisse.

R. Weber. Verhalten und Kennzeichnung der Kathepsine im Schwanz der Xenopuslarve bei spontaner und induzierter Meta- morphose in vitro.

R. Salzmann et R. Weber. Zur Lokalisation der sauren Phosphatase und der Kathepsine im Schwanz der metamorphosierenden Xenopuslarve.

Rev, SUISSE DE Zoot., T. 70, 1963. 17

iw) D SI


No 10. L. N. Ruben, Genève. Lucké carcinoma implants in regenerating and regressing urodele limbs.! (With 4 figures)

Station de Zoologie Expérimentale, Université de Genève,? and Reed College, Portland, Oregon, U.S.A.


I suppose that those of us who work with Amphibian limbs would agree that one of the features of vertebrate regenerating systems that attracted us was the hope that such systems hold for eludication of the properties inherent in the phenomenon of cellular differentiation. How stabile should we consider the differentiated state of cells to be? We know for instance, that many differen- tiated cells possess the ability to perform at least one specialization over and above their normal adult speciality; they can become neoplastic. It would be interesting to know if they can, under unique circumstances, also become metaplastic. The question of the stability or plasticity of differentiated cells is, therefore, of interest to students of both normal and abnormal developmental processes. By virtue of its unique nature, the vertebrate limb regenerating system may someday supply us with this kind of information.

It is my purpose here to review attempts on my part to distin- guish between what might be called morphological dedifferentiation and cellular or functional dedifferentiation. Because of the broad and varied usage of the term dedifferentiation in the literature, it 1s always necessary to define what one means by the term. By morphological dedifferentiation, | mean change in the observable

1 Experiments reported herein were supported in part by Fellowships (C 4167) and grants (C 2913) from the National Cancer Institute of the National Institute of Health, Bethesda, Maryland, U.S.A.

? The author wishes to express his sincere appreciation to Professor M. Fischberg for offering the hospitality and facilities of his laboratory so that this work may be extended. He is further indebted to his colleague M. Balls, for the many stimulating discussions which grew out of his review both of this manuscript and the work presently being performed to extend it.


characteristics of cells such that their organization as tissues is lost. Further, the identity of their tissue of origin is no longer discernible. Nothing is said of cellular potency. Perhaps, disso- ciation or dissaggregation would be appropriate terms here. The term modulation is sometimes used for a similar sequence of events in tissue culture work, but, to me at least, the term modulation seems to have implicit within it, the notion of potential constancy. Cellular or functional dedifferentiation, on the other hand, would entail a possible consequence of morphological dedifferentiation such that a change in potency would occur whereby cells would be able to gain one or more new potencies from which they were re- stricted during progressive differentiation, i.e. a return to a pluri- potent condition followed by metaplasia or redifferentiation along anew path. The most controversial aspects of limb regeneration studies have all centered on this particular issue. The reasons for conflict are apparent when one considers that after limb ampu- tation a blastema is established through the aggregation and proliferation of cells which have come from a variety of stump tissues. These cells which will reform the portion of the lmb which has been removed all look essentially alike. Therefore, since it has not yet been possible to follow the fate of individual cells as they leave their tissue of origin and later differentiate into a tissue of the regenerate, information on this subject has been drawn largely from indirect evidence. At the moment, I favor a view on blastemal cell potency which was originally expressed by Hozrzer, Avery and Hortzer (1954) and which appears to be supported by much of my own work dealing with the induction of accessory limbs in urodeles by foreign implants (RUBEN, 1960, 1963). It seems likely that the cells of the limb blastema have for their primary function the production of skeletal material. If an insufficient amount of regeneration occurs, skeleton is prefe- rentially formed. This occurs regardless of whether the blastema has been established from cells which have come from only soft mesodermal components of the limb, e.g. muscle and connective tissue, or from a cellular population which includes limb skeleton in its tissues of origin. This suggests then a limited metaplasia such that cells which come from non-skeletal mesenchymally derived tissue may form skeleton following morphological dediffe- rentiation. Whether former skeletal cells can form anything but

226 L. N. RUBEN

skeleton in the regenerate is not as yet known. Wiırrıs (1953), in discussing the intermutability of mesenchymal cells in patholo- gical situations, however, has pointed out that the reverse trans- formation, of osteoblast to fibroblast, is a frequent one, displayed in many familiar lesions in which the fibrous replacement of bone takes place, as in the various types of osteitus fibrosa. In these lesions, the fibrous replacement is not effected by the proliferation of pre-existing fibroblasts to fill gaps left by departed bone cells, but by a progressive transformation of bone cells into fibrocytes ”. A variety of tumorous and non-tumorous pathological conditions are known which involve the more usual transformation of fibro- blast to skeleton in heterotopic regions.

One aspect of the experimental approach I shall describe in a moment then, was that it represented an attempt to supply biolo- gically marked cells to a blastema so that one might have the possi- bility of observing metaplasia directly.

The other aspect had to do with the issue of what effect a post-embryonic morphogenetic system might have on a cancer growing in close association with it. This approach was suggested by a hypothesis dealing with the genesis of cancer, expounded by Joseph NEEpHam (1942) which stated that cancerous growths represent morphological escapes from weak or absent individuation fields. He defined an individuation field as a region which causes all the cells within it to form a whole; a whole organism or a whole part of an organism, e.g. an organ. His hypothesis was based upon the observation that mammals, which cannot normally regenerate lost parts, are apparently more susceptible to spon- taneous and experimental cancers than are the lower vertebrates which retain the ability to regenerate. NEEDHAM’s suggestion was then, that the ability to regenerate is an expression of the persistance of individuation fields in the differentiated organism and that such persistance acts as a deterant to cancer formation. He further suggested that cancer be applied to a strong individua- tion field in order to test whether the field could master the cancer and cause it to form structures normal to that field. Such regulation of a cancer might occur in two ways. First, the possi- bility exists that the cancer cells might be like embryonic cells in possessing an array of potentialities, which are realized only upon exposure to some strong director, the individuation field.


On the other hand, since cancer cells are probably in at least a partially differentiated state and as such may be as limited in competence as any other specialized cell type, cellular dedifferen- tiation to some pluripotent condition would become a prerequisite to their transformation.


The first regenerating system utilized in my research was that of the larval urodele limb, which I felt would supply a stronger individuation field than its adult counterpart. Two host species were used, Amblystoma opacum and Amblystoma maculatum, and the donor tissue was the renal adenocarcinoma of Rana pipiens which has been described in detail by BaLpuin LuckE (1934). One can distinguish frog donor cells from salamander host cells by their size, since the frog cells are smaller and by their differential affinity for haematoxylin; frog nuclei are more lightly stained.

The first series of my experiments tested the effect of implan- tation upon the cancerous epithelial structures of the implant. All implants were made subcutaneously on the dorsal forelimb surface between elbow and wrist. The results showed that over a 64 day experimental period, these epithelial elements were unaffected by the foreign environment of the non-regenerating larval limb.

A second series was run having the same aim as the first, how- ever, the antihistamine, Pyribenzamine, was omitted from the operative procedure. Rose and Rose (1952) had found that the carcinoma implants would take in adult urodele limbs when the hosts were exposed to this antihistamine for a short period prior to implantation but that when the Pyribenzamine was omitted, the implants quickly degenerated. The antihistamine served to diminish the initial responses on the part of the host to incompatable donor tissue. It was found, that when these larvae are used as hosts, the Pyribenzamine was unnecessary, since the epithelial elements of the implants maintained their typical morphology throughout the 64 day experimental period, again in a non-rege- nerating limb environment.

The third larval series, dealing with the major problem I have outlined, entailed implanting this carcinoma, using Pyribenzamine, allowing two weeks to make sure that the implant had established


lastitution SEP 27 106:

228 IL. N. RUBEN

itself, amputating the limb through the donor material and looking for any morphological responses on the part of the carcinoma to the various phases of regeneration which had been slowed down by the use of low temperatures (14° C +2). That portion of the limb which was removed by initial amputation through the implant was used as a biopsy to study the condition of the implant tissue prior to its exposure to the regenerative processes. Regeneration of the host limbs was normal in all cases. That the implants remained unaffected by the regenerating system adjacent to it indicated that individuation field strength bears no relationship to the maintenance of the cancerous condition. In the light of this evidence, it seemed unlikely that the carcinoma cells are pluri- potent in the sense of embryonic or cellularly dedifferentiated tissue. This cancerous condition then would appear to represent a differentiated state with respect to this characteristic of compe- tence to react to individuative influences of limb morphogenesis. Assimilation was not achieved by the limb field (Rusen, 1955).

A study by Goopwin (1946) on the regeneration of various age groups of urodela had indicated that larval limbs have less comple- tely differentiated tissues than adults and therefore are able to establish a blastema with greater facility. The level of tissue differentiation then would establish the ease or difficulty in forming a blastema, or to put it another way, the level of differentiation determines the degree of dedifferentiation (at the tissue level at least) necessary before a blastema can be established. Degree of dedifferentiation then becomes a temporal factor. It seemed possible then that an increase in the degree of dedifferentiation to which the cancer was exposed might be useful in achieving the goals of this experimentation. Since the larval experiments demonstrated the unlikelyhood that the cancer cells were them- selves pluripotent, if one hoped to demonstrate any developmental effect on the cancer, it would now be necessary to bring about morphological dedifferentiation of the cancerous tissue. To test the possibility that the duration of dedifferentiation within the host limb might be a factor in determining the ability of the rege- nerating system to alter tissues implanted within it, the carcinoma implants were exposed to three types of experimental situations. Each situation would successively call forth a greater extension of the dedifferentiative phase of regeneration. The three situations


were as follows: 1. The regenerating system in this instance was designated as a simple regenerating system; it was produced in adult urodele limbs in response to a single transverse amputation performed in the usual manner at a level which passed through the donor cancer material. 2. Regeneration as it occurred under the second set of experimental conditions was designated as exar- ticulate regeneration; the method entailed amputation at the elbow, removal of the humerus after its exarticulation at the shoulder, and implantation of a piece of the cancer into the space formerly occupied by the humerus in a position just proximal to the level of amputation. The dedifferentiation phase is of greater duration in this type of system than in simple regenerating systems. 3. The morphological phenomenon produced in accord- ance with the third experimental situation involved regression as a result of denervation in one series and excessive x-radiation in another. In the first instance it was induced to occur, by ampu- tation through the donor cancer in larval host limbs, as in the “simple” system, accompanied by complete serial denervation of the limb by resection of the brachial plexus. Larval urodele limbs will regress subsequent to amputation in the absence of peripheral innervation. These implants were exposed to a rever- sible regressing field; reversible in the sense that after three weekly denervations and some regression, the nerves were allowed to reenter the stump and regeneration proceeded to take place. X-irradiation of amputated larval host limbs prior to cancer implantation producing a totally regressing system was also achie- ved in a separate experiment.

First, the results using a simple regenerating system. The carcinoma implants were made into 70 adult Triturus viridescens hosts following the methods used in the earlier larval work. As before, the implants were placed subcutaneously on the dorsal surface of the limbs between the wrist and elbow. Pyribenzamine was used for implant protection and the amputation cleaved the implant material. The experimental period in this work was from 4 to 70 days post-amputation and the temperature was kept at 20°C. The results indicated once again that the Rana pipiens renal adenocarcinoma implants were refractory to the influences

present in the urodele limb during simple regeneration (RUBEN, 1956a).


230 L. N. RUBEN

Since the remaining experiments have up until now been reported only in abstract form (RuBEN, 1956b, 1958) and one will be reported for the first time, I shall present them with accom-

panying figures.


Eccentric blastema (B) from exarticulate regenerate with a cancer implant occupying a central position (28 days post-amputation). About 80

The dedifferentiation phase in ~ exarticulate regenerating systems includes the post-amputation period from about 10 to 25 days at 20° C, as opposed to 7 to 13 days in simple regene- rating systems at the same temperature. ©“ Exarticulate regene- rating systems were established in both forelimbs of 50 Triturus viridescens adults. The carcinoma implants were made proximal to the level of amputation in the right forelimbs only. The left forelimbs, bearing no implants, regenerated slightly more rapidly than did the right. Nevertheless, blastemata formed in both limbs by 30 days and paddle regenerates, with digital indications


were obtained by 45 days post-amputation. It was of interest that the right forelimbs produced eccentrically placed blastemata. Figure 1 illustrates an early blastema (B) which was eccentric. Note that the cancer implant occupied the central region below the wound epithehum which quickly covered the amputation


Cancer epithelium (E) in direct contact with redifferentiating ulna (U) (45 days post-amputation). About 120 x

surface. As a result of THornton’s recent work (1960) which showed that an eccentric apical cap of the wound epithelium will establish an eccentric blastema below it, I can now by use of hind- sight suggest that the centrally located implants diverted the nerves which normally enter the wound epithelium to establish the cap. This eccentric cap then may act to dissociate, morpholo- gically dedifferentiate, if you prefer, the stump tissues below it. Following Tuornton’s lead one may further suggest that these dissociated cells then are reaggregated into an accumula- tion blastema below the eccentric cap. Proliferation within the reaggregated system would then lead to the type of formation

22. L. N. RUBEN

shown in Figure 1. The cancer epithelium, which had been in contact with this morphogenetie activity, remained intact and was apparently unaffected. The stability of the cancer tissue is demonstrated in a particularly striking fashion in Figure 2, which shows cancer epithelium (E) in direct contact with rediffe-

Biken SX

Cancer implant (I) after 10 days of regression. The implant shows a faw mitoses and no dispersion About 100 x

rentiating ulna (U). Further, it seems obvious that the morpho- logicalogical dedifferentiation which supplied the blastemal com- ponents, which in this case had reformed humeral condyles, as well as more distal structures, failed to dissociate the tumor. No dispersed frog cells could be identified in any preparations studied. The limitations of the technique, however, are such that small numbers of individual cancer cells, which fail to seed a new popu- lation within the host tissues, might not be discerned. That skeleton-less stump can regenerate a new limb portion which is complete is, I think, further support for the idea that all meso- dermally derived limb stump tissues release blastemal cells with


skeletagenous potency. The implant “take” percentage, which in this experiment was 79%, was higher than in larval or adult simple regenerating systems where it is usually about 65%. This effect is most likely correlated with the lack of amputation through the implant in the ~ exarticulate experiments.



Fic. 4

Cancer implant which had been exposed to both regression and regeneration (36 days post-amputation). Note the mitoses in the field. About 320 x

Extension of amputated host limb dedifferentiation by brachial plexus resection was produced in 162 larval Taricha granulosa and 50 larval Amblystoma opacum. The “take” percentage was substantially lower in the denervated groups than in their controls suggesting that perhaps innervation may play a role in trans- plantation success. Only 27% of the implants in denervated unamputated controls survived, as opposed to previous percentages ranging from 65 to 79 percent. Those implants which survived regression and regeneration in the experimental groups exhibited no dispersal of their cellular units and were unaltered by the reversible regression and regeneration occuring about them. Histological

234 L. N. RUBEN

examination of limbs recovered at intervals from zero to 54 days post-implantation indicated that the integrity and stability of the cancerous tissue was maintained (fig. 3). Figure 4 shows an implant which had been exposed to both regression and regeneration. Note that 5 mitotic figures are visible in the field. This mitotic activity is indicative of the healthy state of the cancer.

Finally, 25 larval Amblystoma opacum received 900r x-irra- diation! on the forelimbs only and were implanted 12 hours later with unirradiated Lucké carcinoma. The remainder of the body had been protected with a 34” lead shield. The limbs had all been amputated 6 days prior to irradiation. The implants were all made in the direction of the 6 day irradiated regenerate. X- irradiated limbs of larval urodeles will regress completely to the shoulder if they are amputated either shortly before or subsequent to irradiation. Experimental time was 50 days post-implantation. In all cases, limbs receiving the implant material regressed to a greater extent than the controls. Only one implant was recovered or identified in histological preparations of the implant bearing limbs. This implant though partially necrotic had at least some tubules in the regressing region which were intact. The lack of implant material in all of the other cases may have been due to implant degeneration or implant dissociation. The condition of the one identifiable implant and the absence of dispersed frog calls had suggested that the former was the more likely situation.


The results of the experiments described above demonstrate that on the whole, implants of the Lucké tumor proved to be remar- kably refractory to the forces at work in the immediate host environ- ment during limb regeneration and regression. It is, of course, possible that this reluctance on the part of the Lucké tumor cells to be affected by urodele limb regeneration may be due either to the fact that they are frog in origin in a salamander host or to the foreign nature of the site, since kidney cells, whether normal or

! The author wishes to express his appreciation to Drs. Milton and Selma Hyman, Portland, Oregon, U.S.A. for providing their radiation facilities and their aid. The radiation factors were: Dist. = 21 cm., Time = 54 sec. KV = 220, M.A. 20, no filter, 4% value layer = 5.55 cm., and temp. = 16-180 C.


cancerous, do not usually find themselves as part of a limb system or to both. These results however, are in agreement with those of Breepts (1954) who tested an induced urodele limb sarcoma and SHEREMETIEVA-BRUNST (1955) who tested an Axolot! melanoma in association with urodele limb regeneration.

These failures to demonstrate positive effects on the cancers thus far tested should not be interpreted as denying the potential value of this unique test system. The use of limbs which retain morphogenetic potential for studies of these kinds provides one, I believe, with a potentially superb opportunity to observe inter- actions between cancer and morphogenetic processes in already differentiated organisms. Further, Brervıs (1952) with his results has revealed an unusual demonstration of induction of both normal morphogenesis, in the form of supernumerary limb struc- tures, and transplantable cancer by using the same carcinogenic agent in this system. This suggests to me that a field with mor- phogenetic potential may be capable or organizing cells in their early stages of response to carcinogens into structures which are quite normal for that field. NeEDHAM (1942), you will remember, had suggested that regenerative power and persistance of controll- ing pattern were synonomous and that such persistance acts as a deterant to cancer formation. That BREEDIS induced many more supernumerary limbs than cancers may be indicative of the vali- dity of this part of NeEDHAM's argument. A review of all known occurrences of spontaneous cancers in Amphibia (Batis, 1962) indicates that no tumors of limb tissues which retain morpho- genetic potential have as yet been reported. The second part of NEEDHAM’S suggestion as to whether cancer in an advanced state can be so regulated, remains a controversial matter still open to test.


Experimentation involving the implantation of the renal adenocarcinoma of Rana pipiens into close association with a variety of situations involving urodele limb regeneration and regression is discussed with particular emphasis on the significance of the results as they apply to our knowledge of the stability of cellular differentiation and to the morphological escape hypothesis of cancer.

236 L. N. RUBEN


Batis, M. 1962. Spontaneous neoplasms in Amphibia: A review and descriptions of six new cases. Canc. Res. 22: 1142-1154. Breepis, C. 1952. Induction of accessory limbs and of sarcoma in the newt (Triturus viridescens) with carcinogenic substances. Canc. Res. 12: 861-866. 1954. Effect of temperature on a neoplasm regenerate complex in the newt (Triturus viridescens). Fed. Proc. 13: Abs- tract 1390. Goopwix, P. 1946. A comparison of regeneration rate and metamorphosis in Triturus and Amblystoma. Growth 10: 75-87. Hortzer, H., Avery, G. AND HoLTzER, S. 1954. Some properties of the regenerating limb blastema cells of Salamanders. Biol. Bulls AO tests: Lucké, B. 1934. A neoplastic disease of the kidney of the Leopard Frog. Am. J. Cane. 20: 352-79. NEEDHAM, J. 1942. Biochemistry and Morphogenesis. Cambridge Univ. Press, London, England. Rose, S.M. anp Rose, F.C. 1952. Tumor agent transformations in Amphibia. Cane. Res. 12: 1-12. Rusen, L. N. 1955. The effects of implanting anuran cancer into non- regenerating and regenerating larval urodele limbs. J. Exp. Zool. 128: 29-52. 1956a. The effects of implanting anuran cancer into regenerating adult urodele limbs. I. Simple Regenerating Systems. J. Morph. 93: 389-404. 19565. Anuran cancer implants in urodele exarticulate rege- nerating systems. Anat. Rec. 125: 626-627 (abstr.). 1958. The effect of reversible urodele limb regression upon Lucké carcinoma implants. Anat. Rec. 132: 493-499 (abstr.). 1960. An immunobiological model of implant-induced urodele supernumerary limb formation. Amer. Nat. 94: 427-434. and Srevens, J. 1963. Post-embryonic induction in urodele limbs. J. Morph. (In Press). SHEREMETIEVA-BRUNST, E. A. 1955. Studies on the relationship between neoplastic and regenerative growth. Proc. of the Amer. Assoc. for Canc. Res. 2, No, 1. April. THornton, C. S. 1960. Influence of an eccentric epidermal cap on limb regeneration in Amblystoma larvae. Dev. Biol. 2: 551-569. Wirrıs, R. A. 1953. Pathology of Tumors. The C. V. Mosby Co, St. Louis, Mo. U.S.A.


No 11. M. Balls, Genève. Xenoplastic implantation of amphibian lymphoid tumours.!? (With 5 figures.)

Station de Zoologie expérimentale (Université de Genève), Route de Malagnou 154, Genève.

It has recently been found that the anuran amphibian Xenopus laevis is susceptible to the formation of spontaneous lymphoid tumours (1) and that similar tumours may be induced using methyl- cholanthrene (2) or benzpyrene; both spontaneous and induced tumours are readily transplantable (3). When tumour fragments are placed in adult or immature Xenopus a heightened homograft reaction occurs, followed by, in a very high percentage of cases (97%), the development or lymphoid tumours at the implantation site and in the visceral organs particularly the liver, spleen and kidneys. The use of the dorsal lymph sac permits a clear distinc- tion between tumour growth at the implantation site and at a distance.

This article is concerned with some preliminary xenografts between the anuran Xenopus laevis and the urodele Triturus cris- tatus, which were carried out to take advantage of the difference in cell size as a means of distinguishing between host and implanted cells (see also (4)).

Tumour [LA (3) was induced by placing methylcholanthrene crystals in arachis oil under the abdominal skin of an adult female Xenopus; when the animal was killed 263 days later, lymphoid tumours were found in the liver, spleen and kidneys. Fragments of a liver tumour nodule were implanted into the dorsal lymph sac (DLS) of 6 adult Xenopus (Transfer A), all of which gave positive results. Small fragments of a DLS tumour from one of these individuals were placed in the abdominal cavity of four adult Triturus cristatus (Transfer B), two of which developed lympho-

1 This investigation is being supported by the Fonds national suisse pour la recherche scientifique (No. 2219).

2 The author is grateful to Professor M. Fischberg for his advice, to Profs. A. W. Blackler and L.N. Ruben for their comments on this article, and to Dr. S. Neukomm for the gift of six newts in mid-winter.




sarcomas of liver composed of newt cells (Fig. 1), which are much larger than those of Xenopus and whose nuclei stain differently. Although no spontaneous lymphoid tumours have been reported for Triturus cristatus (1), such tumours have been induced in this species by LEONE (5), while INOUE (6) has found a spontaneous lymphosarcoma in the Japanese newt, Triturus pyrrhogaster.

EIGHT: Liver of Triturus (Tr. B No. 4) bearing a lymphoid tumour (T). L = normal liver tissue containing pigment (P). X 45

In view of the remote possibility that the newt tumours were spontaneous and not connected with the introduction of the Xeno- pus material, fragments of one Triturus tumour (derived from Transfer B) were put into the DLS of 6 immature Xenopus (Trans- fer C). All six recipient frogs subsequently developed DLS tumours which invaded the skin and back muscle, as well as lympho- sarcomas of liver (Fig. 2) and spleen. Three of these animals also bore tumours of the kidney. Both the DLS and visceral neo- plasms were composed of Xenopus cells (Fig. 4) and contained no Triturus cells. One DLS tumour was transferred into a further


six Xenopus (Transfer D), all of which developed invasive tumours in the DLS, liver, spleen and kidneys (Table 1).

Further fragments of the newt liver tumour (Transfer B) used in Transfer C were stored in physiological solution (Niu & Twitty 1/10) at C for one week and then put into the abdominal cavity

IRIs: A.

Liver (L) of Xenopus (Tr. C No. 1) bearing two large lymphoid tumour nodules (T). X 50

of five Triturus cristatus (Transfer E). One host died 10 days later when the swelling of the stomach after feeding pushed part of the liver through the wound. The remaining four were killed 55 days after implantation. All four bore lymphoid nodules on the liver, with increased lymphocytic activity in the cortical region and groups of lymphocytes spread throughout the remaining liver tissue. In each case the kidney contained nodules of lymphocytes infiltrating between the primary tubules. In two cases the spleen bore white nodules of lymphocytes, and part of the implanted mate- rial had been left in the wound (see also (3)) since both showed body wall muscle invasion, and in one the skin was also involved. All the tumour nodules in each animal were composed of Triturus cells.

REV. SUISSE DE Zoot., T. 70, 1963. 18


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i. at distance:



Theoretical pathways of tumour transfer after dorsal lymph sac implantation. The visceral organs pictured are (from left to right) heart, liver, spleen

and kidneys. DLS = dorsal lymph sac, AC = abdominal cavity, I =implant, H = host cells, F = factor.

Theoretically, the transmission of tumours by the transfer of small, cellular fragments could have occurred in two ways (Fig. 3):

a) tumour cell transplantation the transferred cells themselves multiplied and formed growing neoplasms, cells from which

metastasized to other parts of the host.

242 M. BALLS

b) host cell transformation the transferred cells released a sub-cellular factor which transformed the host cells to make them neoplastic. Since lymphoid cells are circulatory there were two further possibilities with the lymphoid tumours:

1) at implantation site host cells moved to the implan- tation site, there became transformed and metastasized to the viscera, while the implant was destroyed and replaced by host cells.

ii) at a distance the factor migrated via the blood or lymph systems from implant to viscera, where the host cells were transformed and produced tumours.

PonteN has worked on this aspect of tumour transplantation using the sex chromosomes to distinguish between host and im- planted cells (7), and has found that, while implanted chicken lymphoid tumour RPL 12 (8) cells grow in the host and metasta- size to the viscera (1.e. type a), implanted Rous sarcoma (9) or chicken erythroleukaemia (10) cells disappear, but tumours com- posed of host cells result (i.e. type b).

The present preliminary series of experiments indicates that the Xenopus lymphosarcoma ILA is transplantable into Triturus cristatus, and that the resulting liver tumours readily grow and spread when transferred to Xenopus or Triturus. Furthermore, the tumour cell type both at the implantation site and in the viscera is that of the host and not that of the implanted material (Figs. 4, 5). Hence, it would seem that, in xenografts at least, the cells of the transplanted tumour do not proliferate to form malignant neo- plasms in the host, but are replaced by transformed host cells, which also make up the visceral tumours (i.e. type b 1 or ii).

A further point of interest from the series of experiments summarized in Table 1 is the very short interval between the DLS implantation of tumour material and the development of skin and back muscle invasion and of advanced visceral tumours. In all six Xenopus used in Transfer D both lobes of the liver, the spleen and kidneys were extensively invaded, although the implantation operation had taken place only 18—23 days before.

It is hoped that a further series of Xenopus-Triturus xeno- grafts, together with other approaches, will result in a more detailed



Detail of Xenopus lymphoid tumour shown in Fig. 2. The Xenopus lymphoid cells are small and the nuclear contents are dense and stain deeply. L = liver cells. X 500


Detail of lymphoid cells of Triturus tumour shown in Fig. 1. The cells are larger than those of Xenopus (above) and the nuclear contents more granular. X 500

244 J. BOVET

knowledge of the aetiology of the Xenopus lymphosarcoma and of any cell-transforming factor which may be involved.


1) Barus, M. 1962. Spontaneous neoplasms in amphibians: a review and descriptions of six new cases. Cancer Res. 22: 1142-1154.

2) Methylcholanthrene-induced tumor in the anuran amphibian Xenopus laevis (in press). 3) Transplantation of spontaneously occurring and chemically induced

lymphoid tumors in Xenopus laevis (in press).

4) Rupen, L. N. 1963. Lucké carcinoma implants in regenerating and

a regressing urodele limbs. Rev. Suisse Zool. 70 (in press).

5) Leone, V. 1957. Tumori da meticolantrene in Tritoni. R.C. Ist. lom- bardo (B) 92: 220-240.

6) Inove, S. 1954. On the transplantable spontaneous visceral tumour in the newt, Triturus pyrrhogaster. Sci. Repts. Tohoku Imp. Univ. 20: 226-236.

7) Pontén, J. 1962. Sex chromosomes as markers in transplanted chicken leukaemia cells. Nature, London 194: 97.

8) 1962. Transplantation of chicken tumor RPLI2 in homologous hosts. J. Nat. Cancer Inst. 29: 1013-1021.

9) 1962. Homologous transfer of Rous Sarcoma by cells. J. Nat. Cancer Inst. 29: 1147-1159.

10) Transmission in vivo of chicken erythroblastosis by cells. J. Cell

& Comp. Physiol (in press).

12. J. Bovet, Lausanne. Etude, par l’analyse du contenu de pelotes de Chouette Effraie (Tyto alba), de fluctuations dans les populations de Micromammi- fères. 1 (Avec une tabelle et une figure dans le texte.)

Institut de Pharmacologie de l’Université de Lausanne.

Le régime alimentaire «normal» de VEffraie est bien connu (cf. UTTENDÖRFER, 1952). Il peut toutefois présenter dans le temps des variations importantes, dont l’interprétation se révèle très

1 Résultats obtenus dans le cadre d’un travail bénéficiant de l’aide du Fonds national suisse pour la recherche scientifique (crédit 1856).


utile pour l’etude des fluctuations des populations des espèces- proies.

En été 1962, nous avons analysé une centaine de pelotes d’Effraie qui provenaient toutes de la méme place-gite, à environ 250 m a l’ouest des bâtiments de la Station biologique de la Tour du Valat, en Camargue !. La moitié de ces pelotes étaient déjà anciennes, de couleur grise, très sèches, et l’autre moitié relativement fraîches, de couleur noire « laquée », très humides. D’après les indications de M. Kowatsk1, les premières dataient de 1961 et les secondes du printemps et du début de l’été 1962; toutes avaient été proba- blement rejetées par un même individu.


Mammifères trouvés dans les pelotes de 1961 et 1962. n= nombres d'individus; % du total = pourcentage du total des Mammifères.