neoteny with goitre in triturus helveticus by j. m....

Neoteny with Goitre in Triturus helveticus

By J. M. DODD AND H. G. CALLAN

(From the Department of Natural History, The University, St. Andrews)

With 2 plates (figs, i and 2)

SUMMARY

A population of newts from a pond near Crail, Fife, was found to contain neotenicand goitrous specimens of Triturus helveticus in 1951 and 1952 but not in 1953 and1954. This is a unique record of goitre among amphibians.

The thyroid glands of normal adult and neotenic non-goitrous T. helveticus arepaired spheroidal or ellipsoidal bodies whose longest dimension varies between 300and 700 fi. They consist of a few large follicles. The thyroid glands of the goitrousneotenic specimens may be as much as 4 mm. long. They are extremely hyperplasticand hyperaemic and for the most part consist of follicles smaller than those charac-teristic of normal newt thyroids. These goitrous thyroids produce major displacementof other structures in the throat region but do not invade other tissues.

It is suggested that the goitres result from the exposure of overwintering newt larvaeto the 'brassica factor' carried to the pond in the faeces of rabbits feeding on turnipsand kale in neighbouring fields.

CONTENTS

PAGE

I N T R O D U C T I O N . . . . . . . . . . . . . I Z I

M A T E R I A L . . . . . . . . . . . . . 1 2 2

M O R P H O L O G I C A L A N D H I S T O L O G I C A L D E S C R I P T I O N O F T H E T H Y R O I D G L A N D S . . - 1 2 3

S T A T U S O F T H E G O I T R E S . . . . . . . . . . . 1 2 5

T H E N E W T P O P U L A T I O N . . . . . . . . . . . 1 2 5

D I S C U S S I O N . . . . . . . . . . . . . 1 2 6

R E F E R E N C E S . . . . . . . . . . . . . 1 2 8

INTRODUCTION

' n r ^ H E phenomenon of neoteny is well known in urodeles, but, so far as weJ. are aware, none of the many records in the literature mentions an accom-

panying goitre. Duchosal and Junet (1926) studied a single neotenic specimenof Triturus ('Triton') alpestris and reported that the thyroid gland did notdiffer in histological appearance from that of a normal specimen. Kuhn (1925)studied the thyroid glands of a single neotenic specimen of Triturus cristatus:he found the glands to be normal in size, though consisting of more numerous,smaller follicles than is usually the case in normal specimens. Hartwig andRotmann (1940), in an extensive study of a partially neotenic population of'Triton taeniatus' (Triturus vulgaris), state that the neotenic specimens whichthey examined had thyroid glands of normal size but showing some histo-logical signs of low activity. They also point out, however, that the histological

[Quarterly Journal of Microscopical Science, Vol. 96, part 1, pp. 121-128, March 1955.]

122 Dodd and Callan—Neoteny with Goitre in Triturus helveticus

appearance of normal newt thyroids varies considerably through the year andthat there is no sharp distinction between neotenic and normal specimens asregards the histological appearance of these glands. Hartwig and Rotmannfurthermore give a comprehensive review of the findings of previous workerson urodele neoteny: the same subject has also been reviewed by Wolterstorffand Freytag (1951) and by Lynn and Wachowski (1951): nowhere has goitrousenlargement of the gland been encountered.

Not only have goitres never been reported from urodeles in nature: experi-mental work testing various goitrogens has shown that both urodeles andanurans are highly resistant to the goitrogenic activity of these substances.Joel, D'Angelo, and Charipper (1949) have pointed out that the changes inamphibian thyroids induced by various goitrogens are both qualitatively andquantitatively less marked than those which occur in birds and mammals as aresult of similar treatment. Adams (1946) kept adult specimens of Triturusviridescens in strong solutions of thiourea for 86 days and found only slighthyperplasia. In view of these findings the occurrence in nature of neotenicnewts with accompanying goitre is of particular interest.

MATERIAL

In a routine collection of newts from a pond near Crail, Fife, on 19 May 1951,two neotenic specimens of T. helveticus Razoumowsky were noticed. One wasa female, length 71 cm., the other a male, length 6-5 cm. Both specimens werein full breeding dress, yet with persistent larval gills and larval head shape.Both specimens died within 3 days of capture, probably owing to their havingbeen kept in damp moss instead of water. Neither of these newts showed evi-dent external signs of goitre and histological examination was not attempted.

In a further collection of newts from the same pond on 14 May 1952, twomore neotenic specimens of T. helveticus were obtained. One animal, length6-7 cm., with well-developed male secondary sexual characters, showed a pro-nounced bilobed pink swelling in the throat region lying below the operculumand extending beyond it posteriorly. A photograph of this animal (specimen A)is shown in fig. 1, A. The swelling was diagnosed as a goitre and subsequenthistological examination confirmed this diagnosis. The other neotenic speci-men from this collection, a male, length 5-2 cm. (specimen B), had no goitre.

Subsequently three further neotenic goitrous specimens (C, D, and E) ofT. helveticus were obtained from the same pond on 11 July 1952. All threeanimals were females, their respective overall lengths being 7-0, 7-5, and7-0 cm. Photographs of specimens D and E are shown in fig. 1, B-D.

During 1953 the pond was visited on numerous occasions and several hun-

FIG. 1 (plate). A, specimen A, showing goitre, persistent gills, and male secondary sexualcharacters.

B, specimen D, profile view.c, specimen D, ventral view showing asymmetral goitre.D, specimen E, ventral view showing smaller bilateral goitre.

• • / * '

mm.

I mm. B

D

FIG. I

J. M. DODD and H. G. CALLAN

Dodd and Callan—Neoteny with Goitre in Triturus helveticus 123

dred newts were examined. Throughout the year 1953 and up to the presenttime in 1954, neither neotenic nor goitrous newts have been found.

All the labelled neotenic specimens were fixed in Bouin's fluid and theentire lower jaw of each was serially sectioned to the level of the posteriormargin of the thyroid glands. The histology of the glands is described in thenext section. Owing to an unfortunate accident the pituitary glands of theseanimals were not subjected to histological examination.

The pond from which these newts were obtained lies in a deep pear-shapedpit in heavy soil. It is approximately 100 feet long by 50 feet wide at the widestpoint. The pit was originally excavated as a stone quarry but the sides, steeplysloping for the most part, are now covered with soil and heavily overgrownwith vegetation. No figures are available as to depth, though this is evidentlyconsiderable and may well reach 15 or more feet. The water surface, whichshows marked fluctuation in level from one season to another, lies some 25feet below the level of the surrounding agricultural land.

A thriving rabbit colony inhabits the sides of the pit and feeds in the sur-rounding fields. These fields are all under cultivation, turnips and kale beinggrown as part of the normal rotation. It is suggested later that this may havesome bearing on the occurrence of the goitre.

MORPHOLOGICAL AND HISTOLOGICAL DESCRIPTION.OF THE THYROID GLANDS

Normal and neotenic non-goitrous specimens

The thyroid glands of normal adult newts are paired structures lying im-mediately anterior to the arterial arches and lateral to the genio-hyoideusmuscles. They are spheroidal or ellipsoidal bodies, the longest dimensionvarying between 300 and 700/z. They consist of a few large follicles and haveno distinct capsule. The follicular epithelium varies through the year fromsquamous to cubical, and the degree of vacuolation of the colloid also varies.

Specimen B is neotenic but non-goitrous. Its thyroid glands (fig. 2, A) liewithin the size range of those of normal newts of similar size (600 by 560/n),but they show signs of low activity. The central section of a complete seriesshows seven large follicles full of eosinophil colloid, which is poorly vacu-olated. The follicular epithelium is low and the cell boundaries are difficultto make out.

Neotenic goitrous newts

The goitres described here have many features in common though theyvary considerably in size, the largest extending from mid-eye region to thelevel of the heart and causing great distension of the entire throat. All aremarkedly hyperaemic, the hyperaemia being readily visible through the oper-culum, whose epidermis, though semi-transparent, is flecked with yellowpigment. The goitres result in major displacement of structures in the throatregion, though there is no invasion of other tissues such as has been found infish thyroid gland tumours. The goitres are not encapsulated, but their


boundaries are well marked and distinct. The follicular epithelium is notfolded: mitotic figures are not common. The colloid is variable in stainingreaction in different parts of the gland: vacuoles are small and few in number.

Specimen A (figs, i, A and 2, B) showed the largest goitre in the series. Thegoitre is symmetrically bilobed, each lobe measuring approximately 4 by3-5 mm. With the exception of the genio-hyoideus muscles and the skeletalstructures it occupies the entire throat region. The follicles are more or lessuniform in size and are smaller than those found in a normal newt thyroid.Cytological details cannot be described since the specimen was fixed afterdeath.

Specimen C also showed a bilobed goitre, the dimensions of each lobe beingapproximately 2 by 2-3 mm. The follicular epithelium varies from squamousto high columnar: there are few large follicles with low epithelium and abun-dant poorly vacuolated colloid, and many small follicles with high columnarepithelium and little or no colloid. The colloid varies in staining reaction. Inthe large follicles it takes eosin and Heidenhain's haematoxylin, whereas in allthe small follicles it takes Heidenhain's haematoxylin exclusively (fig. 2, D).A few cellular inclusions are present in the colloid. The large follicles areirregular in shape and elongated in diverse planes: such follicles are remini-scent of the normal thyroid gland, having low epithelium, indistinct cellboundaries, and colloid with few vacuoles.

Specimen D (fig. 1, B, c; fig. 2, c, E, F). The thyroid gland on the right-handside of this specimen measures 4 by 4-5 mm. and is much larger than that ofthe left-hand side (1-4 by i-6 mm.), though both are goitrous. The largergland contains many small follicles, each with high columnar epithelium andvery small lumen: some regions are non-follicular. There are no cell inclu-sions in the colloid. The follicles of the smaller gland are larger, the follicularepithelium varying between cubical and squamous. A large number of smallperipheral vacuoles are present in the colloid.

Specimen E (fig. 1, D; fig. 2, G). The goitre in this specimen is smaller than

FIG. 2 (plate), A, specimen B, T.S. lower jaw showing thyroid glands of normal size. Bouinfixation; iron haematoxylin and orange G / erythrosin.

B, specimen A, T.S. lower jaw showing massive goitre. The area at top centre consists ofthe genio-hyoideus muscles. Bouin fixation after death; iron haematoxylin and eosin. Theoperculum of this specimen was dissected away before fixation.

C, specimen D, T.S. lower jaw showing asymmetrical goitre. Bouin fixation; ironhaematoxylin and orange G / erythrosin.

D, specimen C, T.S. of part of lower jaw showing goitre. Bouin fixation; iron haematoxylinand eosin. Two regions can be differentiated, one in which the follicular epithelia are low andthe colloid eosinophil, the other with higher epithelia and the colloid stained with haema-toxylin.

E, specimen D, T.S. of non-follicular and microfollicular regions of goitre. Bouin fixation;iron haematoxylin and orange G / erythrosin.

F, specimen D, T.S. of single follicle. Bouin fixation; iron haematoxylin and orangeG / erythrosin.

G, specimen E, T.S. of single follicle showing low epithelium and cellular inclusions in thecolloid. Some of these included cells appear to be in the process of division. Bouin fixation;Mallory's triple stain.

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J. M. DODD awrfH. G. CALLAN


those of the preceding specimens (approximate dimensions of each lobe, 17 byi-8 mm.): it is symmetrically bilobed. Each gland has a relatively normallooking medio-ventral region with large follicles having eosinophil colloid andcubical to squamous epithelium. There are few non-follicular areas. Many ofthe follicles have colloid in which there are large spheroidal vacuolated cellswith nuclei similar to those of the cells of the follicular epithelium itself.

STATUS OF THE GOITRES

As already mentioned in the introduction, previous authors have describedfew or no histological signs of abnormality in the thyroid glands of neotenicnewts. The thyroid glands described in the present paper, on the other hand,are evidently extremely hyperplastic and they appear to have been under theinfluence of an abnormally high level of circulating thyroid-stimulating hor-mone. Though there is no invasion of foreign tissue, the glands are so massiveas to have caused gross displacement of neighbouring structures in the lowerjaw. Marine and Lenhart (1910) have argued at length as to the status of so-called thyroidal carcinomata encountered in trout: in spite of the fact that thetrout tumours invade bone, muscle, and other tissues, these authors have con-cluded that the tumours merely constitute an extreme example of endemicgoitre and that they are not truly cancerous. The newt goitres resemble closelyin histological appearance the spontaneous thyroidal growths described byGorbman and Gordon (1951) in Xiphophorus montezumae, and we considerthat they should in all probability be similarly classed as thyroidal tumoursof a benign nature.

THE NEWT POPULATION

Although we can give no accurate idea of the size of the newt population inthe pond at Crail, this certainly numbers several hundred adults and may wellrun into thousands. The population is mixed and includes both T. helveticusand T. vulgaris. Collections made in March or April include only 10 per cent,or less of vulgaris, but the vulgaris component of the population increasesduring the spring and by June the two species appear to be almost equallycommon. Towards the end of the breeding season the relative abundance ofvulgaris declines. T. vulgaris in most localities in Britain leaves the water atthe end of the breeding season and hibernates terrestrially: clearly the habitsof vulgaris in this pond are those normal for the species.

T. helveticus is considered to be more aquatic in its habits than T. vulgaris(compare Smith, 1951, p. 65). Many, though not all, of the helveticus inhabit-ing the Crail pond overwinter in the water and we have collected them as lateas November in large numbers and in full breeding dress. Collections inMarch, however, although consisting for the most part of fully aquatic speci-mens in good condition, include also a small proportion of specimens whichhave lately returned to the water, these being recognizable by the roughtexture of their skins. Overwintering larvae are common and in spring collec-


tions these show marked variation in size, some being presumably in their firstand some in their second year of life.

The neotenic and goitrous newts which we have collected all belong to thespecies T. helveticus. Such specimens form not more than i or 2 per cent, ofthe total number of helveticus which we have examined. Since most of ourlarge-scale collections have been made in April or May we have handled farfewer vulgaris from this pond. This may account for our failure to find neo-tenic and goitrous specimens of vulgaris: alternatively such animals may notbe present in this population.

DISCUSSION

Many theories have been put forward in attempts to account for the occur-rence of neoteny in urodele Amphibia. We do not propose to review the sub-ject exhaustively in the present paper since extensive surveys have alreadybeen made by Hartwig and Rotmann (1940) and by Lynn and Wachowski(1951). Most theories postulate dysfunction of the thyroid and/or pituitaryglands, due to intrinsic or extrinsic agents.

No student of urodele neoteny has succeeded in providing histological evi-dence of pituitary dysfunction, but several other lines of evidence convergeto implicate the pituitary in this phenomenon. In Anura the importantrole played by the pituitary in metamorphosis has been known for manyyears (Allen, 1916). The experiments of Reineke and Chadwick (1939)on T. viridescens have demonstrated that the urge to enter the water habitatis dependent on a hormone produced by the anterior lobe of the pituitary.Blount (1939) caused the metamorphosis of the normally neotenic Amblystomamexicanum by implantation of pituitary rudiments from the normally meta-morphosing A. tigrinum. As a consequence of this and later work (Blount andBlount, 1947), it has been claimed that two types of thyroid-stimulatinghormone are produced by the pituitary, one being responsible for the produc-tion and storage of thyroid secretion, the other concerned with its release.Blount and Blount suggest that in neotenic forms the releasing hormone isabsent, but the evidence is as yet by no means conclusive. The literature onneotenic newts contains frequent references to specimens which are partialalbinos. Partially albino newts are rare: so are neotenic newts. Clearly thereis a correlation between these two conditions (Smith, 1951) and the pituitarygland may conceivably be the common factor.

Above all, the pituitary gland's connexion with neoteny has been frequentlypostulated on account of the controlling influence which it is known to exertover the thyroid; and knowledge of the implication of the thyroid itself inamphibian metamorphosis dates from the classical experiments of Guder-natsch (1913) and Allen (1916) on Rana spp. and of Jensen (1916) and Huxleyand Hogben (1922) on Amblystoma. The present observations substantiatethis connexion: neotenic newts are rare; goitres have only been observed inneotenic specimens. These two phenomena are clearly correlated with oneanother.


Any attempt to account for neoteny in newts is faced with the problem ofthe coexistence in the same pond of neotenic and normal specimens. Allrecorded cases are similar in this respect. Several authors have consequentlybeen led to postulate a genetic origin of neoteny. In all experiments whereneotenic newts have given rise to offspring, including a successful mating ofa virgin neotenic female with a neotenic male T. vulgaris, the offspring havemetamorphosed as rapidly as have normal control larvae. Although there maybe a genetic component determining a tendency to neoteny, this will be mostdifficult to demonstrate owing to the well-known acceleration of metamor-phosis which regularly attends the raising of newt larvae in captivity.

Several authors have attempted to correlate the occurrence of neoteny withpeculiarities of habitat, but Hartwig and Rotmann, in reviewing the literatureon this subject, were unable to find any significant factors common to pondswith neotenic newt populations. Nevertheless there are two distinct indica-tions that habitat peculiarities may act as causative factors. Zeller (1899) foundneotenic specimens of three different species, T. vulgaris, T. alpestris, andT. cristatus in one and the same quarry pond, while Smith (1950) has describedtwo ponds containing neotenic newts together with giant anuran tadpoles.

Low iodine concentration in the environment is one of the best authenti-cated causes of goitre. In the present instance, however, although no iodinedeterminations have been made for the water of the pond in question, it seemsunlikely that lack of iodine could be the cause since the pond lies within twomiles of the sea and in a district where extensive use is made of sea-weeds asfertilizers. Furthermore the iodine content of Crail drinking water, which istaken from a nearby reservoir, is given as 5 meg. per litre by Murray andothers (1948). This is the second highest iodine concentration recorded indeterminations for 64 Scottish localities.

We are led to postulate that the thyroid goitres here described were pro-duced by the action of a naturally occurring goitrogen, the so-called 'brassicafactor' (see review by Lever, 1951). The goitrogenic action of a diet of cabbageon rabbits was first demonstrated by Chesney and others (1928) and theactive substance isolated and synthesized by Astwood and others (1949). Thelatter authors identified the 'brassica factor' as L-5-vinyl-2-thiob'xazolidone.

Our evidence in favour of this hypothesis is based on four considerations.First, the land surrounding the Crail pond is extensively cultivated: the croprotation practised includes yellow turnip and kale, and these plants weregrown in fields bordering the pond in 1951 and 1952 but not in 1949, 1950,or 1953. Neotenic newts were collected in 1951, neotenic and goitrous newtsin 1952, but neither in 1953 or 1954. Secondly, rabbits which have theirburrows in the slopes leading down to the pond feed in the surrounding fields.Rabbit faeces accumulate in large quantities on the slopes and rainwatercarries faeces and extract of faeces into the pond by drainage. It might reason-ably be expected that this drainage water should contain the 'brassica factor'at times when the rabbits are feeding on turnips and kale. Thirdly, themajority of the larvae and adults of T. helveticus overwinter in the water of the


Crail pond and hence are more exposed to any goitrogens which may bepresent than are newts which habitually overwinter on land. Fourthly, al-though adult amphibians are known to be highly resistant to goitrogens, wedo not know how larval and metamorphosing animals are likely to react. Fromunpublished work on metamorphosing Xenopus larvae in which extensivegoitres have resulted from thyroidectomy (possibly due to the hyperplasia ofsupernumerary follicles) it would appear that the thyroid is in a particularlysensitive state at the time of metamorphosis. Should this be true also of newts,the overwintering helveticus larvae might well be exposed to the postulatedgoitrogen at this critical time.

We have no experimental evidence in support of the above hypothesis.Crude extracts of seeds of Brassica spp. have unfortunately proved highlytoxic to both larval and adult newts and in further work we must test theactivity of the purified 'brassica factor'. In our hands 0-05 per cent, thioureaand c o i per cent. 2-thiouracil have failed to prevent metamorphosis and havealso failed to produce goitres in newt larvae taken from the Crail pond.

This study forms part of a research programme in comparative endo-crinology which is supported by a grant from the Nuffield Foundation. Ourthanks are also due to Mr. D. R. R. Burt and Mr. M. D. B. Burt who intro-duced us to the pond at Crail and who captured the first neotenic specimens.

REFERENCESADAMS, A. E., 1946. Anat. Rec, 94, 532.ALLEN, B. M., 1916. Science, 44, 755.ASTWOOD, E. B., GREER, M. A., and ETTLINGER, M. G., 1949. Ibid., 109, 631.BLOUNT, R. F., 1939. Proc. Soc. exp. Biol. N.Y., 40, 212.BLOUNT, R. F., and BLOUNT, I. H., 1947. Anat. Rec, 97, 380.CHESNEY, A. M., CLAWSON, T. A., and WEBSTER, B., 1928. Johns Hopkins Hosp. Bull., 43, 261.DUCHOSAL, P., and JUNET, W., 1926. Arch. Anat. Strasbourg, 6, 397.GORBMAN, A., and GORDON, M., 1951. Cancer Res., 11, 184.GUDERNATSCH, J. F., 1913. Arch. EntwMech. Org., 35, 457.HARTWIG, H., and ROTMANN, E., 1940. Ibid., 140, :95.HUXLEY, J. S., and HOGBEN, L. T., 1922. Proc. Roy. Soc, B, 93, 36.JENSEN, C. O., 1916. Medd. VetHojsk. Serumlab. Kbh., 44.JOEL, T., D'Angelo, S. A., and CHARIPPER, H. A., 1949. J. exp. Zool., n o , 19.KUHN, O., 1925. Biol. Zbl., 45, 483.LEVER, J., 1951. Experientia, 7, 201.LYNN, W. G., and WACHOWSKI, H. E., 1951. Quart. Rev. Biol., 36, 123.MARINE, D., and LENHART, C. H., 1910. J. exp. Med., 12, 311.MURRAY, M. M., RYLE, J. A., SIMPSON, B. W., and WILSON, D. C, 1948. Medical Research

Council Memorandum No. 18. London (H.M. Stationery Office).REINEKE, E. E., and CHADWICK, C. S., 1939. Proc. Soc. exp. Biol. N.Y., 40, 691.SMITH, M., 1950. Brit. J. Herpetol., 1, 91.

1951- The British amphibians and reptiles. London (Collins).WOLTERSTORFF, W., and FREYTAG, G. E., 1951. Abh. Ber. Naturkd. Vorgesch. Magdeburg, 8,

137-ZELLER, E., 1899. Jh. Ver. vaterl. Naturk. in Wurttemb., 55, 23.

neoteny with goitre in triturus helveticus by j. m....

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