Temperature dependent changes in the thyroid gland of Mertensiella caucasica (Urodela,Amphibia)

Summary The influence of temperature on the thyroid gland of the Caucasian salamander, Mertensiella caucasica has been studied (1) with the light- and electron microscopes, (2) with enzyme histochemical and quantitative methods. Animals which had been kept for years at 16° C were exposed for 48 hrs to 4° C or 27° C.At 4° C the follicular epithelium consisted of cuboidal cells with well-developed rough ER, occasional distended cisternae of the rough endoplasmic reticulum (ER) filling most of the cytoplasm, a relatively small Golgi apparatus and numerous round or oval shaped lysosomes. In animals kept at 27° C the follicular wall was composed of markedly taller epithelial cells, the apices of which bulged into the follicular lumen. Compared to the animals at 4° C, there appeared to be a decrease in the rough ER. The Golgi apparatus did not exhibit clear differences. The lysosomes increased in number and showed various shapes among which elongated ones with pointed ends were particularly striking. In the apical cytoplasm colloid droplets appeared indicating hormone release. The apical microvilli increased in number.The measurements of total T₄ have shown that the hormone level in animals kept at 27° C was about twice as high as in animals kept at 4° C.Dedicated in gratitude to Professor Dr. Drs. h.c. W. Bargmann on the occasion of his 70th birthday.Supported by the Deutsche Forschungsgemeinschaft (We 380/5).     

Fine structural and enzyme histochemical observations on the dorsal tail tubercle of Mertensiella caucasica (Urodela,Amphibia)

Summary Dorsal tubercle and skin of Mertensiella caucasica have been investigated with the electron microscope and enzyme histochemical methods. The epidermis of the tubercle consists of 8–9 cell layers, that of normal dorsal skin of 5–6. The tubercle is filled with large mucous glands which are surrounded by an almost complete layer of smooth muscle cells (myoepithelial cells). Their glandular cells undergo cyclical changes and are characterized by specific secretory granules, which differ from those of the relatively small mucous glands of the normal dorsal skin.In the connective tissue of the tubercle a relatively rich supply of nerve fibres has been found, which in part contain synaptic and dense core vesicles or accumulations of mitochondria. In the normal dorsal skin nerve fibres occur less frequently.The following enzymes have been demonstrated in the mucous glands of the tubercle: SDH, acid phosphatase, unspecific esterases, E 600 resistant esterase.The tubercle seems to stimulate the female cloaca chemically and mechanically.     

Complex secretory products in the oviduct of the plethodontid salamander Bolitoglossa dofleini (Amphibia, Urodela)

The ultrastructure of the secretory granules in the cells of the subdivisions of the oviduct in the neotropical plethodontid salamander Bolitoglossa dofleini was studied by transmission electron microscopy. In addition, we applied the cationic dye Cuprolinic Blue (CB) at different electrolyte concentrations to demonstrate proteoglycans, and the pyrogallol red-copper (PR-C) method to stain proteins at the ultrastructural level. The entire oviduct is lined by a simple epithelium that contains ciliated and microvillous cells in the first subdivision, the aglandular pars recta; microvillous cells show a moderate secretory activity. The following pars convoluta is differentiated into five glandular subdivisions and the aglandular “uterine portion”. Especially in the glandular parts, the epithelium is arranged in longitudinal folds. At their crests ciliated and microvillous cells similar to those in the pars recta occur. Gland cells are crowded with secretory granules that differ in their structural complexity (with and without electron-dense spheres or masses; elaborated, homogeneous or granular matrix; spherical; distorted) along the various subdivisions. Further, as suggested by the CB-technique, the cranial subdivisions contain large amounts of sulphated proteoglycans that decrease in the caudal direction. Carboxylated proteglycans appear to be present in all subdivisions examined. Electron-dense spheres of secretory granules are largely free of CB-precipitates, but stain more or less intensely with PR-C. The ultrastructure of the pars recta, and especially the “uterine portion” indicates transporting capability. The epithelial cells of the “uterus” have coated pits and a considerable amount of lysosome-like bodies.     

Morphological and allozymic variation in Hynobius boulengeri and H. stejnegeri (Amphibia: Urodela: Hynobiidae)

We studied morphological and allozymic variation in populations of Japanese salamanders, Hynobius boulengeri and H. stejnegeri. Adult H. boulengeri showed sexual dimorphism, and juveniles differed greatly from adults in many morphological characters. From the results of multivariate analyses of morphological characters, the populations were divided into four groups: (I) H. boulengeri from Honshu, (II) H. boulengeri from Shikoku, (III) H. boulengeri from the Sobo-Katamuki Mountains of Kyushu and H. stejnegeri, and (IV) H. boulengeri from the Amakusa Islands and the Osumi Peninsula. Phenotypic relationships among the four groups were identical to relationships clarified by allozymic analyses, except for group IV, which was included in group III in the allozyme tree. Some morphometric characters were significantly correlated with environmental variables. We consider H. stejnegeri to be a valid species based on its unique color pattern, morphometric characters, and allelic composition, even though it was nested within group III of H. boulengeri by both morphological and allozymic analyses. We propose that group I from Honshu and group II from Shikoku should be treated as H. boulengeri sensu stricto and H. hirosei, respectively. Resolving the taxonomic status of the remaining populations of groups III and IV from Kyushu requires further study.     

A comparative study of the hyobranchial apparatus in Hynobiidae (Amphibia: Urodela)

The morphology of the adult hyobranchial apparatus has played an important role in understanding the systematics and evolution of urodeles, but the hyobranchial apparatus of hynobiid salamanders has received little attention so far. In this study, the hyobranchial apparatus of eight hynobiid salamanders (Hynobius leechii, Onychodactylus zhangyapingi, Ranodon sibiricus, Batrachuperus pinchonii, Salamandrella keyserlingii, Liua shihi, Pachyhynobius shangchengensis and Pseudohynobius flavomaculatus) is described and compared based on the clearing and double-staining method. The basic elements of the hyobranchial apparatus of the eight species are similar, including one basibranchial, cornua, one pair of radial loops, one pair of ceratohyals, one pair of hypobranchials II, one pair of ceratobranchials II, one urohyal (absent in O. zhangyapingi), one pair of the complex of hypobranchial I and ceratobranchial I (separated in certain species). Although the hyobranchial apparatus is similar among hynobiid salamanders and shows a unique morphological pattern, there are also certain species-specific distinctions that may be used for specific or generic diagnosis. The results of an ancestral state reconstruction of five traits showed that the ossified basibranchial, the presence of a separated hypobranchial I and ceratobranchial I, the absence of a urohyal, the ossified hypobranchial I and the partially ossified ceratohyal are derived traits. The state shown by the traits of each species is consistent with the phylogenetic position of each species. Compared with other Urodela, the hyobranchial apparatus of this group shows certain distinctive features that may represent the diagnostic characters of the family Hynobiidae. The partially ossified ceratohyal is correlated with the habitat and represents an ecological adaptation.     

Ultrastructural and ultracytochemical studies of the gill epithelium in the larvae of Salamandra salamandra (Amphibia,Urodela)

Summary The morphology of Salamandra salamandra (Linné, 1758) larva gills is described by scanning and transmission electron microscopy. Three main cell types comprising the surface of the gill epithelium are described: (a) pavement cell, (b) ciliary cell and (c) mitochondria-rich cell (MRC). Two subtypes of MRC were distinguished by their ultrastructural characteristics: a fibrillar cell and a tubulovesicular cell. K^ü-dependent-nitrophenyl-phosphatase (K-NPPase) activity, indicative of Na^ü-K^ü-ATPase activity was confined to the basolateral membranes of the pavement cells. MRC were devoid of such activity. Possible relationships between structure and function of the different cell types are discussed.     

External fertilisation and paternal care in the paedomorphic salamander Siren intermedia Barnes, 1826 (Urodela: Sirenidae)

Parental care is widespread in the animal kingdom and enhances offspring survival. Amphibians exhibit an extraordinary diversity of care strategies, including guarding, transport and even feeding of young. Among amphibians, females are usually the carers, but here we present a case of male parental care in the aquatic salamander Siren intermedia, accompanied by records of external fertilisation. Sirenids are a phylogenetically distinct group within basal salamanders, of which the precise systematic position has long been debated. Our observations of external fertilisation and paternal care of S. intermedia lead us to conclude that apparently both internal fertilisation and maternal care evolved after the split between the more basal groups (Cryptobranchoidea and Sirenoidea) and all other salamanders (suborder Salamandroidea).     

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela) using the antibiotic filipin

Summary The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin     

The innervation of the uterus in Salamandra salamandra (L.) (Amphibia,Urodela)

The innervation of the uterus in the ovoviviparous urodele Salamandra salamandra was studied. In whole mount preparations of the thin-walled uterus of pregnant females, a dense adrenergic network was demonstrated using a modified glyoxylic acid fluorescence technique. Based on vesicle type and cytochemical reactivity after chromate/dichromate fixation for electron microscopy at least two types of neural process were distinguished and classified as adrenergic and cholinergic. Both types are preferentially situated above or between the smooth muscles of the uterine tissue. Adjacent to the muscles in the walls of arterioles mainly adrenergic fibers are seen. Using high performance liquid chromatography with electrochemical detection in tissue homogenates of uterus a considerable amount of noradrenaline could be identified. The significance of the dense innervation is discussed with respect to the function of the uterus during pregnancy and birth.     

High aquatic niche overlap in the newts Triturus cristatus and T. marmoratus (Amphibia,Urodela)

We studied spatial niche metrics of large-bodied newts (Triturus cristatus and T. marmoratus) in three breeding ponds in western France. Adults and larvae were sampled with underwater funnel traps. Larvae were identified to the species with diagnostic microsatellite DNA markers. The distribution of adult T. cristatus and T. marmoratus across pond regions differed in one out of six cases, no differences were observed between larvae (two ponds studied). Niche overlap and niche breadth indices across resource states defined as pond regions or individual traps were high (Schoener's C: pond regions 0.60–0.98, traps 0.35–0.71; Levins' B: pond regions 0.71–0.98, traps 0.35–0.76). Adults of large-bodied newts significantly differed in resource use from small-bodied newts (T. helveticus). The results are discussed in view of the occurrence of interspecific breeding attempts, and the unpredictable ecological characteristics of newt breeding ponds.     

Observations on the lateral-line sense organs of the Salamander Neurergus crocatus crocatus Cope (Amphibia: Urodela)

The lateral-line system of the subadult and adult Neurergus crocatus crocatus Cope is retained throughout the life. It is constructed of pear-shaped sense organs or neuromasts which in the subadults are confined entirely to the epidermis with their apices opening distally to the exterior at the general level of the epidermal surface whereas in the adults they are embedded proximally more than halfway in the dermis with their distal apices opening into shallow grooves slightly below the regular epidermal surface. Dimensional differences are also observed between these organs in both stages. The neuromasts are constructed of 4 distinct cell types: sense cells, basal cells, sustentacular cells and mantle cells. These cells differ morphologically, structurally, topographically and functionally. The sense cells are clup-shaped, sensory, central in position and couched among the extremely elongated supporting basal and sustentacular cells, all of which are covered laterally by the extremely slender protective mantle cells. In both the mature and larvae, differences are observed among these cells in their size, number, location and arrangement. The sense organs are richly-supplied and well-nourished with vascular and nervous supplies.     

A New Species of the Genus Tylototriton(Amphibia: Urodela: Salamandridae) from Eastern Himalaya

A new species of the genus Tylototriton is described from eastern Himalaya based on molecular and morphological comparisons. The new species is diagnosable from the closely-related species by having light brown colouration in dorsal region in life, flat and blunt snout, greatly separated dorsolateral bony ridges on head and straightthick tailfin. In addition to head morphology, the new species is also morphologically distinguishable from its closelyrelated species Tylototriton shanorum by having 16 dorsal warts and average smaller Snout Vent Length(SVL).     

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.     

Sperm morphology of salamandrids (Amphibia, Urodela): implications for phylogeny and fertilization biology

Mature spermatozoa belonging to four salamander species, Salamandrina terdigitata, Triturus alpestris, Triturus carnifex and Triturus vulgaris, have been investigated by electron microscopy. The sperm ultrastructure of these species was compared with that of previously examined urodeles (36 species and 20 genera) and with that of anurans and caecilians. Many phylogenetic considerations may be inferred as a consequence of comparative spermatology. Urodela appears to be a monophyletic order characterized by three sperm synapomorphies: the acrosomal barb, nuclear ridge and marginal filament. Cryptobranchoidea are confirmed to form a monophyletic suborder having two synapomorphic characters: absence of mitochondria in the tail, and cylindrical shape of the tail axial rod. Within the family Salamandridae, sperm morphology confirms the phylogenetic distance between Salamandrina and Triturus, as already pointed out on the basis of molecular and morphological characters. The very complex ultrastructure of spermatozoa confirms a previous opinion that internal fertilization is the ancestral condition of the Amphibia.     

Multitubular bodies in intestinal cells of Amphiuma means/tridactylum (Urodela): ultrastructural characterization

Summary The jejunal absorptive cells of the salamander Amphiuma, when examined using transmission electron microscopy, were found to possess a unique type of intracellular vacuole containing membranous tubules. These vanoles, tentatively named multitubular bodies, were located in the cytoplasm between the nucleus and the brush-border membrane, and were seen with greatest frequency in the summer and fall. The vacuoles containing multitubular bodies had an average diameter of 0.6 m, and the membranous tubules within had an average diameter of 30 nm. The tubules differed morphologically from the vesicles in the multivesicular bodies, and from the primary lysosomes in the polylysosomal vacuoles. The tubules did not exhibit acid phosphatase activity, and were of similar diameter and membrane thickness as the Golgi saccules. In contrast to the multivesicular bodies, the multitubular bodies did not take up exogenous horseradish peroxidase. Early forms of autophagosomes resembling these vacuoles were often seen in the para-Golgi region of the cell. The multitubular bodies may represent a distinct type of autophagosome. Although the exact origin of the tubules as well as their role in cellular activity is unclear, their seasonal appearance within the multitubular bodies of the absorptive cells suggests a unique means of selective down-regulation of Golgi-like organelles.