Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae).

BACKGROUND: Amphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. METHOD: Using traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). RESULTS: In larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. CONCLUSION: Our results provide a baseline for future comparative research of skin anatomy in salamanders.

Testing the applicability of tagging the Great crested newt (Triturus cristatus) using passive integrated transponders.

Tracking individual animals with small-sized passive integrated transponder tags (PIT tags) has become a popular and widespread method, one which can be used for investigating life history traits, including dispersal patterns of small protected animals such as newts. In this study, we tested the applicability of PIT tag usage for individual marking with the Great crested newt (Triturus cristatus) as a model amphibian species, and to test the detection of the newts in nature using a passive telemetry system. Clove oil was used as an anaesthetic before surgery. We implanted PIT tags under the skin of 140 newts. The survival rate of newts was 98.57%. X-ray images were taken to check the exact positions of the PIT tags. Since approximately 15.71% of the newts were capable of expelling the tag from their bodies, tag loss has to be accounted for in future behavioural studies dealing with newts and other amphibians potentially capable of frequent tag expulsion. Lastly, we detected by passive telemetry 97 individuals out of 100 released into a natural breeding pond. Males had higher activity (13 detected males vs 7 females per hour) than females, thus males could be detected if present with more certainty. The result of the movement behaviour showed that e.g. the male of T. cristatus in a breeding pond can travel up to 20 m in 78 seconds. In summary, this promising method could allow the automatic data collection of marked newts in aquatic as well as in terrestrial biotopes, providing data on their dispersal, diurnal activity and movement behaviour.

Testing the evolutionary constraints of metamorphosis: The ontogeny of head shape in Triturus newts.

In vertebrates with complex, biphasic, life cycles, larvae have a distinct morphology and ecological preferences compared to metamorphosed juveniles and adults. In amphibians, abrupt and rapid metamorphic changes transform aquatic larvae to terrestrial juveniles. The main aim of this study is to test whether, relative to larval stages, metamorphosis (1) resets the pattern of variation between ontogenetic stages and species, (2) constrains intraspecific morphological variability, and (3) similar to the "hour-glass" model reduces morphological disparity. We explore postembryonic ontogenetic trajectories of head shape (from hatching to completed metamorphosis) of two well-defined, morphologically distinct Triturus newts species and their F1 hybrids. Variation in head shape is quantified and compared on two levels: dynamic (across ontogenetic stages) and static (at a particular stage). Our results show that the ontogenetic trajectories diverge early during development and continue to diverge throughout larval stages and metamorphosis. The high within-group variance and the largest disparity level (between-group variance) characterize the metamorphosed stage. Hence, our results indicate that metamorphosis does not canalize head shape variation generated during larval development and that metamorphosed phenotype is not more constrained relative to larval ones. Therefore, metamorphosis cannot be regarded as a developmental constraint, at least not for salamander head shape.

Trunk elongation and ontogenetic changes in the axial skeleton of Triturus newts.

Body elongation in vertebrates can be achieved by lengthening of the vertebrae or by an increase in their number. In salamanders, longer bodies are mostly associated with greater numbers of vertebrae in the trunk or tail region. However, studies on the relative contribution of the length of single vertebra to body elongation are lacking. In this study, we focus on evolutionary and ontogenetic changes in differentiation of the trunk vertebrae and the relative contribution of individual vertebrae to trunk lengthening in Triturus newts, a monophyletic group of salamanders that shows remarkable disparity in body shape. We compared juveniles and adults of the most elongated T. dobrogicus, which has 17 trunk vertebrae, with juveniles and adults of two closely related species (T. ivanbureschi and T. anatolicus belonging to the T. karelinii species complex) representing a stout and robust morphotype with thirteen trunk vertebrae. We show that trunk vertebrae are uniform in size at the juvenile stage of both analyzed morphotypes. In adults, the trunk vertebrae of the elongated T. dobrogicus are largely uniform, while in those of T. anatolicus, the first two vertebrae differ from the remaining trunk vertebrae. There was no difference in the relative contribution of individual vertebrae to body lengthening between species or stages. We conclude that body elongation in Triturus newts is achieved by increasing the number of vertebrae but not their length.

A tongue for all seasons: extreme phenotypic flexibility in salamandrid newts.

Many organisms faced with seasonally fluctuating abiotic and biotic conditions respond by altering their phenotype to account for the demands of environmental changes. Here we discovered that newts, which switch seasonally between an aquatic and terrestrial lifestyle, grow a complex adhesive system on their tongue pad consisting of slender lingual papillae and mucus-producing cells to increase the efficiency of prey capture as they move from water onto land. The adhesive system is reduced again as newts switch back to their aquatic stage, where they use suction to capture prey. As suction performance is also enhanced seasonally by reshaping of the mouth due to the growth of labial lobes, our results show that newts are exceptional in exhibiting phenotypic flexibility in two alternating components (i.e. tongue pad and labial lobes) within a single functional system, and suggest that this form of phenotypic flexibility demands complex genetic regulation.

Description of a new species of crested newt, previously subsumed in Triturus ivanbureschi (Amphibia: Caudata: Salamandridae).

Multilocus molecular data play a pivotal role in diagnosing cryptic species (i.e. genetically distinct but morphologically similar species). A multilocus phylogeographic survey has provided compelling evidence that Triturus ivanbureschi sensu lato comprises two distinct gene pools with restricted gene flow. We conclude that this taxon had better be treated as two distinct (albeit morphologically cryptic) species. The name T. ivanbureschi should be restricted to the western species, which is distributed in western Asiatic Turkey plus the south-eastern Balkan Peninsula. No name is as yet available for the eastern species, which is distributed in northern Asiatic Turkey. We propose the name T. anatolicus sp. nov. for the eastern species and provide a formal species description.

Vertebral shape and body elongation in Triturus newts.

Body elongation in vertebrates is often related to a lengthening of the vertebrae and an increase in their number. Changes in the number and shape of vertebrae are not necessarily linked. In tailed amphibians, a change in body shape is mostly associated with an increase in the number of trunk and tail vertebrae. Body elongation without a numerical change of vertebrae is rare. In Triturus aquatic salamanders body elongation is achieved by trunk elongation through an increase in the number of trunk vertebrae. We used computed microtomography and three-dimensional geometric morphometrics to document the size, shape and number of trunk vertebrae in seven Triturus species. The data suggest that body elongation has occurred more frequently than body shortening, possibly related to a more aquatic versus a more terrestrial locomotor style. Our results show that body elongation is achieved through an increase in the number of trunk vertebrae, and that interspecific differences in vertebral shape are correlated with this pattern of elongation. More gracile trunk vertebrae were found in the more elongated species. The shape differences are such that single trunk vertebrae can be used for the identification of species with a possible application in the identification of subfossil and fossil material.

The ontogenetic origins of skull shape disparity in the Triturus cristatus group.

Comparative studies of ontogenies of closely related species provide insights into the mechanisms responsible for morphological diversification. Using geometric morphometrics, we investigated the ontogenetic dynamics of postlarval skull shape and disparity in three closely related crested newt species. The skull shapes of juveniles just after metamorphosis (hereafter metamorphs) and adult individuals were sampled by landmark configurations that describe the shape of the dorsal and ventral side of the newt skull, and analyzed separately. The three species differ in skull size and shape in metamorphs and adults. The ontogenies of dorsal and ventral skull differ in the orientation but not lengths of the ontogenetic trajectories. The disparity of dorsal skull shape increases over ontogeny, but that of ventral skull shape does not. Thus, modifications of ontogenetic trajectories can, but need not, increase the disparity of shape. In species with biphasic life-cycles, when ontogenetic trajectories for one stage can be decoupled from those of another, increases and decreases in disparity are feasible, but our results show that they need not occur.

A revised taxonomy of crested newts in the Triturus karelinii group (Amphibia: Caudata: Salamandridae), with the description of a new species.

We present a taxonomic revision of the crested newt Triturus karelinii sensu lato. Based on the presence of discrete nuclear DNA gene pools, deep genetic divergence of mitochondrial and nuclear DNA, and no indication of gene flow, we interpret this taxon as comprising two species: one covering the southern Caspian Sea shore, the Caucasus and the Crimea, i.e. the eastern part of the total range and another covering northern Asiatic Turkey and western Asiatic Turkey plus the southeastern Balkan Peninsula, i.e. the central and western part of the total range. We acknowledge that the central/western species should likely be further subdivided into a central and a western taxon, but we prefer to await a more detailed genetic analysis of the putative contact zone, positioned in northwestern Asiatic Turkey. The name T. karelinii (Strauch, 1870) applies to the eastern species as the type locality is positioned along the coast of the Gulf of Gorgan, Iran. The name T. arntzeni has been applied to the central/western species with Vrtovac, Serbia as the type locality. We show that not T. karelinii sensu lato but T. macedonicus occurs at Vrtovac. Hence, the name T. arntzeni Litvinchuk, Borkin, Dzukic and Kalezic, 1999 (in Litvinchuk et al., 1999) is a junior synonym of T. macedonicus (Karaman, 1922) and should not be used for the central/western species. We propose the name T. ivanbureschi sp. nov. for the central/western species and provide a formal species description.

Unraveling the rapid radiation of crested newts (Triturus cristatus superspecies) using complete mitogenomic sequences.

BACKGROUND: The rapid radiation of crested newts (Triturus cristatus superspecies) comprises four morphotypes: 1) the T. karelinii group, 2) T. carnifex - T. macedonicus, 3) T. cristatus and 4) T. dobrogicus. These vary in body build and the number of rib-bearing pre-sacral vertebrae (NRBV). The phylogenetic relationships of the morphotypes have not yet been settled, despite several previous attempts, employing a variety of molecular markers. We here resolve the crested newt phylogeny by using complete mitochondrial genome sequences. RESULTS: Bayesian inference based on the mitogenomic data yields a fully bifurcating, significantly supported tree, though Maximum Likelihood inference yields low support values. The internal branches connecting the morphotypes are short relative to the terminal branches. Seen from the root of Triturus (NRBV = 13), a basal dichotomy separates the T. karelinii group (NRBV = 13) from the remaining crested newts. The next split divides the latter assortment into T. carnifex - T. macedonicus (NRBV = 14) versus T. cristatus (NRBV = 15) and T. dobrogicus (NRBV = 16 or 17). CONCLUSIONS: We argue that the Bayesian full mitochondrial DNA phylogeny is superior to previous attempts aiming to recover the crested newt species tree. Furthermore, our new phylogeny involves a maximally parsimonious interpretation of NRBV evolution. Calibrating the phylogeny allows us to evaluate potential drivers for crested newt cladogenesis. The split between the T. karelinii group and the three other morphotypes, at ca. 10.4 Ma, is associated with the separation of the Balkan and Anatolian landmasses (12-9 Ma). No currently known vicariant events can be ascribed to the other two splits, first at ca. 9.3 Ma, separating T. carnifex - T. macedonicus, and second at ca. 8.8 Ma, splitting T. cristatus and T. dobrogicus. The crested newt morphotypes differ in the duration of their annual aquatic period. We speculate on the role that this ecological differentiation could have played during speciation.

Morphological integration and ontogenetic niche shift: a study of crested newt limbs.

This study deals with the ontogenetic and evolutionary aspects of integration patterns in the limbs of crested newt species, which, like most amphibians, have a biphasic life history with two morphologically distinct stages (larval vs. juvenile and adult) that occupy different environments (aquatic vs. terrestrial). We analyzed the structure and pattern of correlation between limb skeletal elements at three ontogenetic stages (larval, juvenile, and adult) of four closely related species that differ in their preferences of aquatic habitats (more terrestrial and more aquatic). We found dynamic changes in the pattern of morphological integration between successive ontogenetic stages, as well as changes over the course of crested newt phylogeny. Generally, equivalent ontogenetic stages of different species of crested newts show higher concordance in the correlation pattern than successive ontogenetic stages within species. Among species, two opposing correlation patterns were observed: in more terrestrial species, homologous limb elements are less correlated and within-limb elements are more correlated; in aquatic species, the reverse pattern occurs. These results indicate that the function seems to be the covariance-generating factor, which has shaped the patterns of morphological integration of crested newt limbs.

Asymmetric viability of reciprocal-cross hybrids between crested and marbled newts (Triturus cristatus and T. Marmoratus).

Hybridization between divergent lineages often results in reduced hybrid viability. Here we report findings from a series of independent molecular analyses over several seasons on four life stages of F1 hybrids between the newts Triturus cristatus and T. marmoratus. These two species form a bimodal hybrid zone of broad overlap in France, with F1 hybrids making up about 4% of the adult population. We demonstrate strong asymmetry in the direction of the cross, with one class (cristatus-mothered) making up about 90% of F1 hybrids. By analyzing embryos and hatchlings, we show that this asymmetry is not due to prezygotic effects, as both classes of hybrid embryos are present at similar frequencies, implicating differential selection on the two hybrid classes after hatching. Adult F1 hybrids show a weak Haldane effect overall, with a 72% excess of females. The rarer marmoratus-mothered class, however, consists entirely of males. The absence of females from this class of adult F1 hybrids is best explained by an incompatibility between the cristatus X chromosome and marmoratus cytoplasm. It is thus important to distinguish the two classes of reciprocal-cross hybrids before making general statements about whether Haldane's rule is observed.

Chromaffin cells in the amphibian urodele Triturus carnifex show ultrastructural features indicative of a vesicle-mediated mode of cell degranulation.

Recently, suggestive evidence for piecemeal degranulation (PMD), a particulate pattern of cell secretion accomplished by vesicle-mediated extracellular transport of granule-stored material, has been provided by electron microscopy investigations in chromaffin cells of different vertebrate species. In this study, chromaffin tissue from the interrenal gland of the amphibian urodede Triturus carnifex has been investigated by quantitative transmission electron microscopy in search for ultrastructural clues indicative of a vesicle-mediated mode of cell degranulation. Interestingly, a single type of chromaffin cell is recognizable in T. carnifex, which undergoes seasonal variations in its adrenaline and noradrenaline granule ratio according to an annual cycle that matches the trend of changes in secondary sexual characters. In this study, we looked for a series of ultrastructural changes regarded as highly specific for PMD. We calculated the percentage of (i) resting, unaltered granules (type 1 granules); (ii) granules with changes indicative of progressive release of secretory materials, that is, granules with lucent areas in their cores, reduced electron density, disassembled matrices, and residual cores (type 2 granules); and (iii) membrane empty containers (type 3 granules) in chromaffin cells of T. carnifex during the annual cycle. We found a significant increase in type 2 and 3 granules, accompanied by a significant decrease in type 1 granules, in the April and November samples. During the same seasonal periods, the number per net cytoplasmic area of 30-100-nm diameter electron-dense vesicles was found to be significantly augmented, and there was also an augmented percentage of chromaffin granules showing blebs or protrusions in their profiles. These ultrastructural data are indicative of an increased vesicle-mediated transport of chromaffin granule products for extracellular release in the amphibian T. carnifex in accordance with the increased rate of catecholamine release. This vesicle-mediated pattern of cell secretion suits the schema of PMD. In an evolutionary perspective, these findings suggest that PMD is a secretory pathway that has been highly conserved throughout vertebrate classes. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.

Is the prefrontal bone in Alpine newt (Triturus alpestris Laurenti, 1768) of dual origin?

According to current knowledge, only the prefrontal bone (os prefrontale) of the circumorbital series is preserved in the family Salamandridae. However, the exact origin and number of ossification centres creating this bone is unknown. Detailed examination of the prefrontal bone during ontogeny of juvenile and adult specimens of the Alpine Newt (Triturus alpestris) indicates its dual origin (prefrontal and lacrimal). We found that the prefrontal bone originates from four ossification centres, i.e. three prefrontal centres and one posterior lacrimal centre. The anterior lacrimal centre participates in the maxillar ossification. The development of these ossification centres occurs very late in ontogeny (at stage 54), and starts after differentiation of the nasal capsules. The total fusion of the lacrimal ossification centre with the prefrontal bone of T. alpestris is distinct from the fully differentiated lacrimal bone attached to the prefrontal bone of the fossil family Branchiosauridae (Temnospondyly). We propose that heterochrony, observed in the recent species, is a delayed development followed by accelerated ossification that resulted in the fusion of the anterior lacrimal centre with the maxilla and the posterior lacrimal centre with the prefrontal bone.

Structural diversification of the gustatory organs during metamorphosis in the alpine newt Triturus alpestris.

Gustatory organs of the taste bud type occur in the epithelial lining of the oropharyngeal cavity of alpine newt larvae. They resemble the taste buds of bony fish, both in appearance (as revealed by scanning electron microscopy) and in detailed internal structure (seen on transmission electron micropscopy). During metamorphosis, at stage 55 of development, the secondary tongue (i.e. the soft tongue) is well formed and the anlages of taste discs are clearly apparent. Somewhat later, taste discs also appear in the epithelial lining outside the tongue, paralleling the disappearance of the taste buds. Well-developed taste discs of the newt differ from taste buds mainly by their structurally diversified set of 'associate cells' (mucous, wing and glial cells), which have no synaptic contact with nerve fibres. These cells accompany the neurosensory cellular components of the taste disc, i.e. the taste receptor cells and basal cells. This indicates that gustatory organs in metamorphosed newts, regardless of their small dimensions, fulfil the criteria established for taste discs previously defined in other Caudata and Anura species. Therefore, in the development of the newt there are two subsequent types of gustatory organs and two generations of the tongue: primary, in the larvae, and secondary, in metamorphosed animals.

Body size, competitive interactions, and the local distribution of Triturus newts.

1. Pairs of European Triturus newt species of similar size tend not to co-occur syntopically, suggesting that similarity in body size is associated with competitive interactions that prevent coexistence. I tested this hypothesis with an experiment involving larvae of four species in 675-L artificial ponds. 2. There were strong interactions between most species pairs. Even the small T. helveticus had a clear impact on the larger T. alpestris. Pairs of species with different body sizes did not interact less strongly. 3. A standard increase in competitor biomass (c. 2 g mass at metamorphosis) caused 42% lower expected survival from hatching to 1 year of age, regardless of whether the species were of similar or different size. In most cases this resulted from delayed metamorphosis, reduced size at emergence, and slightly lower larval survival. 4. A standard increase in competitor density (0.74 individuals m(-2)) caused a greater reduction in expected 1-year survival when the competitor was larger (18% decline) than when both species were of similar size (6% decline), primarily because the very large T. cristatus consumed the smallest species. 5. These findings suggest that species interactions during the larval stage cannot explain distribution patterns of same- and different-sized Triturus.

Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata).

In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal beta1,3 GalNAc and (GlcNAc beta1,4)(n) oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal beta1-3 GalNAc, Gal-alphaGal, and (GlcNAc beta1,4)(n) oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal beta1,3 GalNAc, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,6 or terminal alpha1,3 or alpha1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-alphaGal, Gal beta1-3 GalNAc and (GlcNAc beta1,4)(n)oligomers; Fuc linked alpha1,2 to Gal, alpha1,3 to GlcNAc in (poly)lactosamine chains and alpha1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H(+)K(+)-ATPase beta-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.

Triturus newts defy the running-swimming dilemma.

Conflicts between structural requirements for carrying out different ecologically relevant functions may result in a compromise phenotype that maximizes neither function. Identifying and evaluating functional trade-offs may therefore aid in understanding the evolution of organismal performance. We examined the possibility of an evolutionary trade-off between aquatic and terrestrial locomotion in females of European species of the newt genus Triturus. Biomechanical models suggest a conflict between the requirements for aquatic and terrestrial locomotion. For instance, having an elongate, slender body, a large tail, and reduced limbs should benefit undulatory swimming, but at the cost of reduced running capacity. To test the prediction of an evolutionary trade-off between swimming and running capacity, we investigated relationships between size-corrected morphology and maximum locomotor performance in females of ten species of newts. Phylogenetic comparative analyses revealed that an evolutionary trend of body elongation (increasing axilla-groin distance) is associated with a reduction in head width and forelimb length. Body elongation resulted in reduced maximum running speed, but, surprisingly, also led to a reduction in swimming speed. The evolution of longer tails was associated with an increase in maximal swimming speed. We found no evidence for an evolutionary trade-off between aquatic and terrestrial locomotor performance, probably because of the unexpected negative effect of body elongation on swimming speed. We conclude that the idea of a design conflict between aquatic and terrestrial locomotion, mediated through antagonistic effects of body elongation, does not apply to our model system.

Expression of PCNA positivity in the brain of normal adult heterothermic vertebrates: further observations.

As part of our study of non-experimentally induced encephalic proliferation in unequivocally adult individuals of several heterothermic Vertebrates (Podarcis sicula, Triturus carnifex, Rana esculenta, Carassius carassius), we deal here with areas not considered in previous investigations, i.e. various encephalic regions (except the telencephalon) in Podarcis sicula, Triturus carnifex and Rana esculenta, the diencephalon and medulla oblongata in Carassius carassius, and the olfactory bulbs in the two Amphibians. In the previous and current research, we have used Proliferating Cell Nuclear Antigen (PCNA) as a marker. PCNA is a ubiquitous intracellular antigen of the cycline family (proteins that regulate the cell cycle), which acts as an auxiliary protein to DNA polymerase delta; it can be detected immunocytochemically with monoclonal antibodies to reveal cell cycle phases that coincide with DNA synthesis. Spontaneous proliferation events, revealed by PCNA positivity, were constantly present in this study, being substantial in the olfactory region and diencephalon, very modest in the mesencephalon and myelencephalon, and absent in the cerebellum. In particular, signs of proliferation were abundant in the epithelium lining the cavities of the olfactory bulbs, while they were of different magnitude in tracts (with multiple and comparatively different sites related to the dorsal and/or ventral thalami) of the ependyma that delimits portions of the III ventricle and also, in all the species examined, at the level of the preoptic and infundibular recesses. Such signs were rare in the ependymal epithelium of the mesencephalic ventricle in Podarcis sicula and the rhombencephalic ventricle in all four species examined. This immunoreactivity was also observed in extra-ependymal areas: in the internal granular layer of the olfactory bulbs in Triturus carnifex and Rana esculenta; in the diencephalic nuclei of the habenula in Podarcis sicula, in both Amphibians and in Carassius carassius; in the mesencephalic tectum in Podarcis sicula and in the two Amphibians. As in our previous studies, the current immunocytochemical picture revealed by PCNA positivity generally agrees with literature reports on the presence of normal proliferation in the areas investigated here. These literature sources consist primarily of the observations of Kirsche (1967), emerging from his preceding experimental investigations, and of confirmatory data from studies in subsequent decades by other researchers obtained with tests different from our marker. Nevertheless, the number of studies that deal with the species considered in the present research, or species closely related to them, is rather limited.

Pepsinogen and H,K-ATPase mediate acid secretion in gastric glands of Triturus carnifex (Amphibia, Caudata).

The gastric glands of Triturus carnifex (Amphibia, Caudata) have been examined by histochemical and immunohistochemical methods with particular regard to hydrochloric acid and pepsinogen secretion. Fundic glands consist of mucous neck cells, endocrine cells and oxynticopeptic cells producing both pepsinogen and hydrochloric acid. The neck cells showed an unexpected distribution pattern which was only observed in the oral fundus, and produced neutral mucins with glycosidic residues of GalNAc and Gal beta1,3GalNAc, and in this respect they differ from the neck cells of anuran amphibians. The secretion of pepsinogen and hydrochloric acid as demonstrated by immunolabelling with anti-H,K-ATPase and with anti-pepsinogen, respectively, seems not to vary significantly along the longitudinal axis of the stomach. The mechanism of gastric acid secretion seems to be mediated by an ATPase, having similar features to the mammalian gastric H,K-ATPase, and is localised in the luminal membrane and in the subapical cytoplasm of the oxynticopeptic cells. Unusually, the same cytoplasmic areas revealed binding specificity for the winged pea lectin (WPA) from Lotus tetragonolobus, even after beta elimination, indicating the presence of fucosyl residues in N-linked oligosaccharidic chains in glycoproteins of beta-H,K-ATPase subunits.