Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata).

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

Immunocytochemical demonstration of visual pigments in the degenerate retinal and pineal photoreceptors of the blind cave salamander (Proteus anguinus).

Visual pigments in the regressed eye and pineal of the depigmented neotenic urodele, the blind cave salamander (Proteus anguinus anguinus), were studied by immunocytochemistry with anti-opsin antibodies. The study included light- and electron-microscopic investigations of both the eye and the pineal organ. A comparison was made with the black pigmented subspecies Proteus anguinus parkelj (black proteus), which has a normal eye structure. In the retina of the black proteus, we found principal rods, red-sensitive cones and a third photoreceptor type, which might represent a blue- or UV-sensitive cone. Photoreceptors in the regressed eye of the blind cave salamanders from the Planina cave contained degenerate outer segments, consisting of a few whorled discs and irregular clumps of membranes. The great majority of these outer segments showed immunolabelling for the red-sensitive cone opsin and only a few of them were found to be positive for rhodopsin. An even more pronounced degeneration was observed in the photoreceptors of the animals derived from the Otovec doline, which are completely devoid of an outer segment, most of them not even possessing an inner segment. Even in some of these highly degenerate cells, the presence of rhodopsin could be detected in the plasma membrane; however, immunoreactions with antibodies recognizing cone visual pigment were negative. In the pineals of all studied animals, the degenerate photoreceptor outer segments were recognized exclusively by the antibody against the red-sensitive cone opsin. The presence of immunopositive visual pigments indicates the possibility of a retained light sensitivity in the blind cave salamander photoreceptors.

Functional morphology of the inner ear and underwater audiograms of Proteus anguinus (Amphibia, Urodela).

Octavolateral sensory organs (auditory and lateral line organs) of cave salamander Proteus anguinus are highly differentiated. In the saccular macula of the inner ear the complex pattern of hair cell orientation and the large otoconial mass enable particle displacement direction detection. Additionally, the same organ, through air cavities within the body, enables detection of underwater sound pressure changes thus acting as a hearing organ. The cavities in the lungs and mouth of Proteus are a resonators that transmit underwater sound pressure to the inner ear. Behaviourally determined audiograms indicate hearing sensitivity of 60 dB (rel. 1 microPa) at frequencies between 1 and 10 kHz. The hearing frequency range was between 10 Hz and 10 kHz. The hearing sensitivities of depigmented Proteus and black Proteus were compared. The highest sensitivities of the depigmented animals (N=4) were at frequencies 1.3-1.7 kHz and it was 2 kHz in black animals (N=1). Excellent underwater hearing abilities of Proteus are sensory adaptations to cave habitat.

The ultrastructure of photoreceptor cells in the pineal organ of the blind cave salamander, Proteus anguinus (Amphibia, Urodela).

We studied ultrastructure of the photoreceptor cells in the pineal organ of blind, depigmented, neotenic cave salamander, Proteus anguinus. Unlike in epigean vertebrates the outer segments of most photoreceptor cells consists of concentrically arranged lamellae, however; in few cells, the outer segments contain 7-9 plasma membrane disks. In both types of photoreceptor cells the outer segments enclose lumps of vesicles of different sizes. The photoreceptor cells of Proteus anguinus are similar to those in other cavernicolous fish species.

Population-specific behavioral electrosensitivity of the European blind cave salamander, Proteus anguinus.

In nine salamanders from different Slovenian populations of the urodele Proteus anguinus, including three specimens of its 'black' variety, P anguinus parkelj, thresholds of an overt avoidance response to electrical field stimuli were estimated as a function of frequency (continuous sine-waves in water). Thresholds down to 0.3V/cm (ca 100 nA/cm2) and up to 2 mV/cm (670 nA/cm2), at 'best frequencies' of around 30 Hz were found. Sensitivity covered a total frequency range of below 1 Hz, excluding DC, up to 1-2 kHz with up to 40 dB higher thresholds. Thresholds and tuning curves are compared with those of a Proteus population raised in captivity for more than 35 years. The biological significance and the apparently still ongoing evolution of the electrical sense in urodeles, ie in the genus Proteus, are interpreted in terms of comparative sensory physiology and ethological ecology as a result of more recent evolutionary diversification during and since glaciation in the Pleistocene.

Some evidence for the ampullary organs in the European cave salamander Proteus anguinus (Urodela, Amphibia).

The multicellular epithelial organs in Proteus anguinus, which Bugnion (1873) assumed to be developing neuromasts, have been analyzed by light- and electron-microscopy. Their fundamental structure consists of single ampullae with sensory and accessory cells with apical parts that extend into the pit of the ampulla, and of a short jelly-filled canal connecting the ampulla pit with the surface of the skin. The organs are located intra-epithelially and are supported by a tiny dermal papilla. The cell elements of sensory epithelium are apically linked together by tight junctions. The free apical surface of the sensory cell bears several hundred densely packed stereocilia-like microvilli whereas the basal surface displays afferent neurosensory junctions with a pronounced round synaptic body. The compact uniform organization of the apical microvillous part shows a hexagonal pattern. A basal body was found in some sensory cells whereas a kinocilium was observed only in a single cell. The accessory cells have their free surface differentiated in a sparsely distributed and frequently-forked microvilli. The canal wall is built of two or three layers of tightly coalescent flat cells bordering on the lumen with branching microvilli. The ultrastructure of the content of the ampulla pit is presented. In the discussion stress is laid on the peculiarities of the natural history of Proteus anguinus that support the view that the morphologically-identified ampullary organs are electroreceptive. The structural characteristics of ampullary receptor cells are dealt with from the viewpoint of functional morphology and in the light of evolutionary hypotheses of ampullary organs.