Long-term starvation in cave salamander effects on liver ultrastructure and energy reserve mobilization.

The morphological alterations of hepatocytes of cave-dwelling salamander Proteus anguinus anguinus after food deprivation periods of one and 18 months were investigated and the concentrations of glycogen, lipids, and proteins in the liver were determined. Quantitative analyses of the hepatocyte size, the lipid droplets, the number of mitochondria, and volume densities of M and P in the hepatocytes were completed. After one month of food deprivation, the cytological changes in the hepatocytes are mainly related to the distribution and amount of glycogen, which was dispersed in the cytoplasm and failed to form clumps typical of normal liver tissue. After 18 months of food deprivation hepatocytes were reduced in size, lipid droplets were less numerous, peroxisomes formed clusters with small, spherical mitochondria, and specific mitochondria increased in size and lost cristae. Lysosomes, autophagic vacuoles, and clear vacuoles were numerous. The liver integrity was apparently maintained, no significant loss of cytoplasmic constituents have been observed. Biochemical analysis revealed the utilization of stored metabolic reserves in the liver during food deprivation. Glycogen is rapidly utilized at the beginning of the starvation period, whereas lipids and proteins are utilized subsequently, during prolonged food deprivation. In the Proteus liver carbohydrates are maintained in appreciable amounts and this constitutes a very important energy depot, invaluable in the subterranean environment.

Cd, Cu, Zn, Se, and metallothioneins in two amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura).

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85 µg/L) for up to 40 days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1 µg/g in kidneys and gills (0.64-0.95 and 0.52-0.76; n = 4), whereas the levels stayed below 0.5 in liver (0.14-0.29; n = 4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn-thioneins in liver and Zn,Cu-thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd-thionein and in pellet extract of kidneys as Zn,Cu,Cd-thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56-5.0 µg/g >0.03-0.72 µg/g; n = 11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r > 0.80; α = 0.05, n = 5), and between Cd and Se in kidney (r = 0.76; n = 5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd-thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.

PCB accumulation and tissue distribution in cave salamander (Proteus anguinus anguinus, Amphibia, Urodela) in the polluted karstic hinterland of the Krupa River, Slovenia.

For over two decades, a manufacturer of electrical capacitors disposed of its waste within the karstic hinterland of the Krupa River (Slovenia) resulting in the surroundings becomming heavily polluted with PCB. Albeit the extent of the contamination has been known since 1983 and the Krupa River has become one of the most PCB polluted river in Europe, the effects on the cave fauna of the region remain unknown. The most famous cave dweller of the Krupa hinterland is the endemic cave salamander Proteus anguinus anguinus. In this study we determine the levels of PCB in the tissues of the Proteus and in river sediments. The total concentration of PCB in individual tissue samples from specimens of the Krupa spring was between 165.59 µg g(-1) and 1560.20 µg g(-1)dry wt, which is at least 28-times higher than those from an unpolluted site. The kidneys contained the lowest concentration, while the highest concentration was in subcutaneous fat and tissues with high lipid contents like visceral fat and liver. Total PCB concentrations in sediment samples from the Krupa River were between 5.47 and 59.20 µg g(-1)dry wt showing that a high burden of PCB still remains in the region. The most abundant PCB congeners in all analyzed samples were di-ortho substituted (PCB #101, #118, #138 and #158), but higher proportion of mono-ortho PCB was present in sediments. The ability of Proteus to survive a high PCB loading in its environment and especially in its tissues is remarkable. Its partial elimination of low chlorinated and mono-ortho substituted congeners is also reported.

Ultrastructure of previtellogene oocytes in the neotenic cave salamander Proteus anguinus anguinus (Amphibia, Urodela, Proteidae).

Oogenesis in the neotenic, cave dwelling salamander Proteus anguinus anguinus has not been studied yet, and this study provides a detailed description of the early growth of the oocytes. Early previtellogene oocytes ranging from 100 to 600 µm in diameter were examined by light and transmission electron microscopy. The oocytes were divided into two stages based on size, color, and histology. Stage I oocytes can be identified by their transparent cytoplasm and a homogenous juxtanuclear mass, composed of numerous lipid droplets and mitochondria. Stage II oocytes are no longer transparent and have increased in diameter to 300- 600 µm, and many cortical alveoli differing in size have appeared. The common and most predominant ultrastructural characteristics of both stages of previtellogene oocytes are extensive quantities of smooth membrane, numerous mitochondria, and lipid droplets, as well as abundant free ribosomes. Myeline-like structures and remarkable annulate lamellae of closely packed membrane stacks are also frequently observed. Previtellogenic oocytes are the most predominant oocytes in the ovaries of Proteus, and while they possess certain structural characteristics typical for other amphibians, some features are unique and could result from adaptation to the subterranean environment.

Isolation and primary culture of Necturus maculosus (Amphibia: Urodela) hepatocytes.

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7 x 10(5) viable hepatocytes per gram body weight with an average viability of 86 +/- 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 degrees C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.

Histology and ultrastructure of the gut epithelium of the neotenic cave salamander, Proteus anguinus (Amphibia, Caudata).

Histological, histochemical, and ultrastructural features of the gut of the European endemic cave salamander Proteus anguinus were studied. The gut is a relatively undifferentiated muscular tube lined with a simple columnar epithelium containing numerous goblet cells. The mucosa and underlying lamina propria/submucosa are elevated into a number of high longitudinal folds projecting into the lumen. The enterocytes are covered apically with uniform microvilli. Irregularity in the arrangement of microvilli was observed. Occasionally, irregular protrusions of the cytoplasm appear between groups of microvilli. Pinocytotic activity occurs at the bases of the intermicrovillous space. Mitochondria are numerous in the apical cytoplasm and basally beneath the nuclei. The supranuclear cytoplasm contains most of the cell organelles. The lateral plasma membranes of adjacent cells interdigitate and are joined by junctional complexes. The periodic acid-Schiff (PAS) reaction, indicating neutral mucosubstances, is positive only in the apical brush border of enterocytes and in goblet cells. The goblet cells also stained with Alcian blue (AB), at pH 2.5, thus revealing the presence of carboxylated glycosaminoglycans. Compact aggregations of AB- and PAS-negative cells are situated directly below the epithelium. Mitotic figures are present in individual clusters of cells. The fine structure of cells in these clusters indicated that these cells could be responsible for renewal of intestinal epithelium. Numerous endocrine-like cells could also be seen. The closely packed smooth muscle cells and amorphous extracellular material with collagen fibrils constitute a net-like structure under the basal lamina that is very closely associated with the epithelium. There are numerous acidophilic granular cells between epithelial cells, in the lamina propria/submucosa, and between cells aggregations. They seem to be associated with nematode infections and possibly constitute a humoral defense mechanism.

Biogenesis of melanosomes in Kupffer cells of Proteus anguinus (Urodela, Amphibia).

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single-membrane-bounded bodies, named as 'clusters of melanosomes' or 'melanosomogenesis centers'. Inside such clusters, different structures are present: (1) filament-like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron-dense melanosomes in different stages of melanization, (3) melanosomes with an electron-dense cortical area and a less electron-dense medullar area, and (4) uniformly highly electron-dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.

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 µPa) 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.

Tectorial structures on the inner ear sensory epithelia of Proteus anguinus (Amphibia, caudata).

The tectorial structures of the inner ear of the proteid salamander Proteus anguinus were studied with transmission and scanning electron microscopy in order to analyze the ultrastructure of the otoconial membranes and otoconial masses of the maculae and the tectorial membrane of the papilla amphibiorum. Both otoconial and tectorial membranes consist of two parts: (1) a compact part and (2) a fibrillar part that joins the membrane with the sensory epithelium. Masses of otoconia occupy the lumina above these membranes. There are two types of calcium carbonate crystals in the otoconial masses within the inner ear of Proteus anguinus. The relatively small otoconial mass of the utricular macula occupies an area no greater than the diameter of the sensory epithelium, and it is composed of calcite crystals. On the other hand, the enormous otoconial masses of the saccular macula and the lagenar macula are composed of aragonite crystals. In the sacculus and lagena, globular structures 2-9 m̈m in diameter were discovered on the lower surfaces of the otoconial masses above the sensory epithelia. These globules show a progression from smooth-surfaced, small globules to large globules with spongelike, rough surfaces. It is hypothesized that these globules are precursors of the aragonite crystals and that calcite crystals develop similarly in the utriculus. The presence of globular precursors in adult animals suggests that the formation of new crystals in the otoconial membranes of the sacculus and lagena of Proteus is a continuous, ongoing process.

Differentiation of the inner ear sensory epithelia of Proteus anguinus (Urodela, amphibia).

The stages of differentiation of the inner ear sensory epithelia of the neotenous cave urodele, Proteus anguinus, was studied with light and electron microscopy. Comparative ultrastructural analysis among specimens of different sizes confirms that new sensory cells may be generated throughout life, particularly along the periphery of the saccular macula. The inner ear of Proteus contains at least four types of sensory cells that differ in their apical ciliary part. The lungs and air-filled buccal cavity may function as transducers of sound pressure in underwater conditions. Sound waves might be transmitted from the buccal cavity to the connected oval window. The very complex orientation of the sensory hair cells of the saccular macula and the large overlying saccular otoconial mass suggest that this macula facilitates orientation of Proteus in its underground aqueous habitat.