Human Fish Research Articles

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 olfaction in Proteus anguinus: a behavioural and cytological study

Life and development in complete darkness for a strictly cavernicolous Urodele species such as Proteus raises the problem of orientation and environmental perception. An experimental study of olfaction in Proteus anguinus was undertaken on two complementary aspects: (a) a anatomical and ultrastructural aspect (photon and electron microscopy); and (b) a behavioural aspect, by testing Proteus’ sensitivity to decreasing amounts of dead prey (chironomid larvae) in a choice chamber. The results show that: (a) Proteus’ olfactory epithelium clearly belonged to the general model established for all vertebrates, with a pseudo-stratified structure mainly composed of the three classical cell types (sensory cells, supporting cells and basal cells). With the unusual thickness of the epithelium common to the Proteidae, a fourth cell type is observed in Proteus; and (b) Proteus shows a well-developed olfactive sense: the sensitivity threshold occurs around 1 g of prey (in a 250 cm 3/min water current). This relatively low detection threshold implies the use of an outstanding sense of smell probably due to the particular necessities of the subterranean environment. The experiments also emphasize the effects of environmental factors on Proteus’ response to experiments, and the role of thigmotactism.

Thyroid hormone receptor genes of neotenic amphibians.

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to alpha and beta genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3',5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRalpha in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRbeta expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRalpha and TRbeta may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis.

Morphometric analysis of black and white European cave salamanders, Proteus anguinus

Morphometric data for the cave salamander, Proteus anguinus, from the Dinaric Karst of south‐eastern Europe are analysed with data for the recently described black and non‐troglomorphic Proteus anguinus parkelj from south‐eastern Slovenia. Proteus a. anguinus and P. a. parkelj are morphologically distinct. Within P. a. anguinus, inland and coastal populations possess distinguishable phenotypes. Mantel permutation tests show that no firm evidence is available that demonstrates the existence of clinal variation. The characters that best describe differences within P. a. anguinus (head, trunk and tail length, the number of costal grooves) also document the differentiation between P. a. anguinus and P. a. parkelj. The claim that P. a. parkelj falls within the (clinal) variation of P. a. anguinus is untenable. Accordingly, its taxonomic status is upheld.

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 0.3 mV/cm ( ca 100 nA/cm 2) and up to 2 mV/cm (670 nA/cm 2), 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.

Tuning of electroreceptors in the blind cave salamander, Proteus anguinus L.

The blind cave salamander's (Proteus anguinus L.) sensitivity for electrical fields was determined by recordings from afferent fibers coming from ampullary electroreceptors. The animals were stimulated with single rectangular or continuous sinusoidal electrical signals passed through the cave water in the experimental tank. Best threshold sensitivities of the fibers for both kinds of signals were in the same range (0.1-0.5 mV/cm = 30-150 nA/cm2). The fibers showed phase-locked responses to rostrally cathodic phases of rectangular or sinusoidal stimuli and suppression to anodal phases. Units were tuned to 'best frequencies' (B.F., sine waves) between 20 and 40 Hz. Any functional, i.e. phase-related, firing or suppression of firing occurs in a total frequency range of less than 0.1 to around 100 Hz. This frequency range of tuning in the primary fibers was the same as found in the behavior, and this therefore reflects the peripheral sensory properties. Electrical sensitivity might be of special value for these cave dwelling animals, as they may detect and recognize prey animals by means of the prey's electrical field spreading into the surroundings, i.e. its electromyogram.

Behavioral sensitivity of the European blind cave salamander, Proteus anguinus, and a Pyrenean newt, Euproctus asper, to electrical fields in water.

In the urodeles Proteus anguinus and Euproctus asper, thresholds of an overt avoidance response to weak electrical field stimuli (continuous sine-waves) were measured as a function of frequency. Thresholds down to 0.1 mV/cm (30 nA/cm2) were found in P. anguinus and 2 mV/cm (600 nA/cm2) in E. asper at 'best frequencies' (B.F.) of 20-30 Hz, but sensitivity covered a total frequency range of below 0.1 Hz to 1-2 kHz, with up to 70 dB higher thresholds. Average thresholds of 1 mV/cm in P. anguinus and 40 mV/cm in E. asper were more than 30 dB apart and significantly different. Both species were sensitive to galvanic DC-pulses, clicks, and noise bursts with intensities of about the same order of magnitude. Specimens of the transparent catfish, Kryptopterus (Siluridae) reacted in the same frequency range as found for Proteus and Euproctus, and had still lower thresholds, down to 0.02 mV/cm (1.5 nA/cm2). The biological significance and possibly still ongoing evolution of the electrical sense in urodeles is interpreted in terms of comparative sensory physiology and more recent, still speculative, evolutionary diversification during and since the Pleistocene.

Cloning and expression of a thyroid hormone receptor alpha 1 in the perennibranchiate amphibian Proteus anguinus.

We demonstrated the presence of thyroid hormone receptor alpha mRNAs in tissues of the perennibranchiate amphibian Proteus anguinus, which is insensitive to thyroid hormone. From P. anguinus muscle we cloned and sequenced the 3' coding and untranslated region of a cDNA corresponding to a thyroid hormone receptor alpha 1. Using cDNA-PCR and in situ hybridization, we showed a tissue-specific expression of thyroid hormone receptor alpha genes, which was not upregulated by thyroid hormone as opposed to that observed in the TH-sensitive species, Xenopus laevis.

A black, non-troglomorphic amphibian from the karst of Slovenia: Proteus anguinus parkelj n. ssp. (Urodela: Proteidae)

A morphologically distinct cavernicolous salamander Proteus anguinus from southeastern Slovenia (Bela Krajina) is described as P. a. parkelj ssp. n. It differs from P. a. anguinus in a dark pigmentation, fully developed eyes, a skull with broader and shorter bones and fewer teeth, a voluminous jaw musculature that gives the head a bulky appearance, a proportionally longer trunk with a higher number of vertebrae, shorter extremities, and a shorter tail. Most of these traits are considered to be plesiomorphic character states. An allozyme analysis over 40 loci has shown the new dark pigmented taxon to be genetically similar to a white and troglomorphic neighbouring population from Stična (DNei = 0.23). Both populations in turn are genetically dissimilar to a geographically more distant population from Postojna (D Nei = 0.49). The observed level of genetic differentiation suggests that western and southeastern Slovenian populations form separate lineages since the uppermost Miocene but conservatively hitherto only a single species is recognised. The new taxon is only known from a small area and may be rare. P. a. parkelj, now under strict legal protection, is threatened by industrial pollution.

Predation in caves: the effects of prey immobility and darkness on the foraging behaviour of two salamanders, Euproctus asper and Proteus anguinus

The behavioural responses of the blind cave salamander Proteus anguinus and the Pyrenean salamander Euproctus asper (a facultative cave dweller) to living and dead chironomids offered in light or in darkness were studied experimentally. Both species were able to detect and locate single prey items positioned at distances of 30 cm. Proteus responded to dead prey in light faster and captured live prey in darkness earlier than E. asper. E. asper captured live prey in light earlier than in darkness. Proteus is well equipped to search for non-visual information and used an active, mechanically and chemically guided approach in all experiments. E. asper showed a more directed, visually dominated approach behaviour with live chironomids in light, but used an active, widely foraging mode with live prey in darkness and dead prey in light. E. asper may forage successfully both in epigean and hypogean habitats.

Thyroidal status and myosin isoenzymic pattern in the skeletal dorsal muscle of urodelan amphibians — The perennibranchiate Proteus anguinus

In the perennibranchiate Proteus anguinus, larval myosin isoforms were shown to coexist for life with the adult isomyosins that appeared at the end of the larval stage. Analysis of the myofibrillar ATPase profile also revealed that a high percentage of immature fibers persisted in adults. A long-term treatment with large amounts of T3 had no effect on juvenile individuals. Applied to subadult animals it promoted a regression of larval myosin isoforms and a reduction in the percentage of immature fiber types. The regulative effect of T3 in the myosin isoenzymic transition may be delayed and depends on metabolic conditions, which suggests it is indirect.

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.

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.

Déterminisme hormonal de la différenciation du muscle strié squelettique chez les amphibiens urodèles

In the urodelan amphibian Pleurodeles waltlii, spontaneous external metamorphosis was correlated with an increase in the serum level of thyroxine (T4). Within the same period, a change occurred in the myofibrillar ATPase profile of the dorsal skeletal muscle; fibres of larval type were gradually replaced by transitional fibres (type IIC), then by adult fibres of the types I, IIA, and IIB. Likewise, a myosin isoenzymic transition was observed. In larval animals, myosin electrophoresis revealed 3 bands corresponding with isoforms having identical heavy chains (MHC), but different light chains (MLC). In the course of metamorphosis, the 3 larval isomyosins were replaced by 3 isoforms having the adult type MHC and different motility. In a related neotenic species, Ambystoma mexicanum, no spontaneous anatomic metamorphosis occurred; at the time it should theoretically take place, the serum T4 level remained low. The ATPase profile was modified, but transitional fibres that replaced the initial larval types appeared to be persistent, and adult fiber types appeared only in a small amount. Myosin isoenzymic transition was also incomplete, larval isoforms were still distinguished in the neotenic adults. Similar persistence of larval characters was observed in adult Proteus anguinus, a perennibranch that never undergoes anatomical metamorphosis. Experimental hypothyroidian Pleurodeles waltlii displayed no external metamorphosis, only the larval fibre types and isomyosins were detected in those animals. External metamorphosis was induced in Ambystoma mexicanum by a triiodothyronine treatment. A complete myosin isoenzymic transition was observed in metamorphosed animals. These results tend to indicate that a moderate increase in the level of thyroid hormones is sufficient to determine the production of the adult type MHC molecules and the differentiation of the corresponding myofibrillar types in the skeletal dorsal muscle of amphibians, while a marked increase would be necessary for repressing the initial larval feature.

Comparative study of the rheotaxis in the cave salamander [formula omitted] and his epigean relative [formula omitted] (Proteidae, Urodela)

The reaction to different water currents of the cave salamander Proteus anguinus and his epigean relative Necturus maculosus has been tested in a choice chamber. Both species showed a positive reaction which started in P. anguinus with a current of 35 cm/min and in N. maculosus 60 cm/min. These thresholds are within the range measured for different species of fish in the absence of visual stimuli. In addition, the rheotactic behaviour is less steady in N. maculosus . It seems that the differences found are not sufficient to be explained by adaptation to cave life.

Persistence of retinal dopamine cells in the degenerated eye of the cave salamander, Proteus anguinus L.

The Proteus anguinus L. is a blind cave perennibranch amphibian whose visual system undergoes an important morphogenetic degeneration in adulthood. The eyeball becomes atrophied and disappears under the fat tissue of the head. However, a retina can still be identified and a photophobic behavior of the animal indicates a remaining photosensitivity. In the oldest animal observed, some photoreceptor cells are still present as well as other types of retinal neurons. Characteristic synapses are observed in both the inner and outer plexiform layers. Dopaminergic amacrine cells, with processes in the inner plexiform layer, can be identified by their tyrosine-hydroxylase immunoreactivity. Taken together, these results indicate a possible functional role of the remaining retina. Since dopamine is especially involved in light adaptation from darkness, the residual retina could act in triggering the turning behavior of Proteus in response to lightening.

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.

Recherches comparatives sur l'ontogénèse et l'évolution de l'appareil hyobranchial de Proteus anguinus L., proteidae aveugle des eaux souterraines

Abstract The hyobranchial skeleton of Proteus anguinus develops 3 branchial arches, whereas the vast majority of urodela undergoing metamorphosis develop 4 branchial arches. More characteristic is the fact that the ossification of the apparatus begins in young animals and continues into the adult stages, whereas ossification usually takes place essentially after metamorphosis. Retention of 3 ossified branchial arches by adult P. anguinus appears to be neither a larval characteristic nor a neoformation; examples of Amphiumidae and urodela fossils would support this. The hyobranchial skeleton of Necturus consists also of 3 branchial arches. The squat form, the individualised hypohyal, the very reduced ossification and denticulation of the ceratobranchials are, however, characteristic comparable to those of the neotenic axolotl. These characteristics indicate that Necturus has diverged further from European perennibranchiate fossils than Proteus.

Influence of temperature on the development of Proteus anguinus (caudata:proteidae) and relation with its habitat in the subterranean world

1. 1.|The development of 144 embryos and 56 young is studied in the temperature range 5–23°C. The duration of embryonic and larval phases (120 and 230 days at 11.4°C) is long and the constants of temperature are high ( Sembryo = 1156 and Slarva = 753 degrees-days). 2. 2.|The upper and lower limit temperatures are moderate, 18 and 6–7°C, respectively. The optimal temperatures, 16 and 18°C, are higher than those of the biotope. 3. 3.|The close fit between the thermal requirements for development and habitat temperature are discussed in the context of the evolution of this subterranean species.

Rhythmic locomotor activity of the Grottenolm(Proteus anguinusLaur.) and the gold fish(Carassiusspec.) measured in a mine

(1977). Rhythmic locomotor activity of the Grottenolm (Proteus anguinus Laur.) and the gold fish (Carassius spec.) measured in a mine. Journal of Interdisciplinary Cycle Research: Vol. 8, No. 3-4, pp. 347-349.