Non-visual sensory physiology and magnetic orientation in the Blind Cave Salamander, Proteus...

© Koninklijke Brill NV, Leiden, 2009 DOI 10.1163/157075609X454971

Animal Biology 59 (2009) 351–384 brill.nl/ab

Non-visual sensory physiology and magnetic orientation in the Blind Cave Salamander, Proteus anguinus (and some other

cave-dwelling urodele species). Review and new results on light-sensitivity and non-visual orientation

in subterranean urodeles (Amphibia) †

Peter A. Schlegel 1 , Sebastian Steinfartz 2 and Boris Bulog 3,*

1 Laboratoire Souterrain, CNRS, F 09200 Moulis - St. Girons, France, and Department Biologie II, Biozentrum der LMU, Grosshadenerstr. 2, D 82152 Planegg-Martinsried, Germany

2 University of Bielefeld, Department of Animal Behaviour, Morgenbreede 45, D-33615 Bielefeld, Germany

3 Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia

Abstract A review is given on several sensory systems that enable troglophile and troglobian urodele species to ori-ent non-visually in their extreme hypogean habitat. A new sense was discovered allowing the animals to orient according to the Earth’s magnetic fi eld, which could serve as a basic and always available reference for general spatial orientation. Moreover, working with permanent magnetic fi eld stimuli off ers a very sensitive experimental method to discover the urodeles’ thresholds for other sensory modalities such as light, sounds, and other stimuli, perhaps in competition or combination with the magnetic one. Proteus’ audition as underwater hearing and light sensitivity due to its partly remaining sensory cells and/or skin sensitivity were studied. Excellent underwater hearing abilities had been demonstrated for Proteus with an acoustic behavioural method. Th e ability of sound pressure registration in Proteus is supposed to be attained by the tight anatomical junction between the ceiling of the oral cavity and the oval window. More generally, all non-visual sensory capabilities may facilitate certain behavioral strategies, compensating for missing visual orientation. Troglobians are more likely than others to own and regularly use the sensorial

†) We would like to dedicate this article to the memory of Prof. Dr. Peter Schlegel and Dr. Wolfgang Briegleb, as a review on several sensory systems that enable troglophile and troglobian urodele species to orient non-visually in their extreme hypogean habitat.

Th e sudden and much too early death of Prof. Schlegel, the leading expert on the research fi eld of sen-sory physiology and ethology of troglophile and troglobian amphibians convulsed us.

Dr. Briegleb died suddenly by accident with his delta dragon. We are grateful to him for his research opus since the late fi fties and early sixties that essentially contributed to the knowledge of Proteus ’ biology, development, sensory physiology, and eco-ethology. *) Corresponding author; e-mail: [email protected]

352 P.A. Schlegel et al. / Animal Biology 59 (2009) 351–384

opportunities of a magnetic sense for spatial orientation. Compared to their epigean relatives, cave animals may have retained phylogenetically older sensorial properties, transformed or improved them, or fi nally acquired new ones which enabled them to successfully survive in dark habitats. Neighbor populations living on surface did not necessarily take advantage of these highly evolved sensory systems and orienta-tion strategies of the troglobian species and may have lost them. E.g. Desmognathus ochrophaeus is partly adapted to cave life and exhibits good magnetic sensitivity, whereas, D. monticula and D. quadrimaculatus are epigean and, although living in rather dark places, did not demonstrate magnetic sensitivity when tested with our method. © Koninklijke Brill NV, Leiden, 2009

Keywords Light sensitivity; skin and rudimentary eyes; non-visual and magnetic orientation; cave urodeles; blind cave salamander; Proteus anguinus

1. Preface

Th is is a review of recent work in multi-sensorial physiology and eco-ethology of Proteus anguinus , contributing to subterranean biology of some other cave-dwelling urodeles with the particular focus on Calotriton = Euproctus asper . Results are hence published and unpublished ones from the authors’ and those of others’ cited to make up this review complete, including all new original details and even own older unpublished fi ndings, essentially retaken from the version published in French (Schlegel et al., 2006 ).

2. Introduction

From an eco-physiological point of view, it is a generally accepted hypothesis that cave dwelling animals have been prompted, among other adaptations, to develop and/or improve non-visual sensory systems in order to orient in and to adapt to permanently dark habitats. Th e mode of cave life and other biological peculiarities of Proteus and other troglobian (= cavernicoles) evoke, among general adaptations, the possibly great importance of underwater hearing, i.e. the sensory role for its inner ear (ref. to the excellent underwater hearing of Proteus , section 4.3, and to Bulog and Schlegel, 2000 ). Moreover, a new sensorial modality, which should improve spatial orientation and navigation in the dark, has been described. Spontaneous alignments to the Earth’s permanent-magnetic or artifi cially deviated fi eld have been evidenced for specimens of at least 3 obligatory or non-obligatory cave-living urodele species, Proteus anguinus, Calotriton (formerly Euproctus ) asper , and Desmognathus ochrophaeus (Schlegel, 1996 ; Schlegel, 2006 , 2008 ; Schlegel et al., 2006 ; Schlegel and Renner, 2006 ). True magnetic navigation, as often suggested by e.g. Philips (1977), Phillips et al. ( 1995 ) and Fischer et al. ( 2001 ), has not yet been made obvious by the techniques to be described. On the other hand, alignments to magnetic fi elds can become disturbed, i.e. deviated, by addi-tionally available visual, acoustical, and other cues as sensory competitors to refer to, but they were successfully eliminated for purely magnetic experiments. Th is means that the animals can effi ciently extract additional orienting information from those occasional or accidental non-magnetic sensorial hints. When, however, those cues are missing and only a (permanent) magnetic fi eld is available the importance of the

P.A. Schlegel et al. / Animal Biology 59 (2009) 351–384 353

primarily magnetic reference for spatial orientation in cave-dwelling animals is clearly demonstrated by our methods (see sections 4.1 and 4.2, and fi gs 2 - 4 , 8 - 12 below). Th is means that for the animals’ strategy of orientation, alignments to the Earth’s magnetic fi eld (Merkel, 1980 ; Wiltschko and Wiltschko, 1995 , 2005 ; compare also Briegleb and Schwartzkopff , 1961 ; Briegleb, 1962 a, b) and alignments according to other, simulta-neously available, external references, seems adequate for all cave dwellers. If landmarks of the substrate itself, and in particular, the presence of thigmotaxic and olfactory cues are experimentally excluded magnetic orientation remains as a last opportunity for blindfolded animals. Any disturbance of the magnetic orientation yields, at the same time, methodologically the opportunity to analyze sensorial tolerance thresholds for other physical cues quite elegantly (compare sections 4.2, 4.3, 4.5, and the primordial magnetic compass orientation of e.g. young inexperienced migrating birds, reviewed by Wiltschko and Wiltschko, 1995 , 2005 ). Further sections deal with the immunocy-tochemistry (section 4.6) of the more or less degenerated photoreceptors of P. angui-nus. Genetic/enzymatic tests should reveal distances between several Calotriton asper populations, using mitochondrial criteria and molecular protein loci (allozyme) tech-niques (section 4.8).

3. Material and methods

Studied animals belonged to the species of Proteus anguinus Laurenti, 1768, European blind cave salamander, which were obtained from the stocks of the breeding colony, raised in Moulis cave (French Pyrenees) since the early sixties, or were taken from the wild effl uents of caves in Slovenia for the eye, ear, and auditory studies. Further on the basically visually oriented Calotriton asper (Pyrenean endemic newt, also raised in Moulis), the American Plethodontid lung-less salamander, Desmognathus ochrophaeus, and two other Desmognathus species ( monticula, and quadrimaculatus ) were tested with the same method. Additionally, axolotl ( Ambystoma mexicanum ) from a laboratory bred tribe , Lissotriton montandoni, Mesotriton alpestris, L. vulgaris (Schlegel and Renner, 2006 ), Pleurodeles waltl , and Notophthalmus viridescens (Schlegel, 2006 ) were included in this study. With the exception of Proteus , these animals are not blind (can “see” pic-tures), but they were all observed under dim infrared light (1-2 Lux), i.e. during the experiments, vision was excluded for all specimens. As an IR-fi lter, cutting at 780 nm (Schott RG 780, also used by other experimenters, Phillips et al., 1995 ) revealed as eventually still disturbing for Proteus ’ alignments to magnetic fi elds (Schlegel, 2007) the Schott-830 and -1000 nm fi lters or IR-diodes, emitting in the far IR-range, were used for registrations with an IR video camera and tape-recorder. Th us, alignments due to any light sensitivity were avoided, as the animals have no opportunity, during the experiments, to “see” by means of the observational IR-light supplied (for statistics, data collection and editing see Schlegel, 2006 , 2007; Schlegel and Renner, 2006 , and Results, below).

Experiments were performed in the Moulis laboratory cave, “Grotte Laboratoire”, on Proteus specimens (see sections 4.1-4.3). Most other experiments on the other species, including some specimens of Proteus transported from Moulis cave, were run


in the basement of the former Zoological Institute in Munich. Th e experimental design is the same as described by Schlegel ( 2006 and 2008 ; Schlegel and Renner, 2006 ), and the sessions under natural and artifi cial magnetic conditions were run at both locations.

Th e animals were introduced into round glass bowls, fi lled with cave or tap water, and observed in the dark under IR light illumination in an “arena-like” situation (Wiltschko and Merkel, 1966 ). All alignment reactions of the animals (head-direction= heading; Wiltschko and Wiltschko, 1995 ) in sequence during a whole session were counted, as the 8 categorized geographical directions (N, NE, E, etc.) over 10-36 hours as a function of the natural or imposed artifi cial magnetic fi eld and were treated with circular statistics (Batschelet, 1981 ). Criteria for a count were: clear stops after a move-ment, i.e. the direction the head of an animal was pointing to (“headings”). Longer straight displacements into one direction were counted as well, but the same direction in sequence was only counted again when the animal fi rst moved but came back to this direction at the end of that movement.

3.1. Immunocytochemistry

For immunolabelling on semithin sections, frontal sections of the eyes and sagittal sec-tions of the pineals (0,7-1,0 μm) were cut on a Reichert-Jung Ultracut and a Reichert Ultracut-S ultramicrotom. Individual sections of a series were placed on separate glass slides numbered consecutively. Th e embedding resin was dissolved with sodium meth-oxide, washed successively with methanol-benzene, methanol and water. Non-specifi c binding sites were blocked by incubating the sections in 1% bovine serum albumin in phosphate-buff ered saline (BSA/PBS) for 30 min. Th e primary antibodies were applied for 1-18 hours at room temperature; the sections were rinsed in PBS and exposed to the biotinylated secondary antibodies for 1.5 hours, diluted 1:250 with BSA/PBS (4 μg/ml). After washes in PBS, biotinyl groups were detected by ABC- solution in a dilution of 1:50 for 45 min. Diaminobenzidine (DAB) was used as chromogene (incu-bation time 6 min at 20°C) in the presence of 0,002% hydrogen peroxide. After rins-ing in water, sections were mounted in glycerol for light microscopic observation and photography in a Zeiss Axiophot I photomicroscope, equipped with phase contrast and diff erential interference contrast (Nomarski) optics. To compare immune reactivi-ties of the same receptor cell, light micrographs were taken from identical regions of consecutive sections, exposed to diff erent primary antibodies (Kos et al., 2001 ).

4. Results

4.1. Animals’ alignments according to the Earth’s magnetic fi eld

Th e main outcome of the original studies was that the animals spontaneously and sig-nifi cantly preferred one individual, hence primarily unpredictable, magnetic direction in the experimental situation (more than 400 sessions with the diff erent species stated have been run: Schlegel, 1996 and 2006 , 2008 ; Schlegel et al., 2006 ). Tests with


artifi cially varied magnetic fi eld conditions, in particular reversals and deviations of 90° of the horizontal component of the Earth’s magnetic fi eld (designated as mN=S/E, W : magnetic North set at geographic S/E/W) demonstrated that the animals defi nitely aligned geographically diff erent from before ( n = natural), i.e. according to the mag-netic fi eld. For instance, the animal of fi g. 2 chose the SE under natural magnetic fi eld conditions (2 sessions, n 1 , 2 ), over all other directions. Accordingly, it preferred the geographical NW when the horizontal fi eld was reversed ( mN=S ). Th e animals do this correctly as long as the appropriate combinations of vertical and horizontal vector strengths, i.e. particular for their local geographical location, were supplied (e.g. mag-netic intensities horizontally 220 miligauss (mG) and vertically 340 mG in Munich and very similar in Moulis: Schlegel, 2007). Choices under altered magnetic vector strengths will be described further on (see below, section 4.4).

Fluctuations of the animals’ angular mean responses around the expected targets were obvious and could be large, up to +/- 90°, but the standard error of the mean was only 6.5°, and circular standard deviation was 45 °. Th e 95% confi dence range was about +/- 13° for e.g. 47 sessions in 6 Proteus and was similar for Calotriton and Desmognathus ( fi g. 1 , up to down; from Schlegel, 2007).

Figure 1. Upper panel: summarizing results from 47 sessions (collected within 1997-2001) with 6 Proteus specimens. Polar plot of the mean angles of all sessions referred to the expected target (“errors” relative to the defi ned target at 0°). Th e mean vector at 1.6° reveals highly signifi cant (Rayleigh-value, 25.1; circular s.d. = 45°). Th e standard error of the mean was 6.5°. Th e 95%- confi dence interval of +/-14.4° is indicated by the broken lines (Oriana 2-plot; c.f. text). Middle panel: same processing for 29 sessions of C. asper ; circular s.d. was 51.4°. Th e Rayleigh-value was 12.9. Te standard error of the mean was 9.6°; vector length, 0.67; 95%- confi dence interval was +/-18°, as indicated and referred to the mean deviation. Lower panel: for Desmognathus ochrophaeus (14 sessions), circular s.d. was 30.8°. Th e Rayleigh-value was 10.5. Th e standard error of the mean was 9.2°; vector length was 0.87. Th e 95% confi dence interval was +/-18° around the median respectively.


4.2. Animals’ alignments according to the Earth’s magnetic fi eld and/or due to non-magnetic cues

When the horizontal magnetic vector was compensated in order to abolish alignments to the natural fi eld lines ( “comp.” : no magnetic compass information is available as the remaining vertical magnetic vector is only pointing vertically down and provides no longer any compass = horizontal information) the animals did not always show ran-dom (even) distributions of their choices as to be expected, but may still preferentially point to one or two non-magnetic directions. It was concluded that possibly other sensorial cues were available, such as light or sounds that have been supplied acciden-tally as competitive reference hints. In fact and in numerous cases, the animals obvi-ously aligned according to both physical parameters together or alternatively (light/sounds and magnetic fi elds, c.f. Schlegel, 2007). Th at means that they did not point just to the mathematically resultant vector direction, but rather alternated between the two targets during the session: magnetic and non-magnetic competitor, or fi nally reacted to the latter exclusively. Hence in those cases, they respected both reference sources during the same session, as revealed by two maxima of the distribution ( fi g. 2 A, B: mN=S+light ; comp.+light and fi g. 3 , diff erent animal: disturbance by noises). In sum, the animals preferred a certain geographic direction regardless of the magnetic information supplied and must have then exclusively been directed according to the non-magnetic cue available.

As stated before, this can best be studied under compensated horizontal magnetic vector conditions ( comp. = compensating horizontal vector). Th e guiding physical nature of the non-magnetic source, now used by the animal as a new reference, became evident in almost all cases. Th is competition off ers the fundamental possibility to fi nd the threshold beyond which the competitor is obviously relevant for orientation, and may even become more indicative for the animals than the magnetic cue (e.g. back-ground sounds or lights, fi gs 2 , 3 ). In other words, the non-magnetic physical cues were perceived as the magnetic ones as well if available and were not ignored with respect to the magnetic fi eld. In detail: when comparing preferences for the magnetic fi eld with some alignments to the interfering light and sound directions alone ( comp. ) it seems plausible that the animal subsequently even “adjusted” its original preference for a magnetic direction into the one to the new non-magnetic target: the altered pref-erence for the magnetic target then coincides with the external sound or light direction as a new reference, possibly a kind of set point adjustment (e.g. fi g. 2 A, n 1,2 and comp.+light ; B, n 1,2 and comp.+light as an avoidance reaction to the light source at SE, negative phototaxis; see also Schlegel, 2006 , 2008 ; Schlegel and Renner, 2006 ). It was sometimes diffi cult to distinguish reactions to the magnetic fi eld from those to other cues. Two peaks or a smeared one can appear (see above and fi g. 2 A, mN=S+light ; B, mN=S+light , unimodal = not /signifi cant and mN=S+light , bimodal, / signifi cant; see also fi gs 5 and 6 ). Th e term bimodal relates to the kind of those distributions of accu-mulated choices of the 8 geographical directions (N, NE, etc.) that shows two peaks, about 180° opposite to each other, which indicates the geographical / magnetic axis, e.g. N/S (c.f. Schlegel, 2006 , 2008 ; Schlegel and Renner, 2006 ). One favoured direction


refers to a unimodal distribution. Consequently, uni- or bimodal mean vector direc-tions appear in the polar plots twice, respectively (a single and a double vector, calcu-lated by circular statistics). Th e axes and directions (head forward) refer to the favoured alignments of the body’s longitudinal axis. Th e animals turned by 180° when the hori-zontal magnetic fi eld was reversed, and no competitor was present anymore (see sec-tion 4.1 and fi g. 2 A, B, mN=S/ mN=S+light ).

As a consequence of sensorial interference, it may arise that the uni- and bimodally calculated vector angles can grossly deviate from the directions to be expected by judg-ing the original distributions, unless being perhaps aligned by chance (compare fi g. 2 A/B, mN=S+light -situation; see also fi g. 3 , n , just below): in several cases, the calcu-lated bimodal vector directions were not pointing to the magnetic nor visual targets but fall in between, i.e. here about perpendicularly ( fi g. 2 B, n 2 ; the unimodal direction not being appreciable/signifi cant). Th e rather meaningless relevance of the overall cal-culated circular vectors between the two competitors became also demonstrated in the case of fi g. 3 A, B. Th e major peaks in the two distributions between the natural ( n ) and reversed condition ( mN=S ) were exactly 180° apart in (A), but the diff erence between the calculated, unimodal vector directions amounted to only 60° ( fi g. 3 B).

Figure 2. Reactions of a P. anguinus to magnetic fi eld conditions and to a light source at SE (run in the ”Salle d’Argile” of Moulis cave). A, distributions of animal’s choices under normal ( n 1 and n 2 , two data sets) and reversed horizontal fi eld vectors ( mN=S ). As data are drawn with approximating spline routines the sinusoidal nature of the functions is visualized (deg. of freedom 6 or 10). In addition, the reaction to a “light” source only, placed at about SE ( comp.+light ) reveals positive phototaxis, while the horizontal magnetic vector was compensated. Diffi cult to interpret is the result of an experiment with the same light “on” while the magnetic fi eld vector was reversed ( mN=S+light ). In B, polar plot, the unimodal circular treatment of that data set did not provide an appreciable favoured direction ( mN=S+light n.s. ), whilst the bimodal treatment yielded a perpendicular appreciable axis: n 1 , Rayleigh-value 9.9; n 2 , R-value, 4.3; mN=S , R-value, 5.9; mN=S+light bimod , R-value 6.6.


Th is is simply due to the circular averaging procedure over the respective double peak distributions, when calculated unimodally. Th e minor peaks at W and NW for the n - and mN=S - distributions, as well as the major peak of the comp. -distribution at NW are clearly caused by the noise coming from NE as an expression of negative phono-taxis (c.f. below, section 4.3 and Schlegel, 2008 ). Th e unimodally calculated vectors of either distribution ( n and mN=S ) are deviated towards the midline between the normal (SW) and the reversed (NE) magnetic peaks, and the two mean vector angles fall rather close to each other between W and N/NW. When, alternatively, applying the bimodal circular treatment for the mN=S -situation, the angular diff erence between the two vec-tors increases to about 140°. Th is produces a better agreement of the two evaluations. On the other hand, calculating the bimodal axis of the n -distribution did not add bet-ter information either as it was, in contrast to the mN=S - bimodal distribution, far from being appreciable ( fi g. 3 ). Hence circular statistical results do not seem always adequate to quantify eff ects when competition between magnetic and non-magnetic alignments is involved. Th erefore, apparent jumps of the animals’ preferences for geo-graphic directions due to an altered position of the magnetic North pole can often be better recognized and evaluated by considering the respective peaks and troughs of the two distributions obtained ( fi g. 3 A) than when treating them with uni- and bimodal circular statistics ( fi g. 3 B and fi g. 4 , see below). On the other hand, when both physi-cal cues, magnetic fi eld and competitor, aligned the animal onto the same axis, and

Figure 3. Negative phonotaxis of another Proteus to a noise source alone (running water pipes, located at about NE and SW, arrows) when the horizontal magnetic vector was compensated ( comp. ) and together with the normal ( n ) and reversed magnetic fi eld ( mN=S ), as labelled in A: the respective peaks laid 180° out of each other superimposed on phonotaxis. In B, neither uni- nor bimodal circular statistics can repro-duce the magnetic reactions of (A) properly even though when averaging over putative bimodal distributions.


hence the magnetic and non-magnetic targets coincide, the calculated preference (vec-tor length and direction) was either reinforced or attenuated, but with a second peak appearing 180° off as shown for a case with acoustic interference (Schlegel, 2007).

We were not able to discover and recognize all deviating sound, light, and else sources, but their direction became always obvious as the animals signifi cantly pointed at these, or away of them, as expression of positive or negative taxis ( comp .-conditions). It was very astonishing how effi ciently Proteus used even the faintest light and non-visual hints for its spatial alignments. Th e disturbing noises, located at e.g. NE/SW in the experimental room (laboratory in Munich: fi gs 3 and 4 , same animal) were due to water running through pipes and were just noticeable to the human observer and ranged between 30-40 dB SPL, measured in air. It became possible to suppress the reactions to these faint competing sounds by delivering continuous white noise from straight above (around and beyond 80 dB SPL and up to 3 kHz, measured in air at the place of the animal). Th e masker was defi nitely eff ective and provoked a random dis-tribution of choices when run under comp. - conditions ( fi g. 4 A, B, comp. ( n.s ) ; Rayleigh-value 2.6<3: not yet signifi cant/appreciable). Again this disturbance by sounds demonstrates the excellent directional hearing abilities of these animals, as reported by Bulog and Schlegel ( 2000 and fi g. 7 , below, section 4.3). When the natural ( n ) or the reversed horizontal magnetic fi eld was applied ( mN=S ) the animal then chose only according to the horizontal magnetic vector ( fi g. 4 ). Only a small, superim-posed double characteristic at NE/SW is still visible, although below signifi cance level, in particular under mN=S -condition and located at the original disturbing negative

Figure 4. A/B, reactions of the same animal as in fi g. 3 to the normal n , mN=S , and comp. magnetic conditions when originally disturbing noises were jammed/masked by low frequency continuous noise of around 80 dB SPL from straight above. Remaining minor comp. - peaks are no longer uni- or bimodally appreciable/signifi cant (arrows at NE/SW).


noise reaction (NE/SW, compare with fi g. 3 , same animal). Th is showed again very clearly how much such animals rely on any available reference cue for orientation and navigation when “vision” is not possible.

4.3. More details on photo- and phonotaxis

In sum, depending upon the intensity of the competitive reference cues (in particular light or sounds), the animals oriented towards, positively, or away, negatively, with respect to the source: i.e. they showed phonotaxis and/or phototaxis, as seen in the examples of fi gs 2 , 3 , 4 . Light in the range of 1 Lux elicited a positive reaction, but more than 10 Lux often led to negative photo taxis (see below).

Under water sounds are characterized by changes in pressure of the medium through which the sound waves pass. Such pressure oscillations may produce pulsations of a gas-fi lled cavity (i.e. buccopharyngeal cavity and lungs in amphibians or swim bladder in teleost fi shes) within the body of an animal, which in turn may result in displace-ments that stimulate and excite the hair cells in the inner ear (Popper and Fay 1999). A little is known about the hearing capability of urodeles. Th eir buccal cavity fi lled with air, and probably also the air-infl ated lungs, may transduce sound pressure in underwater conditions, according to the evidence of physiological studies on larval and adult Ambystoma tigrinum (Hetherington and Lombard, 1983 ). Wever ( 1985 ) sup-posed that sound passing through the water will rich the columella in the oval window through the mass of peripheral lateral tissues in the otic region of Necturus maculosus .

In addition to functional lungs (Sojar, 1980 ), Proteus also has a large buccopharyn-geal cavity (Istenic and Bulog, 1979 ). Th e air-fi lled mouth cavity adjacent to the inner ear and the lungs perhaps may act as under water pressure transducers. Th is ability of sound pressure registration in Proteus is supposed to be attained by the tight anatomical junction between the ceiling of the oral cavity and the oval window ( fi g. 5 ). Proteus has functional lungs, which may also serve as a transducer of under water sound pressure. Sounds rather attracted the animals, but sometimes also repelled them (e.g. fi gs 3 and 4 , see before). In particular, natural noises, typical for caves and the aquatic habitat, i.e. in our case purling, and dribbling water, which attracted Proteus within sound levels of estimated and measured 30-40 dB SPL (laboratory in Munich, measured in air: Sound Pressure Level referred to 20 μ Pascal= 0 dB SPL) and perhaps less, i.e. just above human thresholds. However, these values have to be judged as “above tolerance” and not as absolute thresholds. Excellent underwater hearing abilities had been demonstrated for Proteus with an acoustic behavioral method before (Bulog and Schlegel, 2000 ).

Th e thresholds given in the original paper were, however, largely underestimated due to an over-conservative assumptions for the earlier calibration applied. Th e cor-rected curve is presented on fi g. 6 : thresholds were at 40-50 dB rel.1 μPascal instead of the originally stated 60-70 dB rel.1 μPa. Th is is almost the lowest underwater threshold reported for any aquatic animal species falling in the human audiogram range. Only some cave fi sh are almost as good as Proteus but in a lower, more restricted frequency range: Astyanax (Hawkins, 1981 ; Popper and Coombs, 1980 ). Dolphins are as good or a bit better, but only in the ultrasonic range.


Clicks of relays and noises from the turntable device, although faint and attenuated for the animals by a lid covering the experimental dish, and other artifi cial (man-made) noises in the laboratory (running/purling water through pipes, fans of electronic equip-ment), general diurnal noises outside the set-up from the laboratory building, includ-ing vibrations due to subway trains as only not running between 1 to 5 a.m., as well as background scattered light were apparently eff ective enough to sometimes attract the animals to those sources rather than to the magnetic fi elds applied (r.f. again to fi gs 2 and 3 ; see also Briegleb and Schatz, 1974 ).

Moreover, the very good directional sensitivity for underwater sounds interfering with our magnetic experiments has pointed to the special adaptive value of its otic laby-rinth, suggested by a diverse and very complex orientation of the hair cell cilia of the saccular macula ( fi g. 7 ). Th e hair cells orientation in the saccular macula is more hetero-geneous from those of other urodelans and anurans studied. In amphibians and in some other vertebrates there are two vertically oriented groups of hair cells in the saccular macula (Lewis et al. 1985 ). Proteus has four groups of hair cells with diff erent orienta-tion similar to the pattern in many fi shes (Bulog, 1989a , 1989b ; Popper 1977 , 1981). Th ese fi shes have developed a potential for sound source registration (Popper and Northcutt 1983 ). Th e complex pattern of orientation together with the overlaying

Figure 5. Cross section of the otic labyrinth in the region of the sacculus and saccular macula. Proposed sound waves transmission (�) from the ceiling of the oral cavity (OC) through the fi brillar connective tissue (Ct) to the columella (Co) of the oval window. Th e propagation of the sound waves (�) would continue through the periotic cistern (PC) to the sacculus (S) and then proceed to the individual sensory epithelia. Bar = 500 μm


Figure 6. Under water audiograms of specimens of white (* - white, best specimen; � - average over whites) and black Proteus of Slovenia (�). Approximating lines calculated as “kernel” smoothing and drawn on the base of the measured points (Axum 6 graphic program). Two lines on top of the graph indicate the maximal pressure levels available (two calibrations, — and ¸: ordinate given in dB relative 1 μPascal) from the under water loud speaker at the position of the animal driven with 0 dB attenuation; x-axis given in log of frequency (kHz).

Figure 7. Complex functional-morphological orientation pattern (arrow heads �, �, �, ) of sensory hair-cells in the saccular macula of Proteus anguinus. Bar = 100 μm


otoconial mass allows the animal to analyze sound directions by virtue of the directional sensitivity-characteristic of inner ear hair cells. Diff erent bundles’ various directionali-ties function as detectors for sound waves’ particle velocity within the otoconial mass displacements ( fi g. 7 ). Proteus could detect diff erent sounds produced in its under-ground water habitat owing to the sudden rises of water level during rainy periods. Th is could be an excellent protecting mechanism to prevent ejection of specimens to the surface waters where diff erent potential predators would threaten them. It would be also of adaptive value in caves with no vision available, to profi t from underwater hearing by recognizing of particular sounds like localization of prey and other sound sources.

4.4. Long term stability of magnetic preferences and eff ects of magnetic stimulus combinations with diff erent (inexperienced) vertical vector strengths, compared to the natural, home-related, ones

Th e consistency of spontaneous choices according to the magnetic fi eld vectors was studied when the same animal was tested again and again over longer periods of time (up to months). Typically the same preferences with fl uctuations (Schlegel, 2007) could be observed over time: this particular animal’s choices were rather stable. However, it altered its preference transiently for a few days by 180° or into bimodal, after a particular sequence of magnetic conditions had been supplied (to be followed up in fi gs 8 - 12 : numbers of days referred to the fi rst “comp.”[1]- condition of fi g. 8 , see below). After these transitions, the animal went back to its original choice after days, going through epochs of fi rst purely bimodal, then staying still slightly bimodal with two uneven peaks, but ended up with an unimodal choice as originally (SE for n , fi g. 12 , n 27 , see below).

Figure 8. Sequence of reactions of a Proteus to diff erent magnetic conditions as labelled and described in the text; numbers in boxes refer to days after the fi rst experiment ( comp. [1] ). “Kernel” (interpolating) spline smoothing (see text).


Th is animal chose SE, under normal fi eld conditions ( n [3] ) and NW accordingly, under reversed conditions one day later ( mN=S [4] , fi g. 8 ). It again preferred the SE when switching to the condition ” mN=S+mN up ” ( fi g. 8 , mN=S+mN up ( 1 ) [5] and ( 2) [6] ) but produced a second minor peak at NW already (slightly bimodal choices and unimodal as well, mN=S+mN up(1+2) [5+6] , both data sets together). Th e animal switched to purely bimodal choices when the vertical vector was removed (c.f. fi g. 8 , vert.=0° [7] ) and did that even when total intensity was supplied horizontally alone ( fi g. 9 , vert.=0° + 430mGauss [8] ). When the next day, the n -condition was reintroduced but with the same reinforced horizontal vector strength ( fi g. 9 , n 2(+430mG)[11] and n 1+2(+430mG)[10] ) the animal chose slightly bimodal ( n 2[11] ) but overall still SE (n 1+2[10] ). If however, again the next day ( fi g. 9 , n 3 [12 1 ] ), natural horizontal and vertical fi eld strengths were supplied the animal switched to clearly preferring NW instead of SE as if mN=S had been applied ( fi g. 9 , n 3 [12 1 ] ). When the next day, the vertical vector was reversed ( fi g. 9 , mN up [12 2 ] ) the animal fi nally showed a bimodal choice as two days before and as under both vert.=0° -conditions supplied earlier ( fi g. 8 , vert.=0°[7] and fi g. 9 , vert.=0°(+430 mG)[8] ). Th e new magnetic reference now seems to be NW instead of originally SE ( fi g. 9 , n3[12 1 ] ). By contrast, especially if the individual animal was not returned back to its home aquarium but into another container (or presuma-bly even in a natural environment) it may alter its magnetic preference due to added non-magnetic cues (c.f. Schlegel, 2006 , 2008 ; Schlegel and Renner, 2006 ).

Th is means that not only non-magnetic references can alter the animals alignments and eventually correct them, but also the history of magnetic situations, supplied fol-lowing in series when inexperienced physiological magnetic conditions as mN up, vert.=0° , or n+ 430 mGauss are given one after the other for some time (10 consecutive

Figure 9. Reactions following fi g. 8 : further magnetic conditions as labelled. Spline smoothing (10 deg. of freedom) was chosen to roughly demonstrate basic mathematical functions (single or double sine waves).


situations observed during 12 days, fi gs 8 - 10 ). When supplying normal ( n ) continu-ously during the next days ( fi g. 10 , n [12 1 ] and [13, 14-18] ) the animal gradually came back to its original choice ( fi g. 10 , compare n [2] , then n[12 1 ] ) and fi nally n [13, 14-18] , after an intermezzo of a reinforced n -fi eld and reversed vertical vector, n+430 mG(10, 11) and mN up[12 2 ] ). Th is was then almost exactly alike the choice taken two weeks earlier.

Th is demonstrates again that inexperienced vector combinations can deviate, reverse, or weaken the animal’s choices, but the primordial preferences seem to be very stable over longer periods of time. At least the main axis remained extremely stable in this animal ( fi g. 11 ), despite the fact that transiently (one or two days) or even for longer times, it altered its preference between NW to SE as described when inexperienced vector combinations were supplied ( fi g. 11 vert.=0° , mN up , mN=S+mN up ). Th erefore, the choices after such conditions, again with naturally possible/reasonable vector com-binations ( n ; mN=S ) fell fi rst opposite, but then went gradually back to original, i.e. SE with the n -situation ( fi g. 12 , labels with indices apply to the days of the series the condition was changed as stated; n 2 , not plotted and n 17 are almost equal, but the choice obtained with mN=S 23 swapped into a preference for SE as under n -conditions but now under the mN=S+mN up 24 -condition). When then switching back to natural ( n 26 ) the animal again chose NW as if mN=S was supplied, but then returned to its SE-preference as originally ( fi g. 12 , n 27 , compare before). In sum, under all cases with inexperienced magnetic vector conditions, the animal transiently chose bimodally even with naturally possible conditions when coming from inexperienced fi eld conditions (see lowest n -curve in fi g. 11 and in fi g. 12 , n [14] ). Several other animals showed the same stable axis characteristics over longer times (not shown, but c.f. Schlegel, 2006 , and 2008 ).

Figure 10. Further experiments after the series of fi gs 8 /9 showing deviations of choices when unnatural magnetic vector combinations were supplied in sequence ( n+430 mG; mN up [10, 11], and *); note the gradual return from n (2, ¾) over n (12 1 , �) and n (13, —) back to n (14-18, �) and mN up (12 2 , ¸); Kernel (interpolating) spline smoothing.


Figure 11. Combined polar plot (A) and distributions (B) of choices of the same animal in series ( fi gs 8 - 12 ) of experiments with magnetic vector conditions as labelled. Note very stable choices within the SE/NW-axis. Kernel (interpolating) spline smoothing.

Figure 12. Stable and transient preferences of the same animal to the 3 standard magnetic conditions (days of the experiments as labelled by indices). Note the transient choices n 14 (¿), n 15 (�), and n 26 (). See text (data partly redrawn from fi gs 8 - 11 ). Approximating spline smoothing (10 deg. of freedom) was used for the stable results ( mN=S+mN up 24 (˜), mN=S 23 (�) , n 17 (¾) , n 27 (*) and Kernel (interpolating) smoothing for the transient distributions (n 14, 15, 26 ).


In another context, we tried intentionally to alter the choices in one longer experi-ment where two rather young animals (4-6 years) were kept under mN up -conditions for 3 weeks, but similarly as seen just before, the animals, then suddenly reintroduced into n -conditions, showed rather insecure bimodal choices and later on went back to their original preferences, but this has to be confi rmed by repeating with more animals. Further experiments indicated that and how the spontaneous magnetic choices can eff ectively and persistently be altered by hiding tubes (c.f. Schlegel and Renner, 2006 ). Adjustments in form of altering the magnetic target set point between an originally spontaneous magnetic preference and the newly accepted external, non-magnetic ref-erence had been observed occasionally (see also above, sections 4.2, 4.3).

4.5. Possible competition between alignments to dish-related markings or landmarks as further competing external references

In contrast to external references, dish-related landmarks or markings, such as olfac-tory and gustatory ones, generally considered as very eff ective orienting cues for cave animals (Briegleb, 1962 a; Durand and Parzefall, 1987 ; Durand et al., 1981 , 1983 ; Guillaume, 1999 ; Parzefall et al., 2000 ; Richard et al., 1983 ), did not play a major role in the context of our study. Possible alignments to chemicals in the experimental dish or even to given landmarks in the form of scratches and failures in the glass bowl did not interfere with magnetic cues. Turntable experiments were conducted to eliminate these possible infl uences but fi nally revealed as not necessary and relevant if not coun-terproductive as a continuous or intermittent turning of the substrate may be disturb-ing by itself and needs readjustments to the magnetic reference by the animal itself (as seen by Schlegel, 2006 , 2007). Th ere seemed to be even a tendency of the animals to counteract the deviating turns when beginning their own movement, presumably due to vestibular and mechanical inputs, i.e. animals frequently turned visibly left when the experimental bowl had been turned right and vice versa.

On the other hand, frequent spontaneous displacements of the animals or at least movements of the head were perhaps due to the somewhat stress situation in the arena with no external or dish-related references available. Th is was, taken overall, probably the main motivation for the animals to align so frequently and continuously over days and weeks. Th e experiments had shown that, compared with surface-living animals, troglophile species ( Desmognathus ochrophaeus ) and troglobionts in general, as Proteus , align most consistently, i.e. without major fading, under the experimental conditions of this study (refer to the terms in textbooks of biospeology, such as those by e.g. Culver, 1982 ; Juberthie and Decu, 1994 ). Th e former epigean species/populations showed either no alignment to the magnetic fi eld or aligned more vaguely and there-fore not appreciably, i.e. randomly and maybe due to eventual habituation.

Proteus also use chemical signals as directional cues for homing and also for social behaviour and chemical signals may attract conspecifi cs (Guillaume, 2000 ). Surface dwelling aquatic amphibians do not always use chemical cues carried by water in order to meet their mates (Guillaume, 1999 ). Cave dwelling Proteus shows signifi cant and surface dwelling relative, Necturus only weak preference for water which passes living prey before entering a choice chamber (Durand et al., 1982 ).


It seems also a general observation that these tend to routinely move much, presum-ably to fi nd food and mates with better chances than when only waiting. On the other hand, Proteus uses to stay in its shelter even for months. Th at fi nding corroborates typi-cal observations by Juberthie-Jupeau ( 1983 ) on cave beetles, which apparently do not walk and wander around purposefully, following olfaction among other sensory modal-ities, but run continuously and seemingly at random until they fi nd food or mates. Th is may be due to a general olfactory excitement in those experiments, but it is abso-lutely striking how much cave beetles, as do hypogean urodeles, move around in the cave habitat all the time regardless of the illumination present, as we have defi nitely observed in caves. Th e same was to be noticed on all video-recordings from “cave”-urodeles during this study.

4.6. Experiments with respect to Proteus ’ light sensitivity

4.6.1. Anatomical fi ndings on rudimentary eyes and the pineal organs in Proteus. Although the rudimentary structures of the photoreceptors in Proteus suggest that they are non-functional, at least some of the eyes investigated retained traces of light sensitivity. Th e presence of immunopositive visual pigments was demonstrated with anti-opsin antibodies in the regressed eye and pineal. Th is indicates the possibility of a certain preserved light sensitivity in the endemic cave salamander Proteus . To investigate diff erent grades of degeneration, we recently studied the photoreceptors in the rudimentary eye in diff erent populations of Proteus . Th e ultrastructural analysis brings interesting results about the various levels of structural degeneration of photoreceptor cells in diff erent populations in Slovenia (Bulog, 1992 ; Kos and Bulog, 1996 ; Kos et al., 2001 ).

Th e immunocytochemical analysis of visual pigments gives a new insight into the functionality of these photoreceptors. Photoreceptors in the regressed eye of unpig-mented Proteus anguinus anguinus , from the Planina cave have degenerated outer seg-ments, consisting of a few whorled disks and irregular clusters of membranes (Kos et al., 2001 ). Most of these outer segments showed immunolabelling for the red-sensi-tive cone opsin, and some of them were found to be positive for rhodopsin. An even more remarkable degeneration was observed in the photoreceptors of unpigmented specimens from the Otovec doline, which are completely without of an outer segment, and majority of them do not even possess an inner segment.

Th e predominance of COS-1 positive outer segments in the retina of Proteus from the Planina cave reveals that there is a strong shift in the cone/rod ratio from pig-mented P. anguinus parkelj to unpigmented P. anguinus anguinus in behalf of the red–sensitive cones. Th is trend is even more explicit in the pineals of all animals studied, where the sole visual pigment present was the COS-1 positive (red-sensitive) cone pig-ment, which was localized in rudimentary outer segments. COS-1 is a mouse mono-clonal antibody recognizing the C-terminal portion of the red/green cone opsins from amphibians up to primates (Röhlich and Szél, 1993 ). On the contrary, animals from another population (Otovec doline) with higher stage of retinal degeneration showed rhodopsin immune reactivity in some of the cells’ soma, while COS-1 reaction was not observed.


Th e eyes of Proteus anguinus parkelj show the general characteristics of the vertebrate eye (Bulog 1992 ). Immunocytochemical labelling with diff erent anti-opsin antibodies allows the classifi cation of photoreceptor types. Th e majority of g specifi c for the long-wavelength-sensitive visual pigments.

In the retina of the black Proteus we found:

• Principal rods, labelled with anti- rhodopsin antibody AO,• Red-sensitive cones, labelled with mAb COS-1 and B2, antibodies specifi c to the long-

wavelength sensitive visual pigments in a large number of vertebrate species, and• A photoreceptor type, which might represent a blue- or UV-sensitive cone, labelled

with anti-rhodopsin mAb K55-75C (Kos et al., 2001 and see fi g. 13 ).

In amphibians, two types of rods and three types of colour-specifi c cones were described, the latter assumed to represent red-, blue, and UV-sensitive cones ( Xenopus : Röhlich et al., 1989 ; Röhlich and Szél, 2000 ; Witkovsky, 2000 ; Zhang et al., 1994; tiger salamander: Sherry et al., 1998 ).

Th erefore visual pigments are still expressed in the degenerate photoreceptors of the eyes and the pineal of the “blind” cave salamander. Th is fi nding cannot defi nitely answer the question of Proteus’ light sensitivity, since the visual pigments are only the fi rst members of the phototransduction cascade. But it provides an additional support to the hypothesis of retained light sensitivity in Proteus , which has been ascertained in previous physiological, morpho-functional, and behavioral studies. Th e electrophysi-ological investigations revealed that retinal photoreceptor cells of P. a. anguinus are still responsive to light (Gogala et al., 1965 ; Zener, 1973 ). Th e optic nerve is always present (Hawes, 1946 ; Durand, 1971 ) so that light stimuli could basically be transmitted to the central nervous system, but this remains even questionable in view of the poorly positive reaction of eye-transplanted Proteus in opto-motoric tests (Uiblein et al., 1993 ).

Our fi nding, that some visual pigment is still expressed even in the highly regressed eyes of Proteus is in accordance with the results of developmental studies on the eye of the cave fi sh Astyanax fasciatus whose photoreceptors undergo similar regression as in Proteus . In situ -hybridization experiments on the transcription of the opsin- gene revealed that a structural gene required for the morphogenesis of the eye is still expressed, although its relevant structures, the outer segment membranes, are not nor-mally diff erentiated (Langecker et al., 1993 ). Th is result supports the hypothesis that the cavefi sh eye regression is caused by mutations of developmental control genes rather than by the loss of functioning structure genes (Wilkens, 1988 , Langecker et al., 1993 ), which could also be true for the degenerated eye of Proteus .

4.6.2. Th e pineal organ of amphibians. Th e pineal organ of amphibians is known to contain well-developed photoreceptors (Vigh and Vigh-Teichmann, 1999 ). A larger and a smaller rod type as well as two, immunocytochemically distinct cone types were described by electron microscopy. Th e larger cone was COS-1 positive, while the smaller cone type was negative with any of the antibodies used (Vigh and Vigh-Teichmann, 1999 ). Comparing these fi ndings with the pineal of Proteus , the only


Figure 13. Immunolabelled serial semithin sections of the retina in the Proteus anguinus parkelj (modifi ed after Kos, Bulog, Szél, and Röhlich, 2001 ). A / mAb OS2 stains all photoreceptor cells (�), B / mAb B2 stains red-sensitive cones (�), C / Anti-rhodopsin antibody AO labels rod outer segments (�). Bars = 50 μm


surviving rudimentary photoreceptors seem to be the red-sensitive cones. Th e skin area above the pineal of Proteus is paler in comparison to other parts of skin due to the absence of melanophores (Kos and Bulog 1996 ). We applied the light stimulus with a fi bre optic light source when illuminating this area (the brain being rather small anyway). Th e potent photo-phobic reaction was observed in animals.

4.6.3. Light-sensitivity of the skin. Th e staining of cells in the basal layer of the skin epithelium of non-black Proteus with mAb COS-1 indicates the possibility that there is also a photosensitive pigment present that could be involved in the photosensitivity of Proteus’ skin. Recently there was an opsin, (melanopsin), in photosensitive dermal melanophores of Xenopus laevis identifi ed (Provencio et al., 1998 ). Its conformation is similar to all known opsins, especially with invertebrate opsins. Th e amino acid sequence shares 39% uniqueness with octopus rhodopsin and 30% correspondence with vertebrate opsins. Regardless of its vertebrate origin, it is more homologous to invertebrate opsins, especially with octopus rhodopsin and less to typical vertebrate opsins. Phylogenetic analysis indicates that melanopsin and the invertebrate opsins branched from a common ancestor. Melanopsin’s anatomical distribution in melanophores, the iris, and the deep brain suggests that it is used by non-visual photoreceptors (Provencio et al., 1998 ). Deep within the retina of the vertebrate eye are also photoreceptors that bind light to regulate non-visual processes such as the narrowing and widening of the pupil. Th ese special cells are called intrinsically photoreceptive retinal ganglion cells with pigments melanopsins. Deep brain photoreceptors are known to regulate circadian rhythms and photoperiodic responses in many non-mammalian vertebrate species. Th ese responses have been best characterized in birds and reptiles but are also found in amphibians.

4.6.4. Behavioural observations concerning Proteus light sensitivity. We have retaken and extended older experiments concerning the eye-less classical cave-dwelling Proteus (Briegleb, 1962 a, b, who had referred to a study by Hawes, 1946 ). We were interested in whether the sensitivity of the skin to light is restricted to certain wavelengths of the visible spectrum for human, i.e. in the thresholds at diff erent wavelengths, and if and how the peripheral sensory-reactions to light are projected to the CNS. We therefore stimulated the head region of about 50 years old animals in order to distinguish between reactions of the reduced, degenerated eyes and, eventually, of the pineal organ, and reactions when only stimulating the skin. Again we observed under overall IR and/or faint red light. We stimulated with small, distinct light spots with diameters of 0.5 to 2 cm projected on a leg or the tail of an otherwise hidden Proteus, or else on the head of the same animal when not hiding. We obtained, as Briegleb (1962 b), that the animals moved the limb or tail away from the light spot after a few seconds of lighting, and that in a directive way, i.e. negative phototactically (s had been calculated as 3.66 originally: out of 230 trials in total, the animals had correctly avoided the light source 143 times).

As typically known for Proteus as the classical cavernicole, the animals did not habit-uate (as to all other modalities) to either head or skin stimulation when repeated, but sometimes ended up by completely going out of the stimulated area by eventually


hiding the body completely under the next shelter available in their familiar and there-fore well-known 3.5 by 5.5 m home basin (depth 8 cm down to 75 cm). However, one 7 years old Proteus , “hand-risen” and fully habituated to light since being a larva, did not react to any kind of light.

In four other specimens, we were able to estimate reaction thresholds with 7 diff er-ent colour glasses of the range between 400 and 680 nm and found that animals always ceased to react below 0.5-1 Lux brightness, which corroborated with older qualitative observations ( fi g. 14 and Briegleb, 1962 b; J. Durand, pers. com.).

About the same values were found when lighting the head area, but in this case, the animals showed often their typical “alert” reaction, lifting up the head, or even bit at the light spot in form of a “suction bite” involving the whole body, as described by Brie gleb (1962 a) and also mentioned for the auditory threshold reactions (“ audiogram”, fi g. 6 ) by Bulog and Schlegel ( 2000 ). If the entire animals were illuminated for some seconds they did not move at all. When the light was turned off the animals fl ed imme-diately with their typical turning reaction (Hawes, 1946 ). Th e threshold values stated here are certainly not absolute, the sensitivity may be even better (see below). Th is may be rather considered as the animals’ irritation threshold as long as they are held in dark-ness. However, the animals loose obviously their irritation threshold for light if they are held in a natural day/night cycle with light intensities in the 500-1000 Lux range (Nusbaum, 1907 ; supported by own observations, see above, and compare with run-ning experiments in the Moulis cave; C. Juberthie and J. Durand, pers. com.).

On the other hand, some animals tested in Moulis cave (Salle d’Argile) revealed extreme sensitivity for a much fainter light source. Again in magnetic alignment exper-iments, two animals directed themselves signifi cantly towards SE regardless of the magnetic situation and even under horizontally comp. -magnetic fi eld-conditions. After

Figure 14. “Spectral sensitivity” of Proteus anguinus . Th e graph shows roughly the tolerance thresholds (Lux, ordinate) of Proteus as a function of wavelength (abscissa).


some search, it became clear that these animals reacted to a far away (40-50 m) 60 W reddish heating bulb at a higher position of the Salle d’Argile (compare section 4.1 and fi g. 2 A, B, mN=S+comp. ). Th is bulb was just visible to the dark-adapted human observer as a light spot but not bright enough to eventually see any objects with its illumination. Th e brightness, not measurable at the position of the experimental set-up with a very sensitive optometer, was evaluated successfully at a distance of about 2 m away from the bulb, and showed just above the threshold of that meter, which lays at 0.01 Lux (= 0.001 FTCD) when removing the photometric fi lter. Th e content in wavelengths of that bulb was between orange and red, but the light-meter cuts off at 900 nm, beyond which apparently was the main energy content of it (IR). Th is was evident as the same light was easily recognizable on the video-screen when the IR CD-camera was placed 50 m away from the bulb. It seems not unlikely that the ani-mals are sensitive enough to recognize far IR of high intensity. Whether the observed reaction is possible by virtue of the few rudimentary rods of the degenerated eyes still present in animals of that population of Proteus (see above), which the breeding colony of Moulis belongs to, is not yet clear. Th e latter is originally derived and descendent of the Pivka/Planina cave system population. Alternatively, it may be due to the pineal organ (compare above) or just skin sensitivity, which is not clear yet for the moment. In the experiments with the light spots, we were not able to test the animals’ sensitivi-ties to even IR, as described above because we did not recognize, by means of the IR-camera, the IR-spots produced (830-1000 nm) at the depth of the water of 50-70 cm where the animals were located. IR is so much absorbed by even shallow water that the IR-intensities available cannot be controlled, neither was it possible to decide whether the animals reacted to the faint IR-spots at all. In above described experiments in the Salle d’Argile, water in the experimental aquarium was shallow, and the dim red/IR-light came from the side so that it was presumably easily recognizable for the ani-mals. When fi nally the disturbing bulb was covered with black metal foil the animals no longer oriented to it but now, exclusively, to the magnetic fi eld ( fi g. 2 , n and comp. see also Schlegel, 2007, fi g. 3 ).

We still do not know whether and how the animals are able to determine light direc-tion by using their eyes or pineal organ, but we are sure they could do it via skin sensi-tivity by a central nervous comparison of the light intensities perceived from an asymmetrically illuminated part of the body. Briegleb (1962 b, page 308) found a sig-nifi cant (s= 3,66, see before) photo taxis for the light avoiding reaction of the distal tail, i.e. movements away from the direction of light incidence. How the sensory infor-mation is relayed to the CNS is an open question, too, as it is not known whether the information is basically conveyed by the lateral line system or through the general somato-sensory innervation of the skin via the spinal cord’s dorsal root ganglia. Th e sensitivity of the skin to light is very common in the animal kingdom (e.g. Millott, 1968 ; de la Motte, 1964 ; Steven, 1963 ), but is not so obvious in higher vertebrates, including reptiles, birds, and mammals, where mainly protection against UV-light might be of biological relevance.

If cave animals happen to suddenly get into daylight (actively or passively) the classical interpretation was that the observed light avoiding reaction of Proteus


(Hawes, 1946 ; see below) had developed in the cave as a protection against predators and/or parasites as in epigean creeks and brooks. In this context, we propose an alter-native hypothesis: high sensitivity for light might have been an ancient property of the epigean ancestors of Proteus , which obviously were neotenic already (Herre, 1939), but not fl avistic and may have lived in brooks like the recent gender of Necturus (Proteidae) in North America, which may be also skotophile. Th e eyes and skin melanophores of Proteus evidently have disappeared in the dark. However, our studies of Proteus integument have confi rmed the presence of melanophores. Both forms of Proteus (black and white subspecies) have stellate pigment cells with pig-ment granules in the dermis, but the processes of the melanophores beneath the basement lamella are more numerous in pigmented (black) specimens (Kos, 1992 ; Kos and Bulog, 1993 ).

Only in this way, as an ecological niche, the cave secondarily facilitated degenerative and regressive evolution. Skotophily doesn’t prevent from being hunted by e.g. crayfi sh and Salmonids, as in Pivka Jama (cave) near Postojna (Slovenia) but this is only due to nutritional and topological separation (Briegleb, 1962 a, b; Briegleb and Schwartzkopff , 1961 ).

A similar process can be observed recently in the hypogean aquatic habitat of the Pivka Jama, eroded by the river Pivka, concerning an endemic crayfi sh population ( Astacus spec. ). Th ere is a dense population in the whole Pivka fl uviatile cave, down-stream gradually loosing the dark pigments, resulting in a red and blue colorization. Proteus has never been found here despite the fact that the main habitat of Proteus of this area is hydrologocally densely connected with the underground river Pivka (the so-called ecotope = habitat, defi ned as “Kluftsohlengewässer” by Briegleb, 1962 a, b). As a conclusion: negative photo taxis alone does not prevent from being eaten (see above). By contrast, in epigean water biotopes without predators and parasites, Proteus is dwelling defi nitely and successfully.

In this context, the idea could be raised that the originally neotenic Proteus , as well as Astacus , may have recently (during the last interglacial before “Würm” and see below) invaded available caves in the dinaric Karst. As possible prey available, Proteus found populations of more ancient taxa (e.g. crustaceans: Troglocaris , Niphargus, Assellus ) together with aquatic worms (e.g. Turbellaria). Proteus became able to settle down in these special underground ecological niches, which are most of the year separated from the underground rivers. Concerning its sensory apparatus, and in particular the eyes, Proteus secondarily adapted to these conditions and developed in the form of regressive evolution.

Negative photo taxis of Proteus may switch over into a positive one in the natural habitat: this means that the priority of a light avoidance reaction (light: Hawes, 1946 , cited by Briegleb, 1962a) changes into an escape reaction: light plus e.g. massive mechanical stimulation, dissimilar to normally running water, provoke a decision of the animal to switch from the light avoiding reaction to a topological orientated escape reaction by virtue of a perfectly memorized topography of the underwater riffl e concerned.


4.7. General behaviour of Proteus in the dark

After being released in an unknown habitat, Proteus , similar to other vertebrates, walks along the outer boarder line for about 20 min (thigmotaxic/topologic learning). It fol-lows a searching of the central parts until fi nding a hiding place (e.g. shelter between stones). After the searching walks, the shelter is then actively regained and not changed for several months or even years. A walk along the boarder line was then no longer observed. In a known habitat, Proteus frequently scans the substrate, apparently at random, searching for prey with the snout in the sediment detritus and clay. Prey cap-ture is then fi nally successful by combining mechano-, chemo-, and eventually electro-perception (Schlegel, 1997 ). Besides capturing the typical cave prey animals (see before), Proteus lurks for smaller fi sh, frog, and urodele larvae. Such prey has no chance to escape. Furthermore, Proteus performs „neutral search walks“ in its known territory, resulting in e.g. intra-specifi c, even hostile, contacts, and also being prepared to explore unknown areas as outlined before. Almost always, an inter-individual contact results in an escape of one or both of the participants. Th ere are courtship behaviour and fi ghts of rivals and even hierarchies among males (Briegleb, 1962 b). Adult Proteus in a rela-tively small aquarium (35x20x20 cm) put together may end up in damaging fi ghts with the death of one participant.

If we place a freshly caught Proteus onto wet clay on land the animal starts to walk with the body lifted. In the laboratory, a particular Proteus spent weeks on a fl at wet ramp of concrete. It looked as if the animal was hunting for Assellidae or had escaped from being dominated by other individuals. It can be concluded that Proteus occasion-ally changes diff erent locations of its primarily aquatic habitat even outside the water (own, Briegleb, and further observations by M. Aljančič in Planina cave, personal comm.).

Th e observation that Proteus are alerted and directed to dim light sources (positive photo taxis as shown e.g. in fi g. 2 and Schlegel, 2007, fi g. 3 ) may corroborate with reports that Proteus come occasionally out in numbers from fl ow off openings of certain caves/siphons in Slovenia. Moreover, they do this eventually under full moon conditions, as described and documented on video tapes by Bulog and Parzefall (1997 – video tapes documentation, personal comm.). Th ere might be then good chances for them to get more abundant food from outside the cave or at the entrance and also to meet conspecifi cs to mate with (a possible synchronization of behaviour, triggered by important external events).

4.8. Genetic characterization of cave and surface Calotriton asper-populations

It was established during the experiments on animals’ alignments to magnetic fi elds that only cave animals, i.e. Proteus , Desmognatus ochrophaeus , and those C. asper from a cave population and not specimens from a geographically separated surface popula-tion demonstrated their capability to align according to the Earth’s permanent- magnetic fi eld and its polarity (Schlegel, 2006 , 2007; Schlegel and Renner, 2006 ). Th is, at least qualitative, diff erence is corroborated by a quantitative one, concerning the sensitivity


for electrical stimuli, as the cave population was more homogenous and had up to 30 dB better thresholds than the surface population ( fi g. 15 ; compare also with electri-cal sensitivity in Proteus : Roth and Schlegel, 1988 ; Schlegel, 1997 ; Schlegel and Bulog, 1997 ). Th is characteristic seems not only to be plastic, concerning seasonal external physical conditions, but could also, to a certain degree, be genetically controlled. In order to fi nd out whether gene pools of surface and cavernous C. asper are already sepa-rated, we analyzed several individuals from both habitats for the neutrally evolving mitochondrial “Control Region” (D-loop) and for 22 nuclear coding protein loci (19 allozyme loci and 3 plasma proteins; for technical references, see Steinfartz et al., 2000 ; Steinfartz et al., 2002 ). Animals were sampled from one cave population (“Grotte de Siech” near Saurat) and two surface populations, namely the Aulus brook (ca. 20 km distant by air and topologically/hydrologically separated from the “Grotte de Siech”) and from the geographically more distant Ravin de Castelmouly near the Tourmalet-pass, about 150 km West of above locations. C. asper from this location had been for-merly described as C. a. castelmouliensis Wolterstorff ( 1925 ). On the basis of 597 base pairs of the mitochondrial D-loop, one diagnostic character can be assigned to the animals from the “Grotte de Siech” ( fi g. 16 ). All protein loci but the Albumin locus were identical for C. asper from “Grotte de Siech” and the Aulus brook, thus providing another diagnostic character of genetic diff erentiation of gene pools. Th e low but sig-nifi cant number of genetic diff erences both for the mitochondrial and nuclear genome suggests that the “Grotte de Siech” population must have underwent its on evolution with respect to the surface population (the Aulus brook population) probably staring

Figure 15. Sensitivity (tuning) of diff erent species and populations (as labelled on the right margin) to continuous sinusoidal electrical stimuli, delivered into the water, as a function of frequency (abscissa, Hz, log.-scale). Ordinate, threshold amplitudes for just noticeable behavioral reactions in rel. dB (attenuation; 0 dB att.= 1V/cm; -80 dB att.= 0.1 mV/cm pp and = μA eff . /cm 2 as indicated at the left and right margins).


36 000 years ago at the end of the Würm glaciations in the Pyrenees. In our view, the splitting and separation of gene pools between cave and surface populations might be the result of environmental specifi c adaptations on surface and cave conditions. Although no habitat-specifi c external morphological characters have been evolved so far, the ability to align to magnetic parameters is solely restricted to C. asper -individuals from two cave habitats studied. Th e second cave population (Cueva de los Moros, T.M. de Fanlo, Spain) was magnetically sensitive but was not yet studied genetically. Th at the adaptation to habitats can drive population separation and genetic diff erentia-tion within short time frames has been recently shown for another salamandrid species, the fi re salamander ( Salamandra salamandra ), in Germany (Weitere et al., 2004 ; Steinfartz et al., 2007b ).

5. Discussion, concluding remarks, outlook

Th e ability to align to the Earth’s magnetic fi eld can be seen as a key adaptation of Calotriton for cave life resulting in genetic diff erentiation of populations. However, the decaying tendency of thresholds in two separate sensory systems ( fi g. 15 , electric, Proteus and Calotriton and magnetic, diff erent Calotriton - populations and other species

Figure 16. a) Variable sites of Calotriton=Euproctus asper populations from one cave (“Grotte de Siech”) and two surface populations (“Aulus” and “Ravin de Castelmouly”) present in 597 bp of the mitochon-drial Control Region (D-loop) taking Triturus marmoratus for outgroup comparison. Th e only diagnostic site for C. asper from the “Grotte de Siech” is marked by an arrow. b) Bootstrap consensus tree based on 1000 bootstraps replicates for C. asper with Triturus marmoratus as an outgroup. Numbers above nodes indicate percentage support for this node and its corresponding terminal taxa.


stated in the text, abstract and below) of presumably the same phylogenetic origin (trigeminus nerve) may be pure analogy or coincidence, but could also demonstrate parallel losses in sensory systems when populations switch from a hypogean to an epigean habitat or vice versa . Th e ability to orient or not according to the Earth’s mag-netic fi eld seems even critical in Calotriton animals from cave populations when kept in a normal day/night cycle for longer periods of time. After about one year, when again tested for magnetic sensitivity, the animals weren’t able any longer to align mag-netically (fading out). On the other hand, C. asper , taken originally from a surface brook (Aulus) more than 15 years ago, then inbred in captivity in Moulis cave up to now, were no more able to align under magnetic conditions either, although the ani-mals had lived individually, for their whole life, in the permanent dark of the cave except when being fed once per week under illumination. Apparently the ability to align in a magnetic fi eld is missing in this inbred colony and seems not possible to be recruited or activated de novo under permanent dark conditions.

Similarly, we found that e.g. the closely related and often sympatric Mesotriton alpestris and Lissotriton vulgaris show a quite good ( alpestris ) or no ( vulgaris ) magnetic sensitivity (Schlegel and Renner, 2006 ). T. alpestris very likely uses this ability for seasonal or otherwise necessary migrations and demonstrated an inborn common magnetic preference, as never seen in hypogean species or populations studied here (Steinfartz et al., 2002 ).

On the other hand, a recent experience with adaptation of a migration behaviour of blackcaps birds ( Sylvia atricapilla ) showed a still more rapid genetic fi xation of a new migrating strategy when selecting and raising the animals (Berthold, 2001 ; Pulido and Berthold, 2001 ; Pulido et al., 2001 ).

All major or minor observations and arguments, stated before and made along with an extensive study on magnetic orientation in cave urodeles, have demonstrated that animals, deprived of vision due to their permanent subterranean habitat, use all their sensory systems, including the no more visual eye, pineal, and dermal light sensitivity. Among special sensitivities for magnetic fi elds or electric fi elds in water (Schlegel, 1997 ), they take profi t of these adaptations for orientation (spatial displacements, nav-igation, topologic knowledge) in their habitat. In this context and concerning the putative mechanisms of the magnetic sense, it should be pointed out and had been made most unlikely already that any sensitivity to the Earth’s magnetic fi eld could be perceived using the electro-sensory system (Schlegel, 1996 ). Two principles of sensing/measuring permanent magnetic fi eld strengths are claimed and discussed actually, i.e. a magnetically modulated visual receptor cell transduction cascade or magnetite con-taining mechanoreceptors belonging to the trigeminal nerve. Th is would be controlled by magnetic forces at magneto-mechanical transduction receptor cells or nerve endings (Kirschvink and Gould, 1981 ; Kirschvink et al., 1985 ; Kirschvink et al., 2001 ; Deutschlander et al., 1999 ; Diebel et al., 2000 ; Hanzlik et al., 2000 ; Winklhofer et al., 2001 , Fleissner et al., 2003 ; Fleissner and Stahl, 2005 ; Stahl et al. 2006a, b; Stahl and Fleissner, 2007 ).

We disregard here, although basic and certainly relevant, olfaction and taste as other orienting factors (e.g. Briegleb, 1962 b; Durand et al., 1981 , 1982 , 1983 ; Richard


et al., 1983 ; Guillaume, 1999 ; Papi and Ioalè, 1988 ; Parzefall et al., 2000 ) and also the conventional (touch, including kinesthetic/idiothetic abilities) mechanical senses widely as we do not make a point to these (refer for e.g. rheotaxis: Durand and Parzefall, 1987 ). Anyway, the abilities found, and presumably improved to a high degree during relatively recent evolution, allowed the candidates to successfully colonize and use the subterranean caverns as their new permanent habitat, which they are well, if not yet optimally, adapted to. Finally, orientation and navigation in the environment (compare especially e.g. Phillips et al., 1995 ; Fischer et al., 2001 ;) is, as mentioned, always and everywhere based on a neural integration of all available sensory information, includ-ing the excellent space memory within the ”own” habitat. All these sensorial fl uxes are used for adequate motor-controlled behaviours by an adaptive CNS as a necessary condition for best fi tness to survive even in such an extreme habitat as a cave.

Discussion is going on when the taxon Proteus has most likely colonized the hypo-gean habitat and has then subsequently lost vision and functional eyes. New arguments came up as a subspecies of Proteus ( P. anguinus parkelj , the eyed “black” Proteus ) was fi rst discovered in 1986 and was cited and described by Sket and Arntzen ( 1994 ). It was claimed that the origin of the taxon could have been as early as in the late (upper) Miocene/early (lower) Pliocene at least 1.1 million years or up to 9 million years ago, before recent karstifi cation, and that animals only invaded the Karst just before or dur-ing the Pleistocene (glaciations). Th e allozyme genetic diff erentiation tests among white and black Proteus populations (Sket and Arntzen, 1994 ) tend rather to show that loss of vision and separation of the populations could have been even more recent dur-ing the last glaciations (Würm) which terminated only 15,000 years ago in Slovenia. However, at least the defi nite degeneration of the individual eyes of each specimen within the “white” Proteus populations seems already fi xed genetically and rather speaks for a bit longer time since the loss of eye sight, maybe between or during the Mindel- and Riss- glaciations more than 300,000 years ago.

Th e variable eye structure, observed within a population of the blind cave salaman-der, is related to only weak selection pressure on the biologically functionless features in the caves. Th e variability of regressive traits is common in cave animals and was observed also in the eyes of a cavefi sh, Astyanax fasciatus (Wilkens, 1988 ). Th e remark-able structural and immunocytochemical diff erences between populations from the Planina cave and from the Otovec doline (both in Slovenia) must be genetically deter-mined. Th ese animals belong to two genetically distinct groups of populations, as shown by allozyme analysis (Sket and Arntzen, 1994 ).

Diff erent environmental conditions are not responsible for the diff erences between these two populations because, in animals from the Otovec doline, having occasional contacts with weak light, the photoreceptors are much more degenerated than in ani-mals from the Planina cave, never exposed to any light. In a cave animal living in per-manent darkness, one would expect a retina comparable to nocturnal animals or those that live underground or in murky conditions. Th eir retinas contain mostly rods, which operate in dim light. Th e frequency of cones in the retina and their sole occurrence in the pineal of the blind cave salamander Proteus anguinus anguinus is surprising. It appears that during the regressive evolution of Proteus , rods degenerated sooner than cones.


Th e study of alozyme variability in a few Slovenian populations (Sket and Arntzen, 1994 ), and a geographically broader analysis of mitochondrial control region (CR) DNA sequences (Gorički, 2004 ) unveiled that the currently recognized non-troglomorphic subspecies P. a. parkelj and the implicated P. a. anguinus are not sister clades. Th e species is split into at least six deeply separated clades of CR haplotypes, from (1) Th e Istra Peninsula, (2) Lika and Gorski Kotar, (3) Dalmacija and Hercegovina, (4) Bosanska Krajina, (5) SW Slovenia, and (6) SE Slovenia. Th e pigmented subspecies is nested within the SE Slovenian clade (Gorički 2006 ).

6. Acknowledgements

Th e stays of one author (P.S.) in Moulis were partly sponsored by the program PROCOPE (German federal government for foreign aff airs) and grants of the “Bayrisch-Französisches Hochschulzentrum”. He is very much indebted to the French counterpart of these exchange programs, the directors of the “Laboratoire Souterrain”, Drs Ch. Juberthie, J. Durand, A. Mangin, and J. Clobert (CNRS-Moulis) who invited him for these stays and helped essentially with their background knowledge and the facilities off ered of the laboratory and by its staff . For discussions and valuable sugges-tions, I thank Drs J. Durand, M. Beblo (who also provided two Helmholtz-coils for the laboratory cave and valuable earthmagnetic and geophysical knowledge), G. and G. Fleissner, R. and W. Wiltschko for valuable comments. Th e specimens of Desmognathus were obtained by courtesy from Prof. Dr. G. Roth, Dr. U. Dicke, and Dr. W. Grunwald, Univ. of Bremen. I fi nally thank M. Diesener, M. Dupuis, and K. Schmidt for kind and professional animal care taking and interesting hints concern-ing biology and ecology of some of the species dealt with in this study.

Functional-morphological, ethological and immunocytochemical studies of Proteus’ sensory organs performed on the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia in collaboration with Zoologisches Institut der L.- M. - Universität, D 80333 München, Germany and Department of Human Morphology and Developmental Biology, Medical Faculty, Semmelweis University, Budapest, Hungary were supported by Ministry of Higher education Science and Technology, Republic of Slovenia.

Th ese studies and all stays of Prof. P. Schlegel in Ljubljana, Slovenia were also sup-ported by VW foundation, Germany, and by some companies from Slovenia: Hyundai Avto Trade d.o.o., Krka, d.d., Eles, d.o.o., BTC, d.d., Debitel, d.d., Svema co d.o.o., Mladinska knjiga Založba d.d.

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Non-visual sensory physiology and magnetic orientation in the Blind Cave Salamander, Proteus...

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