Human Fish Research Articles

A molecular test for cryptic diversity in ground water: how large are the ranges of macro‐stygobionts?

Summary1. Various groundwater habitats have exceptionally high levels of endemism caused by strong hydrographical isolation and low dispersal abilities of their inhabitants. More than 10% of macro‐stygobiotic species nevertheless occupy relatively large ranges, measuring from some hundred to over 2000 km in length. These species represent a challenge because their distributions disregard hydrographical boundaries, and their means to disperse and maintain long‐term gene flow are unknown.2. Based on mitochondrial and nuclear gene sequences, we examined the phylogeographic structure of six formally recognised stygobiotic species (Niphargus virei,N. rhenorhodanensis,Troglocaris anophthalmus,T. hercegovinensis,Spelaeocaris pretneri,Proteus anguinus) and searched for cryptic lineage diversity in a genus‐wide phylogeny ofNiphargus. Using tree‐based criteria as well as comparative divergence measures, we identified cryptic lineages, which may tentatively be equated with cryptic species.3. Fourteen analysed nominal stygobiotic species with large ranges emerged as highly diversified, splitting into 51 tentative cryptic lineages. The degree of divergence was within the range or larger than the divergence of other related pairs of sister species. A substantial part (94%) of the cryptic lineages had ranges <200 km in length. One half of them were recorded at single sites only. The largest range recorded was that of a crypticN. vireilineage (700 km), while none of the very large traditional ranges (e.g.Niphargus aquilex– 2300 km,N. tauri– 1900 km) could be corroborated.4. These data suggest that small ranges of macro‐stygobionts are the rule, and ranges over 200 km are extremely rare.5. The implications of this result for groundwater biodiversity assessment and conservation include a considerable increase in overall diversity at the regional and continental scale and a strong decrease in faunal similarities among regions, coupled with greater endemism.

High anoxia tolerance in the subterranean salamander Proteus anguinus without oxidative stress nor activation of antioxidant defenses during reoxygenation

The present study describes a high anoxia tolerance in an amphibian at high temperature. Indeed, the subterranean salamander Proteus anguinus survived 12 h under anoxia at 12 degrees C. Surprisingly, such experimental conditions did not affect P. anguinus oxidative status while muscles and liver antioxidant enzymes activities decreased under 8 h anoxia and only return to basal level during reoxygenation. To test if such adaptation is common in Urodels, equivalent experimentations have been conducted on another newt: the stream-dwelling Calotriton asper. This latter species exhibited only 1.5 h survival under anoxia in spite of higher antioxidant enzymes activities than P. anguinus. Furthermore, aerobic recovery after 1 h anoxia induced a 30% increase of oxidative damage partly explained by SOD and CAT activities that did not return to control values during reoxygenation, demonstrating a lower capacity to counteract ROS overproduction than P. anguinus. In addition, uncoupling protein (UCP) transcript was for the first time detected, partly sequenced and quantified in amphibian muscles and liver. UCP may be considered as a ROS production attenuator by mediating a discharge of the proton gradient generated by the respiratory chain. The putative role of UCP in post-anoxic oxidative status of both species is discussed.

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)

Abstract A review is given on several sensory systems that enable troglophile and troglobian urodele species to orient non-visually in their extreme hypogean habitat. A new sense was discovered allowing the animals to orient according to the Earth's magnetic field, which could serve as a basic and always available reference for general spatial orientation. Moreover, working with permanent magnetic field stimuli offers 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. The 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 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 finally 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 orientation 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.

Mechanisms of pigmentation loss in subterranean fishes

Troglobitic (exclusively subterranean) organisms usually present, among their apomorphies related to the subterranean life (troglomorphisms), the regression of eyes and melanic pigmentation. The degree of regression varies among species, from a slight reduction to the complete loss of eyes and dark pigmentation, without a taxonomic correlation. While mechanisms of eye reduction have been intensively investigated in some troglobites such as the Mexican blind tetra characins, genus Astyanax, and the European salamander, Proteus anguinus, few studies have focused on pigmentation. The Brazilian subterranean ichthyofauna distinguishes not only by the species richness (23 troglobitic fishes so far known) but also by the variation in the degree of reduction of eyes and pigmentation. This study focused on Brazilian fishes completely devoid of melanic pigmentation: the characiform Stygichthys typhlops (Characidae) and the siluriforms Ancistrus formoso (Loricariidae), Rhamdiopsis sp.1 (Heptapteridae; from caves in the Chapada Diamantina, Bahia) and Rhamdiopsis sp. 2 (cave in Campo Formoso, Bahia). In order to investigate if such depigmentation is the result of blockage in some step in the melanogenesis, in vitro tests of administration of L-DOPA were done, using caudal-fin fragments extracted from living fish. Except for Rhamdiopsis sp. 2, all the studied species were DOPA(+), i.e., melanin was synthesized after L-DOPA administration. This indicates these fish do have melanophores but they are unable to convert L-tyrosine to L-DOPA. On the other hand, Rhamdiopsis sp. 2, like the albino specimens of Trichomycterus itacarambiensis previously studied (which correspond to one third of the population), are DOPA(-), either because the block of melanin synthesis occurs downstream in melanogenesis, which is probably the case with T. itacarambiensis (monogenic system in view of the phenotypic discontinuity), or because the so-called albinos do no possess melanophores. The physiological loss in the ability to synthesize melanin, apparently caused by different genetic processes in DOPA(+) and in DOPA(-) fishes, may co-exist in subterranean populations with a decrease in the density of melanophores, as observed in the pigmented two thirds of T. itacarambiensis population, a morphological reduction apparently controlled by polygenic systems producing a continuous phenotypic variation.

Hepatic pigment cells of Proteidae (Amphibia, Urodela): A comparative histochemical and ultrastructural study

AbstractAmphibian liver pigment cells, also known as Kupffer cells, contain various amounts of melanin, haemosiderin and lipofuscin. We used different histochemical and ultrastructural methods to analyse and compare the level of hepatic pigmentation and the structure of hepatic pigment cells in the livers of three representatives of the family Proteidae; two subspecies of the hypogean Proteus anguinus (depigmented Proteus a. anguinus and pigmented Proteus a. parkelj) and the epygean Necturus maculosus. Our analysis revealed differences at histological and ultrastructural level. While the percentage of the pigmented area and ultrastructural characteristics are similar in both subspecies of P. anguinus, great differences occur in the amount and structure of the pigment cells between P. anguinus and N. maculosus. Pigment cells are more numerous and compose larger pigmented clusters in P. anguinus. They are structurally more heterogeneous and contain a larger amount of haemosiderin when compared to N. maculosus. Our results confirm a high degree of variation in hepatic pigmentation among different amphibian species. Because many factors influence the level of hepatic pigmentation in poikilotherms, differences among species from different habitats and also among individuals of the same species are expected but are not easily explained. We propose two possible explanations for the large amount of iron present in Proteus anguinus: (1) accumulation of pigments due to the very low metabolic rate and extended lifetime; (2) large iron storage capacity as an adaptation to a low and discontinuous food supply in caves.

Taxonomy and biogeography of Niphargus steueri (Crustacea: Amphipoda)

Niphargus steueri (Niphargidae) comprises a complex of four subspecies (N. s. steueri, N. s. kolombatovici, N. s. subtypicus, and N. s. liburnicus), the morphology and distribution of which have been poorly known until now. New diagnostic characters of the species and its four subspecies are presented and illustrated. The species is distributed along the major part of the Dinaric Karst, between Slovenia in the northwest and Bosnia and Herzegovina in the southeast. The distribution of the four subspecies approximately resembles the distribution of the evolutionary lineages of the subterranean amphibian Proteus anguinus and the Dinaric lineage of the cave shrimp Troglocaris agg. anophthalmus. Niphargus s. steueri is restricted to the Istran Peninsula; N. s. subtypicus is distributed in southeast Slovenia and northwest Croatia; N. s. liburnicus is known from two disjunctive localities, one on the island of Krk (Croatia) and the other in Gorizia (Italy); and N. s. kolombatovici is restricted to Dalmacija and Herzegovina. The somewhat variable putative synapomorphies of N. steueri probably suggest that the group is old and that the present distribution pattern is a result of historical events, possibly events in the Miocene Dinaride Lake system. Two populations of N. s. kolombatovici and one population of N. s. subtypicus deviate from the general distributional pattern and may belong to cryptic taxa that cannot be distinguished on the basis of morphology. Both hypotheses corroborate with the estimated times of divergence and with the number of independent lineages in the similarly distributed but unrelated stygobionts Proteus and Troglocaris.

Structure and evolution of the mitochondrial control region and flanking sequences in the European cave salamander Proteus anguinus

The European cave salamander Proteus anguinus Laurenti 1768 is one of the best-known subterranean animals, yet its evolutionary history and systematic relationships remain enigmatic. This is the first comprehensive study on molecular evolution within the taxon, using an mtDNA segment containing the control region (CR) and adjacent sequences. Two to seven tandem repeats of 24–32 bp were found in the intergenic spacer region (VNTR1), and three, four or six repeats, 59–77 bp each, in the 3′ end of the CR (VNTR2). Different molecular mechanisms account for VNTR2 formation in different lineages of Proteus. The overall CR variation was lower than that of the spacer region, the 3′ end of the cytb gene, or the tRNA genes. Individual genes and the concatenated non-repetitive sequences produced similar, well resolved maximum likelihood, Bayesian inference and parsimony trees. The numbers of repeat elements as well as the genealogy of the VNTR2 repeat units were mostly inconsistent with the groupings of the non-repetitive sequences. Different degrees of repeat array homogenization were detected in all major groups. Orthology was established for the first and the second VNTR2 elements of some populations. These two copies may therefore be used for analyses at the population level. The pattern of CR sequence variation points to strong genetic isolation of hydrographically separated populations. Genetic separation of the major groups of populations is incongruent with the current division into subspecies.

Underground and unknown: updated distribution, ecological notes and conservation guidelines on the Olm Proteus anguinus anguinus in Italy (Amphibia, Proteidae)

Proteus anguinus reaches the extreme western border of its distribution in Venezia Giulia, a narrow area of Italian territory in the provinces of Trieste and Gorizia. An alien population of P. anguinus is nowadays well acclimatised near Oliero Valstagna (Vicenza). The ancestors of diis population were introduced from Postojna (Slovenia) and could therefore be genetically well distinct from the Italian ones in Venezia Giulia. Proteus anguinus is autochthonous in 26 different localities from the Carso Triestino plateau, were it inhabits hypogeal aquifers situated around sea level, but sometimes more than 300 m under the ground level of the karstic plateau. Specimens that occur in springs, pools or wells in big caves near the surface, seem to do so only to feed. Eggs, larvae, breeding and territorial behaviour have never been observed in nature. Further studies need to be addressed to better understand ecology and genetic characterisation of P. anguinus, focusing mainly on the naturalised population of Valstagna and on the olms naturally occurring under the village of Gradisca. Conservation efforts need to be made to maintain P. anguinus habitats, stopping aquifer pollution and regulating caves and karst exploitation.

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

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

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

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

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

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12 000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

TRACE ELEMENT CONCENTRATIONS IN THE TISSUES OF PROTEUS ANGUINUS (AMPHIBIA, CAUDATA) AND THE SURROUNDING ENVIRONMENT

The concentrations of some essential (Cu, Zn, Se) and some toxicelements (Hg, As) were determined in tissues (liver, kidneys, integument, and muscle) of the endemic cave salamander, Proteus anguinus Laurenti 1768 and Proteus anguinus parkelj ssp.n. captured in the Planina Cave, Kompolje Cave, and Jelsevnik, all situated within the Dinaric Karst in Slovenia. The highest amounts of selenium and mercury were found in the liver of Proteus anguinus specimens from all three localities. Some of the animals from the Planina Cave showed higher concentrations of copper in the liver and muscle. Pigmented subspecies of Proteus from Jelsevnik showedincreased values of arsenic in the integument and of zinc in theliver and integument. The liver of the animals contained the highest amounts of the metals analysed and therefore may be considered as a target organ. Metal levels were also measured inthe cave water and sediments. The waters were not polluted withmetals. However, the metals were relatively high in the sediments, but not to levels considered contaminated. In additionto our study of metal concentrations in Proteus tissues andhabitats, several water quality parameters were measured in the water of each locality. The main differences between the threelocalities concerned dissolved oxygen concentration and saturation, while all other physical and chemical parameterswere characteristic of the aquatic environment in the calcareousKarst region.

The ventral skin glands, new additional cloacal glands in Proteus anguinus (Caudata, Proteidae). II. Male

Guillaume, O. 2000. The ventral skin glands, new additional cloacal glands in Proteus anguinus (Caudata, Proteidae). II. Male. —Acta Zoologica (Stockholm) 81: 223–234 Cloacae from male Proteus anguinus were examined for the first time by light and electron microscopy. Male P. anguinus possess anterior and posterior ventral glands, dorsal and lateral pelvic glands, vent glands and Kingsbury’s glands as do Necturus males. However, male P. anguinus also possess the new additional cloacal glands found in females and which the author has called the ventral skin glands. Furthermore, the number, the length and the diameter of these tubular glands are higher in males. Therefore the inference is that these glands are sexually dimorphic glands.

The ventral skin glands, new additional cloacal glands in Proteus anguinus (Caudata, Proteidae). I. Female

Guillaume, O. 2000. The ventral skin glands, new additional cloacal glands in Proteus anguinus (Caudata, Proteidae). I. Female. —Acta Zoologica (Stockholm) 81: 213–221 Cloacae of female Proteus anguinus were examined by light and electron microscopy. Females possess spermathecae and anterior ventral glands such as Necturus females. However, female P. anguinus possess new additional cloacal glands of unknown homology. These tubular glands secrete on the ventral epidermis of the cloacal area. Considering these characteristics, it is proposed that they will be called the ventral skin glands.

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

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

Metabolism and circadian rhythms of the European blind cave salamander Proteus anguinus and a facultative cave dweller, the Pyrenean newt (Euproctus asper)

Comparisons of circadian rhythmicity, behavior, and metabolism between surface- and cave-dwelling salamanders allow evolutionary trends in these processes to be elucidated. The proteid Proteus anguinus, an obligate cave-dweller, showed no apparent daily rhythm of activity or resting metabolic rate. In contrast, the salamandrid Euproctus asper, a surface-dweller/facultative cave-dweller, had a circadian resting metabolic rate and activity cycle. These circadian rhythms had an endogenous component. The lives of both studied salamanders were characterized by long periods of inactivity punctuated by bouts of foraging or exploratory/predatory behavior. Proteus anguinus had reduced resting metabolic and spontaneous activity rates (considerably lower than those of most surface-dwelling amphibians), and therefore appears to be a good example of a vertebrate as a low-energy system. The low metabolic and activity rates of P. anguinus are interpreted as adaptations to a subterranean environment, where a poor and discontinuous food supply and (or) intermittent hypoxia may be present for long period

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

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

Role of chemical communication and behavioural interactions among conspecifics in the choice of shelters by the cave-dwelling salamander Proteus anguinus (Caudata, Proteidae)

Non-sexually active males and females of the cave-dwelling salamander Proteus anguinus always aggregate in the same shelters, either under stones or in fissures. However, it was not known if this behaviour results from chemical communication and (or) behavioural interactions among conspecifics. I tested the hypothesis that this is the case by using two-choice tests. The results showed that salamanders re-located their own shelter even when it was displaced. However, they preferred a shelter that belonged to a conspecific or contained faecal pellets of a conspecific to their own or a control "blank" shelter. When two salamanders were simultaneously given the choice between two blank shelters, they cohabited. When they were simultaneously given the choice between their own shelter and each other's, they used them indiscriminately and frequently cohabited. These data provide evidence that P. anguinus use chemical signals as directional cues for homing and also for social behaviour. Chemical signals may attract conspecifics, which subsequently may exhibit gregarious behaviour by occupying common shelters.

Role of chemical communication and behavioural interactions among conspecifics in the choice of shelters by the cave-dwelling salamander <i>Proteus anguinus</i> (Caudata, Proteidae)

Role of chemical communication and behavioural interactions among conspecifics in the choice of shelters by the cave-dwelling salamander <i>Proteus anguinus</i> (Caudata, Proteidae)

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

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