Human Fish Research Articles

Morphology and differential counts of blood cells as important health indicators in the olm, Proteus anguinus

Abstract Haematological investigation is an important component of stress and immune response assessment and holds great potential for evaluating amphibian welfare. The olm (Proteus anguinus) is a neotenic, cave-dwelling salamander endemic to the Dinaric Karst and classified as a vulnerable species by the IUCN. The aim of the present study was to gain basic knowledge about the haematology of this endangered species and to establish reference values of leucocyte differential counts. Blood smears were obtained from recently captured animals and compared to smears from the same animals in long-term captivity (up to five years). The elongated erythrocytes are nucleated and are among the largest in vertebrates, which is also reflected in their lower concentration per millilitre of blood. The circulating blood can contain mitotically active, immature erythrocytes (erythroblasts), indicating that erythrocytes of the olm can undergo proliferation and maturation in circulating blood. Olms show a typical urodele leucocyte profile, with lymphocytes predominating, followed by neutrophils, although the proportion of lymphocytes is higher than in other urodeles. Eosinophil and monocyte counts were relatively low, while basophils were nearly absent. We found no statistically significant differences in leucocyte counts or in neutrophil to lymphocyte ratio neither between recently captured olms and same animals during captivity nor between the sexes, except for lower neutrophil count in captive females. Thus, no differences in stress levels between recently captured olms and captive olms were detected via haematological investigations. The significance of these results is discussed in the context of detecting stress and immune responses in olms.

Generation of genome-wide SNP markers from minimally invasive sampling in endangered animals and applications in species ecology and conservation.

High-density genotyping methods have revolutionized the field of population and conservation genetics in the past decade. To exploit the technological and analytical advances in the field, access to high-quality genetic material is a key component. However, access to such samples in endangered and rare animals is often challenging or even impossible. Here, we used a minimally invasive sampling method (MIS) in the endangered cave salamander Proteus anguinus, the olm, to generate thousands of genetic markers using ddRADseq for population and conservation genomic analyses. Using tail clips and MIS skin swabs taken from the same individual, we investigated genotyping data properties of the two different sampling types. We found that sufficient DNA can be extracted from swab samples to generate up to 200,000 polymorphic SNPs in divergent Proteus lineages. Swab and tissue samples were highly reproducible exhibiting low SNP genotyping error rates. We found that SNPs were most frequently (~50%) located within genic regions, while the rest mapped to mostly flanking regions of repetitive DNA. The vast majority of DNA recovered from swabbing was host DNA. However, a fraction of DNA recovered from swabs contained additional ecological information on the species, including eDNA from the surrounding environment and bacterial skin fauna. Most exogenous DNA recovered from swabs were bacteria (~80%), followed by vertebrates (~20%). Our results demonstrate that MIS can be used to (i) generate tens of thousands of ddRADseq markers for conservation and population genomic analyses and (ii) inform on the species health status and ecology from exogenous DNA.

Microplastic pollution calls for urgent investigations in stygobiont habitats: A case study from Classical karst

Microplastic pollution in karst systems is still poorly studied, despite the presence of protected species and habitats, and important water reserves. Vulnerable key species hosted in these habitats could consume or assimilate microplastics, which can irreversibly damage management efforts, and thus ecosystems functionality. This can be particularly true for subterranean water habitats where microplastic pollution effects on wildlife management programs are not considered. The aim of this study is to provide a case study from the Classical Karst Region, which hosts peculiar habitats and key species protected at European level, such as the olm Proteus anguinus. As this area has been deeply exploited and modified over time, and is adjacent to highways, roads and railways, which could contribute to pollution within the karst system, threatening the ecosystems, it provides a perfect model system.In this study we collected and investigated water and sediment samples from aquatic environments of surface and subterranean habitats hosting several subterranean environment-adapted organisms. Examined particles were counted and characterized by size, color and shape via visual identification under a microscope, with and without UV light. Furthermore, spectroscopic analyses were carried out in order to identify microplastics typology. Microplastics were found in all examined habitats. In water, microplastics concentration ranged from 37 to 86 items/L, in sediments from 776 to 2064 items/kg. Fibre-shape was the main present, followed by fragments and beads, suggesting multiple sources of pollution, especially textile products. Most of the particles were fluorescent under UV light and were mainly transparent, while not-fluorescent ones were especially black, blue or brown. Samples contained especially polyesters and copolymers. These results highlight intense MP pollution in karst areas, with significant impacts on water quality, and potential effects on subterranean environment-dwelling species. We stress the importance of monitoring pollution in these critical environments for biodiversity and habitat conservation: monitoring in karst areas must become a priority for habitat and species protection, and water resources management, improving analyses on a larger number of aquatic surface and subterranean habitats.

New locality of Proteus anguinus in the area of the Tabor Ridge near Grosuplje, Central Slovenia

New locality of Proteus anguinus in the area of the Tabor Ridge near Grosuplje, Central Slovenia

Attempted predation of a cave olm Proteus anguinus by a dice water snake Natrix tessellata, in Bosnia and Herzegovina

Attempted predation of a cave olm Proteus anguinus by a dice water snake Natrix tessellata, in Bosnia and Herzegovina

Hydrogeological characterization of karst springs of the white (Proteus anguinus anguinus) and black olm (Proteus anguinus parkelj) habitat in Bela krajina (SE Slovenia)

The springs west of Črnomelj, in SE Slovenia, are the habitat of the black (Proteus anguinus parkelj) and the white olm (Proteus anguinus anguinus). Some of these springs are also the only known habitat in the world of endemic species of black olm. A steady decline in olm populations has been observed in this area over the past decades. Owing to the rapid runoff and groundwater flow high-resolution monitoring is essential in providing better insight into the hydrogeological characterization of the catchment area of springs. Specific factors and critical parameters of water behind said olm degradation have not yet been defined. Because the olm’s environment is largely aquatic, one potential critical parameter could be the higher water temperatures (>12 °C) or higher nitrate concentration (>9.2 mg/l). The six-month observation of the springs (July – December 2021) point to water temperature as a potential critical parameter since the water temperature of the springs exceeded 12 °C in months July and August. Nitrate concentrations could also be a second critical parameter in the degradation of the olm’s habitat. Maximum nitrate concentrations above 9.2 mg/l throughout much of the observation period (except for Dobličica spring). Due to less agricultural activity in December in the spring catchment area and a higher dilution rate due to reduced evapotranspiration and increased effective precipitation during this time of the year, the nitrate concentrations are decreased. The results of the measured parameters of groundwater could show the hydrogeological connection between the Otovski and Pački breg springs and between Šotor, Jamnice and Dobličica. The Obršec spring has an independent catchment area. A detailed estimation of the springs catchment area is possible due to a detailed geologic map. It is necessary to determine the origin of the nitrate (nitrate isotope analysis), to quantify the threshold values of the critical parameters, to define precisely all the causes of the olm deterioration, and to make proposals for appropriate measures to limit or even stop the decline of the olm population.

Temperature-independent telomere lengthening with age in the long-lived human fish (Proteus anguinus).

Despite a number of studies showing a negative relationship between age and telomere length, the universality of this pattern has been recently challenged, mainly in ectothermic animals exhibiting diverse effects of age on telomere shortening. However, data on ectotherms may be strongly affected by the thermal history of the individuals. We thus investigated the age-related changes in relative telomere length in the skin of a small but long-lived amphibian living naturally in a stable thermal environment over its entire life, allowing comparison with other homeothermic animals like birds and mammals. The present data showed a positive relation between telomere length and individual age, independent of sex and body size. A segmented analysis highlighted a breakpoint in the telomere length-age relationship, suggesting that telomere length reached a plateau at the age of 25 years. Further studies focusing on the biology of animals that live much longer than expected based on body mass will contribute to our better understanding of how ageing processes evolved and may also bring innovation for extending human health span.

Multiple transitions between realms shape relict lineages of Proteus cave salamanders.

In comparison to biodiversity on Earth's surface, subterranean biodiversity has largely remained concealed. The olm (Proteus anguinus) is one of the most enigmatic extant cave inhabitants, and until now little was known regarding its genetic structure and evolutionary history. Olms inhabit subterranean waters throughout the Dinaric Karst of the western Balkans, with a seemingly uniform phenotypic appearance of cave-specialized traits: an elongate body, snout and limbs, degenerated eyes and loss of pigmentation ("white olm"). Only a single small region in southeastern Slovenia harbours olms with a phenotype typical of surface animals: pigmented skin, eyes, a blunt snout and short limbs ("black olm"). We used a combination of mitochondrial DNA and genome-wide single nucleotide polymorphism data to investigate the molecular diversity, evolutionary history and biogeography of olms along the Dinaric Karst. We found nine deeply divergent species-level lineages that separated between 17 and 4 million years ago, while molecular diversity within lineages was low. We detected no signal of recent admixture between lineages and only limited historical gene flow. Biogeographically, the contemporaneous distribution of lineages mostly mirrors hydrologically separated subterranean environments, while the historical separation of olm lineages follows microtectonic and climatic changes in the area. The reconstructed phylogeny suggests at least four independent transitions to the cave phenotype. Two of the species-level lineages have miniscule ranges and may represent Europe's rarest amphibians. Their rarity and the decline in other lineages call for protection of their subterranean habitats.

Cultivable microbiota of Proteus anguinus from underground habitats and animals accidently washed to the surface in Croatia

Proteus anguinus (olm) is an amphibian endemic to underground cave systems of the Dinaric karst of Central Europe, adapted to a life in complete darkness. Recent studies have demonstrated the global decline of amphibians due to poorly understood microbiological diseases, emphasizing just how little is known about the natural microflora of amphibians. Not much is known about the olm’s microbiota in their cave habitats, and nothing is known about the microbiology of animals washed to the surface. This observational study describes the microbiological analysis of the skin, cloaca and oral cavity of six olms collected from their cave habitats, and 16 collected after being washed out by flows from two springs, to learn more about the olm’s normal microbiota and possible changes after contact with the different environmental conditions on the surface. Standard microbiological procedures, MALDI-TOF and Real-Time PCR were used for microbiological species identification. All animals tested negative for Ranavirus, Batrachochytrium dendrobatidis, Batrachochytrium salamandrivorans and Chlamydia spp. The most abundant fungi isolated were Penicillium spp. and Cladosporium spp. The washed out animals showed a higher diversity of bacterial flora than those from cave habitats, with Acinetobacter johnsonii, Aeromonas hydrophila, Bacillus spp. and Janthinobacterium lividum as the most frequently identified isolates.

First sighting of 'white' and 'black' olms (Proteus anguinus) in the same spring

First sighting of 'white' and 'black' olms (Proteus anguinus) in the same spring

Preliminary study on differences characterizes the populations of olm (Proteus anguinus) in cave and those found in epigean environment

The olm (Proteus anguinus) is considered the most representative example of all the stygobiont fauna. Nevertheless, it is also reported in some spring habitats of Friuli Venezia Giulia (North-eastern Italy). Springs are intriguing habitats that border two strongly distinct environments: surface and underground. They may provide distinct environmental pressures to stygobiont animals promoting new adaptations. The aim of this study is to verify if there are differences in the structural morphology of the head and in the presence of melanin in the skin between the olms that are found in caves compared to the individuals found in the external environment. During 2021, 28 olms, 18 from underground habitats and 10 from external sites, were photographed in standard conditions with a metric reference. For each individual, only the cephalic region has been considered. The pictures were processed using TPSdig software. For each picture we placed 2 landmark homologues, matching the emergence of the gills branches, and 48 semi-landmark to define the contour of the head. With regard to melanin 2 mm of skin were removed from the tail of the olm and analyzed through a spectrophotometer. The results indicate a significant effect of the environment on the shape of the olm’s head, in fact the shape of the head appears more elongated in the front and narrower in the parietal region in the olms coming from the hypogean environment. In addition, the olms that use the epigean environment show higher melanin levels in their skin. These findings provide a first evidence of differences occurring between olm populations/sub-populations living in caves and those exploiting surface habitats.

The cave salamander Proteus anguinus in captivity in Moulis, France for the scientific research.

Proteus anguinus fascinated the most famous savants like Lamarck, Cuvier and Darwin. However, the knowledge about this species was improved little because very few specimens were available, even less alive. To face this situation, The French National Center for Scientific Research built in 1948 the cave-laboratory at Moulis and started to breed in P. anguinus. The first hatchings date from 1959 (Vandel and Bouillon 1959, Juberthie et al. 1991). Since then, more than 160 egg-layings producing more than 4,600 eggs has occurred. The availability of a certain number of specimens and the continuity of this breeding for more than 60 years have made possible to better understand the biology of this species, for instance, the development (sexual maturity reached at 15 years old, lifespan estimated from 68 to 100 years old, see Voituron et al. 2010). The aim of this presentation is to make a review of the latest and prospective research conducted with partners on captive P. anguinus in Moulis.

Cultivable Skin Mycobiota of Healthy and Diseased Blind Cave Salamander (Proteus anguinus).

Proteus anguinus is a neotenic cave salamander, endemic to the Dinaric Karst and a symbol of world natural heritage. It is classified as “vulnerable” by the International Union for Conservation of Nature (IUCN) and is one of the EU priority species in need of strict protection. Due to inaccessibility of their natural underground habitat, scientific studies of the olm have been conducted mainly in captivity, where the amphibians are particularly susceptible to opportunistic microbial infections. In this report, we focused on the diversity of cultivable commensal fungi isolated from the skin of asymptomatic and symptomatic animals obtained from nature (20 specimens) and captivity (22 specimens), as well as from underground water of two karstic caves by direct water filtration and by exposure of keratin-based microbial baits and subsequent isolation from them. In total 244 fungal isolates were recovered from the animals and additional 153 isolates were obtained from water samples. Together, these isolates represented 87 genera and 166 species. Symptomatic animals were colonized by a variety of fungal species, most of them represented by a single isolate, including genera known for their involvement in chromomycosis, phaeohyphomycosis and zygomycosis in amphibians: Acremonium, Aspergillus, Cladosporium, Exophiala, Fusarium, Mucor, Ochroconis, Phialophora and Penicillium. One symptomatic specimen sampled from nature was infected by the oomycete Saprolegnia parasitica, the known causative agent of saprolegniosis. This is the first comprehensive report on cultivable skin mycobiome of this unique amphibian in nature and in captivity, with an emphasis on potentially pathogenic fungi and oomycetes.

First sighting of ‘white’ and ‘black’ olms (Proteus anguinus) in the same spring

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Oviparity, viviparity or plasticity in reproductive mode of the olm Proteus anguinus: an epic misunderstanding caused by prey regurgitation?

Abstract Cave animals are biological models of fast evolutionary change induced by transition to extreme subterranean environments. But their concealed lifestyle makes it inherently difficult to study life-history changes. Therefore, currently very little is known on the reproduction of cave species, and even less is known on general patterns and potentially shared reproductive strategies. Theory predicts that the cave environment favours the production of a few well-developed offspring and live birth. For one of the most enigmatic cave animals, the olm (Proteus anguinus), it has been debated fiercely whether they reproduce by live birth (viviparity), egg-laying (oviparity) or facultatively. While successes in captive breeding after the 1950s report oviparity as the single parity mode, some historically older observations claimed viviparity. The controversial neo-Lamarckist Paul Kammerer even claimed to have induced changes in parity mode by altering environmental conditions. Here, we report on the feeding and regurgitation of fire salamander (Salamandra salamandra) larvae by olms. The salamander larvae showed clear teeth marks and other injuries on the head caused by the olm, yet one larva was still alive after regurgitation. We suggest that historical reports of olm viviparity could have been misled by regurgitated salamander larvae. Our data bring additional indications that at least some of Kammerer’s experiments were fraudulent.

Oviparity, viviparity or plasticity in reproductive mode of the olm Proteus anguinus: an epic misunderstanding caused by prey regurgitation?

Oviparity, viviparity or plasticity in reproductive mode of the olm Proteus anguinus: an epic misunderstanding caused by prey regurgitation?

Living in darkness: Exploring adaptation of Proteus anguinus in 3 dimensions by X-ray imaging.

BackgroundLightless caves can harbour a wide range of living organisms. Cave animals have evolved a set of morphological, physiological, and behavioural adaptations known as troglomorphisms, enabling their survival in the perpetual darkness, narrow temperature and humidity ranges, and nutrient scarcity of the subterranean environment. In this study, we focused on adaptations of skull shape and sensory systems in the blind cave salamander, Proteus anguinus, also known as olm or simply proteus—the largest cave tetrapod and the only European amphibian living exclusively in subterranean environments. This extraordinary amphibian compensates for the loss of sight by enhanced non-visual sensory systems including mechanoreceptors, electroreceptors, and chemoreceptors. We compared developmental stages of P. anguinus with Ambystoma mexicanum, also known as axolotl, to make an exemplary comparison between cave- and surface-dwelling paedomorphic salamanders.FindingsWe used contrast-enhanced X-ray computed microtomography for the 3D segmentation of the soft tissues in the head of P. anguinus and A. mexicanum. Sensory organs were visualized to elucidate how the animal is adapted to living in complete darkness. X-ray microCT datasets were provided along with 3D models for larval, juvenile, and adult specimens, showing the cartilage of the chondrocranium and the position, shape, and size of the brain, eyes, and olfactory epithelium.Conclusions P. anguinus still keeps some of its secrets. Our high-resolution X-ray microCT scans together with 3D models of the anatomical structures in the head may help to elucidate the nature and origin of the mechanisms behind its adaptations to the subterranean environment, which led to a series of troglomorphisms.

Inferring predator\u2013prey interaction in the subterranean environment: a case study from Dinaric caves

Predator–prey interactions are among the most important biotic interactions shaping ecological communities and driving the evolution of defensive traits. These interactions and their effects on species received little attention in extreme and remote environments, where possibilities for direct observations and experimental manipulation of the animals are limited. In this paper, we study such type of environment, namely caves of the Dinarides (Europe), combining spatial and phylogenetic methods. We focused on several species of Niphargus amphipods living in phreatic lakes, as some of them use the dorsal spines as putative morphological defensive traits. We predicted that these spines represent a defense strategy against the olm (Proteus anguinus), a top predator species in the subterranean waters. We tested for spatial overlap of the olm and Niphargus species and showed that spined species live in closer proximity to and co-occur more frequently with the olm than non-spined species. Modeling of the evolution of the spines onto Niphargus phylogeny implies coevolution of this trait in the presence of olm. We conclude that these spines likely evolved as defensive traits in a predator–prey arms race. Combining multiple analyses, we provide an example for a methodological framework to assess predator–prey interactions when in-situ or laboratory observations are not possible.

Toward the massive genome of Proteus anguinus-illuminating longevity, regeneration, convergent evolution, and metabolic disorders.

Deciphering the genetic code of organisms with unusual phenotypes can help answer fundamental biological questions and provide insight into mechanisms relevant to human biomedical research. The cave salamander Proteus anguinus (Urodela: Proteidae), also known as the olm, is an example of a species with unique morphological and physiological adaptations to its subterranean environment, including regenerative abilities, resistance to prolonged starvation, and a life span of more than 100 years. However, the structure and sequence of the olm genome is still largely unknown owing to its enormous size, estimated at nearly 50 gigabases. An international Proteus Genome Research Consortium has been formed to decipher the olm genome. This perspective provides the scientific and biomedical rationale for exploring the olm genome and outlines potential outcomes, challenges, and methodological approaches required to analyze and annotate the genome of this unique amphibian.

Toward the massive genome of Proteus anguinus — illuminating longevity, regeneration, convergent evolution, and metabolic disorders

Toward the massive genome of Proteus anguinus — illuminating longevity, regeneration, convergent evolution, and metabolic disorders