Pigmentary system of the adult alpine salamander Salamandra atra atra (Laur., 1768).

The pigmentary system of the skin from adult specimens of the black alpine salamander Salamandra atra atra was investigated by light microscope, electron microscope, and biochemical studies. Results were compared with those obtained in previous study of the subspecies Salamandra atra aurorae. Unlike Salamandra atra aurorae, which presents epidermal xanthophores and iridophores, Salamandra atra atra is completely melanized, presenting only epidermal and dermal melanophores. The melanosomes in both the epidermis and the dermis appear to derive from a multivesicular premelanosome similar to that in the goldfish, and the epidermal melanosomes are smaller than those in the dermis. Premelanosomes with an internal lamellar matrix were not observed. The biochemical results have shown that in the ethanol extracts obtained from the skin in toto and from the melanosomes, pteridines and flavins are always present and are the same as those extracted from the black skin areas of Salamandra atra aurorae.     

Pigmentary system of the adult alpine salamander Salamandra atra aurorae (Trevisan, 1982)

The pigmentary system of skin from adult specimens of the amphibian urodele Salamandra atra aurorae was investigated by light microscope, electron microscope, and biochemical studies. Yellow (dorsum and head) and black (flank and belly) skin was tested. Three chromatophore types are present in yellow skin: xanthophores, iridophores, and melanophores. Xanthophores are located in the epidermis whereas iridophores and melanophores are found in the dermis. Xanthophores contain types I, II, and III pterinosomes. Some pterinosomes are very electron-dense. Black skin has a single type of chromatophore: the melanophores. Some melanophores are located in the epidermis. In contrast to the dermal melanophores, these present, in addition to typical melanosomes, organelles with different morphology and vesicles having a limiting membrane and containing little amorphous material. Both skin types present some pteridines and flavins, though they are qualitatively and quantitatively more abundant in yellow skin extracts.     

Diversity among peripheral populations: genetic and evolutionary differentiation of Salamandra atra at the southern edge of the Alps

Separate populations at the edge of a species range are receiving great attention and have been shown to be often different from populations in the core area. However, it has rarely been tested whether neighboring peripheral populations are genetically and evolutionarily similar to each other, as expected for their geographical proximity and similar ecological conditions, or differ due to historical contingency. We investigated isolation and differentiation, within‐population genetic diversity and evolutionary relationships among multiple peripheral populations of a cold‐adapted terrestrial salamander, Salamandra atra, at the southern edge of the species core range. We carried out population genetic, phylogeographic, and phylogenetic analyses on various molecular markers (10 autosomal microsatellite loci, three mitochondrial loci with total length >2,100 bp, two protein‐coding nuclear genes) sampled from more than 100 individuals from 13 sites along the southern Prealps. We found at least seven isolated peripheral populations, all highly differentiated from the remaining populations and differentiated from each other at various levels. The within‐population genetic diversity was variable in the peripheral populations, but consistently lower than in the remaining populations. All peripheral populations along the southern Prealps belong to an ancient lineage that is also found in the Dinarides but did not contribute to the postglacial recolonization of the inner and northern Alps. All fully melanistic populations from the Orobian mountains to the southern Dinarides represent a single clade, to the exclusion of the two yellow‐patched populations inhabiting the Pasubio massif and the Sette Comuni plateau, which are distinguished as S. atra pasubiensis and S. atra aurorae, respectively. In conclusion, multiple populations of S. atra at the southern edge of the species core area have different levels of differentiation, different amount of within‐population genetic diversity, and different evolutionary origin. Therefore, they should be regarded as complementary conservation targets to preserve the overall genetic and evolutionary diversity of the species.     

Genetic variation in an endemic salamander, Salamandra atra, using amplified fragment length polymorphism

The pattern of genetic differentiation of the endemic alpine salamander, Salamandra atra, has been studied using amplified fragment length polymorphism (AFLP) from 11 populations throughout the range of the two currently recognized subspecies, atra and aurorae. Five different primer combinations produced 706 bands and were analyzed by constructing a phylogenetic tree using NJ and principal component analysis. Significant genetic variation was revealed by AFLP between and within populations but, our results show a lack of genetic structure. AFLP markers seems to be unsuitable to investigate complex and recent diversification.     

Phylogeography of the Alpine salamander, Salamandra atra (Salamandridae) and the influence of the Pleistocene climatic oscillations on population divergence

Fifty individuals of the endemic Alpine salamander, Salamandra atra, representing 13 populations throughout the range of the two currently recognized subspecies, atra and aurorae, were examined for sequence variation in a large portion (1050 bp) of the mitochondrial cytochrome b gene. We revealed a large number of mitochondrial DNA (mtDNA) haplotypes (10). Interpopulation sequence divergence was very low, ranging from 0 to 3.1%. The relationships among haplotypes were poorly resolved. The divergence time estimate between several mtDNA haplotypes suggested a pre-Pleistocene differentiation approximately 3 million years ago. Moreover, the impact of the Pleistocene glaciations on the phylogeographical patterns appears to have been secondary, although a somewhat reduced genetic variability was found in populations living in areas that were directly affected by the glaciation.     

Inhibition of fungi,actinomycetes and bacteria by Stachybotrys atra

The effect ofStachybotrys atra on fungi, actinomycetes and bacteria was studied in agar culture. Of the 27 genera and 52 species of fungi, 6 species of actinomycetes and 5 genera and 10 species of bacteria tested,S. atra was found to inhibit 95.7 % fungi, all the actinomycetes and 83.3 % of bacteria.Aspergillus ustus, 2 unidentified species ofFusarium, Rhizobium trifolii andPseudomonas spp. were however not inhibited. In experiments in soil,S. atra inhibited the growth ofTrichoderma viride. S atra was ineffective whereT. viride andS. atra were inoculated simultaneously or whereT. viride was added 7 days earlier. Besides,S. atra was not found to controlMacrophomina phaseoli infection on cotton. Soil culture ofS. atra showed phytotoxic effect on cotton seedlings.S. atra produced a dark brown to black thermostable toxic metabolite in the medium. The culture filtrate retarded the growth ofM. phaseoli in agar culture. Despite the importance ofS. atra as a fungistatic agent with wide antimicrobial spectrum, the use ofS. atra as a protectant against plant pathogens seems to be limited because of its phytotoxicity on cotton.     

Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% ± 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering.     

Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% ± 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering. Supported by the Major Technology Program of Beijing Municipal Science & Technology Commission (Grant No. H060920050130) and the Major State Basic Research Development Program of China (Grant No. 2005CB522702)     

Ultrastructural features of skin pigmentation in the lizard Heloderma suspectum with emphasis on xanto‐melanophores

The morphological origin of the dark and pink‐orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark‐grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta‐layer contributes to increase the black intensity. In the orange‐pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto‐melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid‐rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto‐melanophores localized in the pink‐orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.     

Ultrastructure of normals and castrates and the effects of testosterone and ultraviolet (UVL-B) irradiation on scrotal skin of rats.

The ultrastructure of the testosterone dependent epidermal melanocyte system of the scrotal skin of normals and castrates, with and without testosterone replacement therapy, and UVL-B (280-315 nm) radiation in black Long Evans rats is reported. UVL-B increases melanocyte activity, melanosome forming apparatus, (size of Golgi zone and RER, and quantity of cytoplasmic vesicles, dendrites, and stages of melanosomes) in normals and in castrates. Testosterone replacement therapy to castrates is not a prerequisite for stimulation by UVL-B, but it enhances the effects of UVL-B without restoring normalcy as melanosome packaging into complexes predominates. After UVL-B stimulation of normals or castrates, melanocyte dendrites are observed more often. Melanocyte dendrites of skin of castrated rats are observed less often than in normals, but with testosterone replacement therapy, the dendrites become more numerous. Melanosomes donated to keratinocytes are mostly located as singles in normals and as complexes in castrates. After UVL-B, castration, or testosterone replacement therapy, the melanosomes are packaged in keratinocytes in complexes larger than in normals. In the epidermis of long term castrates (9-109 days), non-specific clear cells are observed and Langerhans cells containing melanosomes; we did not observe them in normals. Melanocytes of castrates have a reduced melanosome forming apparatus. The dermis of castrates contains many dermal melanocytes in the superficial dermis with melanosomes in several stages of formation. These cells are not apparent in normals at this location in the dermis. Testosterone replacement therapy and/or UVL-B administered to castrates does not restore the epidermal melanocyte system nor the dermis to precastration ultrastructural appearance; castration has a permanent altering effect as melanosomes are packaged into complexes.     

A variation of pigmentation in the glabrous skin of dogs

The usual pigmentation pattern in mammalian skin consists of fixed melanocytes in the basal layer of the epidermis, supplying keratinocytes with melanosomes. We observed that the glabrous skin (rhinaria and footpads) of dogs deviates from this pattern. In dogs, melanocytes are found in both the dermis and epidermis. The epidermal melanocytes are situated in the intercellular spaces of the basal and spinous layers. They are characterized by a quantity of cytoplasm containing a centriole, also developing melanosomes, and in some cases annulate lamellae. There is a high frequency of closely apposed melanocytes in the epidermis. Melanosomes in different stages of formation are also abundant. The morphology of the glabrous skin of dogs suggests transport of melanocytes from the dermis into the epidermis and formation of melanosomes in the epidermis. A distributed and intense pigment formation may be necessary to achieve the black noses of many dog breeds and wild canids, as well as dark footpads despite heavy abrasion and rapid skin renewal.     

Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians

Alibardi, L. 2011. Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians. —Acta Zoologica (Stockholm) 00 :1–11. The cytology and distribution of chromatophores responsible for skin pigmentation in chelonians is analyzed. Epidermal melanocytes are involved in the formation of dark spots or stripes in growing shelled and non‐shelled skin. Melanocytes rest in the basal layer of the epidermis and transfer melanosomes into keratinocytes during epidermal growth. Dermal melanophores and other chromatophores instead remain in the dermis and form the gray background of the skin. When dermal melanophores condense, they give origin to the dense spots or stripes in areas where no epidermal melanocytes are present. In the latter case, the epidermis and the corneous layer are transparent and reveal the dermal distribution of melanophores and other chromatophores underneath. As a result of this basic process of distribution of pigment cells, the dark areas visible in scales can have a double origin (epidermal and dermal) or a single origin (epidermal or dermal). Xanthophores, lipophores, and a cell containing both pterinosomes and lipid droplets are sparse in the loose dermis while iridophores are rarely seen in the skin of chelonians analyzed in the present study. Xanthophores and lipophores contribute to form the pale, yellow or oranges hues present among the dark areas of the skin in turtles.     

Zur Evolution der Embryonal- bzw. Larvalentwicklung bei Salamandra1

In the genus Salamandra occur mainly two kinds of propagation. The european spotted salamander (Salamandra salamandra) is ovoviviparous and lays a great number of larvas into the water, whereas the alpine salamander (Salamandra atra) gives birth to completely transformed gilless young ones. There are, however, several races of spotted salamander which are also viviparous. The development of such a variety, Salamandra salamandra bernardezi, is described and compared with the generation of the alpine salamander. The emancipation of the development of the larvas from their life in the water has certainly happened in several cases independent of each other under various oecological circumstances. Besides a race of spotted salamander was said to exist in a mountain lake in central Spain at about 6000 ft. This assertion proved to be wrong. The development of the embryo and the larva takes place in exactly the same way as with our native species. The results of the experiments of Mr. Kammerer are dicussed on the basis of the facts at our disposal.     

Climate change drives speciation in the southern rock agama (Agama atra) in the Cape Floristic Region, South Africa

Aim Vicariance has played a major role in the evolution of the southern rock agama, Agama atra (Reptilia: Agamidae), and it is hypothesized that habitat shifts will affect small‐scale patterns of gene flow. The Cape Floristic Region (CFR) is known for high levels of diversity and endemism; thus we set out to investigate whether genetic structuring of CFR populations of A. atra corresponds to regional environmental shifts. Location Cape Fold Mountains and the Cape Floristic Region of South Africa. Methods The phylogeographical structure of 116 individuals of A. atra was determined by making use of 988 characters derived from two mitochondrial DNA fragments (control region and the NADH dehydrogenase subunit 2 coding region, ND2). Most animals originated from the CFR, but to gain a better understanding of the processes and patterns of dispersal within the species, 17 additional specimens from outside the CFR were also included and analysed in a phylogenetic context. Results Parsimony and Bayesian analyses revealed four distinct CFR clades (Cape clades) associated with geography. Phylogenetic analyses suggest that populations of A. atra in the CFR region are not entirely isolated from other populations, because some individuals from outside the CFR were nested within the four main Cape clades. The combined mitochondrial DNA data set revealed 59 distinct haplotypes in the CFR. Analysis of molecular variance (amova ) confirmed the high degree of genetic structure among the Cape clades, with more than 75% of the genetic variation found among the geographical areas. A spatial amova suggested that a ‘central clade’ originally defined as one of the four Cape clades may contain several additional populations. The main cladogenesis of A. atra within the CFR is estimated to have taken place c. 0.64–2.36 Ma. Main conclusions Agama atra shows at least four distinct genetic provinces within the CFR region, which highlights the conservation importance of this biologically diverse area. The dates of separation among the clades coincide well with the documented Pleistocene climate fluctuations, which might have contributed towards the isolation among lineages; the congruent genetic structure of A. atra with other CFR taxa further supports vicariance as a main isolating factor.     

Host location by Gelis festinans, an eggsac parasitoid of the linyphiid spider Erigone atra

Interactions were investigated between the spider Erigone atra (Blackwall) (Araneae, Linyphiidae) and the insect parasitoid Gelis festinans (Fabricius) (Hymenoptera, Ichneumonidae), both found in an agricultural wheat field surrounded by grass edges. The searching behaviour of G. festinans was studied in laboratory experiments. Odours from detached wheat and grass leaves were preferred by male and female parasitoids when tested against clean air in Y-tube olfactometer experiments. Female parasitoids, collected from the field, and their lab-reared female offspring, preferred wheat odours when given the choice between wheat vs. grass odours. Male parasitoids showed no significant preference for wheat or grass leaves. Both field-captured and lab-reared female parasitoids responded to silk from the spider E. atra with an increased searching behaviour, while showing no response to silk from the spiders Lepthyphantes tenuis (Blackwall), Bathyphantes gracilis (Blackwall), Oedothorax retusus (Westring) and O. fuscus (Blackwall) (Araneae, Linyphiidae). Female parasitoids spent significantly less time on surfaces that were pre-searched by themselves or conspecific females than on unsearched areas, even in the presence of E. atra webbing. Parasitized eggsacs were distinguished from unparasitized ones; superparasitization was not observed.     

Cultures of Skin Fragments of Salamandra salamandra salamandra (L.) Larvae

As part of a study on the pigmentary system of Salamandra salamandra salamandra (L.), we cultured skin fragments of 7–10-day-old larvae in order to examine the expression of molecules implicated in cellular adhesion and migration and in regulating cell-cell relationships. Keratinocytes, fibroblasts, Leydig cells, xanthophores, and melanophores migrated from the fragments and were observed in the outgrowth. Keratinocytes and fibroblasts organized into an epidermal layer and an underlying “dermal portion.” The chromatophores were always located below the epithelial cells, often with fibroblasts. We examined by immunocytochemistry the expression of fibronectin, β₁-integrin, L-CAM, and A-CAM in the cultures. Many keratinocytes, fibroblasts, and Leydig cells expressed all the signal molecules tested. Xanthophores and melanophores were only immunoreactive to the anti-adhesion molecules antisera. Since the molecules tested are known to play a role in cell adhesion, growth, and spreading, as well as in regulating tissue differentiation and in maintaining normal tissue morphology, we may hypothesize that in Salamandra salamandra salamandra fibronectin, β₁-integrin, L-, and A-CAMS concertedly act to stabilize the architecture of the outgrowth and regulate the relationships between chromatophores and those between chromatophores and the other elements of the skin culture.     

Xanthophore migration from the dermis to the epidermis and dermal remodeling during Salamandra salamandra salamandra (L.) larval development

During larval development of Salamandra salamandra salamandra chromatophores organize to form the definitive pigment pattern constituted by a black background with yellow patches that are characterized by epidermal xanthophores and dermal iridophores. Simultaneously the dermis undergoes remodeling from the larval stage to that typical of the adult. In the present study we ultrastucturally and immunocytochemically examined skin fragments of S. s. salamandra larvae and juveniles in order to investigate the modalities of xanthophore migration and differentiation in the context of dermal remodeling from the larval to adult stage. Semithin and thin sections showed that the dermis in newly born larvae consists of a compact connective tissue (basement lamella), to which fibroblasts and xanthophores adhere, and of a loose deep collagen layer. As larval development proceeds, fibroblasts and xanthophores invade the basement lamella, skin glands develop and the adult dermis forms. At metamorphosis, xanthophores reach the epidermis crossing through the basal lamina. We examined immunocytochemically the expression of signal molecules, such as fibronectin, vitronectin, beta1-integrin, chondroitin sulfate, E-cadherin, N-cadherin and plasminogen activator, which are known to be involved in regulating morphogenetic events. Their role in dermal remodeling and in pigment pattern formation is discussed.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long‐term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore‐fibroblast co‐culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast‐derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane‐enclosed melanosomes in a process that was upregulated by α‐melanocyte‐stimulating hormone (α‐MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane‐proteins seemed to be of importance, as the cluster‐like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long‐term colour changes and for studies of factors that affect pigmentation.     

Depigmenting Action of Phenylhydroquinone,an o‐Phenylphenol Metabolite,on the Skin of JY‐4 Black Guinea‐Pigs

The effects of o‐phenylphenol (OPP) and its metabolite, phenylhydroquinone (PHQ) on the skin of JY‐4 black guinea‐pigs were studied. Topical application of 1 or 5% PHQ on the black skin of the back caused marked depigmentation and hypopigmentation of the skin after 5 weeks, whereas OPP applied at the same concentrations had little effect. Depigmented skin had an increased L* (lightness) value in the CIE‐L*a*b* color system. This corresponded with a decreased number of melanocytes and melanosomes in the melanocytes and keratinocytes, the disruption of melanosomes in the melanocytes, and destruction of the membranous organelles of the melanocytes. These morphological and numerical changes in epidermal melanocytes indicate that selective melanocyte toxicity occurred. Furthermore, application of PHQ to the skin of white guinea‐pigs caused skin irritation, as shown by a colorimetric increase in a* value (redness) and by histological observation of inflammation. This study confirmed that OPP, which is a reported depigmenter, has little depigmenting action, while its metabolite, PHQ, is a potent depigmenter preferentially affecting melanocytes.     

Volume Changes of Individual Melanosomes Measured by Scanning Force Microscopy

Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G‐protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three‐dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P<0.05, n=200). The average ellipsoidal volume was 0.14±0.01 (aggregated) and 0.17±0.01 μm³ (dispersed), a difference of 18%. The average major diameter was 810±20 and 880±20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non‐destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.