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PubMed1982

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.

Animals↗
PubMed2002

Caiman periodontium as an intermediate between basal vertebrate ankylosis-type attachment and mammalian "true" periodontium.

The teeth of many fish, amphibia, and reptiles are attached to the alveolar bone via ankylosis. In contrast, mammalian periodontia are characterized by a gomphosis, an attachment of the tooth root in the alveolar bone socket via periodontal ligament fibers. Among the reptiles, the crocodilians are the only group featuring a gomphosis-type connection between tooth root and alveolar bone, while in other reptiles tooth-root and jawbone are connected via ankylosis. The purpose of the present study was to compare several key features of the crocodilian periodontium with those of the mammalian and noncrocodilian reptile periodontium. As experimental models for our study we chose the periodontium of newborn geckos (Hemidacylus turcicus), juvenile caimans (Caiman crocodilus crocodilus), and 10-day-postnatal Swiss-Webster mice (Mus musculus) as representative models for noncrocodilian reptiles, crocodilian reptiles, and mammals. The caiman periodontium emerged as an intermediary between the mineral-free mouse ligament and the mineralized gecko ankylosis-type attachment. Caiman ligament fibers were less organized than mouse ligament fibers but featured distinct fasciae surrounding ligament fiber bundles. Caiman Hertwig's epithelial root sheath (HERS) was similarly perforated as mouse HERS and distinctly different from the continuous gecko HERS. Both caiman and mouse HERS covered the entire tooth root length, while in the gecko HERS was limited to the coronal portion of the root, allowing for cementoid-mediated ankylosis at the apical tip of the root. We interpret our data to indicate distinct differences in mineral distribution, periodontal ligament fiber organization, and HERS distribution between noncrocodilian reptiles, crocodilian reptiles, and mammals. Mineral deposits in the caiman ligament may reflect an evolutionary position of the caiman periodontium between ankylosis and gomphosis.

Alligators and Crocodiles↗
PubMed2003

Immunohistochemical localization of S100-like protein in non-mammalian kidney.

The immunolocalization of S100-like protein was investigated in the kidney of saltwater fishes (Dicentrarchus labrax; Coris julis; Serranus cabrilla; Scorpaena porcus), amphibia (Rana aesculenta), reptiles (Lacerta viridis), and aves (Gallus domesticus; Strutio camelus). S100-like immunoreactivity was detected in the juxtaglomerular cells of all saltwater fishes studied. No immunoreactivity was observed in other tracts of the nephron or in the interstitial tissue. In frog kidney, S100-like immunoreactive cells were localized in the proximal tubule, singly distributed or placed side by side in clusters of two or three cells. S100-like immunoreactive cells were distributed in the distal and in the collecting tubules in lizard, chicken, and ostrich kidney. In the distal tubule of lizard kidney, S100-like immunoreactive cells were numerous and uniformly distributed. In lizard collecting tubules, S100-like immunoreactive cells showed less intense immunoreactivity than in the distal tubule, except for a cluster of cells at the junction with the initial collecting duct. In chicken and ostrich kidney, S100-like immunoreactive cells of the distal tubules were closely packed together. In the collecting tubules, S100-like immunoreactive cells were alternate to negative cells. These results indicate the high conservation degree of S100 proteins through phylogenesis and suggest a functional role for these proteins in the vertebrate kidney.

Animals↗
PubMed1992

Fine structural aspects of secretion and extrinsic innervation in the olfactory mucosa.

The mucus at the surface of the olfactory mucosa constitutes the milieu in which perireceptor events associated with olfactory transduction occur. In this review, the ultrastructure of olfactory mucus and of the secretory cells that synthesize and secrete olfactory mucus in the vertebrate olfactory mucosa is described. Bowman's glands are present in the olfactory mucosa of all vertebrates except fish. They consist of acini, which may contain mucous or serous cells or both, and ducts that traverse the olfactory epithelium to deliver secretions to the epithelial surface. Sustentacular cells are present in the olfactory epithelium of all vertebrates. In fish, amphibia, reptiles, and birds, they are secretory; in mammals, they generally are considered to be "non-secretory," although they may participate in the regulation of the mucous composition through micropinocytotic secretion and uptake. Goblet cells occur in the olfactory epithelium of fish and secrete a mucous product. Secretion from Bowman's glands and vasomotor activity in the olfactory mucosa are regulated by neural elements extrinsic to the primary olfactory neurons. Nerve fibers described in early anatomical studies and characterized by immunohistochemical studies contain a variety of neuroactive peptides and have several targets within the olfactory mucosa. Ultrastructural studies of nerve terminals in the olfactory mucosa have demonstrated the presence of adrenergic, cholinergic and peptidergic input to glands, blood vessels, and melanocytes in the lamina propria and of peptidergic terminals in the olfactory epithelium. The neural origins of the extrinsic nerve fibers and terminals are the trigeminal, terminal, and autonomic systems.

Animals↗
PubMed1995

Spermatogenesis in nonmammalian vertebrates.

Spermatogenesis appears to be a fairly conserved process throughout the vertebrate series. Thus, spermatogonia develop into spermatocytes that undergo meiosis to produce spermatids which enter spermiogenesis where they undergo a morphological transformation into spermatozoa. There is, however, variation amongst the vertebrates in how germ cell development and maturation is accomplished. This difference can be broadly divided into two distinct patterns, one present in anamniotes (fish, amphibia) and the other in amniotes (reptiles, birds, mammals). For anamniotes, spermatogenesis occurs in spermatocysts (cysts) which for most species develop within seminiferous lobules. Cysts are produced when a Sertoli cell becomes associated with a primary spermatogonium. Mitotic divisions of the primary spermatogonium produce a cohort of secondary spermatogonia that are enclosed by the Sertoli cell which forms the wall of the cyst. With spermatogenic progression a clone of isogeneic spermatozoa is produced which are released, by rupture of the cyst, into the lumen of the seminiferous lobule. Following spermiation, the Sertoli cell degenerates. For anamniotes, therefore, there is no permanent germinal epithelium since spermatocysts have to be replaced during successive breeding seasons. By contrast, spermatogenesis in amniotes does not occur in cysts but in seminiferous tubules that possess a permanent population of Sertoli cells and spermatogonia which act as a germ cell reservoir for succeeding bouts of spermatogenic activity. There is, in general, a greater variation in the organization of the testis and pattern of spermatogenesis in the anamniotes compared to amniotes. This is primarily due to the fact there is more reproductive diversity in anamniotes ranging from a relatively unspecialized condition where gametes are simply released into the aqueous environment to highly specialized strategies involving internal fertilization. These differences are obviously reflected in the mode of spermatogenesis and this is particularly true of the stage of spermiogenesis where the morphology of the species-specific spermatozoon is determined. Moreover, unlike amniotes, many anamniotes display a spermatogenic wave manifest, depending upon the species, either at the level of the cyst or seminiferous lobule. This variation in the organization of the testis makes certain anamniotes perfect models for investigating germ cell development and maturation. For instance, the presence of a spermatogenic wave provides an opportunity to manually isolate discrete germ cell stages for analysis of specific Sertoli/germ cell interactions. Furthermore, for many anamniotes, germ cells mature in association with a morphologically poorly developed Sertoli cell. This seeming independence of Sertoli cell regulation allows the in vitro culture of isolated germ cells of some species of anamniotes through several developmental stages. Thus, due either to the anatomical organization of the testis, or structural simplicity of the germinal units, nonmammalian vertebrates can provide excellent experimental animal models for investigating many basic problems of male reproduction.

Animals↗
PubMed1981

Cyclic AMP in mammalian follicle cells and oocytes during maturation.

Intact ovarian follicles and isolated oocytes were cultured for 30 sec to 18 hr in the presence of gonadotrophins, and the cyclic AMP content in the follicle and oocyte was measured. The basal content of cyclic AMP in ovine oocytes before gonadotrophic stimulation was 6.3 +/- 0.7 fmol/oocyte or 8 microM. There was no fall in oocyte cyclic AMP concentration as an immediate response to the gonadotrophins, but at 1, and 12-18 hr after stimulation, the concentration in both the oocytes and follicle cells was considerably elevated. There was no comparable increase in intracellular cyclic AMP in oocytes denuded of follicle cells before culture, even when both gonadotrophins and phosphodiesterase inhibitors were included in the medium. We conclude that the signal which initiates oocyte maturation in mammals differs from that of amphibia, where an early fall in intracellular cyclic AMP is essential for the resumption of meiosis. Moreover, cellular interactions within the mammalian follicle are necessary for the characteristic periods of increased cyclic AMP in oocytes during maturation.

Animals↗
PubMed1984

Stump currents in regenerating salamanders and newts.

We report here that a variety of salamanders and newts from differing habitats all drive a steady ionic electric current out of the forelimb stump tip after forelimb amputation. Several hours after amputation the density of this stump current ranges from about 10 to 100 microA/cm2 in most species, and declines with time. In most cases, the magnitude of the stump current is dependent on the concentration of Na+ in the external medium (an artificial pondwater), suggesting that the well-known Na+ -dependent transcutaneous voltage described in amphibia (particularly frogs) is the EMF for this stump current. These measurements add to those previously reported for the North American red spotted newt (Notophthalmus viridescens), and suggest that electrical changes following amputation of urodele limbs are widespread among members of this group.

Amputation, Surgical↗
PubMed1986

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates.

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.

Animals↗
PubMed1992

Relationship between cell size and nuclear volume in nucleated red blood cells of developmentally matched diploid and tetraploid mouse embryos.

Analysis of control diploid and polyploid amphibia indicated that cell and nuclear volumes were closely related to their ploidy, so that an increase in ploidy was generally associated with an increase in cell size. This relationship is also believed to occur in mammalian polyploids. However, since the latter are only rarely encountered spontaneously, or only occasionally following experimental manipulation, no detailed morphometric studies have been carried out to date to confirm whether such a relationship exists. In this study, the cellular and nuclear volume of primitive red blood cells was analyzed in carefully developmentally matched control diploid mouse embryos and tetraploid embryos produced by the technique of electrofusion. All of the cells and/or nuclei studied had a characteristic spherical shape which greatly simplified the morphometric analysis. A defined and predictable relationship between ploidy and cellular and/or nuclear volume was observed in the red blood cells between 8.25 and 14.5 days of gestation. During this period the primitive red blood cells are gradually replaced by the definitive erythrocytes. The ratio of control values to tetraploid values was found to be close to the theoretical value of 1:2 when comparable cells and/or their nuclei were analyzed in carefully developmentally matched material.

Animals↗
PubMed1995

Defensins and other endogenous peptide antibiotics of vertebrates.

Gene-encoded peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Their de novo synthesis or release from storage sites can be induced rapidly, which makes them particularly important in the initial phases of resistance to microbial invasion. The endogenous antimicrobial peptides of animals are products of single genes and are synthesized as preproproteins. Multistep processing yields the mature peptide, which generally acts by inducing microbial membrane permeabilization. Several families of antimicrobial peptides have been identified that differ with respect to the presence of disulfide linkages, amino acid composition, structural conformation, and spectrum of activity. The arginine-rich three disulfide-containing beta-sheet defensins are remarkably abundant and widely distributed in animals and plants. The antibiotic peptides of higher eukaryotes merit further study for their role in natural immunity and their potential as novel therapeutic compounds.

Amino Acid Sequence↗
PubMed2004

Development of the nasal chemosensory organs in two terrestrial anurans: the directly developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), and the metamorphosing toad, Bufo americanus (Anura: Bufonidae).

Nearly all vertebrates possess an olfactory organ but the vomeronasal organ is a synapomorphy for tetrapods. Nevertheless, it has been lost in several groups of tetrapods, including aquatic and marine animals. The present study examines the development of the olfactory and vomeronasal organs in two terrestrial anurans that exhibit different developmental modes. This study compares the development of the olfactory and vomeronasal organs in metamorphic anurans that exhibit an aquatic larva (Bufo americanus) and directly developing anurans that have eliminated the tadpole (Eleutherodactylus coqui). The olfactory epithelium in larval B. americanus is divided into dorsal and ventral branches in the rostral and mid-nasal regions. The larval olfactory pattern in E. coqui has been eliminated. Ontogeny of the olfactory system in E. coqui embryos starts to vary substantially from the larval pattern around the time of operculum development, the temporal period when the larval stage is hypothesized to have been eliminated. The nasal anatomy of the two frogs does not appear morphologically similar until the late stages of embryogenesis in E. coqui and the terminal portion of metamorphosis in B. americanus. Both species and their respective developing offspring, aquatic tadpoles and terrestrial egg/embryos, possess a vomeronasal organ. The vomeronasal organ develops at mid-embryogenesis in E. coqui and during the middle of the larval period in B. americanus, which is relatively late for neobatrachians. Development of the vomeronasal organ in both frogs is linked to the developmental pattern of the olfactory system. This study supports the hypothesis that the most recent common ancestor of tetrapods possessed a vomeronasal organ and was aquatic, and that the vomeronasal organ was retained in the Amphibia, but lost in some other groups of tetrapods, including aquatic and marine animals.

Animals↗
PubMed2006

The corneal epithelial surface in the eyes of vertebrates: environmental and evolutionary influences on structure and function.

The smooth optical surface of the cornea is maintained by a tear film, which adheres to a variety of microprojections. These microprojections increase the cell surface area and are thought to improve the movement of oxygen, nutrients, and metabolic products across the outer cell membranes. However, little is known of these structural adaptations in vertebrates inhabiting different environments. This field emission scanning electron microscopic study examined the cell density and surface structure of corneal epithelial cells across 51 representative species of all vertebrate classes from a large range of habitats (aquatic, amphibious, terrestrial, and aerial). In particular, we wished to extend the range of vertebrates to include agnathans and some uniquely Australian species, such as the Australian lungfish (Neoceratodus forsteri), the Australian galah (Eolophus roseicapillus), the Australian koala (Phascolarctos cinereus), and the rat-tailed dunnart (Sminthopsis crassicaudata). Epithelial cell densities ranged from 28,860 +/- 9,214 cells mm(-2) in the flathead sole Hippoglossoides elassodon (a marine teleost) to 2,126 +/- 713 cells mm(-2) in the Australian koala (a terrestrial mammal), which may indicate a reduction in osmotic stress across the corneal surface. A similar reduction in cell density occurred from marine to estuarine to freshwater species. The structure and occurrence of microholes, microplicae, microridges, and microvilli are also described with respect to the demands placed on the cornea in different environments. All species that spend significant periods out of an aquatic environment possess microvilli and/or microplicae. These include all of our species of Mammalia, Aves, Reptilia, Amphibia, and even one species of Teleostei (Australian lungfish). Well-developed microridges occur only in teleosts in high osmolarity environments such as marine or estuarine habitats. Clear interspecific differences in corneal surface structure suggest a degree of adaptive plasticity, in addition to some phylogenetic trends.

Animals↗
PubMed1976

The development and replacement of teeth in viviparous caecilians.

Tooth development and replacement in fetal and adult viviparous caecilians (Amphibia: Gymnophiona) are described and analyzed according to current theories of tooth succession. The fetal dentition differs from that of the adult in morphology, position, and function. Teeth are used by fetuses to scrape the oviducal epithelium, thus stimulating the secretion of a nutrient substance. Fetal dentitions vary in morphology and position in different species. The ontogeny of teeth of several species is described and the patterns of addition of loci and of replacement are analyzed. Loci are added both posteriorly along the jaw and between existing loci as the jaw grows prior to ossification; subsequently addition is restricted to the posterior part of the jaw. Tooth replacement is alternate. The several rows and patches of teeth are the result of retention of replacement series on the dentigerous elements. Tooth development and replacement in a series of juveniles and adults of different sizes in a single species are also considered. Post-fetal patterns of development and replacement are similar to those seen in larvae and adults of oviparous species. Variation in numbers of teeth and proportions of teeth at particular stages occurs ontogenetically and among individuals of the same size, though proportions occur in a similar pattern throughout the series. The general pattern of tooth replacement in fetuses and adults can be explained by either Edmund's Zahnreihen theory or by Osborn's Tooth Family theory, but replacement in fetal tooth patches and the fetal-adult dentitional transition are explained by neither.

Amphibians↗
PubMed1990

Metamorphosis and fish vision.

Many species of fish exhibit metamorphosis in which dramatic external transformations occur as a consequence of coordinated changes in gene expression within an organism. Because postembryonic development and change appears to be the rule rather than the exception in teleost fish species, we view metamorphosis as one of many developmental strategies in fish which have continued plasticity as a common theme. Metamorphic changes are manifested in the visual system by modification of photoreceptor peak sensitivity, rod photoreceptor cell addition, and retinal reorganization. These changes correspond to significant changes in the natural habitat of the animal and in its visual capabilities as demonstrated behaviorally. Thyroxine is the main metamorphic hormone as has also been found in amphibia. The sequence of metamorphic events occur in all teleosts, but they are compressed in time in direct developing animals suggesting that such animals might prove useful for understanding the evolution of metamorphosis in fish. It seems likely that rod photoreceptors may have evolved in conjunction with the change from larval to juvenile stage through metamorphosis in indirect developing fishes. During evolution, the contraction and/or loss of the larval stage has resulted in earlier appearance of rod photoreceptors during development although they always arise later than cone photoreceptors. This ontogenetic developmental sequence supports Walls's (1942) proposal that cones are phylogenetically older than rods and suggests that rods may have evolved several times.

Animals↗
PubMed2002

Implantation of MNNG crystals into a Triturus intact limb affects mitotic and labeling indices, regeneration rate, and morphogenesis in the contralateral, regenerating limb.

Experimental administration of chemical carcinogens to various mammals is highly effective in inducing malignant tumors. In contrast, treatment of regeneration-competent animals even with much higher doses of the same drugs only exceptionally leads to tumor-like growth. Usually, carcinogenic materials implanted or injected into a regenerating limb of urodele amphibia interfere with the regenerative process and frequently lead a). to growth retardation or arrest of regeneration, b). to development of a great variety of abnormal regenerates, and c). to generation of accessory, limb-like structures. Autonomous or experimental incidence of carcinogenesis is extremely low in animals endowed with strong regenerative capabilities. Of exceptional biological significance is the fact that such induced tumors usually regress spontaneously. This unique property of the regeneration-competent animals to resist carcinogenesis provides opportunities to compare non-cancerous alterations in the differentiated state of adult cells to those occurring in neoplasia. The mode of action of the chemical carcinogens on limb regeneration has not yet been clarified with certainty at the cellular and the molecular level. Several scientists claim that the above-mentioned effects might be attributed to local toxic influences of the drugs; therefore the present study was designed to investigate whether the administration of the carcinogen MNNG can affect cell proliferation, histogenesis, and morphogenesis at a region distant from the site of its implantation, even after a relatively long time period. To this end, 40 animals of the species Triturus cristatus had their right hindlimb surgically removed at the distal zeugopod. Then, a small microcrystal (approximately 5 micro g) of MNNG was inserted under the ventral aspect of the skin of the left tarsus in 20 of these animals (groups T and A; see below). Two months later, nine of the MNNG-treated animals were injected intraperitoneally with tritiated thymidine. After 2 h, six of these animals had their right hindlimb amputated at the distal zeugopod, whereas the rest were left to regenerate. The results were evaluated by camera lucida drawings, clearing in methyl benzoate, classical histology, and autoradiography. It was revealed that administration of MNNG at a somatic region (left hindlimb) reduces DNA synthesis and mitosis at a distant place (right hindlimb) even 2 months after MNNG implantation. Despite this, the rate of limb elongation is not substantially reduced. Classical histology revealed normal tissue structure throughout. All regenerated limbs displayed several teratogenic abnormalities.

Amputation, Surgical↗
PubMed1997

Identification and characterization of MARCKS in Xenopus laevis.

MARCKS proteins are widely distributed in mammalian cells and subserve an important role as probes in the examination of signal transduction processes because they are specific endogenous phosphoreceptors for activated protein kinase C. Experiments were performed to determine whether MARCKS proteins are present in amphibia and to show their usefulness as substrates for stimulated PKC activation, using cultured renal epithelial cells (A6) derived from Xenopus laevis as an experimental model.

Animals↗
PubMed1994

An inhibitory effect of Xenopus gastrula ectoderm on muscle cell differentiation and its role for dorsoventral patterning of mesoderm.

In Amphibia, mesoderm cells such as notochord, muscle, and blood are formed as a result of mesoderm induction, the first known inductive interaction during the embryonic development of Vertebrates. Recent evidence shows, however, that, in addition to mesoderm induction, further cell-cell interactions during gastrulation also play an important role in the differentiation of mesoderm cells in Xenopus embryos. Here we report the existence of an inhibitory effect of gastrula ectoderm on muscle cell differentiation. When placed in contact with a muscle progenitor cell population, ectoderm from the ventral side of a gastrula embryo exerts an inhibitory effect on MyoD expression and muscle differentiation. This inhibitory effect is not observed on notochord differentiation. Dorsal ectoderm shows the same extent of inhibitory effect if it is isolated at the early-gastrula stage but gradually loses its effect during gastrulation as it is induced to become neural tissue. Cell mixing experiments have shown that this inhibitory effect, clearly seen in ventral ectoderm, is not observed in ventral mesoderm cells. We propose that the ectoderm emits a signal counteracting dorsalization and the community effect and that this signal plays an important role in the correct dorsoventral patterning of mesoderm.

Animals↗
PubMed1994

Pantropic retroviral vector-mediated gene transfer, integration, and expression in cultured newt limb cells.

Limb regeneration is a unique developmental phenomenon restricted to certain urodeles in which limb cells dedifferentiate and produce the blastema and then redifferentiate into the tissues that compose the missing part. Genetic modification of the blastema cells would greatly facilitate understanding the programmed gene expression that results in the reconstitution of the limb. To test whether pantropic retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein could mediate gene transfer into blastema cells, we infected a stable newt limb cell line and demonstrated integration and expression of the provirus. Thus, pantropic retroviral vectors offer a new tool for the study of limb regeneration and other developmental phenomena in amphibia.

Animals↗