Bufo Ictericus Research Articles

Expression pattern of glycoconjugates in the integument of Bufo ictericus (Anuran, Bufonidae): Biochemical and histochemical (lectin) profiles

Mucous consists of glycoproteins and proteoglycans produced by specific secretory cells (mucocytes). In anurans the cutaneous mucous is produced by intradermal glands and displays both mechanical and chemical protection functions. Indeed, mucous maintains the integument moist and facilitates gas exchange (cutaneous respiration). In this work, the carbohydrate moiety distribution was investigated in the integument of Bufo ictericus using conventional and lectin histochemistry to describe the pattern of cutaneous glycoconjugate expression, including both secretory and structural proteoglycans. As a preliminary step, the descendent chromatography in Whatmann 1 MM paper was undertaken to prepare the histochemical trials involving the lectins. In B. ictericus, the integument exhibits the basic morphological structure found in lower terrestrial vertebrates: the epidermis is a keratinized squamous stratified epithelium supported by spongious and compact layers. The spongy dermis contain secretory portion of both mucous and serous (or poison) glands. The paper chromatography identified galactose, fucose and mannose as characteristic sugar residues. The secretory cells of the mucous gland in the dermis, as well as the interstice between the stratum corneum and the subjacent stratum spinosum in the epidermis exhibit α- l-fucose and α-galactose residues. The serous glands give no reaction. The α-mannose residue was detected in the extracellular matrix of spongious dermis, but not in the dermal glands. The different glycoconjugate location reflects in two glycoconjugates categories: the secretory which participate in the water flow regulation, and the structural which is involved in the dermal maintenance.

ACANTHOCEPHALA IN AMPHIBIANS (ANURA) AND REPTILES (SQUAMATA) FROM BRAZIL AND PARAGUAY WITH DESCRIPTION OF A NEW SPECIES

In a survey of 1,732 amphibians and reptiles collected across São Paulo Province, Brazil, and 7 provinces in Paraguay, 26 species were found infected with acanthocephalans. Of 1,510 anurans, 14 anurans, representing 11 species, were infected with cystacanths of Centrorhynchus spp. and 1 anuran with cystacanths of Oligacanthorhynchus sp. Of 107 lizards, 1 lizard was infected with cystacanths of Centrorhynchus sp. and 1 lizard with cystacanths of Oligacanthorhynchus sp. Acanthocephalus caspanensis was found in 3 anurans (3 species) and Acanthocephalus lutzi in 3 anurans (2 species) and 2 snakes (2 species). The systematic position of A. lutzi cannot be resolved using presently available morphological data. Acanthocephalus saopaulensis n. sp. was found in a single individual of Bufo ictericus. The new species can be differentiated from all its congeners except A. caspanensis in having a sigmoid-shaped male posterior end and from A. caspanensis in having a proboscis armature of 16 rows of 5-7 hooks rather than 18-19 rows of 6-7 hooks and larger eggs. The status of Acanthocephalus and Pseudoacanthocephalus continues to be problematic.

Morphological re-evaluation of the parotoid glands of Bufo ictericus (Amphibia, Anura, Bufonidae)

Multicellular glands in the amphibian integument represent a significant evolutionary advance over those of fishes. Bufonids have parotoid glands, symmetrically disposed in a post-orbital position. Their secretion may contribute to protection against predators and parasites. This study provides a re-evaluation of the morphology of the Bufo ictericus parotoid glands. The parotoid gland integument of the medial surface shows rounded depressions with small pores that connect with the duct openings of the larger granular glands. Under light microscopic evaluation the integument is constituted by typical epidermis, supported by dermis subdivided into a spongious dermis, a reticular dermis, and a compact dermis. The Eberth-Katschenko layer is identified as a basophilic material scattered throughout the superficial spongious dermis. The parotoid gland is an integument region, in which three exocrine glandular types occur: mixed glands, smaller granular glands and larger granular glands. The mixed gland is formed by mucous and serous cells while the small granular glands contain a homogene acidophilic intake. The larger granular glands produce a basophilic and alcianophilic material, and are responsible for the macroscopic protuberances designed as parotoid glands. Thus, the end product released by the parotoid glands is a mix of secretions produced by the three glands.

Dermatan sulfate is the major metachromatic glycosaminoglycan in the integument of the anuran Bufo ictericus

Glycosaminoglycans from the ventral and dorsal integuments of the anuran Bufo ictericus were characterized based on biochemical and histochemical methods. Dermatan sulfate is the major metachromatic glycosaminoglycan found in these tissues, but small amount of heparan sulfate was also detected. The average molecular mass of the dermatan sulfate is ∼ 20 kDa, similar to the glycosaminoglycan isolated from mammalian skin. In addition, the amphibian integument contains high amounts of hyaluronic acid, especially in the ventral area. We also observed that the glycosaminoglycans occur in the anuran integument as irregular deposits through the spongious dermis and in the mast cells, as revealed by histochemical analysis using Alcian blue, dimethylmethylene blue and toluidine blue stains. The concentration and composition of glycosaminoglycans found in the amphibian integument resemble those from mammalian skin except for the higher concentration of hyaluronic acid in the amphibian tissue. Possibly, this observation indicates that the function of the sulfated glycosaminoglycan in these tissues has been preserved during evolution, although the amphibian integument and the human skin have their own particular physiology.

Hyaluronan in the epidermal and the dermal extracellular matrix: Its role in cutaneous hydric balance and integrity of anuran integument

New researches have revealed that hyaluronan (HA) is not a passive molecule. HA has being pointed out to participate in many processes, such as cell interactions, proliferating and migrating cell events and function as hydrate agent. The present study was undertaken to localize HA in Bufo ictericus integument providing a better understating for the role of cutaneous HA. Paraffinized sections were stained with haematoxylin-eosin and with 1% Alcian blue 8GX at pH 1.0 and 2.5. Alcianophilic reaction was visualized in both spongious dermis and Eberth-Katschenko layer. The mucus cells of mucus glands were also stained with AB methods. Besides these histological techniques, the localization of HA was performed using the FITC-labeled HA probe labeled with fluorescein isothiocyanate. In the extracellular matrix of spongious dermis, the reaction for HA was evident, being less intense in hypodermis and in pericellular keratinocyte matrix of the cornified tubercles regions. Thus, since HA was localized in the pericellular epidermal matrix and in the spongious dermis of anuran integument, it plays an important role in hydric balance, and is essential for integument integrity and functionality.

Expression pattern of glycoconjugates in the Bidderian and ovarian follicles of the Brazilian toad Bufo ictericus analyzed by lectin histochemistry

The Bidder's organ and ovary of the Brazilian toad Bufo ictericus were studied by light microscopy, using hematoxylin-eosin (HE) and periodic acid Schiff (PAS) staining. The expression and distribution of carbohydrate moieties was analyzed by lectin histochemistry, using 8 lectins with different carbohydrate specificities: Ulex europaeus (UEA I), Lens culinaris (LCA), Erythrina cristagalli (ECA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Aleuria aurantia (AAA), Triticum vulgaris (WGA), and Glycine maximum (SBA). The results showed that the Bidderian zona pellucida presented alpha-mannose, alpha-L-fucose, beta-D-galactose, N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. The Bidderian follicular cells showed the presence of beta-D-galactose and N-acetyl-D-glucosamine. In the extracellular matrix, alpha-mannose and alpha/beta-N-acetyl-galactosamine residues were detected. The ovarian zona pellucida showed alpha-L-fucose, N-acetyl-D-glucosamine, alpha/beta-N-acetyl-galactosamine residues, and alpha-mannose and N-acetyl-D-glucosamine residues were detected in the follicular cells. Thus, the zona pellucida in both organs contains N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. alpha-L-fucose residues were detected in the zona pellucida of both organs, using different lectins. Considering that beta-D-galactose residue was absent from ovary but present in the Bidder's organ, this sugar residue may play an important role in follicle development, blocking the Bidderian follicles and preventing further development of the Bidder's organ into a functional ovary.

Dermal collagen organization in Bufo ictericus and in Rana catesbeiana integument (Anuran, Amphibian) under the evaluation of laser confocal microscopy

Collagen structural organization plays an important role in the mechanical property of the vertebrate integument. Bufo ictericus and Rana catesbeiana integument was investigated by light microscopy and laser confocal microscopy. Collagenous elements of the dermis were statistical analyzed. The integument is formed by the keratinized squamous stratified epidermis supported by the dermis that is subdivided into the spongious layer with a loose arrangement, and the compact layer formed by collagenous fibers arranged compactly in a criss-crossed manner. Thick collagenous columns have a perpendicular trajectory, and are formed by the assembling of alternating collagenous lamellae in both animals. Short intercolumns of collagenous fibrils connecting collagenous lamellae obliquely or transversally are observed in R. catesbeiana dorsal integument. The present study provides evidences that B. ictericus and R. catesbeiana integument has well-organized compact dermis, constituted by collagenous lamellae in a plywood manner. The integument organization is in contrast to the literature in some aspects. This dermal arrangement is important to the biomechanical property of both anuran integuments.

Circadian rhythm of physiological color change in the amphibian Bufo ictericus under different photoperiods

Ectothermic vertebrates can exhibit chromatic adaptation to the environment. The aim of this work was to characterize the rhythm of color change of the amphibian Bufo ictericus, submitted to different photoperiodic regimens, as quantified by skin reflectance values. Adult males were maintained under a 12 : 12 Light/Dark (LD) cycle during seven days before every experiment. During the experiments, animals were kept in individual boxes for 8 days, under the following photoperiodic regimens: LD 12 : 12, LD 14 : 10, DD and LL. In the last 3 days of the treatments, the reflectance of the toad dorsal skins was measured at 3-h intervals, with the aid of a reflectometer. A 3-day time series consisting of 8 data points per day was obtained, which was analyzed by the Cosinor method. The analysis demonstrated that the reflectance values exhibited significant circadian oscillations in the regimens LD 12 : 12, LD 14 : 10 and DD, suggesting that the specie B. ictericus shows an effective circadian rhythm of color change. The reflectance values did not exhibit a significant circadian rhythm in the LL regimen showing that this is a condition not permissive for the expression of the color change rhythm.

Morphology of Bufo ictericus integument (Amphibia, Bufonidae)

Bufo ictericus integument was investigated by stereoscopic, low vacuum scanning electron microscopy, transmission electron microscopy and light microscopy. The studies revealed, that the dorsal integument surface is rougher than ventral. Three types of projections are visualized: larger rounded verrucae, smaller conical cornified tubercles, and conical short spines. Prominent verrucae are observed on the dorsal surface, being flatter on the ventral surface. The tubercles are visualized only on the dorsal surface. The verrucae are separated by grooves that may contribute spreading and retention of the glandular secretion upon the integument. The pattern of the epidermal grooves is also important for water distribution, protecting the animal against desiccation. The epidermis is composed of a stratified epithelium with intraepithelial blood vessels, where keratinocytes predominate, but flask cells, and Merkel cells also occur. In the spongious dermis, cutaneous glands are visualized. The compact dermis is a series of alternating layers of bundles of collagenous fibers, and between spongious and compact dermis there are basophilic areas that correspond to Eberth-Katschenko layer. The dorsal and the ventral surfaces of B. ictericus are morphologically distinct. The integument structure is related to the physiology of each surface and represents an adaptation to habitat, reflecting a lifestyle of the animal.

Structural aspects of an unusual choroidal polarized hyaline cartilage in the eyeball of Bufo ictericus and Rana catesbeiana (Amphibia, Anura): morphological and physiological significance

Amphibian eyes play an important role in vision and in several physiological processes, such as food capture and breathing. To maintain the integrity of the eyeball there is a unique cuplike hyaline cartilage as a supporting tissue. In Bufo ictericus and Rana catesbeiana the cartilage layer is located between the retina and the choroids, being designated as choroidal cartilage, important to visual performance. On the retinal surface, there is no perichondrium, and the pigmented epithelium exhibits an intimate relationship to the cartilage layer.

Structural aspects of the Eberth-Katschenko layer of Bufo ictericus integument: histochemical characterization and biochemical analysis of the cutaneous calcium (Amphibian, Bufonidae)

Anuran, such as Brazilian toad Bufo ictericus Spix, 1824, possess a calcified dermal layer called the Eberth-Katschenko (EK) layer located between the stratum spongiosum and the stratum compactum. It has been regarded as a protective barrier against desiccation or involved in storage and mobilization of calcium, or a barrier for the interchange of substances between the animal's internal and external environment. The B. ictericus integument was removed from the ventral and dorsal regions and examined by light microscopy, using histochemical techniques, and also submitted to biochemical calcium analysis. The intermediate layer is strongly basophilic and metachromatic. In the EK layer Alcian blue at different critical electrolytic concentration revealed hyaluronic acid, which coexists with the dermal calcium. We conclude that the EK layer of B. ictericus is a discontinuous layer and is formed of hyaluronic acid and a sulfated GAG different from chondroitin sulfate, heparin and heparan sulfate or keratan sulfate. The amount of cutaneous calcium is independent of region and sex. GAGs may play an important role in dermal hydric balance, protecting the animal against desiccation, and hyaluronic acid is probably able to anchor dermal calcium. Thus, all compounds of the EK layer are essential for integument integrity and functionality.

Bidder’s organ in the female of Bufo ictericus : morphological characterization

Bidder’s organ has been cited as a structure present only in males of the toad family Bufonidae, and is used as a systematic characteristic. In this study, we examined females of Bufo ictericus in order to ascertain whether this structure also occurs in females. Macroscopic observations and light microscopy technique for paraffin embedding were performed. This study reveals that females of Bufo ictericus can have a Bidder’s organ with typical morphology, and in close spatial relationship with the ovary. This suggests that the Bidder’s organ is not an exclusive structure for male toads, but that it may also occur in active females.

Comparative cytogenetic studies of Bufo ictericus, B. paracnemis (Amphibia, Anura) and an intermediate form in sympatry

Specimens of Bufo ictericus, Bufo paracnemis and a third type, considered an intermediate subgroup between these species, were cytogenetically studied by conventional Giemsa staining, C-banding and staining of the nucleolus organizer region (NOR). The nuclear DNA content and seroproteins were also analyzed to characterize these species, and verify the possibility of hybridization between them. Karyotypes and cytogenetic markers were essentially equal on the basis of the methods used. The DNA nuclear content found was 6.25 ± 0.30 pg/DNA in Bufo ictericus; 7.57 ± 0.40 pg/DNA in Bufo paracnemis and 7.04 ± 0.29 pg/DNA in the intermediate subgroup. Eletrophoresis of total blood serum in Bufo ictericus, Bufo paracnemis and the intermediate specimens revealed a remarkable difference in the patterns of the protein bands whose molecular weight corresponded to that of albumin. While the parental species presented two different bands, the intermediate form presented 4. However, only three of these bands were seen in each specimen. The results obtained pointed to a high probability for natural hybridization between Bufo ictericus and Bufo paracnemis in the site and specimens studied.

Infestação de Amblyomma rotundatum (Koch) (Acari, Ixodidae) em sapos Bufo ictericus (Spix) (Amphibia, Bufonidae): novo registro de hospedeiro

Bufo ictericus Spix, 1824 toad population (N = 125) foraging in lighted areas in the Corupa Municipality, Santa Catarina State, was surveyed to evaluate the prevalence (percentage of infestation) and the mean infestation intensity of Amblyomma rotundatum Koch, 1844 (Acari, Ixodidae) ticks. The prevalence was of 19.2% and the mean infestation intensity was 7.4 ticks per infested toad. For the first time B. ictericus as host of A. rotundatum is reported.

Bidder's organ of Bufo ictericus: a light and electron microscopy analysis

Male toads of the Bufonidae Family have rudimentary ovaries designated Bidder's organs, and if the testes are removed this organ develops into a functional ovary, representing a morphological strategy for the reproduction of the species. The Bidder's organ of Bufo ictericus was examined using routine and histochemical techniques by light microscopy and transmission electron microscopy. Each Bidder's organ presented a typical ovarian morphology, being composed of a cortex and a medulla. Bidderian follicles in different stages of development were visualized in the cortex, where they are better developed. The germ cells exhibit a large oocyte with a round-shaped nucleus. The Bidderian follicles are supported by a loose net of reticular fibers. In the medullar region, collagen fibers were immersed in the matrix rich in blood vessels that also contained a small quantity of neutral glycoproteins rich in hexose and/or sialic acid and carboxylated polymers with a characteristic distribution of glycosaminoglycans. The oocyte and the follicular cells were separated by a narrow space containing microvilli. The oocyte exhibit a well developed smooth endoplasmic reticulum, a poorly developed Golgi apparatus, and occasional lysosomes. Concentric cisternal complexes are often visualized; however, their morphological significance remains unclear. The peroxisomes display a fine granular matrix without a crystalline core, with a weak 3,3′-diaminobenzidine-reaction. Intimate association between peroxisomes, peroxisomes and lipid inclusions was observed in the oocyte, suggesting its participation in yolk metabolism.

MITOCHONDRIAL KINETICS DURING AMPHIBIAN ERYTHROPOIESIS RELATED TO HAEME SYNTHESIS

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation–reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.

Comparative study of immature erythroid cells of the diploid Bufo ictericus and the tetraploid Odontophrynus americanus (Amphibia, Anura): ultrastructural cytochemical detection of nucleic acids and polysaccharides, and mapping of the element phosphorus

Comparative study of immature erythroid cells of the diploid Bufo ictericus and the tetraploid Odontophrynus americanus (Amphibia, Anura): ultrastructural cytochemical detection of nucleic acids and polysaccharides, and mapping of the element phosphorus

Morphology of the large granular alveoli of the parotoid glands in toad ( Bufo ictericus) before and after compression

The parotoid glands of toads are constituted by an accumulation of venom-producing granular alveoli. Parotoid glands of Bufo ictericus were manually compressed and immediately excised. Normal glands (not submitted to compression) were also excised and processed for histological and ultrastructural observations. The glandular body of large granular alveoli in parotoid glands is formed by a myoepithelial layer, internally covered by a syncytial secretory layer. In the normal parotoid gland this layer is very electron dense, thus preventing clear visualization of organelles. Large secretion granules are located in the main secretion mass, near the syncytium. In the compressed parotoid gland, the myoepithelium shows a number of alterations, including presence of myelin figures and foci of degradation in the cytoplasm. The organelles of the syncytial cytoplasm are easily observed. The mitochondria are swollen; the cristae are shortened or absent. Syncytial secretion granules and lengthened structures linked to the syncytial matrix are to be seen. A large amount of smooth endoplasmic reticulum is present, while the rough endoplasmic reticulum is reduced. This large quantity of smooth endoplasmic reticulum suggests enhanced synthesis of the cardiotoxic steroids of the venom. Alterations in the mitochondria and the presence of myelin figures and degenerative foci in the cytoplasm may probably be regarded as consequences of the cellular injury provoked by compression.

Hydrins, hydroosmotic neurohypophysial peptides: osmoregulatory adaptation in amphibians through vasotocin precursor processing.

From neurointermediate pituitary glands of Xenopus laevis and Rana esculenta, previously unreported peptides termed hydrins, active on water permeability of frog urinary bladder and frog skin (Brunn or "water-balance" effect), have been isolated and sequenced. These peptides seem to be derived from the pro-vasotocin-neurophysin precursor. Hydrin 1, found in Xenopus, has been identified as vasotocin C-terminally extended with the Gly-Lys-Arg sequence; hydrin 2, found in Rana, has been identified as vasotocin C-terminally extended with glycine. Hydrin 2 has been detected in several Ranidae (R. esculenta, Rana temporaria, Rana pipiens) and Bufonidae (Bufo bufo, Bufo ictericus) and appears to have a large distribution in terrestrial or semiaquatic anurans. Hydrins, in contrast to vasotocin, are not active on rat uterus or rat blood pressure. They are absent from other vasotocin-bearers such as birds and could be involved specifically in water-electrolyte regulation of amphibians.

The action of parotoid venom on the heart of the toad ( Bufo Ictericus Ictericus SPIX 1824) and its effects on the inhibition caused by vagal stimulation

1. The administration of crude venom of the parotoid glands of the toad Bufo ictericus ictericus to the in situ (via abdominal vein) or isolated heart of this anuran causes both chronotropic and inotropic effects. 2. While under action of parotoid venom, the heart of the animal is insensitive to vagus nerve stimulation. 3. This blocking of vagal action is dose dependent and it is suggested that it results from a functional antagonism between the venom constituents and the acetylcholine liberated by the nerve endings on stimulation. 4. The venom constituents probably involved in this antagonism are catecholamines (adrenaline and noradrenaline), tryptamine derivatives (serotonin and bufotenidin) and genins (bufagin and bufotoxin), possibly also ATP. 5. Adrenaline, noradrenaline and serotonin, or a mixture of the three, mimic, at least partially, the blocking of vagal action caused by crude venom. 6. The blocking action of crude venom can be prevented by previously or simultaneously adding acetylcholine to the infused crude venom. This prevention is dose dependent. 7. The blocking action persists in the boiled venom and in the material dialysed from crude venom.