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Comparative study of vitellogenesis in the anuran amphibians Ceratophrys cranwelli (Leptodactilidae) and Bufo arenarum (Bufonidae).

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.

Animals↗

Vasculature of the parotoid glands of four species of toads (bufonidae: bufo).

The parotoid glands of toads (Bufonidae) consist of large aggregations of granular glands located between the otic region of the skull and the scapular region. To determine the circulatory pattern of these glands, we perfused the vascular systems of Bufo alvarius, B. marinus, B. terrestris, and B. valliceps with either India ink or Microfil, a fine latex. The perfused glands were studied by gross dissection, microscopic examination, and histology. The vascular patterns of the parotoid glands were compared to the arrangement of vessels in the dorsal skin of Rana sphenocephala (Ranidae), a frog that lacks parotoid glands. The parotoid glands of the four species of toads are supplied with blood by the lateral and dorsal cutaneous arteries and are drained by one or more branches of the internal jugular vein. The dorsal cutaneous artery supplies most of the blood to the parotoid glands in B. terrestris and B. valliceps. In B. alvarius and B. marinus, both the lateral and dorsal cutaneous arteries serve major roles in the blood supply of the glands. These patterns of blood flow have not been described previously for parotoid glands and conflict with earlier accounts for B. alvarius and B. marinus. The arteries and veins associated with the parotoid glands of toads are present in R. sphenocephala, but are arranged differently. In R. sphenocephala, the lateral cutaneous artery supplies the dorsal and lateral skin posterior to the shoulder region, whereas the dorsal cutaneous artery supplies the skin of the shoulder region. In toads, both the lateral and dorsal cutaneous arteries supply the skin of the shoulder region and ramify into subcutaneous capillaries that surround the secretory units of the parotoid glands. Extensive vasculature presumably is important for delivering cholesterol and other precursor molecules to the parotoid glands, where those compounds are converted into toxins.

Animals↗

Molecular phylogeny and biogeography of West Indian toads (Anura: Bufonidae).

Toads of the Bufo peltocephalus Group (Anura: Bufonidae) occur throughout the Greater Antilles (Cuba to the Virgin Islands), a geographic region of relatively high endemicity. Previous morphological and immunological studies suggested that the West Indian toads are a monophyletic lineage derived from Neotropical Bufo but were unable to clarify relationships within the group. We examined the evolutionary relationships and biogeography of this group of frogs by collecting approximately 2 kb of mitochondrial DNA sequence data from eight West Indian species and selected non-West Indian species from the New World and the Old World. Our analyses support the monophyly of native West Indian toads and a New World origin for the group. Relationships among the West Indian species are less certain, but a Cuban lineage is defined in most analyses.

Animals↗

Occurrence of skin alkaloids in non-dendrobatid frogs from Brazil (Bufonidae), Australia (Myobatrachidae) and Madagascar (Mantellinae).

Several taxa of small frogs from the southern hemisphere contain alkaloids similar or identical to compounds previously known only from neotropical poison frogs of the family Dendrobatidae. Skin of the Brazilian toad Melanophryniscus moreirae (family Bufonidae) contains a new alkaloid 8-hydroxy-8-methyl-6-(5'-hydroxy-2'-methyl-hexylidene)-1-azabicycl o-[4.3.0] nonane (C16H29NO2), which is designated pumiliotoxin 267C. Such a structure is typical of the pumiliotoxin-A class of dendrobatid alkaloids. Melanophyryniscus moreirae contains smaller quantities of an alkaloid (C19H33NO3) identical in chromatographic and mass spectral properties to the dendrobatid alkaloid allopumiliotoxin 323B. Allopumiliotoxin 323B and an isomer of 267C occur with unidentified alkaloids in skin of the Australian frog Pseudophryne semimarmorata (family Myobatrachidae) and also in the skin of the Madagascan frog Mantella aurantiaca (family Ranidae, subfamily Mantellinae). In addition to new compounds, Mantella aurantiaca and M. madagascariensis also contain other alkaloids (e.g. histrionicotoxin and pumiliotoxin B) that were known previously only in dendrobatid frogs. Such alkaloids have not been detected in a phylogenetically wide array of other anuran amphibians, and the dendrobatid alkaloids thus become an evolutionary enigma. Certain of these compounds may have arisen convergently from new biosynthetic pathways in several families of frogs, or these alkaloids may represent parallel expression of shared-primitive pathways that are unexpressed or lost in related frogs.

Alkaloids↗

Lack of bufadienolides in the skin secretion of red bellied toads, Melanophryniscus spp. (Anura, Bufonidae), from Uruguay.

The South-American red bellied toads (Melanophryniscus spp.) belonging to the Bufonidae family contain toxic alkaloids in their skin, predominantly of the pumiliotoxin group. Whole animal methanolic extracts of individual specimens of three species (Melanophryniscus atroluteus, M. devincenzii, and M. montevidensis) were analyzed for the presence of toad specific bufadienolides and indolalkylamines (serotonin derivatives) by HPLC-electrospray (ESI)-MS-TOF. No bufadienolides, but few bufotenines, mainly dehydrobufotenine, were detected in the extracts in variable amounts. The concentration of the dehydrobufotenine in the extracts seems to be species specific. Whereas M. atroluteus and M. montevidensis contain very low or trace amounts, M. devincenzii specimens exhibit high concentrations of this indolalkylamine. In comparison, analysis of extracts from Bufo arenarum (Uruguay) and from B. bufo (Germany) confirmed the presence of bufadienolides as well as of bufotenine derivatives. Tadpoles of both species exhibited a different pattern: extracts from B. arenarum tadpoles contained only dehydrobufotenine, but those from B. bufo tadpoles bufotoxin and two alkylamines. Melanophryniscus toads appear not to be able to compensate the high variability of toxic skin alkaloids by producing defensive bufadienolides.

Amphibian Venoms↗

Serum protein profile and blood cell counts in adult toads Bufo arenarum(Amphibia: Anura: Bufonidae): effects of sublethal lead acetate.

Lead is a multiple-source pollutant, well known for its toxicity, of great risk both for the environment and human health. The main target organs of lead are the hematopoietic, nervous, and renal systems; there are also reports in support of its impairment effects on the reproductive and immune systems. It is well known that most of the metal is accumulated in the blood cells and that many of the deleterious effects are related to its circulating concentrations. These adverse effects have been described not only in humans but also in a number of other vertebrates such as fish and birds. The purpose of the present work was to evaluate the effects of weekly administration of sublethal Pb (as acetate, 50 mg x kg(-1)) during 6 weeks on the profile of the serum proteins and blood cell counts of the adult South American toad, Bufo arenarum (Anura: Bufonidae). The electrophoretic patterns of serum proteins pointed out the presence of four fractions; the metal provoked a significant decrease in both total proteins and albumin fraction; among the globulin fractions, the G3 resulted augmented. These findings may be related to the impact of lead on the toads' hepatic cells and immune system. The number of total red blood cells (RBC) showed a tendency to decrease after the injections of the metal, whereas the number of white blood cells (WBC) increased significantly; the differential leukocyte counts showed a statistically significant increase in the absolute number and in the relative percentage of blast-like cells. The decrease in RBC was attributed to the negative impact of the metals on the hemoglobin synthesis. The increasing of the WBC counts may be interpreted as a consequence of the induction of proliferation of pluripotential hematopoietic cells.

Animals↗

Serous gland dimorphism in the skin of Melanophryniscus stelzneri (Anura: Bufonidae).

Two serous gland types (I and II) in the skin of the Argentine toad Melanophryniscus stelzneri were discovered using light and electron microscopy. Glands of the two types differ in several traits: features of the products (both mature and immature), organelles involved in biosynthesis, and paths of serous maturation. No consistent differences, however, were detected between the myoepithelial sheaths encircling the secretory units. Type I glands manufacture vesicles containing a single dense body with a repeating substructure and conform to the fundamental secretory line of bufonid skin, a secretory line involved in biosynthesis of steroids. Type II glands synthesize granules of varying densities and seem to belong to a line of glands that secrete proteinaceous products. The occurrence of the two serous gland types in Melanophryniscus stelzneri is discussed in a comparison with current literature on the morphofunctional characteristics of anuran poison glands, which perform both regulative and defensive roles. It is suggested that di- or polymorphism in serous glands is an adaptive trait that allows differential release of active molecules on the body surface.

Animals↗

Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae).

This study deals primarily with the morphology and ultrastructure of the pronephros in the green toad Bufo viridis during prometamorphosis when the pronephros and the developing mesonephros function simultaneously. Furthermore, the mesonephros was studied during pro- and postmetamorphosis with emphasis on the distal segments of the nephron. The paired kidneys consist of two cranial pronephroi immediately behind the gill region and two more caudal elongated mesonephroi. Each pronephros consists of a single convoluted tubule which opens into the coelom via three nephrostomes. This tubule is divided into three ciliated tubules, three proximal tubule branches, a common proximal tubule and a distal tubule, which in turn continues into the nephric duct. No intermediate segment is present. The length of the pronephric tubule is 12 mm, including the three branches of the ciliated tubules and proximal tubules. Primary urine is formed upon filtration from an external glomerulus, which is a convoluted capillary lined by podocytes, a specialization of the coelomic epithelium. From the coelom the filtrate is swept into the ciliated tubules. In the collecting duct system of the developing mesonephric nephron epithelial cells with conspicuous, apical osmiophilic granules appear in larvae of 9-10 mm. Heterocellularity of mixed intercalated (mitochondria rich) cells and principal cells is observed in the collecting duct system and nephric duct from a larval body length of 14 mm. As the proliferation of mitochondria-rich cells proceeds, the osmiophilic granules disappear and are completely absent from the adult amphibian mesonephros.

Animals↗

The anatomy of the parotoid gland in Bufonidae with some histochemical findings. II. Bufo alvarius.

The gross and microscopic anatomy of the venom producing parotoid glands of Bufo alvarius has been studied by light and electron microscopy. Histochemical reactions for the presence of venom constituents and of components in biochemical pathways in the synthesis and release of venom were performed. The gland is composed of numerous lobules. Each lobule is an individual unit with a lumen surrounded by a double cell layer. Microvilli of the outer layer interdigitate with microvilli of the inner layer. Cells of the outer layer resemble smooth muscle cells, are rich in adenosine triphosphatase and glucose-6-phosphatase, and contain numerous pinocytotic vesicles, glycogen granules and various organelles. These organelles include "crystalloids" of what seem to be highly organized agranular reticulum. These outer layer cells probably function in some aspects of venom synthesis, active cellular transport and contraction in the discharge of the secretory product. The inner cell layer demonstrates a positive chromaffin reaction, contains steroid material, various organelles, some pinocytotic vesicles and glycogen granules, and appears devoid of a plasmalemma on its inner surface. This layer is probably involved in venom formation and release via an apocrine type of secretion. Bufo alvarius parotid gland shows significant morphological and histochemical differences from that of B. marinus and more nearly resembles a typical steroid producing organ.

Animals↗

Histological examination of the effects of corticosterone in larvae of the western toad, Bufo boreas (Anura: Bufonidae), and the Oriental fire-bellied toad, Bombina orientalis (Anura: Discoglossidae).

The effects of corticosterone (CORT)-treatment on various tissues were examined in two species of anuran larvae, the discoglossid Bombina orientalis, and the bufonid Bufo boreas. Corticosterone was administered directly into aquarium water for 15 days. After treatment, histological analyses were conducted on skin, gut, spleen, thymus, and neural and muscle tissue. Corticosterone treatment prevented sloughing of the skin, which resulted in a build-up of stratum corneum, and inhibited the development of gland nests and the subsequent formation of dermal granular and mucous glands in both species. Corticosterone treatment also decreased epithelial folding in the gut and caused vesiculation of the gut epithelial cells. The thymus of CORT-treated animals was significantly reduced in size (P < .05) and cell density (P < .05), and the spleen of CORT-treated animals was completely involuted. The brain and pituitary of CORT-treated animals had a decreased cell density (P < .05) and many pyknotic cells. An examination of muscle revealed that muscle fibers of CORT-treated animals had a decreased cross-sectional area (P < .05). The dose of CORT used (1.1 microM) was within the range used in other studies in the literature and resulted in tissue levels within the range experienced by larvae at metamorphic climax. Thus, this study is appropriate to address the histological effects of CORT in experimental manipulations and the role of increasing CORT at metamorphic climax. The data suggest that increasing endogenous CORT at metamorphosis may be involved in degeneration of larval tissue, prior to regeneration, which is stimulated by thyroid hormones.

Animals↗

The ontogeny of kin recognition in tadpoles of the toad Bufo melanostictus (Anura; bufonidae).

The ontogeny of kin recognition and influence of social environment on the development of kin recognition behaviour was experimentally investigated in tadpoles of Bufo melanostictus that lived in aggregations and showed low larval dispersion. Embryos and tadpoles of the toad were reared as (i) kin only, (ii) with kin and non-kin (separated by a mesh screen), and (iii) in isolation. They were tested for the ability to discriminate between (i) familiar siblings and unfamiliar non-siblings, (ii) familiar siblings and familiar non-siblings and, (iii) unfamiliar siblings and unfamiliar non-siblings. All tadpoles were fed on boiled spinach before conducting trials. Preference of test tadpoles to associate near the end compartments whether empty or containing members of specific stimulus groups was assessed using a rectangular choice tank. When tested in tanks with empty end compartments, the test tadpoles showed random distribution and thus no bias for the apparatus or the procedure. In the presence of kin/non-kin in the end compartments a significantly greater number of test tadpoles spent the majority of the time near familiar or unfamiliar kin rather than near familiar or unfamiliar non-kin. Kin discrimination ability persisted throughout larval development. Familiarity with siblings is not required for discriminating kin from non-kin, and kin discrimination ability is not modified following exposure to non-kin. Also, involvement of dietary cues is unlikely to be the prime mechanism of kin recognition in B. melanostictus unlike in some other anurans.

Animals↗

Neuroepithelial bodies in the lung of Melanophryniscus stelzneri stelzneri (Anura, Bufonidae).

Electron microscopy of the lungs of Melanophryniscus stelzneri stelzneri (Anura) revealed the presence of a complex pattern of corpuscular cells (CCs). The respiratory surface over the septa presents small areas where the CCs are grouped forming neuroepithelial bodies (NEBs). These corpuscular structures can also be localized in the inner layer of the lung wall. Although in both cases NEBs protrude slightly into the airway lumen, they are separated from the airway lumen and the basal connective tissue by thin apical and basal cytoplasmic processes of neighbouring pneumocytes. Ultrastructurally, the CCs possess a large nucleus, clear cytoplasm and vesicles of variable morphology and size, containing an electron dense material surrounded by a lucent space in some cases. The size of these dense-core vesicles (DCVs) ranged from 40 to 100 nm. The NEBs are associated with afferent and efferent terminal nerves. These types of nerve endings are located between the CCs and in the basal part of the NEBs. The location of the NEBs in strategic positions on the septa and in the wall of the lung, the presence of the DCVs in the basolateral region of the CCs, the occurrence of synaptic contacts between nerve endings and the CCs and the occurrence of capillaries close to the NEBs, suggest a receptosecretory function for NEBs in the lung of M.s. stelzneri.

Animals↗

Further report of the occurrence of tetrodotoxin in Atelopus species (family: Bufonidae).

In alcoholic extracts from museum samples of the toads Atelopus subornatus and A. peruensis tetrodotoxin and its analogues, 4-epitetrodotoxin and 4,9-anhydrotetrodotoxin were detected by fluorometric HPLC analysis, extending the list of amphibians containing this toxin. However, the toxin was not detected in samples of Melanophryniscus stelzneri, Dendrophryniscus minutus and Oreophrynella sp.

Animals↗

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.

Animals↗