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Corythomantis greeningi is a tree-frog endemic of the Brazilian semi-arid (Caatinga), mainly characterized by the flat, mineralized and spiny head, which is associated with phragmotic habits. It is already known that the skin secretion of this amphibian from both head and body is quite toxic and is used as an efficient chemical defence against predators. However, the biochemical characteristics and pharmacological effects of this secretion are still very little studied. We have tested the crude skin secretion, as well as the ten major fractions obtained by RP-HPLC for nociceptive and edema activity and for in vitro cytotoxicity using murine models. SDS-PAGE analyses demonstrated that the majority of proteins ranging through the gel lie between 55 and 30 kDa. LC-MS analysis showed multiple low molecular mass molecules (200–500 Da), which are consistent with masses of alkaloids and steroids. The crude skin secretion was able to induce fast and persistent edema accompanied by intense dose-dependent nociception. From the 10 tested fractions, five induced both edema and nociception, six fractions were able to induce only edema (80–170% control), and seven fractions induced only nociception (15–30 times compared to control). In addition, inhibition of cell growth (IC50) was demonstrated in murine fibroblasts and melanoma cells. From the data obtained, we confirmed that the skin secretion of C. greeningi is very toxic and is rich in compounds able to directly provoke local inflammation and nociception. Such characteristics are important as part of the chemical defensive repertory of this species.

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The rudimentary characteristic of the eyes of fossorial animals raises some questions regarding its evolution and functionality. Would these eyes result from atrophy or from stagnated development? How would its visual function work? Anatomical investigations of these organs are the fundamental preamble to answer those questions, which are still little explored by the literature. in this article we have studied anatomical aspects of the eyes of three species of fossorial reptiles, within the suborder Amphisbaena (Amphisbaena alba, Amphisbaena mertensi, Leposternon infraorbitale), as well as a species within the ophidian suborder (Typhlops brongersmianus). the minuscule eyes (1-2 mm diameter) were visualized through a scale, a translucent area which corresponds to the spectacle. This spectacle is a thinner and transparent scale, covering a conjunctival sac. the retrobulbar space was filled with the harderian gland. the eyes of Typhlops presented an oval shape, whereas Amphisbaena specimens presented cup-shaped eyes. in Amphisbaenian sclera is comprised of cartilage, while the thin sclera of Typhlops consists of connective tissue and striated muscle fibers. the retina presented all the typical layers found in vertebrates, regardless the species. the characteristics involved in the fossil adaptation of these species include: reduced size of the eyeball, rudimentary cornea, absence of the anterior chamber, presence of a complex iris-ciliary body, and lens with amorphous nucleate cells. the analysis of the eye morphology of these animals suggests that there might be a specific function concerning light perception. Anat Rec, 293:351-357, 2010. (C) 2009 Wiley-Liss, Inc. Universidade Federal de São Paulo, Disciplina Anat Descrit & Topog, Dept Morfol & Genet, Escola Paulista Med, BR-04023900 São Paulo, Brazil Inst Butantan, Lab Biol Celular, São Paulo, Brazil Universidade Federal de São Paulo, Disciplina Anat Descrit & Topog, Dept Morfol & Genet, Escola Paulista Med, BR-04023900 São Paulo, Brazil Web of Science

Centipedes are widely distributed over all the continents. As they are well adapted to urban areas they can often cause accidents to humans by injecting venom produced in the glands located inside their maxillipeds. The fine morphology of the centipede venom glands is practically unknown. This present study is the first comparative report on the histology, histochemistry and ultrastructure of the venom glands of the centipede species responsible for the majority of accidents to humans in Brazil: Scolopendra viridicornis, Cryptops iheringi and Otostigmus pradoi. In all species the glands are basically composed of columnar secretory cells radially disposed side by side, individually opening through pores in a central chitinous duct. Each secretory cell is covered by striated muscular fibres. The secretion has the form of small PAS positive granules and large hyaline secretory bromophenol blue positive vacuoles, indicating the presence of neutral polysaccharides and protein. The secretion is conducted through the secretory cell necks to the pores, which open into the central chitinous duct. The results indicate a great similarity both in morphology and primary chemical composition of the venom among the studied species, except for the size of the glands, which is proportional to the body dimensions of each species.

Amphibian skin secretions are a source of potential new drugs with medical and biotechnological applications. Rich in peptides produced by holocrine-type serous glands in the integument, these secretions play different roles, either in the regulation of physiological skin functions or in the defense against predators or microorganisms. The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of Phyllomedusa nordestina. In order to achieve this goal, the crude skin secretion of this frog was pre-fractionated by solid phase extraction and separated by reversed-phase chromatography. The fractions were screened for low-molecular-mass peptides and sequenced by mass spectrometry. It was possible to identify three novel bradykinin-related peptides, namely: KPLWRL-NH2 (Pnor 3), RPLSWLPK (Pnor 5) and VPPKGVSM (Pnor 7) presenting vascular activities as assessed by intravital microscopy. Pnor 3 and Pnor 7 were able to induce vasodilation. On the other hand, Pnor 5 was a potent vasoconstrictor. These effects were reproduced by their synthetic analogues.

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.

Amphibian skin secretions contain several bioactive compounds such as biogenic amines, alkaloids, steroids, proteins and peptides; being peptides a continuously growing field of interest. This work aims to describe the main physiopathological properties of the tree frog Phyllomedusa hypochondrialis skin secretion, obtained by manual stimulation of the dorsal skin surface. Intravenous skin secretion administration provoked lethal effect in mice after 5 min. Low doses induced significant systemic and local effects like edema and nociception in mice and topic administration induced myonecrosis in the endothelium of cremaster mice. The presence of phospholipase A2 activity, proteolytic activity and creatine kinase activity (in the plasma of treated mice) are reported and are very likely to be related to the physiopathological (edematic and myotoxic) activities observed. These data provide in vivo evidence of the complex toxic effects of the P. hypochondrialis skin secretion as well as possible mechanisms of action for these effects.

The antiparasitic and antifungal activities of nine amphibian skin secretions were studied in different in vitro models. Seven secretions presented a considerable antiprotozoan activity and one showed promising results against Candida sp . These results can be the basis for the development of new drugs, especially for neglected parasitic diseases.

Many lizards have epidermal glands in the cloacal or femoral region with semiochemical func- tion related to sexual behavior and/or territorial demar- cation. Externally, these glands are recognized as a row of pores, opening individually in the center of a modified scale. In many species the pores are used as systematic characters. They form a glandular cord or, in some spe- cies, a row of glandular beads below the dermis, and are connected to the exterior through the ducts, which con- tinuously liberate a solid secretion. Dead cells, desqua- mated from the secretory epithelium, constitute the secretion, known as ''a secretion plug.'' The present work focusses on the morphology of the femoral glands of the teiid lizard Ameiva ameiva, correlating it to the way in which the secretion is deposited in the environment. The results here obtained are compared to those available for other lizards and amphisbaenians. We observed that the diameter of the glandular pores did not show signifi- cant differences between males and females. The glands comprise germinative and secretory cells, which pass through at least three stages of differentiation, during which an accumulation of cytoplasmic granules, with a glycoprotein content, occurs. The cells eventually die and desquamate from the secretory epithelium, forming a secretory plug mostly constituted by juxtaposed non- fragmented secretory cells. Because of the arrangement of the rosette-like scales surrounding the femoral pores, we suggest that when the animal is in a resting position, with its femoral regions touching the ground, these scales may be involved in the breakage of their respec- tive plugs, depositing tiny portions on the substrate. In this manner, it seems that the method for signal disper- sion in this species involves specifically adapted struc- tures and does not simply involve the chance breakage of the plug, as the gland secretes it. Signal dispersion must also be intimately associated with the animal's movement within its territory. J. Morphol. 00:000-000, 2007. 2007 Wiley-Liss, Inc.

El presente estudio consiste en el establecimiento de las competencias para el personal del Departamento de Experticia Vehicular Automotriz, DEVA que forma parte de la División Policía Técnica y Científica, DPTC adscrita a la Policía Nacional Civil de El Salvador, institución que se encuentra en un proceso de implementación de un Sistema Integrado de Gestión de la Calidad basado en normas internacionales de la calidad ISO/IEC 17025:2005 e ISO 9001:2008.

We investigated the morphology of the skin and the biochemistry of the lipids in the skin secretion of Bokermannohyla alvarengai, a montane treefrog that is known to bask regularly, motionless in full sunlight for extended periods of time. Our primary goal was to identify structural and biochemical modifications that might assist this frog species to accommodate the conflicting demands for heat exchange and water balance while basking. The modulation of heat exchange in basking B. alvarengai involves changes in skin coloration. We found that this response was supported by a prominent monolayer of large iridophores, whose light reflectance property is adjusted by the response of intervening melanophores. Mucosubstances and lipid compounds, mainly consisted of saturated fatty acids and presumably secreted from granular glands, were detected on the skin of B. alvarengai. These compounds formed an extra-epidermal layer over the animal's dorsal surface that might assist in the prevention of excessive water loss through evaporation. Additionally, we found well-developed skin folds at the ventral region of the frogs that lead to an increment of surface area. This feature combined with the extensive hypervascularization, also noticed for the skin of B. alvarengai, may play an important role in water reabsorption. The suite of structural and biochemical modifications identified for the integument of B. alvarengai seems to conjugate aspects relevant to both, heat exchange and water balance, allowing for this species to explore basking as an efficient thermoregulatory strategy.

A new species of siphonopid caecilian, Microcaecilia butantan sp. nov., is described based on four specimens from Belterra, in the State of Pará, Brazil. The new species differs from all other Microcaecilia in having a combination of more than 135 primary annuli and long premaxillary-maxillary tooth series that extend posteriorly beyond the choanae. Some specimens were dug from soil in a cupuaçu (Theobroma grandiflorum) plantation suggesting that this form of agriculture provides an environment suitable for at least some caecilians.

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by 'de novo' mass spectrometry and grouped into Dermaseptins (DPh-1).

A highly unusual form of parental care has been identified in a mysterious group of tropical vertebrates. In the oviparous amphibian Boulengerula taitanus, the young use specialized teeth to peel and eat the outer layer of their mother's modified skin, thereby ensuring a good start in life. This bizarre behaviour can be seen as an evolutionary link paving the way for fetal feeding on the oviduct lining of related viviparous amphibians. The movies, in the Supplementary Information on http://www.nature.com , are pretty gory. Although the initial growth and development of most multicellular animals depends on the provision of yolk, there are many varied contrivances by which animals provide additional or alternative investment in their offspring1. Providing offspring with additional nutrition should be favoured by natural selection when the consequent increased fitness of the young offsets any corresponding reduction in fecundity2. Alternative forms of nutrition may allow parents to delay and potentially redirect their investment. Here we report a remarkable form of parental care and mechanism of parent–offspring nutrient transfer in a caecilian amphibian. Boulengerula taitanus is a direct-developing, oviparous caecilian3, the skin of which is transformed in brooding females to provide a rich supply of nutrients for the developing offspring. Young animals are equipped with a specialized dentition, which they use to peel and eat the outer layer of their mother's modified skin. This new form of parental care provides a plausible intermediate stage in the evolution of viviparity in caecilians. At independence, offspring of viviparous and of oviparous dermatotrophic caecilians are relatively large despite being provided with relatively little yolk. The specialized dentition of skin-feeding (dermatophagous) caecilians may constitute a preadaptation to the fetal feeding on the oviduct lining of viviparous caecilians.

Amphibian skin is characterized by the presence of mucous glands, related to cutaneous breathing, reproduction and water balance, and granular glands, related to the production of toxins used in defence. In some species the granular glands can form accumulations in certain regions of the body. This is the case for inguinal macroglands of the leptodactylid frog Physalaemus nattereri, where these structures form a pair of black discs associated with deimatic behaviour. The morphology of the inguinal macroglands and their secretion were studied in this species and correlated to deimatic behaviour. The inguinal macroglands are formed from elongated granular glands that, in contrast with the granular glands of the rest of the skin, have small spherical granules with a proteinic content. In the dermis of the whole body, except for the inguinal macroglands and the inguinal region, a well-developed calcified dermal layer is observed. During deimatic behaviour these macroglands discourage a potential predator from attacking, but if visual cues are insufficient and the predator persists in the attack, a toxic secretion is eliminated in its mouth. This elimination is favoured by the absence of a calcified dermal layer in the macroglands, which makes the dermal region softer than the rest of the dorsal skin.

Some anurans have a peculiar casqued head with the skin co-ossified with the underlying bones. This type of skull usually is associated with phragmosis, a protective behaviour in which the animal enters a hole and closes it with the head. Although co-ossification of the head in lissamphibians frequently has been associated with water economy, recent studies of Corythomantis greeningi, a casque-headed tree frog from semi-arid areas in north-eastern Brazil, suggest that cranial co-ossification contributes little to conservation of water in the frog. Instead, during phragmotic behaviour, the co-ossified head protects the animal against predators and indirectly enhances water balance. Thus, the primary role of co-ossification is defence, a hypothesis that is the focus of this study, which describes the morphology of the head of C. greeningi with an emphasis on the co-ossification and the venom glands. We report on behavioural features and on the toxicity of the cutaneous secretion produced by the abundant venom glands that are associated with large spicules on the skull.

The Brazilian tree-frog Corythomantis greeningi inhabits the Caatinga, a semi-arid habitat. This species exhibits putative adaptations to a dry environment, including a flat head, co-ossification of the cranial skin, and phragmotic behaviour (retreat to crevices using the head to seal the entrance). Real frogs were compared with agar models to study water balance under simulated Caatinga conditions and to investigate the ability of C. greeningi to find small water sources. The benefits of skin co-ossification were also studied by comparing agar models with and without nail polish on the head. Tree-frogs repeatedly preferred a humid test tube out of 10 possible choices, and adopted phragmotic behaviour inside it. In both agar models and tree-frogs, total water evaporation increased with body size, but relative water loss was higher in smaller individuals. The presence of nail polish on agar models barely reduced water loss, suggesting that cranial skin co-ossification has a small role in water economy. Although both models and tree-frogs evaporated about 90% more water when exposed than when phragmotic, tree-frogs evaporated about 50% less water than agar models at any test condition. We conclude that reduced skin permeability, phragmotic behaviour and a keen capacity to identify humid crevices, explain the success of this species in a semi-arid habitat.

A comparative approach is potentially useful for understanding the role of mammal innate immunity role in stimulating adaptive immunity as well as the relationship between these two types of immune strategies. Considerable progress has been made in the elucidation of the co-ordinated events involved in plant perception of infection and their mobilisation of defence responses. Although lacking immunoglobulin molecules, circulating cells, and phagocytic processes, plants successfully use pre-formed physical and chemical innate defences, as well as inducible adaptive immune strategies. In the present paper, we review some shared and divergent immune aspects present in both animals and plants.

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella. Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity, Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine. Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action. Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use. Electronic supplementary material The online version of this article (doi:10.1186/1678-9199-20-45) contains supplementary material, which is available to authorized users.

Toads have a pair of parotoid macroglands behind the eyes that secrete poison used in passive defence against predators. These macroglands are composed of juxtaposed alveoli, each one bearing a syncytial gland, all connected to the exterior by ducts. When the parotoids are bitten, the poison is expelled on the predator oral mucosa in the form of jets, causing several pharmacological actions. After poison release, the empty secretory syncytia immediately collapse in the interior of their respective alveoli and gradually start refilling. After parotoid manual compression, simulating a predator's bite, we studied, by means of morphological methods, the replacement of the poison inside the alveoli. The results showed that after compression, a considerable number of alveoli remained intact. In the alveoli that were effectively affected the recovery occurs in different levels, from total to punctual and often restrict to some areas of the syncytia. The severely affected alveoli seem not recover their original functional state. The fact that only a part of the parotoid alveoli is compressed during an attack seems to be crucial for toad survival, since the amphibian, after being bitten by a predator, do not lose all its poison stock, remaining protected in case of new attacks.

The facial pits of rattlesnakes, copperheads, lanceheads, bushmasters and other American and Asian pitvipers (Crotalinae) are highly innervated and densely vascularized infrared (IR) receptor organs. For over a century, studies have focused on a small sample of model species from North America and Asia. Based on an expanded survey of Central and South American crotalines, we report a conspicuous accessory structure composed of well-defined papillae that project from the anterior orbital adnexa. The papillae are continuous with the inner chamber of the IR receptor organ and our histological and ultrastructural data suggest that they possess a well-developed nervous network and extensive vascularization; however, they lack the characteristic IR-sensitive terminal nerve masses found in the IR-receptive pit membrane. The function of the IR receptor organ papillae is unknown.

Some species of anuran amphibians pos- sess a calcified dermal layer (the Eberth-Kastschenko layer) located between the "stratum spongiosum" and the "stratum compactum." This layer consists of calcium phos- phate deposits, proteoglycans, and glycosaminoglycans. Although regarded as a protective layer against desicca- tion, a calcium reservoir, or possibly a remnant of a der- mal skeleton present in anuran ancestors, very little is known about its origin, structure, and function. Thus, we studied the structure and composition of the mineralized dermal layer of Corythomantis greeningi, a peculiar hylid from the Brazilian semiarid region (caatinga), using con- ventional and cryosubstitution methods combined with transmission, scanning, and analytical electron micros- copy. Results show that the dermal layer consists of dense, closely juxtaposed, globular structures. Although the elec- tron opacity of the globules was variable, depending on the type of preparation, crystal-like inclusions were present in all of them, as confirmed by dark field microscopy. Elec- tron probe X-ray microanalysis showed calcium, phospho- rus, and oxygen, and electron diffraction revealed a crys- talline structure comparable to that of a hydroxyapatite. J. Morphol. 248:56 - 63, 2001. © 2001 Wiley-Liss, Inc.

Opossums are considered natural predators of snakes and possess resistance to the venom of some viperids. The resistance of Didelphis to Crotalus venom has been demonstrated through biochemical and immunological assays. However, systematic observations on the behavior of adult Didelphis preying on venomous snakes have never been conducted. In this study the predatory and defensive behaviors of Didelphis marsupialis and Crotalus durissus, respectively, were analyzed in captivity. Defensive strategies showed by snakes included immobility, flight attempts, coiling, cocking, rattling, and counterattack with strikes and bites. The most common defensive behavior of the rattlesnakes was immobility. The way the opossums attacked was classified in three categories, depending on the defensive reactions presented by the snakes. On all occasions when the opossums were bitten, the injection of venom apparently did not affect the predation. The great ability in capturing and handling Crotalus durissus together with the apparent great tolerance to the venom shown by Didelphis marsupialis when preying on these snakes confirms the existent biochemical and immunological data about the resistance of opossums to crotalic venoms. In this way our data strongly reinforce the supposition that this species is an effective snake predator in nature.

The femoral or cloacal region of many species of lizards and amphisbaenians exhibits epidermal glands. The pores of these glands are plugged with holocrine solid secretions that serve as semiochemical sources. Many authors assume that these glands are mainly associated with reproduction and demarcation of territory. The structure of precloacal glands in Amphisbaena alba was previously studied by Antoniazzi et al. (Zoomorphology 113:199-203, 1993; J. Morphol. 221:101-109, 1994). These authors suggested that as the animal moves inside tunnels, the secretion plugs are abraded against the substrate, releasing a secretion trail. Some aspects of the plug were difficult to interpret in fine sections due to the dense and brittle nature of the plug. The morphology of the trail, and the manner of deposition on the substrate, have never been reported. This study presents a primarily scanning electron microscopic description of A. alba precloacal glands and of the secretion plugs. It also demonstrates experimentally the formation of the trail and its fine morphology. The results show that when the plugs scrape against the substrate, their constitution helps them to fragment into tiny pieces, which are spread on the ground, thus forming a trail. Each one of the fragments corresponds to a secretion granule of the precloacal gland's secretory cells. In this way, the trail might have an extensive area for volatilization of semiochemicals, constituting an efficient means of intraspecific communication inside the tunnels.

The Gymnophiona are the least well known amphibian order, an unfortunate situation because these animals can clarify many aspects of amphibian natural history, vertebrate evolution, and the physiology of viviparity and fossorial life. Our aims are to assemble a number of topics about the current knowledge of Caecilian physiology and morphology, and to integrate our own observations on the morpho-physiology of a Brazilian species (Siphonops annulatus). We start with a brief discussion of the evolution of the group and some general aspects of its ecology and behavior. Then we cover the external anatomy, emphasizing morpho-physiological aspects of skeleton, skull, teeth and integument. Then we cover ventilation and gas exchange, blood and circulation, and some aspects of water exchange and sensory organs, stress the fascinating aspects of physiology and morphology associated with viviparity, and finish with a brief discussion about Caecilian energetics. We hope that this review will extend the interest in the group and become both a helpful reference and a source of insight for scientists in search of new questions.

Corythomantis greeningi , a casque-headed tree frog, was studied in the field and in captivity. This species belongs to a monotypic genus, and together with other species of anurans, possesses a differentiated head which is flat, very rough and presenting cranial co-ossification with the skin. These are characteristics that are usually associated with phragmotic behaviour which is supposed to have a role in water saving and protection against predators. Observations were focused on the characteristics of the species which make it adapted to life in the Brazilian semi-arid (“caatinga”). Data about the body and external anatomy of the head, integument morphology, life, reproduction, interest for sheltering and defense against desiccation is presented. Finally, it is proposed that adaptation of this species to the dry environment is probably the result of the synergism of a number of different strategies against water loss, including phragmosis.

Amphisbaena alba when in a defensive position adopts a horseshoe posture, raises its head and tail and opens its mouth. The tail is rigid; the dorsal surface often has non-pigmented areas which resemble scars. Leposternon microcephalum lives sympatrically with A. alba; it has a soft tail which does not appear to be involved in defensive display. Both species have been observed in captivity. The height to which A. alba raises head and tail during defensive display was measured. Morphometrics and morphology of the integument structure in both species were studied in the body and in the tail from the collagen bundles organization viewpoint. The results reveal a mechanical resistance of the tail in A. alba, but not in L. microcephalum. It is possible that this structure, formed of a dense piece of collagen, serves as a ‘biting block’ ; lesions caused by potential predators could cause no great harm. The skin of the remainder of the body in A. alba (but not in L. microcephalum) is also very resistant; the animal is practically covered by a flexible armour.

Amphibians have many skin poison glands used in passive defense, in which the aggressor causes its own poisoning when biting prey. In some amphibians the skin glands accumulate in certain regions forming macroglands, such as the parotoids of toads. We have discovered that the toad Rhaebo guttatus is able to squirt jets of poison towards the aggressor, contradicting the typical amphibian defense. We studied the R. guttatus chemical defense, comparing it with Rhinella marina, a sympatric species showing typical toad passive defense. We found that only in R. guttatus the parotoid is adhered to the scapula and do not have a calcified dermal layer. In addition, in this species, the plugs obstructing the glandular ducts are more fragile when compared to R. marina. As a consequence, the manual pressure necessary to extract the poison from the parotoid is twice as high in R. marina when compared to that used in R. guttatus. Compared to R. marina, the poison of R. guttatus is less lethal, induces edema and provokes nociception four times more intense. We concluded that the ability of R. guttatus to voluntary squirt poison is directly related to its stereotyped defensive behavior, together with the peculiar morphological characteristics of its parotoids. Since R. guttatus poison is practically not lethal, it is possibly directed to predators' learning, causing disturbing effects such as pain and edema. The unique mechanism of defense of R. guttatus may mistakenly justify the popular myth that toads, in general, squirt poison into people's eyes.

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25-0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50=34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX(®) Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.

The parotoid macroglands of toads (bufonids) and leaf frogs (hylids) are used in passive defence against predators. The parotoids release poison when the amphibian is bitten by a predator. Despite the apparent similarity, the anatomical and histological structure of these macroglands in hylids is poorly studied when compared with those of bufonids. In this paper, we focused on the morphology of the macroglands of P. distincta, a leaf frog endemic to the Brazilian Atlantic rainforest, comparing their structure with those of bufonids. In addition, we compared the macrogland morphology of P. distincta with those from major clades of Phyllomedusa. All results revealed a macrogland morphology in leaf frogs distinct from that of toads, suggesting that the term parotoid should be used only for those of bufonids.

The amphibian order Gymnophiona is poorly known, and studies about their reproduction are mainly comparative and descriptive, focusing on the structure of testes, ovaries and oviducts. However, to understand the reproductive processes, including those of the oviparous dermatophagic species, it is important to know the dynamics of storage and mobilization of energetic substrates to gonads and skin during the reproductive cycle of males and females, as well as the endocrine control associated. For the present study, total lipids and proteins were measured during the annual cycle in the plasma, liver, muscle, testes, ovaries and skin of Siphonops annulatus in captivity. Plasma levels of gonadal steroids (estradiol, testosterone and progesterone) were quantified by radioimmunoassay. Histological analyses of ovaries and testes were performed to classify the maturation stages of the animals during the reproductive cycle. Gonadal maturation in males and females of S. annulatus was accompanied by metabolic changes in reserve tissues, which supported gonadal development and prepared the females' epidermis for skin feeding by the offspring. Even in confinement conditions, females and males synchronized the reproductive period. However, due to the absence of environmental cues in captivity inadequate levels of the hormones responsible for gamete release were triggered, leading to a lack of reproductive success.

Ultrastructural changes of organelles involved in hemoglobin (Hb) biosynthesis were studied in immature peripheral erythrocytes of Amphisbaena alba (Amphisbaenia: Reptilia). Iron uptake (probably via transferrin) and accumulation results in formation of siderosomes, which serve as an iron source for heme biosynthesis. Differentiation of the mitochondrial inner chamber results in a lamellated body, which then produces a lateral expansion; this expansion retains ferruginous compounds and globin polypeptides and, later, folds upon itself to form a prehemosomal vesicle. Vesicle content condenses, and iron in an amorphous state associates with the partially coiled vesicle membrane. Invagination of the membrane gives rise to a prohemosome; narrowing of the broad intralamellar spaces and reduction of overall diameter results in formation of a hemosome. Stages of hemosomegenesis in maturing erythroid cells of Amphisbaena alba are similar to those in other vertebrate groups; the significant difference is the periodical and temporary transverse alignment of Hb molecules in the organelle matrix of some of the hemosomes. Electrophoresis reveals a similar light Hb migration rate pattern in both organellar and cytoplasmic fractions; the cytoplasmic fraction also shows a heavy Hb pattern with a lower migration rate.

Through gradual acquisition of a set of adaptations, both morphofunctional and behavioural, the first amphibians were able to spend longer periods in a terrestrial environment. The multicellular exocrine glands of the skin represent one such adaptation. Two fundamental types of cutaneous glands are to be observed in the amphibians: mucous and granular (serous or venom) glands. The mucous glands produce a mucus which plays a part in a variety of functions: cutaneous respiration, reproduction, thermoregulation and defense. The granular glands produce a toxic or repellent secretion with an effect on various vertebrate species; this is one of the principal elements in amphibian defense. The granular glands are generally made up of a gland alveolus formed by a secretory layer covered externally by a myoepithelial layer. This contractile layer is related with the extrusion of glandular products. Although they are responsible for the production of noxious or toxic substances with a variety of pharmacological effects, the granular glands may also have other functions, so far not recognized. The granular glands in the Gymnophiona and the Urodela are made up of a secretory layer with, in some cases, well-individualized cells. In the course of the secretory cycle, these cells usually lose their limits and turn into a syncytium. The venom of the Gymnophiona is chemically and pharmacologically unknown. Of note in the venom of the Urodela are tetrodotoxin and steroidal alkaloids such as samandarine. In the Anura, the granular glands have a syncytial secretory layer. Depending on the chemical composition of the venom secreted, the organelles of this syncytium vary in type and concentration. In general, the venoms contain peptides, guanidine derivatives, biogenic amines, steroids, and alkaloids. In terms of pharmacological effects, these substances are cardiotoxic, haemotoxic, neurotoxic, myotoxic, hypotensive, hypertensive and anaesthetic. In a number of amphibian species, clusters of granular glands are to be observed in certain regions of the body. These are known as macroglands, and may be divided into the following types: parotoid, paracnemid, lumbar, hedonic, and pectoral. They are fundamentally related to reproduction and defense. Parotoid macroglands occur in a number of urodelan and anuran species. They consist principally of a large concentration of granular alveoli, which contain the venomous secretion. In the toads, in general, this secretion basically contains steroids and biogenic amines. In certain species, the venom contains non-bioactive substances such as glycosaminoglycans and catalase; the functional significance of these is as yet unknown. The paracnemid macroglands are cutaneous gland clusters situated around the level of the tibia in certain anurans. In Bufo, these glands are histologically similar to the parotoid macroglands. In some leptodactylids, the gland clusters are situated immediately posterior to the sacrum, and are known as lumbar or inguinal macroglands. Their function is supposedly defense. Their form, pigmentation and position in the body, furthermore, makes them look like two large eyes; this suggests a role in the intimidation of potential enemies. Substances with an antimicrobial action, such as samandarine, spinaceamine and the magainins were identified in the integument of amphibians. Certain elements of the venom have a cytotoxic effect; they inhibit the cellular respiratory process, or provoke haemolysis, or cause injuries to the organelles of the muscle fibers and the neurones. The amphibians have also developed a set of defensive behavioural activities in association with the secretion of venom. In the salamanders, rigid antipredator corporal positions are to be observed; these act synergistically with the cutaneous venom. As is the case with the anurans, certain postures permit the exposure of a larger area of concentrations of granular glands to an enemy. The parotoid and inguinal macroglands would seem to play an important part in these defensive postures.

The ultrastructure of the resting thrombocytes of the snake Waglerophis merremii and the process of ADP aggregation are studied. These thrombocytes are nucleate, ellipsoidal, and contain a marginal band. Endoplasmic reticulum and ribosomes are few. An open canalicular system, Golgi complex, granules resembling the alpha-granules in human platelets, and structures similar to secondary lysosomes are also present. The activation response of W. merremii thrombocytes to ADP is examined and compared to the morphological alterations in human platelets. The thrombocytes, normally ellipitical in shape, become spheroid with cytoplasmic protrusions, and adhere to one another. The open canalicular system undergoes dilation and the granules cluster at the centre of the thrombocytes. The release reaction of these granules occurs after stimulation by ADP, similar to what happens in human platelets. This similarity may suggest a process of evolutional specialization since thrombocytes in the majority of non-mammal vertebrates are not aggregated by ADP.

Phylogenetic hypotheses of species of Leptodactylus have been proposed but relationships often consider few species and high-level groups are supported by few, homoplasious morphological characters. Similarities between the reproductive biology of species of the L. marmoratus (formerly Adenomera) and those of the L. fuscus group may represent homoplasies or, as previously suggested, shared derived features pointing to their closer relationship. We determined the presence of buccal foaming glands in tadpoles of three species of Leptodactylus (L. furnarius, L. labyrinthicus and a member of the L. marmoratus group) by histological preparations. The presence of these glands and other seven characters were mapped onto two alternative topologies in order to understand the relationships among Leptodactylus species groups and the evolution of their reproductive features. Two sets of foaming glands were found in all species studied: 1) rows of secretory ridges and 2) secretory pits. Mapping the nine characters on the currently recognized phylogeny (emphasizing closer relationship among L. latrans, L. fuscus and L. labyrinthicus) resulted in sixteen steps (CI = 56; RI = 22); in the alternative hypothesis (closer relationship between L. marmoratus group and Leptodactylus of the L. fuscus groups) it resulted in eleven steps (CI = 82; RI = 78). Our evidences support that species in the L. marmoratus group are not the sister group to the remainder of Leptodactylus, but probably a subset of the L. fuscus group.

In amphibians, secretions of toxins from specialized skin poison glands play a central role in defense against predators. The production of toxic secretions is often associated with conspicuous color patterns that warn potential predators, as it is the case of many dendrobatid frogs, including Ameerega picta. This species resembles the presumably nontoxic Leptodactylus lineatus. This study tests for mimicry by studying the morphology and distribution of skin glands, components of skin secretion, and defensive behavior. Dorsal skin was studied histologically and histochemically, and skin secretions were submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed phase high performance liquid chromatography and assays for proteolytic activity. We found that poison glands in A. picta are filled with nonprotein granules that are rich in carbohydrates, while L. lineatus glands present protein granules. Accordingly, great amounts of proteins, at least some of them enzymes, were found in the poison of L. lineatus but not in that of A. picta. Both species differ greatly on profiles of gland distribution: In L. lineatus, poison glands are organized in clusters whose position coincides with colored elements of the dorsum. These regions are evidenced through a set of displays, suggesting that poison location is announced to predators through skin colors. In contrast, A. picta presents lower densities of glands, distributed homogeneously. This simpler profile suggests a rather qualitative than quantitative investment in chemical defense, in agreement with the high toxicity attributed to dendrobatids in general. Our data suggest that both species are toxic or unpalatable and transmit common warning signals to predators, which represents a case of Mullerian mimicry.

Amphibian defence mechanisms commonly rely on cutaneous toxins produced in either isolated or clustered glands, such as toad parotoid macroglands. In contrast to the passive mechanism of poison liberation in other amphibians, we discovered that the Amazonian toad Rhaebo guttatus is unique because it can voluntarily squirt jets of poison from its parotoids. Amphibians commonly use toxic, cutaneous se- cretions produced by glands spread over the body as a defence against predators (Duellman and Trueb, 1994). In bufonids, these glands are grouped behind the eyes, forming a pair of pos- torbital structures, known as parotoid macrog- lands (Toledo and Jared, 1995; Tyler, Burton and Bauer, 2001). Amongst amphibians, toads provide a classic example of passive defence, based on contact between a predator's oral mu- cosa and cutaneous poisonous secretion. When threatened, they react by inflating their bodies in such a way that the parotoids become turgid and primed. When an aggressor bites a toad, it becomes the agent of its own poisoning by trig- gering the macroglands and, consequently, re- ceiving jets of poison in its mouth (Jared et al., 2009).

Female Pipa carvalhoi incubate their eggs in the skin of the dorsum where the embryos develop until they emerge. Behavioral and morphological aspects of this reproductive mode were studied through courtship until the tadpoles emerged. Samples of the female skin were collected beginning a few hours after egg deposition and through subsequent phases of larval development and examined using standard histology, histochemistry and immunohistochemistry. The females' dorsal skin structure changes during egg implantation and development. These changes may be mediated by hormones and enzymes in a manner paralleling that of trophoblast implantation in mammal endometrium. The lack of vittelum and the extensive vascularization in the female after the 14th day of egg implantation suggest interaction through blood between the female and embryos. This hypothesis could be better accessed, if comparisons could be made with similar species such as Pipa pipa and Pipa arrabali, in which the embryos hatch only after...

Pre-cloacal glands occur in some species of amphisbaenians. Although these glands are important in systematics, their biology and chemistry are little known. The pre-cloacal glands of Amphisbaena alba are of the holocrine type. They are made up of a glandular body and a duct. The glandular body is conical to elongate and is formed of clongatc lobules separated one from another by collagen septa. Each lobule is composed, at its periphery, of germinative cells, and within of polyhedral secretory cells, of different degrees of differentiation. The germinative cells, set on a basal lamina, are basophilic and their cytoplasm is fairly electron dense. The polyhedral cells display bulky cytoplasm, filled with spherical granules, wrapped in membranes and differing in their electron densities. Towards the lumen of the gland, these granules are increasingly eosinophilic and have an affinity for orange G. The secretion is discharged into the duct leading to the pore, which is situated in the central region of the scale. This secretion shows positive histochemical results for mucopolysaccharides and proteins. The similarity between the epidermal glands of lizards and those of A. alba raises the suggestion that the glands have equivalent functions, possibly in the course of intra- or interspecific communication.

1. 1. The integument of the amphibians is, in general terms, not adapted to a strictly terrestrial life, in that it does not serve as a barrier to a bidirectional water flow. 2. 2. One of the major problems which thus faces the terrestrial amphibians is dehydration. 3. 3. To reduce water loss, or to absorb water through the skin, some amphibians have developed morphological and behavioural adaptations capable of acting together to maintain the hydric balance. 4. 4. Skin texture and sculpturing seem to be involved with water absorption. A granular ventral skin absorbs more efficiently than a smooth skin; the occurrence of cutaneous grooves in the ventral and lateral regions of the body in the salamander and the bufonids permits the transfer of water to other parts of the body by capillarity. 5. 5. The arrangement of the superficial epidermal cells, sometimes united, sometimes separated by ample intercellular spaces, and also, in some cases, the concentration of cutaneous lipids, influence the permeability of the skin to water. 6. 6. Some amphibian species form a cocoon composed of various layers of stratum corneum; this covers the body and reduces water loss considerably during the dry season. 7. 7. It has been observed that the hydration capacity in some anurans is related to the cutaneous vascularization in the ventral pelvic region, and that, in general, species of terrestrial habit have a greater degree of vascularization than those of aquatic habit. 8. 8. A number of substances, including hormones such as vasotocin, oxytocin, thyroxin and prolactin, have some influence on the permeability of the skin to water. 9. 9. The cutaneous mucus produced by the amphibians has great functional diversity. In some anuran species it has a role in thermoregulation and water economy. 10. 10. The anuran genera Phyllomedusa and Litoria wipe the skin with the legs so as to spread the lipid secretion from the cutaneous glands over the body surface. This secretion causes a marked reduction in the loss of body water. 11. 11. The hydric balance in some anuran species is probably associated with the secretions of the granular glands, and in particular with the glycosaminoglycans found in granular alveoli in the parotoid and paracnemid glands of bufonids.

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.

Species of the genus Brachycephalus, have a snout-vent length of less than 18 mm and are believed to have evolved through miniaturization. Brachycephalusephippium, is particularly interesting; because its entire skull is hyperossified, and the presacral vertebrae and transverse processes are covered by a dorsal shield. We demonstrate in this paper that, at the macroscopic level, a completely hyperossified skull and dorsal shield occur only in B. ephippium, but not in B. ferruginus, B. izechsohni, B. pernix, B. pombali, B. brunneus, B. didactylus, and B. hermogenesi. An intermediate condition, in which the skull is hyperossified but a dorsal shield is absent, occurs in B. vertebralis, B. nodoterga, B. pitanga, and B. alipioi. The microscopic structure of hyperossification was examined in skulls of B. ephippium and B. pitanga, revealing a complex organization involving the presence of Sharpey fibers, which in humans are characteristic of periodontal connections. J. Morphol., 2009. © 2009 Wiley-Liss, Inc.

When toads (Rhinella) are threatened they inflate their lungs and tilt the body towards the predator, exposing their parotoid macroglands. Venom discharge, however, needs a mechanical pressure onto the parotoids exerted by the bite of the predator. The structure of Rhinella jimi parotoids was described before and after manual compression onto the macroglands mimicking a predator attack. Parotoids are formed by honeycomb-like collagenous alveoli. Each alveolus contains a syncytial gland enveloped by a myoepithelium and is provided with a duct surrounded by differentiated glands. The epithelium lining the duct is very thick and practically obstructs the ductal lumen, leaving only a narrow slit in the centre. After mechanical compression the venom is expelled as a thin jet and the venom glands are entirely emptied. The force applied by a bite of a potential predator may increase alveolar pressure, forcing the venom to be expelled as a thin jet through the narrow ductal slit. We suggest that the mechanism for venom discharge within all bufonids is possibly similar to that described herein for Rhinella jimi and that parotoids should be considered as cutaneous organs separate from the rest of the skin specially evolved for an efficient passive defence.

S. M. Carneiro , V. R. Pinto , C. Jared , L. A. B. M. Lula , F. P. Faria and A. Sesso . Morphometric studies on venom secretory cells from Bothrops jararacussu (Jararacucu) before and after venom extraction. Toxicon29, 569–580, 1991.—A comparative morphometrical analysis was carried out on secretory cells from Bothrops jararacussu venom glands, before manual extraction of the venom (milking) and 4 and 8 days after milking. At the 8th day after milking, the cytoplasmic volume increased by 160%. The rough endoplasmic reticulum (RER) volume density increase, up to the 8th day after milking, is mainly due to widening of the intra-scisternal space. The total volume and membrane surface of the RER, Golgi apparatus and subcomponents, secretory vesicles and mitochondria, increased during the experimental period while the volume and surface densities of these organelles, with the exception of the RER, did not vary. The numerical density of Golgi-associated microvesicles per Golgi volume unit also increased. The greatest relative increments in these parameters occurred within the first 4 days. These results are compatible with an increased rate of membrane synthesis and transport in the milked glands and suggest that the membrane biogenesis, degradation and circulation that takes place in the first week after milking is achieved through coordinated cellular mechanisms that maintain the rate between total membrane surface and total cytoplasmic volume unaltered.

Maternal dermatophagy, the eating of maternal skin by offspring, is an unusual form of parental investment involving co-evolved specializations of both maternal skin and offspring dentition, which has been recently discovered in an African caecilian amphibian. Here we report the discovery of this form of parental care in a second, distantly related Neotropical species Siphonops annulatus, where it is characterized by the same syndrome of maternal and offspring specializations. The detailed similarities of skin feeding in different caecilian species provide strong evidence of its homology, implying its presence in the last common ancestor of these species. Biogeographic considerations, the separation of Africa and South American land masses and inferred timescales of amphibian diversification all suggest that skin feeding is an ancient form of parental care in caecilians, which has probably persisted in multiple lineages for more than 100 Myr. These inferences support the hypotheses that (i) maternal dermatophagy is widespread in oviparous direct-developing caecilians, and (ii) that viviparous caecilians that feed on the hypertrophied maternal oviduct evolved from skin-feeding ancestors. In addition to skin-feeding, young S. annulatus were observed to congregate around, and imbibe liquid exuded from, the maternal cloacal opening.