Cutaneous glands of the striped toad, Rhinella crucifer (Wied-Neuwied, 1821) (Amphibia: Bufonidae): Histological study and bioactivities of glandular secretions.

This study investigated the morphology of Rhinella crucifer cutaneous glands, as well as the protein/peptide profiles and bioactivities of body gland secretions (BGS) and parotoid macrogland secretions (PS). The parotoid as well as dorsal and ventral skin fragments of male and female individuals were processed for histological analysis. The protein and peptide profiles of male and female gland secretions were evaluated. Male secretions were also assessed for proteolytic, trypsin inhibiting, hemagglutinating, hemolytic, antimicrobial, and anticoagulant activities. The R. crucifer skin structure presented protuberances that are clearly visible and formed by the integument, which has cutaneous glands throughout the body. An average of 438 and 333 glands were identified in males in females, respectively. No significant differences were observed in the distribution of glands across the body as well as for area and perimeter of glands. Differences were observed in protein composition between the PS and BGS from males and females, and secretions from animals collected from undisturbed and anthropogenically disturbed areas. Proteins with similarities to catalase and elongation factor 1-alpha were detected in the PS. Zymography revealed proteolytic activity in both male BGS and PS. Male BGS showed antibacterial activity against Enterococcus faecalis and Escherichia coli and anticoagulant activity, being able to prolong prothrombin time by 6.34-fold and activated partial thromboplastin time by 2.17-fold. Finally, male PS and BGS caused a maximum hemolysis degree of 1.4%. The data showed that the cutaneous secretions of R. crucifer are potentially promising for biotechnological prospecting.

Morphology of the Cutaneous Poison and Mucous Glands in Amphibians with Particular Emphasis on Caecilians (Siphonops annulatus).

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

Morphology of the cutaneous poison and mucous glands in amphibians with particular emphasis on caecilians (Siphonops annulatus)

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

Distribution of major toxins in Rhinella marina parotoid macroglands using Desorption-Electrospray-Ionization mass spectrometry imaging (DESI-MSI).

Amphibian cutaneous glands secrete toxins used in different vital functions including passive defense. Through Desorption Electrospray Ionization-Imaging we analyzed the distribution of the major toxins of the toad Rhinella marina parotoid macroglands. Alkaloids and steroids showed characteristic distribution and intensity within the glands and were also present at lower levels on the skin surface. A comprehensive overview of toxins distribution in toads' skin might help to understand their full biological role within the amphibians.

Distribution of major toxins in Rhinella marina parotoid macroglands using desorption-electrospray-ionization mass spectrometry imaging (DESI-MSI)

Amphibian cutaneous glands secrete toxins used in different vital functions including passive defense. Through Desorption Electrospray Ionization-Imaging we analyzed the distribution of the major toxins of the toad Rhinella marina parotoid macroglands. Alkaloids and steroids showed characteristic distribution and intensity within the glands and were also present at lower levels on the skin surface. A comprehensive overview of toxins distribution in toads’ skin might help to understand their full biological role within the amphibians.

Morphological Changes in Skin Glands During Development in Rhinella Arenarum (Anura: Bufonidae).

Avoiding predation is critical to survival of animals; chemical defenses represent a common strategy among amphibians. In this study, we examined histologically the morphology of skin glands and types of secretions related to chemical skin defense during ontogeny of Rhinella arenarum. Prior to metamorphic climax the epidermis contains typical bufonid giant cells producing a mucous substance supposedly involved in triggering a flight reaction of the tadpole school. An apical layer of alcianophilic mucus covers the epidermis, which could produce the unpleasant taste of bufonid tadpoles. Giant cells disappear by onset of metamorphic climax, when multicellular glands start developing, but the apical mucous layer remains. By the end of climax, neither the granular glands of the dorsum nor the parotoid regions are completely developed. Conversely, by the end of metamorphosis the mucous glands are partially developed and secrete mucus. Adults have at least three types of granular glands, which we designate type A (acidophilic), type B (basophilic) and ventral (mucous). Polymorphic granular glands distribute differently in the body: dorsal granular glands between warts and in the periphery of parotoids contain protein; granular glands of big warts and in the central region of parotoids contain catecholamines, lipids, and glycoconjugates, whereas ventral granular glands produce acidic glycoconjugates. Mucous glands produce both mucus and proteins. Results suggest that in early juveniles the chemical skin defense mechanisms are not functional. Topographical differences in adult skin secretions suggest that granular glands from the big warts in the skin produce similar toxins to the parotoid glands.

Análise bioquímica e biológica da secreção cutânea do anuro Dermatonotus muelleri / Biochemistry and biological analysis of skin secretion of anura Dermatonotus muelleri

Poisons and secretions animals are among the most complex and rich sources of biological materials, ie new molecules with potential biotechnological applications or pharmaceutical. Accordingly, molecules isolated from amphibian skin secretions may be used as alternatives for the development of new biotechnology tool, such as those target to combat resistant pathogens or develop new industrial products. This project aimed to identify and characterize molecules in the skin secretion of Dermatonotus muelleri (only specie in the genus of the family Microhylidae), through the isolation and biochemical characterization. The secretions were filtered through 10 kDa cut-off membranes and the retained fraction was analyzed by SDS-PAGE, followed by a proteomic approach. A zymographic assay was also performed. The low molecular mass filtrate was fractionated by HPLC and analyzed by mass spectrometry and nuclear magnetic resonance. Gel electrophoresis showed protein content of various molecular masses. Proteomics analysis found relevant matches for proteins distributed in the electrophoresis gel, including proteolytic enzymes. On the other hand, the < 10kDa fraction showed to contain sugars, according to the interpretation of the NMR data. The ESI-IT-TOF analyses revealed low molecular mass molecules (<800 Da) throughout the chromatographic separation, and tryptophan could be identified among the molecules. The filtered fractions were assayed for antibiotic activity but no inhibition of bacterial growth could be observed. Interestingly, one fraction exhibited a significant increase in the growth of Staphylococcus aureus. The proteomic analysis was pertinent, demonstrating the presence of several proteins related to the animal's biology and defense mechanism. From the chemical point of view, one amino acid was detected and induced bacterial growth, which could be related to alternative mechanisms to the absence of low molecular mass antibiotic compounds.

Venenos e secreções animais estão entre as mais ricas e complexas fontes de materiais biológicos, ou seja, novas moléculas com potencial aplicação biotecnológica ou mesmo farmacêuticas. Nesse sentido, moléculas isoladas da secreção da pele de anfíbios podem ser utilizadas como alternativas biotecnológicas destinadas aos novos medicamentos, como por exemplo, o combate de patógenos resistentes ou no desenvolvimento de novos produtos de utilidade industrial. Assim, esse projeto teve como objetivo identificar e caracterizar moléculas presentes na secreção cutânea de Dermatonotus muelleri (única espécie no gênero da família Microhylidae), por meio do isolamento e caracterização bioquímica. As secreções foram filtradas em membranas de corte de 10 kDa e o material retido foi analisado por SDS-PAGE e em seguida por uma abordagem proteômica. Também foi realizado um ensaio de zimografia. O filtrado de baixa massa foi fracionado por HPLC e analisado por espectrometria de massas e ressonância magnética nuclear. A eletroforese em gel apresentou conteúdo proteico de diversas massas moleculares. A análise proteômica encontrou correspondências relevantes para as proteínas distribuídas no gel de eletroforese, inclusive enzimas proteolíticas. Já os compostos filtrados na membrana de corte de 10kDa, demonstraram conter açúcares de acordo com a interpretação dos dados de RMN. Na análise por ESI-ITToF verificou-se moléculas de baixa massa molecular (<800 Da) ao longo de toda a amostra, sendo identificado o aminoácido triptofano entre elas. As frações filtradas foram ensaiadas para a atividade de antibiótica mas não foi observada inibição do crescimento bacteriano para estas frações, entretanto uma fração apresentou um significativo aumento do crescimento para Staphylococcus aureus. A análise proteômica foi pertinente, tendo demonstrando a presença de diversas proteínas relacionadas a biologia do animal e seu mecanismo de defesas. Do ponto de vista químico detectou-se um aminoácido que apresentou atividade de indução ao crescimento bacteriano, que pode estar relacionada a mecanismos alternativos à ausência atividade antimicrobiana nos compostos de baixa massa molecular.

Functional assessment of toad parotoid macroglands: a study based on poison replacement after mechanical compression.

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 forelimb swellings of Pygoderma bilabiatum (Chiroptera: Phyllostomidae) / The forelimb swellings of Pygoderma bilabiatum (Chiroptera: Phyllostomidae)

Pygoderma bilabiatum is a medium-sized short-faced stenodermatine bat (Chiroptera: Phyllostomidae) with prominent doughnut-shaped glandular tissue masses surrounding the eyes, among other distinctive characters. In this paper, we describe gross external morphology of newly discovered swellings of Pygoderma, probably enclosing glandular tissue found in the dorsal forelimbs of certain male individuals and discuss intra-specific variation in their development. All male individuals observed with large forelimb swellings also had largely developed periorbital and submandibular glands thus suggesting that all male skin glands/swellings in this species develop more or less concordantly. The episodic chronological distribution of male Pygoderma specimens with developed forelimb swellings suggests a relationship with reproductive cycles.

Pygoderma bilabiatum is a medium-sized short-faced stenodermatine bat (Chiroptera: Phyllostomidae) with prominent doughnut-shaped glandular tissue masses surrounding the eyes, among other distinctive characters. In this paper, we describe gross external morphology of newly discovered swellings of Pygoderma, probably enclosing glandular tissue found in the dorsal forelimbs of certain male individuals and discuss intra-specific variation in their development. All male individuals observed with large forelimb swellings also had largely developed periorbital and submandibular glands thus suggesting that all male skin glands/swellings in this species develop more or less concordantly. The episodic chronological distribution of male Pygoderma specimens with developed forelimb swellings suggests a relationship with reproductive cycles.

The forelimb swellings of Pygoderma bilabiatum (Chiroptera: Phyllostomidae) / The forelimb swellings of Pygoderma bilabiatum (Chiroptera: Phyllostomidae)

Pygoderma bilabiatum is a medium-sized short-faced stenodermatine bat (Chiroptera: Phyllostomidae) with prominent doughnut-shaped glandular tissue masses surrounding the eyes, among other distinctive characters. In this paper, we describe gross external morphology of newly discovered swellings of Pygoderma, probably enclosing glandular tissue found in the dorsal forelimbs of certain male individuals and discuss intra-specific variation in their development. All male individuals observed with large forelimb swellings also had largely developed periorbital and submandibular glands thus suggesting that all male skin glands/swellings in this species develop more or less concordantly. The episodic chronological distribution of male Pygoderma specimens with developed forelimb swellings suggests a relationship with reproductive cycles.

Pygoderma bilabiatum is a medium-sized short-faced stenodermatine bat (Chiroptera: Phyllostomidae) with prominent doughnut-shaped glandular tissue masses surrounding the eyes, among other distinctive characters. In this paper, we describe gross external morphology of newly discovered swellings of Pygoderma, probably enclosing glandular tissue found in the dorsal forelimbs of certain male individuals and discuss intra-specific variation in their development. All male individuals observed with large forelimb swellings also had largely developed periorbital and submandibular glands thus suggesting that all male skin glands/swellings in this species develop more or less concordantly. The episodic chronological distribution of male Pygoderma specimens with developed forelimb swellings suggests a relationship with reproductive cycles.