A atividade secretória cutânea em pererecas: uma abordagem morfológica / Cutaneous secretory activity in tree frogs: a morphological approach

The skin in amphibians is an organ involved in several functions, such as breathing and chemical defense against predators and microorganisms. These functions are related to different skin components, such as cutaneous glands of two basic types, the mucous glands and the poison glands. Among anurans, in special in tree frogs, calls attention the fact that, in stressful situations, while some species can release large amounts of secretion, usually thick and sticky, others do not modify their secretory mode, making use of behavioral strategies to escape of danger. These differences, at first sight, may be related to greater or lesser capacity to control myoepithelial cells that involve the skin glands, isolated or together with other cutaneous morphological characteristics. This work aims at the morphological and morphometric study of the skin of different tree frogs capable of release abundant secretion, Trachycephalus mesophaeus and Corythomantis greeningi, in comparison with a little secretory tree frog, Itapotihyla langsdorffii. We tried to focus on the main characteristics of the cutaneous glands, mainly with regard to the myoepithelial cells, but we also analyzed other specific characteristics, such as the thickness of the calcified dermal layer and the number of pillar cells scattered in the dermis. Samples of dorsal skin of adults (n = 3) from T. mesophaeus, I. langsdorffii and C. greeningi obtained from animals previously fixed in buffered paraformaldehyde solution, were processed for histology in historesin. The sections (2 µm) were stained with toluidine blue-fuchsin. The images were analyzed in an Olympus BX51 light microscope, equipped with a digital camera and the Olympus Cellsens 1.17 software. The measurements were performed in triplicate and the media was calculated. A statistical analysis was performed using unidirectional ANOVA and the differences were significant when P ≤ 0.05. The results showed that in all analyzed species, in some cases, both the poison and the mucous glands, did not show differences in thickness, contrarily of what was expected. In relation to the other structural characteristics, called attention the number of pillar cells that were apparently more numerous in I. langsdorffii and in C. greeningi, fact that was not initially expected, in addition to the thicker calcified layer in T. mesophaeus and C. greeningi. Our results demonstrate that the cutaneous morphological structures do not seem to perform a predominant individual role in any of the analyzed species. All structures seem to participate of a more complex system of skin secretion release, together with other factors of phylogenetic, physiological and behavioral origin.

A pele nos anfíbios é um órgão envolvido em diversas funções vitais, como respiração e defesa química contra predadores e microrganismos. Essas funções estão relacionadas a diferentes componentes da pele, tais como as glândulas cutâneas de dois tipos básicos, as mucosas e as de veneno. Entre os anuros, em especial nas pererecas, chama a atenção o fato que, em situações de estresse, enquanto algumas espécies conseguem liberar imediatamente grandes quantidades de secreção, geralmente espessa e pegajosa, outras não modificam seu modo secretório cutâneo, fazendo uso de estratégias comportamentais para escapar do perigo. Essas diferenças, à primeira vista, deveriam se relacionar com a maior ou menor capacidade de contração das células mioepiteliais que envolvem as glândulas da pele, agindo isoladamente ou em conjunto com outras características morfológicas cutâneas. Este trabalho objetivou o estudo morfológico e morfométrico da pele de diferentes pererecas capazes de liberar secreção abundante, Trachycephalus mesophaeus e Corythomantis greeningi, em comparação com uma perereca pouco secretora, Itapotihyla langsdorffii. Procuramos focar nas principais características das glândulas cutâneas, principalmente no que se refere às células mioepiteliais, mas também analisamos outras características estruturais, como a espessura da camada dérmica calcificada e o número de células pilares espalhadas na derme. Amostras de pele dorsal de indivíduos adultos (n = 3) de T. mesophaeus, I. langsdorffii, C. greeningi obtidas de animais previamente fixados em solução de paraformaldeído tamponada, foram processadas para histologia em historesina. Os cortes (2 μm) foram corados com azul de toluidina-fucsina. As imagens foram obtidas e analisadas em um microscópio de luz Olympus BX51, equipado com uma câmera digital e o software Olympus Cellsens 1.17. As medições foram realizadas em triplicata e as médias calculadas. A análise estatística foi realizada através da ANOVA one-way e as diferenças foram consideradas significantes quando P ≤ 0,05. Os resultados mostraram que, em todas as espécies analisadas, os 9 mioepitélios, tanto das glândulas de veneno como das mucosas, não apresentaram diferenças significativas em suas espessuras, contrariado o que era inicialmente esperado. Em relação às outras características estruturais, chama a atenção o número de células pilares aparentemente mais numerosas em I. langsdorffii e C. greeningi, fato que também não era esperado inicialmente, além da camada calcificada mais espessa em T. mesophaeus e C. greeningi. Nossos resultados demonstram que as estruturas morfológicas cutâneas analisadas aparentemente não possuem um papel individual predominante em uma ou outra espécie. Todas devem participar de um sistema mais complexo de liberação da secreção cutânea, conjuntamente com outros fatores de origem filogenética, fisiológica e comportamental.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion.

BACKGROUND: Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. METHODS: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. RESULTS: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. CONCLUSIONS: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)