Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Background:Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied.Methods:The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line.Results:The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis.Conclusions:Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)

Distribuição geográfica de Scinax crospedospilus (Anura, Hylidae), incluindo novos registros na Mata Atlântica de Minas Gerais (Brasil) / Geographic distribution of Scinax crospedospilus (Anura, Hylidae), including new records in the Atlantic Forest of Minas Gerais (Brazil)

A perereca Scinax crospedospilus foi descrita com base em síntipos coletados no vale do rio Paraíba do Sul e posteriormente foi registrada em várias localidades na Mata Atlântica dos estados do Rio de Janeiro, São Paulo e Minas Gerais. Apresenta-se um amplo mapeamento da espécie, baseado em revisão bibliográfica, análise de coleções científicas e amostragens em campo, incluindo novos registros na Mata Atlântica de Minas Gerais. Scinax crospedospilus foi coletado em algumas localidades de Mariana, na borda oriental do Quadrilátero Ferrífero, mostrando-se raro na região. Com a análise de coleções, foram localizados exemplares procedentes de 13 municípios localizados em diversas unidades de relevo. Em campo se observou que S. crospedospilus possui certa tolerância a perturbações do hábitat, realiza reprodução em pequenas represas na borda de floresta alterada e os machos vocalizam à noite sobre árvores e arbustos na margem d’água e, em período vespertino, no dossel de árvores baixas, inclusive dentro de bromélias. Os dados levantados permitiram concluir que S. crospedospilus é endêmico da Mata Atlântica e possui uma ampla distribuição pelo bioma no Sudeste do Brasil.

The tree frog Scinax crospedospilus was described based on syntypes collected from the Paraíba do Sul River valley and later was recorded in various localities in the Atlantic Forest of the Rio de Janeiro, São Paulo and Minas Gerais states. We present a wide mapping of the species, based on bibliographic review, analysis of scientific collections and field samplings, including new records in the Atlantic Forest of Minas Gerais. Scinax crospedospilus was collected in some localities of Mariana, on the eastern edge of the Quadrilátero Ferrífero, showing itself to be rare in the region. With the analysis of collections, we found specimens of S. crospedospilus from 13 municipalities located in several relief units. In the field, we observed that S. crospedospilus has a certain tolerance to habitat disturbances, breeds in small dams on the edge of altered forest, and the males vocalize over trees and shrubs on the water's edge at night, and in the canopy of low trees, including inside bromeliads, in the afternoon. The data collected allowed us to conclude that S. crospedospilus is endemic to the Atlantic Forest and has a wide distribution in the biome in Southeastern Brazil.

Distribuição geográfica de Scinax crospedospilus (Anura, Hylidae), incluindo novos registros na Mata Atlântica de Minas Gerais (Brasil) / Geographic distribution of Scinax crospedospilus (Anura, Hylidae), including new records in the Atlantic Forest of Minas Gerais (Brazil)

A perereca Scinax crospedospilus foi descrita com base em síntipos coletados no vale do rio Paraíba do Sul e posteriormente foi registrada em várias localidades na Mata Atlântica dos estados do Rio de Janeiro, São Paulo e Minas Gerais. Apresenta-se um amplo mapeamento da espécie, baseado em revisão bibliográfica, análise de coleções científicas e amostragens em campo, incluindo novos registros na Mata Atlântica de Minas Gerais. Scinax crospedospilus foi coletado em algumas localidades de Mariana, na borda oriental do Quadrilátero Ferrífero, mostrando-se raro na região. Com a análise de coleções, foram localizados exemplares procedentes de 13 municípios localizados em diversas unidades de relevo. Em campo se observou que S. crospedospilus possui certa tolerância a perturbações do hábitat, realiza reprodução em pequenas represas na borda de floresta alterada e os machos vocalizam à noite sobre árvores e arbustos na margem dágua e, em período vespertino, no dossel de árvores baixas, inclusive dentro de bromélias. Os dados levantados permitiram concluir que S. crospedospilus é endêmico da Mata Atlântica e possui uma ampla distribuição pelo bioma no Sudeste do Brasil.(AU)

The tree frog Scinax crospedospilus was described based on syntypes collected from the Paraíba do Sul River valley and later was recorded in various localities in the Atlantic Forest of the Rio de Janeiro, São Paulo and Minas Gerais states. We present a wide mapping of the species, based on bibliographic review, analysis of scientific collections and field samplings, including new records in the Atlantic Forest of Minas Gerais. Scinax crospedospilus was collected in some localities of Mariana, on the eastern edge of the Quadrilátero Ferrífero, showing itself to be rare in the region. With the analysis of collections, we found specimens of S. crospedospilus from 13 municipalities located in several relief units. In the field, we observed that S. crospedospilus has a certain tolerance to habitat disturbances, breeds in small dams on the edge of altered forest, and the males vocalize over trees and shrubs on the water's edge at night, and in the canopy of low trees, including inside bromeliads, in the afternoon. The data collected allowed us to conclude that S. crospedospilus is endemic to the Atlantic Forest and has a wide distribution in the biome in Southeastern Brazil.(AU)

Novos registros geográficos, variação morfológica e notas de história natural de Scinax tripui (Amphibia, Anura, Hylidae) na Mata Atlântica de Minas Gerais (Brasil) / New geographic records, morphological variation, and natural history notes of Scinax tripui (Amphibia, Anura, Hylidae) in the Atlantic Forest of Minas Gerais (Brazil)

O anuro Scinax tripui permanecia pouco conhecido. A partir de amostragens em campo no Quadrilátero Ferrífero e análises de coleções científicas são apresentados um expressivo número de novos registros geográficos da espécie na Mata Atlântica do estado de Minas Gerais, observações de uso de habitat e variações morfológicas. Exemplares de S. tripui foram coletados em 40 localidades e em coleções foram levantadas mais 19 localidades. Observou-se que a espécie distribui-se pelas serras do Quadrilátero Ferrífero e porção norte da Serra da Mantiqueira, no centro-sudeste de Minas Gerais e sul do Espírito Santo, com ocorrência frequente nas matas do Quadrilátero. Constatou-se que S. tripui é restrito a córregos e riachos perenes em ambiente florestal, sendo associado a microhabitats mais íntegros, e exibe atividade reprodutiva ao longo do inverno e início da primavera (julho a setembro). A coloração em vida de S. tripui variou principalmente na região inguinal e partes anterior e posterior da coxa, que podem ser verde-claras, azul-claras, amarelo-claras ou brancas; e no padrão de coloração dorsal, que pode ser cinza, oliva, palha ou marrom-claro. Foram aferidas sete medidas morfométricas de adultos, sendo obtida expressiva variação do tamanho (comprimento rostro-cloacal). As variações de coloração, medidas e porte foram recorrentes ao longo do Quadrilátero Ferrífero, sendo constatado que S. tripui exibe expressiva variação intrapopulacional.

The tree frog Scinax tripui is still little known. From field collections in the Quadrilátero Ferrífero, and analysis of scientific collections, we present here a large number of new geographic records for the species in the Atlantic Forest of Minas Gerais State, as well as observations of habitat use and morphological variation. We collected specimens of S. tripui in 40 localities, and obtained 19 further locations from scientific collections. We found that the species is distributed throughout the mountains of the Quadrilátero Ferrífero and northern portion of Mantiqueira mountain range, in the central-southeast of Minas Gerais and south of Espírito Santo States, and it is frequent in the forests of the Quadrilátero. Field studies showed that individuals of S. tripui are restricted to permanent streams in forested areas, associated with preserved microhabitats, and reproduce during local winter and early spring (July to September). We observed in life color variation for S. tripui, mainly of the inguinal region and anterior and posterior parts of the thigh (which may be light green, light blue, light yellow or white); and in the dorsal color pattern (which may be grey, olive, straw or light brown). Seven morphometric measurements of adults were taken, and significant variation was obtained for snout-vent length. Color variations, measurements and size were present along the Quadrilátero Ferrífero and S. tripui exhibits large intrapopulational variation.

Novos registros geográficos, variação morfológica e notas de história natural de Scinax tripui (Amphibia, Anura, Hylidae) na Mata Atlântica de Minas Gerais (Brasil) / New geographic records, morphological variation, and natural history notes of Scinax tripui (Amphibia, Anura, Hylidae) in the Atlantic Forest of Minas Gerais (Brazil)

O anuro Scinax tripui permanecia pouco conhecido. A partir de amostragens em campo no Quadrilátero Ferrífero e análises de coleções científicas são apresentados um expressivo número de novos registros geográficos da espécie na Mata Atlântica do estado de Minas Gerais, observações de uso de habitat e variações morfológicas. Exemplares de S. tripui foram coletados em 40 localidades e em coleções foram levantadas mais 19 localidades. Observou-se que a espécie distribui-se pelas serras do Quadrilátero Ferrífero e porção norte da Serra da Mantiqueira, no centro-sudeste de Minas Gerais e sul do Espírito Santo, com ocorrência frequente nas matas do Quadrilátero. Constatou-se que S. tripui é restrito a córregos e riachos perenes em ambiente florestal, sendo associado a microhabitats mais íntegros, e exibe atividade reprodutiva ao longo do inverno e início da primavera (julho a setembro). A coloração em vida de S. tripui variou principalmente na região inguinal e partes anterior e posterior da coxa, que podem ser verde-claras, azul-claras, amarelo-claras ou brancas; e no padrão de coloração dorsal, que pode ser cinza, oliva, palha ou marrom-claro. Foram aferidas sete medidas morfométricas de adultos, sendo obtida expressiva variação do tamanho (comprimento rostro-cloacal). As variações de coloração, medidas e porte foram recorrentes ao longo do Quadrilátero Ferrífero, sendo constatado que S. tripui exibe expressiva variação intrapopulacional.(AU)

The tree frog Scinax tripui is still little known. From field collections in the Quadrilátero Ferrífero, and analysis of scientific collections, we present here a large number of new geographic records for the species in the Atlantic Forest of Minas Gerais State, as well as observations of habitat use and morphological variation. We collected specimens of S. tripui in 40 localities, and obtained 19 further locations from scientific collections. We found that the species is distributed throughout the mountains of the Quadrilátero Ferrífero and northern portion of Mantiqueira mountain range, in the central-southeast of Minas Gerais and south of Espírito Santo States, and it is frequent in the forests of the Quadrilátero. Field studies showed that individuals of S. tripui are restricted to permanent streams in forested areas, associated with preserved microhabitats, and reproduce during local winter and early spring (July to September). We observed in life color variation for S. tripui, mainly of the inguinal region and anterior and posterior parts of the thigh (which may be light green, light blue, light yellow or white); and in the dorsal color pattern (which may be grey, olive, straw or light brown). Seven morphometric measurements of adults were taken, and significant variation was obtained for snout-vent length. Color variations, measurements and size were present along the Quadrilátero Ferrífero and S. tripui exhibits large intrapopulational variation.(AU)

A New Species of Scinax Wagler, 1830 (Anura: Hylidae) From the Middle Amazon River Basin, Brazil

A new species of the hylid genus Scinax is described and illustrated. The new taxon was found in the Amazonian rainforest of northern Brazil, municipalities of Maués and Careiro da Várzea, state of Amazonas. The new species is characterized by its moderate size (male mean snout-vent length 36.3 mm); body robust; large, orange, black-bordered axillary and inguinal spots; and bilobate vocal sac. This new species was found in primary and secondary forest on branches of shrubs or trees in, or next to, permanent ponds and flooded areas.

A New Species of Scinax Wagler, 1830 (Anura: Hylidae) From the Middle Amazon River Basin, Brazil

A new species of the hylid genus Scinax is described and illustrated. The new taxon was found in the Amazonian rainforest of northern Brazil, municipalities of Maués and Careiro da Várzea, state of Amazonas. The new species is characterized by its moderate size (male mean snout-vent length 36.3 mm); body robust; large, orange, black-bordered axillary and inguinal spots; and bilobate vocal sac. This new species was found in primary and secondary forest on branches of shrubs or trees in, or next to, permanent ponds and flooded areas.(AU)