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.Fil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Cajade, Rodrigo. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología. Laboratorio de Herpetología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Piñeiro, Jose Miguel. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología. Laboratorio de Herpetología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Ana M.. Universidad Nacional del Nordeste; ArgentinaFil: da Silva, Igor R. F.. Universidade Estadual de Campinas; BrasilFil: Hyslop, Stephen. Universidade Estadual de Campinas; BrasilFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Pimenta, Daniel C.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Bustillo, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentin

Growth-Mark Formation and Chronology of Two Neotropical Anuran Species

In amphibians from tropical or subtropical regions well-expressed growth marks in bone could be expected because of the existence of strong dry/wet seasons, but little evidence is available. Additionally, temporal patterns of growth-mark formation may be a function of genetic and environmental factors. We used skeletochronology and a laboratory experiment to tease apart both causal sources, establishing direct correspondence between an observed pattern of bone growth and the actual age of Ceratophrys cranwelli and Dermatonotus muelleri from the subtropical Argentinean Chaco. Also, we describe the growth by application of the von Bertalanffy model. Growth rates decreased in both species, tending to reach asymptotic values at age 8¿10 months after metamorphosis. Both species clearly exhibited haematoxylinophilic lines of arrested growth (LAG) after one year growing in captivity. These results reinforce the hypothesis that in subtropical climates the LAG formation is ultimately caused by a general intrinsic (genetic) control.Peer Reviewe

The phylogeny of the Casque‐headed Treefrogs (Hylidae: Hylinae: Lophyohylini)

The South American and West Indian Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini) include 85 species. These are notably diverse in morphology (e.g. disparate levels of cranial hyperossification) and life history (e.g. different reproductive modes, chemical defences), have a wide distribution, and occupy habitats from the tropical rainforests to semiarid scrubland. In this paper, we present a phylogenetic analysis of this hylid tribe based on sequence fragments of up to five mitochondrial (12S, 16S, ND1, COI, Cytb) and six nuclear genes (POMC, RAG-1, RHOD, SIAH, TNS3, TYR). We included most of its species (> 96%), in addition to a number of new species. Our results indicate: (i) the paraphyly of Trachycephalus with respect to Aparasphenodon venezolanus; (ii) the nonmonophyly of Aparasphenodon, with Argenteohyla siemersi, Corythomantis galeata and Nyctimantis rugiceps nested within it, and Ap. venezolanus nested within Trachycephalus; (iii) the polyphyly of Corythomantis; (iv) the nonmonophyly of the recognized species groups of Phyllodytes; and (v) a pervasive low support for the deep relationships among the major clades of Lophyohylini, including C. greeningi and the monotypic genera Itapotihyla and Phytotriades. To remedy the nonmonophyly of Aparasphenodon, Corythomantis, and Trachycephalus, we redefined Nyctimantis to include Aparasphenodon (with the exception of Ap. venezolanus, which we transferred to Trachycephalus), Argenteohyla, and C. galeata. Additionally, our results indicate the need for taxonomic work in the following clades: (i) Trachycephalus dibernardoi and Tr. imitatrix; (ii) Tr. atlas, Tr. mambaiensis and Tr. nigromaculatus; and (iii) Phyllodytes. On the basis of our phylogenetic results, we analyzed the evolution of skull hyperossification and reproductive biology, with emphasis on the multiple independent origins of phytotelm breeding, in the context of Anura. We also analyzed the inter-related aspects of chemical defences, venom delivery, phragmotic behaviour, co-ossification, and prevention of evaporative water loss