Differential Mitochondrial Genome Expression of Four Hylid Frog Species under Low-Temperature Stress and Its Relationship with Amphibian Temperature Adaptation.

Extreme weather poses huge challenges for animals that must adapt to wide variations in environmental temperature and, in many cases, it can lead to the local extirpation of populations or even the extinction of an entire species. Previous studies have found that one element of amphibian adaptation to environmental stress involves changes in mitochondrial gene expression at low temperatures. However, to date, comparative studies of gene expression in organisms living at extreme temperatures have focused mainly on nuclear genes. This study sequenced the complete mitochondrial genomes of five Asian hylid frog species: Dryophytes japonicus, D. immaculata, Hyla annectans, H. chinensis and H. zhaopingensis. It compared the phylogenetic relationships within the Hylidae family and explored the association between mitochondrial gene expression and evolutionary adaptations to cold stress. The present results showed that in D. immaculata, transcript levels of 12 out of 13 mitochondria genes were significantly reduced under cold exposure (p < 0.05); hence, we put forward the conjecture that D. immaculata adapts by entering a hibernation state at low temperature. In H. annectans, the transcripts of 10 genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2 and ATP8) were significantly reduced in response to cold exposure, and five mitochondrial genes in H. chinensis (ND1, ND2, ND3, ND4L and ATP6) also showed significantly reduced expression and transcript levels under cold conditions. By contrast, transcript levels of ND2 and ATP6 in H. zhaopingensis were significantly increased at low temperatures, possibly related to the narrow distribution of this species primarily at low latitudes. Indeed, H. zhaopingensis has little ability to adapt to low temperature (4 °C), or maybe to enter into hibernation, and it shows metabolic disorder in the cold. The present study demonstrates that the regulatory trend of mitochondrial gene expression in amphibians is correlated with their ability to adapt to variable climates in extreme environments. These results can predict which species are more likely to undergo extirpation or extinction with climate change and, thereby, provide new ideas for the study of species extinction in highly variable winter climates.

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae).

Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.

Experimental evaluation of how biological invasions and climate change interact to alter the vertical assembly of an amphibian community.

While biotic-abiotic interactions are increasingly documented in nature, a process-based understanding of how such interactions influence community assembly is lacking in the ecological literature. Perhaps the most emblematic and pervasive example of such interactions is the synergistic threat to biodiversity posed by climate change and invasive species. Invasive species often out-compete or prey on native species. Despite this long-standing and widespread issue, little is known about how abiotic conditions, such as climate change, will influence the frequency and severity of negative biotic interactions that threaten the persistence of native fauna. Treefrogs are a globally diverse group of amphibians that climb to complete life-cycle processes, such as foraging and reproduction, as well as to evade predators and competitors, resulting in frog communities that are vertically partitioned. Furthermore, treefrogs adjust their vertical position to maintain optimal body temperature and hydration in response to environmental change. Here, utilizing this model group, we designed a novel experiment to determine how extrinsic abiotic and biotic factors (changes to water availability and an introduced predator, respectively) interact with intrinsic biological traits, such as individual physiology and behaviour, to influence treefrogs' vertical niche. Our study found that treefrogs adjusted their vertical niche through displacement behaviours in accordance with abiotic resources. However, biotic interactions resulted in native treefrogs distancing themselves from abiotic resources to avoid the non-native species. Importantly, under altered abiotic conditions, both native species avoided the non-native species 33 %- 70 % more than they avoided their native counterpart. Additionally, exposure to the non-native species resulted in native species altering their tree climbing behaviours by 56 % - 78 % and becoming more vertically dynamic to avoid the non-native antagonist. Our experiment determined that vertical niche selection and community interactions were most accurately represented by a biotic-abiotic interaction model, rather than a model that considers these factors to operate in an isolated (singular) or even additive manner. Our study provides evidence that native species may be resilient to interacting disturbances via physiological adaptations to local climate and plasticity in space-use behaviours that mediate the impact of the introduced predator.

Hybridization despite elaborate courtship behavior and female choice in Neotropical tree frogs.

The mechanisms of hybridization can be elucidated by analyzing genotypes as well as phenotypes that could act as premating barriers, as the reproductive interactions among heterospecifics can alter the evolutionary history of species. In frogs, hybrids typically occur among species that reproduce explosively (in dense aggregations) with few opportunities for mate selection but are rare in species with elaborate courtship behaviors that may prevent erroneous mating. Using 21 microsatellite markers, we examined hybridization in the prolonged-breeding tree frogs Bokermannohyla ibitiguara and B. sazimai sampled within a contact zone in the Brazilian savanna (72 tadpoles; 74 adults). We also compared acoustic and morphological data. We confirmed both parental species genetically; STRUCTURE results confirmed 14 hybrids, 11 of which were second-generation according to NEWHYBRIDS, all with intermediate values of genetic dissimilarities compared to the parentals. Morphological and acoustic analyses revealed that hybrids showed variable but not necessarily intermediate phenotypes. Moreover, 2 hybrids exhibited call types different from parentals. The reproduction of B. ibitiguara involves territorial and aggressive males, elaborate courtships with acoustic and tactile stimuli, choosy females, and opportunistic strategies. Our study uncovers a rare case of viable hybridization among closely related frogs with such a combination of complex courtship behaviors and mate choice. We discuss the likely directionality and mechanisms behind this phenomenon, and highlight the importance of investigating hybridization even in species that show elaborate reproduction and female choice to advance our understanding of animal diversification.

High diversity of 5S ribosomal DNA and evidence of recombination with the satellite DNA PcP190 in frogs.

Distinct types of 5S rDNA repeats, differing by size and composition of its non-transcribed spacer (NTS), have been found in diverse taxa. Both concerted evolution and birth-and-death evolution have proven to play important roles in the evolution of the 5S rDNA family. In anurans, however, this subject has been underexplored as only a few anuran species had their 5S rDNA characterized and evolutionary analyzed to date. In the present study, we characterized the 5S rDNA sequences from species of two anuran families using classical molecular biology techniques and bioinformatic approaches. Based on the NTS, more than one type of 5S rDNA was identified in each analyzed species, which suggests that birth-and-death processes take part in the evolution of these sequences along the Anura tree of life. In addition, closely related species shared the same types of sequences, in accordance with the model of concerted evolution. We also found evidence of recombination between 5S rDNA and PcP190 satellite DNA, a repetitive sequence that is derived from the 5S rDNA. The interplay between 5S rDNA and PcP190 satellite DNA might favor the maintenance of the latter in the genome and respond to its presence in several species of frogs. The analysis of 5S rRNA transcripts confirmed the type I 5S rDNA of leiuperine as a functional 5S rRNA gene. Finally, the chromosome mapping of 5S rDNA sequences allowed some inferences of chromosome homology in Leiuperinae. In conclusion, our study provides additional information about the organization, differentiation and functionality of 5S rDNA in anuran species, revealing the potential participation of satellite DNA in the evolution of this family of rDNA.

Sequencing of laser captured Z and W chromosomes of the tocantins paradoxical frog (Pseudis tocantins) provides insights on repeatome and chromosomal homology.

Pseudis tocantins is the only frog species of the hylid genus Pseudis that possesses highly heteromorphic sex chromosomes. Z and W chromosomes of Ps. tocantins differ in size, morphology, position of the nucleolar organizer region (NOR) and the amount and distribution of heterochromatin. A chromosomal inversion and heterochromatin amplification on the W chromosome were previously inferred to be involved in the evolution of this sex chromosome pair. Despite these findings, knowledge related to the molecular composition of the large heterochromatic band of this W chromosome is restricted to the PcP190 satellite DNA, and no data are available regarding the gene content of either the W or the Z chromosome of Ps. tocantins. Here, we sequenced microdissected Z and W chromosomes of this species to further resolve their molecular composition. Comparative genomic analysis suggests that Ps. tocantins sex chromosomes are likely homologous to chromosomes 4 and 10 of Xenopus tropicalis. Analyses of the repetitive DNA landscape in the Z and W assemblies allowed for the identification of several transposable elements and putative satellite DNA sequences. Finally, some transposable elements from the W assembly were found to be highly diverse and divergent from elements found elsewhere in the genome, suggesting a rapid amplification of these elements on the W chromosome.

Morphometry of two cryptic tree frog species at their hybrid zone reveals neither intermediate nor transgressive morphotypes.

Under incomplete reproductive isolation, secondary contact of diverged allopatric lineages may lead to the formation of hybrid zones that allow to study recombinants over several generations as excellent systems of genomic interactions resulting from the evolutionary forces acting on certain genes and phenotypes. Hybrid phenotypes may either exhibit intermediacy or, alternatively, transgressive traits, which exceed the extremes of their parents due to epistasis and segregation of complementary alleles. While transgressive morphotypes have been examined in fish, reptiles, birds, and mammals, studies in amphibians are rare. Here, we associate microsatellite-based genotypes with morphometrics-based morphotypes of two tree frog species of the Hyla arborea group, sampled across a hybrid zone in Poland, to understand whether the genetically differentiated parental species also differ in morphology between each other and their hybrids and whether secondary contact leads to the evolution of intermediate or transgressive morphotypes. Using univariate approaches, explorative multivariate methods (principal component analyses) as well as techniques with prior grouping (discriminant function analyses), we find that morphotypes of both parental species and hybrids differ from each other. Importantly, hybrid morphotypes are neither intermediate nor transgressive but found to be more similar to H. orientalis than to H. arborea.

Phylogenetic analysis of adaptation in comparative physiology and biomechanics: overview and a case study of thermal physiology in treefrogs.

Comparative phylogenetic studies of adaptation are uncommon in biomechanics and physiology. Such studies require data collection from many species, a challenge when this is experimentally intensive. Moreover, researchers struggle to employ the most biologically appropriate phylogenetic tools for identifying adaptive evolution. Here, we detail an established but greatly underutilized phylogenetic comparative framework - the Ornstein-Uhlenbeck process - that explicitly models long-term adaptation. We discuss challenges in implementing and interpreting the model, and we outline potential solutions. We demonstrate use of the model through studying the evolution of thermal physiology in treefrogs. Frogs of the family Hylidae have twice colonized the temperate zone from the tropics, and such colonization likely involved a fundamental change in physiology due to colder and more seasonal temperatures. However, which traits changed to allow colonization is unclear. We measured cold tolerance and characterized thermal performance curves in jumping for 12 species of treefrogs distributed from the Neotropics to temperate North America. We then conducted phylogenetic comparative analyses to examine how tolerances and performance curves evolved and to test whether that evolution was adaptive. We found that tolerance to low temperatures increased with the transition to the temperate zone. In contrast, jumping well at colder temperatures was unrelated to biogeography and thus did not adapt during dispersal. Overall, our study shows how comparative phylogenetic methods can be leveraged in biomechanics and physiology to test the evolutionary drivers of variation among species.

Positive-to-negative behavioural responses suggest hedonic evaluation in treefrog mate choice.

Sexual competition hinges on the ability to impress other conspecifics, to drive them away or attract them. In such cases, the selective environment may be hedonic or affective in nature, as it consists of the evaluations of the individuals making the decisions. This may contribute to the power of sexual selection because evaluations may range from positive to negative rather than simply from positive to neutral. Selection due to mate choice may therefore be stronger than currently appreciated. Further, change in preferred mate types can occur simply by changes (flips) in the evaluation of similar display features, adding to the dynamism of sexual selection as well as its strength. We tested the hypothesis of positive-to-negative behavioural responses in mate choice with a playback experiment using two treefrog species with 'mirror image' structures in their advertisement and aggressive calls. Female treefrog responses ranged from approach to evasion, and the presence of an aversive stimulus tainted evaluation of an attractive stimulus. Further, females in the two species showed flips in approach/evasion of stimuli with comparable signal structure. These results suggest that hedonic evaluation may have an important role in mate choice and showcase how mechanistic analysis can help understand evolutionary processes.

Buccopharyngeal morphology of the tadpoles of Scinax v-signatus/, with comments on larval characters of the S. perpusillus/ species group (Amphibia: Anura: Hylidae).

The Neotropical genus Scinax Wagler currently comprises 127 species of small treefrogs distributed from southern Mexico to Argentina and Uruguay, including some islands such as Trinidad and Tobago, and St. Lucia (Frost 2020). Two major clades are recognized within Scinax, the S. catharinae and the S. ruber clades. The former is composed of two species groups, the S. catharinae and the S. perpusillus groups (Faivovich 2002; Faivovich et al. 2005). The S. perpusillus species group currently comprises 13 species: S. alcatraz (Lutz); S. arduous Peixoto; S. atratus (Peixoto); S. belloni Faivovich, Gasparini Haddad; S. cosenzai Lacerda, Peixoto Feio; S. faivovichi Brasileiro, Oyamaguchi Haddad; S. insperatus Silva Alves-Silva; S. littoreus (Peixoto); S. melloi (Peixoto), S. peixotoi Brasileiro, Haddad, Sawaya Martins; S. perpusillus (Lutz Lutz); S. tupinamba Silva Alves-Silva; and S. v-signatus (Lutz). These species are endemic of the Brazilian Atlantic Forest and are recognized by their intimate association with bromeliads (Fig. 1), in which adults breed and lay their eggs, and tadpoles develop (Peixoto 1987, 1995; Alves-Silva Silva 2009).

A New Species of Cosmocercoides (Ascaridida: Cosmocercidae) Parasitic in Tree Frogs from Southern Amazonia.

Cosmocercoides meridionalis n. sp. is described from the large and small intestines of 5 species of tree frogs belonging to the families Hylidae and Phyllomedusidae collected from 2 localities in Southern Amazonia. The new species is allocated to the genus Cosmocercoides Wilkie, 1930, due to the presence of papillae in rosettes, which are complex caudal papillae surrounded by punctuations. Cosmocercoides meridionalis n. sp. differs from its congeners by a combination of the following characters: the absence of the gubernaculum, the total length of the female (twice the size of the male), and the presence of 26 rosette papillae with a unique arrangement and distribution pattern: 11 pre-cloacal pairs, 1 ad-cloacal pair, and 1 post-cloacal pair. This is the first species of the genus described from the Amazonia region.

Phylogenetic relationships of the Boana pulchella Group (Anura: Hylidae).

In this paper we present a phylogenetic analysis of the treefrogs of the Boana pulchella Group with the goals of (1) providing a rigorous test of its monophyly; (2) providing a test of relationships supported in previous studies; and (3) exploring the relationships of the several species not included in previous analyses. The analyses included>300 specimens of 37 of the 38 species currently included in the group, plus 36 outgroups, exemplars of the diversity of Boana and the other genera of the hylid tribe Cophomantini. The dataset included eight mitochondrial genes (12S, 16S, CytB, COI, ND1, tRNAIle, tRNALeu, and tRNAVal) and five nuclear genes (RHO, TYR, RAG-1, CXCR4, SIAH1). The phylogenetic analyses recover the monophyly of the B. pulchella Group with lower support than previous studies, as a result of the inclusion of the B. claresignata Group, which is recovered as its sister taxon. Within the B. pulchella Group, the inclusion of almost all species of the group had little impact on previous notions of its phylogeny, except for the rejection of the hypothesized B. polytaenia Clade (B. goiana and B. phaeopleura are nested in the clade here called the B. prasina Clade), which is redefined. Phylogenetic support is strong for five major clades, which collectively include all but three of the species sampled: the B. balzani Clade (B. aguilari, B. balzani, B. gladiator, B. melanopleura, B. palaestes), the redefined B. polytaenia Clade (B. botumirim, B. buriti, B. cipoensis, B. jaguariaivensis, B. leptolineata, B. polytaenia, B. stenocephala, and two undescribed species), the B. prasina Clade (B. bischoffi, B. caingua, B. cordobae, B. goiana, B. guentheri, B. marginata, B. phaeopleura, B. prasina, B. pulchella, and one undescribed species), the B. riojana Clade (B. callipleura, B. marianitae, B. riojana), and the B. semiguttata Clade (B. caipora, B. curupi, B. joaquini, B. poaju, B. semiguttata, B. stellae, and two undescribed species). The monophyly of the B. prasina + B. riojana Clades, and that of the B. polytaenia + B. semiguttata Clades are well-supported. The relationships among these two clades, the B. balzani Clade, B. ericae + B. freicanecae, and B. cambui (representing the deepest phylogenetic splits within the B. pulchella Group) are recovered with weak support. We discuss the phenotypic evidence supporting the monophyly of the B. pulchella Group, and the taxonomy of several species, identifying three new synonyms of Boana polytaenia, one new synonym of Boana goiana, and one new synonym of B. riojana.

Intra- and interindividual variation in the advertisement call of Dendropsophus banner (Cochran, 1948) (Anura: Hylidae).

Vocalization is one of the main behaviors of anurans (Köhler et al. 2017). Calls emitted by these animals display several social and ecological functions. For example, they can be used for attraction of conspecific females (Toledo et al. 2015) and territorial defense (Wells 2010). Advertisement calls are emitted predominantly by males (Kelley 2004) and constitute a key-factor for species identification (Köhler et al. 2017).

The advertisement call of Boana freicanecae (Carnaval amp; Peixoto, 2004) (Anura: Hylidae), an endemic and poorly known treefrog from northeastern Brazil.

Boana freicanecae is a medium-sized hylid (males SVL= 37.3-42.2 mm) belonging to the B. pulchella group (Carnaval Peixoto 2004). This species is associated with streams in Atlantic montane forests (450-750 m elevation) located from the Pernambuco Biogeographic Sub-Region (Silva Casteleti 2003), in the states of Pernambuco and Alagoas, northeastern Brazil (Carnaval Peixoto 2004; Cardoso et al. 2006). Currently, B. freicanecae is classified as 'Data Deficient' according to the Brazilian Red List of Threatened species (ICMBio 2018) and many aspects of its natural history (e.g., advertisement call) are poorly known. Here we describe the advertisement call of this treefrog based on calls obtained from two populations.

The advertisement call of Scinax funereus (Cope, 1874) (Anura: Hylidae) from Ecuadorian Amazon.

Scinax Wagler currently harbors 72 treefrog species distributed throughout the Neotropical region, occurring from Mexico to Argentina and Uruguay, and also in some Caribbean islands (Frost et al. 2020). The genus has a high level of cryptic diversity, with its actual number of species currently considered to be underestimated (Fouquet et al. 2007, Ferrão et al. 2016). Scinax funereus (Cope) is a medium-sized species described from Moyobamba, San Martín Department, northern Peru, with occurrences reported from the Peruvian and Ecuadorian Amazon (Duellman Wiens 1993), southern Colombia (Lynch 2005), and from the Brazilian states of Acre (Bernarde et al. 2011) and Amazonas (Ramalho et al. 2016). Duellman (1978) reported that the advertisement call of S. funereus "consists of a moderately long, rather high-pitched, single note", but as no call recording was obtained at the time, it was not described. Although Read (2000) published the species' call in the audio form in a compact disc (CD), it was not formally described. Given this, herein we provide the first formal description of the advertisement call of S. funereus.

Climatic breadth of calling behaviour in two widespread Neotropical frogs: Insights from humidity extremes.

Climate change is severely altering precipitation regimes at local and global scales, yet the capacity of species to cope with these changes has been insufficiently examined. Amphibians are globally endangered and particularly sensitive to moisture conditions. For mating, most amphibian species rely on calling behaviour, which is a key weather-dependent trait. Using passive acoustics, we monitored the calling behaviour of two widespread Neotropical frogs in 12 populations located at the humidity extremes but thermal mean of the species distribution. Based on 2,554 hr of recordings over a breeding season, we found that both the aquatic species Pseudis paradoxa and the arboreal species Boana raniceps exhibited calling behaviour at a wide range of relative humidity. Calling humidity was significantly lower in conspecific populations subjected to drier conditions, while calling temperature did not differ between populations or species. Overall, no variation in climatic breadth was observed between large and small choruses, and calling behaviour was scarcely detected during the driest, hottest and coldest potential periods of breeding. Our results showed that calling humidity of the studied species varies according to the precipitation regime, suggesting that widespread Neotropical anurans may have the capacity to exhibit sexual displays in different climatic environments. Regardless of the underlying mechanism (plasticity or local adaptation), which should be determined by common garden experiments, a wide and population-specific climatic breadth of calling behaviour may assist species to deal with changing humidity conditions. To our knowledge, this is the first study to explore the response capacity of anurans to perform calling behaviour under contrasting precipitation regimes.

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.

A grave drum at rocky springs: the vocalization of Bokermannohyla alvarengai (Anura: Hylidae) from the Espinhaço Range, southeastern Brazil, with notes on its natural history.

Communication in anurans is mediated mainly by acoustic signals, which are relevant for mating, territoriality, and other social interactions (Wells 2007). Although these signals are usually conspicuous and relatively stereotyped, some Neotropical frogs display noticeable diversity and complexity in their calls. For instance, Bokermannohyla, a genus of gladiator treefrog from the Atlantic Forest, Cerrado, and Caatinga Biomes of Brazil, often have complex vocal repertoires (Carvalho et al. 2012; Nali Prado 2014), whilst some congeners have simple and stereotyped calls emitted continuously (Carvalho et al. 2013; Taucce et al. 2015; Giaretta et al. 2016; Rocha et al. 2016).

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.

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)