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The karyotype of Litoria (L.) paraewingi (Watson et al., 1971) (Big River State Forest, Victoria) is described here for the first time. It is prepared following tissue culture of toe clipping macerates, cryopreservation, reculture and conventional 4',6-diamidino-2-phenylindole (DAPI) staining. The L.paraewingi karyotype is then compared to similarly processed IUCN (International Union for the Conservation of Nature) least concern members L.ewingii (Duméril et Bibron, 1841) (southern Victoria) and L.jervisiensis (Duméril et Bibron, 1841) (Myall Lakes National Park, New South Wales), all members of the same L.ewingii complex/group. The L.paraewingi diploid number is 2n = 26, the same as for the other two species. Litoriaparaewingi chromosomes 1, 2, 6 and 7 are submetacentric, chromosomes 3 and 5 are subtelocentric and the remainder are metacentric. No secondary constriction or putative nucleolus organiser region (NOR) was readily identifiable following conventional DAPI staining in any scored L.paraewingi metaphase spread. Conversely, a putative NOR was readily identifiable on the long arm of chromosome 1 in all examined metaphase spreads for the other two species. The karyotypes of L.ewingii and L.jervisiensis here further differ from L.paraewingi with chromosome 1 being metacentric and chromosomes 8 and 10 being submetacentric for both former species. The L.jervisiensis karyotype differs from those of L.ewingii and L.paraewingi by DAPI staining with: (i) apparent relative length inversion of subtelocentric chromosome 3 and metacentric chromosome 4 and (ii) chromosome 6 being metacentric rather than submetacentric. All three species have a highly conserved chromosome morphology with respect to chromosomes 2, 5, 7, 9, 11, 12 and 13. The greatest gross morphological difference karyotypically is observed between L.paraewingi and L.jervisiensis. These karyotype data support the previous phylogenetic separation of these three species based upon genetic compatibility and behavioural, biochemical and molecular genetic analyses.

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Clinostomum spp. are common parasites of piscivorous birds. Metacercaria are typically observed in the muscles or just under the skin of fish and rarely amphibians. We describe an unusually severe case of Clinostomum marginatum infection in an adult female green tree frog (Hyla cinerea) from Georgia (USA). The frog was found in November 2015 with a high number (>250) of widely disseminated, raised, subcutaneous nodules. The frog died in December. At necropsy, it was emaciated, and the skin was covered in raised uniform, tan-green, subcutaneous, ∼2-3 mm diameter nodules. Each nodule contained 1-3 C. marginatum metacercariae. Microscopically, high numbers of trematodes were within subcutaneous tissues and in coelomic and oral cavities, lung, liver, kidney, ovary, orbit and calvarium. Small to large numbers of lymphocytes and melanomacrophages were in connective tissues and epidermis. A 732 bp region of COI was 98.8-99.8% similar to numerous sequences of C. marginatum and, phylogenetically it grouped with these C. marginatum sequences. The ITS-1 region was 100% similar to a C. marginatum sample from a great egret (Ardea alba) from Mississippi. This report represents a novel finding of severe trematodiasis in a free-ranging amphibian with C. marginatum infection.

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For adult anuran amphibians, the kidney and urinary bladder play important osmoregulatory roles through water reabsorption. In the present study, we have examined ontogenetic expression of aquaporins, i.e., AQP2, AQPamU (AQP6ub, AQPa2U), and AQP3, in these organs using the Japanese tree frog, Dryophytes japonicus. Immunohistochemistry using the metamorphosing larvae at stages 40-43 localized AQP2 protein to the collecting ducts in the dorsal zone of the mesonephric kidney. At prometamorphic stages 40 and 41, labelling of AQP2 protein was observed in the apical/ subapical regions of the collecting duct cells. At climax stages 42 and 43, labels for AQP2 and AQP3 became observed in the apical/subapical regions and basolateral membrane of the collecting duct cells, respectively, as seen in the adults. As for the urinary bladder, immuno-positive labels for AQPamU were localized to the apical/subapical regions of granular cells in the mucosal epithelium at stages 40-43. On the other hand, AQP3 immunoreactivity was hardly observed in the urinary bladder at stage 40, and weakly appeared in many granular cells at stage 41. Thereafter, labels for AQP3 became evident along the basolateral membrane of granular cells at stages 42 and 43, together with AQPamU in the apical/subapical regions. These results suggest that the kidney and urinary bladder might be capable of water reabsorption, via AQP2, AQPamU, and AQP3, at stage 42, contributing to the acclimation of the tree frogs to terrestrial environments.

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The tree frog is a prominent amphibian among terrestrial vertebrates known for its ability to adhere to various surfaces through the capillary forces of water in the microchannels between micropillars on its disc-shaped toe pads, a phenomenon known as wet adhesion. However, the secretion pattern of mucus on the attachment surface of living tree frog toe pads and the distribution of active mucus pores (AMPs) have not yet been fully elucidated. In this study, we utilized synchrotron X-ray micro-computed tomography and interference reflection microscopy to obtain the spatial distribution of the entire population of ventral mucus glands on the toe pads of living tree frogs and the real-time mucus secretion patterns from the ventral mucus pores on the contact surface under different environmental conditions. We observed that the number and secretion frequency of AMPs on the toe pad are regulated according to environmental conditions. Such dynamic mucus secretion on the tree frog's toe pad could contribute to the understanding of capillary force regulation for wet adhesion and the development of adhesive surfaces by mimicking the mucus-secreting toe pad.

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Temperature is essential for the survival and development of eggs. Some anurans have evolved and developed foam nesting traits, with thermal insulation considered to be among their functions. Foam-nesting frogs tend to exhibit reproductive plasticity. For example, they oviposit on both trees and the ground. How such plasticity affects foam nest function is of major relevance and is likely related to the adaptation of foam nesting frogs. However, this has not been well studied. In this study, we examined the interaction between foam nest site, foam nest function, and egg fate using the Japanese green tree frog, Zhangixalus arboreus, and analysed how nest site differences (arboreal or terrestrial) affect the thermal function of foam nests. We compared the thermal functions of foam nests between arboreal and terrestrial oviposition sites of Z. arboreus. We artificially replaced half of the arboreal nests with terrestrial environments and recorded temperature in and outside of the experimental terrestrial nest and original arboreal nests. We also examined egg survival and hatching rates for all the nests. The results indicated superior heat insulation in terrestrial nests, with warmer temperatures inside than outside the nests, especially at night, which led to a high egg survival rate. Therefore, terrestrial ovipositing should be valid under cold weather conditions. This may be related to the evolutionary history of oviposition site plasticity of this genus, which originally had an arboreal oviposition trait but evolved into terrestrial site use owing to global cooling. Our novel insights into the evolution and adaptivity of foam nesting and oviposition site use in Z. arboreus make an important contribution to animal ecology.

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Due to excessive use or abuse in the food industry, agriculture, and medicine, many pathogens are developing resistance against conventional antibiotics. Antimicrobial peptides (AMPs) hold promise as effective therapeutic options for the treatment of bacterial infections. Herein, a novel cathelicidin antimicrobial peptide (Zs-CATH) was identified from the tree frog Zhangixalus smaragdinus. Zs-CATH mainly adopted an amphipathic ß-sheet structure in a membrane-mimetic environment. It showed broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria in vitro and significantly protected mice from lethal infections induced by Gram-negative bacteria Escherichia coli ATCC 25922 or Gram-positive bacteria Staphylococcus aureus ATCC 25923 in vivo. In addition, Zs-CATH exerted a strong anti-inflammatory effect by neutralizing lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and promoting macrophage M2 polarization, thus inhibiting the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) and enhancing the production of M2 macrophage markers IL-10, IL-4, and CD206. The MAPK and NF-κB inflammatory signaling pathways and transcriptional activator 6 (STAT6) were involved in this effect. In mice, Zs-CATH rapidly recruited neutrophils and monocytes/macrophages to the abdominal cavity but not T and B lymphocytes. Zs-CATH did not exhibit a direct chemoattractant effect on phagocytes but significantly promoted phagocyte migration in the presence of macrophages. Zs-CATH stimulated macrophages to secrete chemokines CXCL1, CXCL2, and CCL2, which mediated the recruitment of phagocytes. Furthermore, Zs-CATH promoted the production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs), which are oxygen-dependent and oxygen-independent mechanisms of the microbicidal activity of neutrophils, respectively. Zs-CATH exhibited no toxic side effects on mammalian cells and mice. These findings show that in addition to direct antibacterial activity, Zs-CATH also possesses the ability to modulate immune and inflammatory processes during bacterial infection, showing potential for development as anti-infective and/or anti-inflammatory agents.

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Magnetic fluid shock absorbers (MFSAs) have been successfully utilized to eliminate microvibrations of flexible spacecraft structures. The method of enhancing the damping efficiency of MFSAs has always been a critical issue. To address this, we drew inspiration from the tree frog's toe pads, which exhibit strong friction due to their unique surface structure. Using 3D printing, we integrated bionic textures copied from tree frog's toe pad surfaces onto MFSAs, which is the first time to combine bionic design and MFSAs. Additionally, this is also the first time that surface textures have been applied to MFSAs. However, we also had to consider practical engineering applications and manufacturing convenience, so we modified the shape of bionic textures. To do so, we used an edge extraction algorithm for image processing and obtained recognition results. After thorough consideration, we chose hexagon as the shape of surface textures instead of bionic textures. For theoretical analysis, a magnetic field-flow field coupling dynamic model for MFSAs was built for the first time to simulate the magnetic fluid (MF) flow in one oscillation cycle. Using this model, the flow rate contours of the MF were obtained. It was observed that textures cause vortexes to form in the MF layer, which produced an additional velocity field. This increased the shear rate, ultimately leading to an increase in flow resistance. Finally, we conducted vibration reduction experiments and estimated damping characteristics of the proposed MFSAs to prove the effectiveness of both bionic texture and hexagon surface textures. Fortunately, we concluded that hexagon surface textures not only improve the damping efficiency of MFSAs but also require less MF mass.

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Some frog species have a unique skeletal element, referred to as the intercalary element (IE), in the joints between the terminal and subterminal phalanges of all digits. IEs are composed of cartilage or connective tissue and have a markedly differ shape than the phalanges. IEs are highly related to the arboreal lifestyle and toe pads. The IE is found only in neobatrachian frogs among anurans, suggesting that it is a novelty of Neobatrachia. IEs are widely distributed among multiple neobatrachian lineages and are found in the suborders Hyloides and Ranoides (the two major clades in Neobatrachia). However, it is unclear whether the IEs found in multiple linages resulted from convergent evolution. Therefore, in this study, we aimed to examine how similar or different the developmental trajectories of the IEs are between Hyloides and Ranoides. To that end, we compared the osteological and histological developmental processes of the IEs of the hyloid frog Dryophytes japonicus and the ranoid frog Zhangixalus schlegelii. Both species shared the same IE-initiation site and level of tissue differentiation around the IE when it began to form in tadpoles, although the IE developments initiated at different stages which were determined by external criteria. These results suggest that similar mechanisms drive IE formation in the digits of both species, supporting the hypothesis that the IEs did not evolve convergently.

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The strong adhesion on dry and wet surfaces and the durability of bioinspired hierarchical fibrillar adhesives are critical for their applications. However, the critical design for the strong adhesion normally depends on fine sub-micron structures which could be damaged during repeat usage. Here, we develop a tree frog-inspired gradient composite micropillars array (GP), which not only realizes a 2.3-times dry adhesion and a 5.6-times wet adhesion as compared to the pure polydimethylsiloxane (PDMS) micropillars array (PP), but also shows excellent durability over 200 repeating cycles of attachment/detachment and self-cleaning ability. A GP consists of stiffer tips and softer roots by incorporating gradient dispersed CaCO3 nanoparticles in PDMS micropillar stalks. The modulus gradient along the micropillar height facilitates the contact formation and enhances the maximum stress during the detaching. The study here provides a new design strategy for robust adhesives for practical applications in the fields of robotics, electronics, medical engineering, etc.

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Phyllomedusa bicolor (Phyllomedusidae), popularly known as the kambô in Brazil, is a tree frog that is widely distributed in South American countries and is known for producing a skin secretion that is rich in bioactive peptides, which are often used in indigenous rituals. The biological effects of the skin secretion were observed in the first studies with indigenous communities. Over the last six decades, researchers have been studying the chemical composition in detail, as well as the potential pharmacological applications of its constituents. For this reason, indigenous communities and health agents fear the misuse of the kambô, or the inappropriate use of the species, which can result in health complications or even death of users. This article seeks to provide a transdisciplinary review that integrates knowledge regarding the biology of P. bicolor, ethnoknowledge about the ritual of the kambô, and the chemistry and pharmacology of the skin secretion of this species, in addition to medical aspects of the indiscriminate use of the kambô. Furthermore, this review seeks to shed light on perspectives on the future of research related to the kambô.

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Glycerol and aquaporin 9 (aquaglyceroporin) are known to be involved in freeze tolerance in the Japanese tree frog Hyla japonica. However, the regulatory mechanisms of freeze tolerance in this species have not been fully elucidated. In the present study, we focused on the inter- and intracellular dynamics of glucose to analyze the role of glucose and glucose-related proteins such as transporter and metabolic enzymes in freeze tolerance. Serum glucose concentrations were compared among the frogs that were nonhibernating, hibernating, and thawed after freezing at -4°C for 6 hr. Serum concentrations of glucose in thawed frogs were significantly higher than those in hibernating and nonhibernating, active frogs. Periodic acid-Schiff staining showed that the accumulation of glycogen in the hepatocytes increased before hibernation and decreased after freezing and thawing. Quantitative RT-PCR analysis using the liver showed that, compared with active frogs, the type 2 glucose transporter gene (glut2) was upregulated in frozen frogs, the liver glycogen phosphorylase gene (pygl) was upregulated in frozen or thawed frogs, and the type 2 glycogen synthase gene (gys2) was upregulated in hibernating frogs. Immunohistochemistry of liver sections showed that, compared with nonhibernating frogs, Glut2 proteins were clearly increased most likely on the plasma membrane of hepatocytes in hibernating frogs and further increased by freezing, then decreased after thawing. These results suggest the possibility that glucose acts as a cryoprotectant in H. japonica.

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Grey tree frogs (Dryophytes versicolor) have the remarkable ability to endure full-body freezing over the winter, with up to 42% of total body water converted into extracellular ice. Survival is aided by metabolic rate depression that greatly reduces tissue energy costs over the winter. Post-transcriptional controls on gene expression which include miRNA regulation of gene transcripts can aid implementation of the reversible changes required for freeze tolerance, since miRNAs are ideal for facilitating the rapid metabolic reorganization needed for this process. The energy cost for synthesizing new miRNAs is low, and miRNAs' ability to target more than one mRNA transcript (and vice versa) allows a wide versatility in their capability for metabolic restructuring. Western immunoblotting was used to examine protein expression levels of members of the miRNA biogenesis pathway in D. versicolor liver, skeletal muscle, and kidney. Four of these proteins (Dicer, Drosha, Trbp, Xpo5) were upregulated in liver of frozen frogs, suggesting enhanced capacity for miRNA biogenesis, whereas expression of four proteins in frozen muscle (Ago1, Ago2, Dgcr8, Xpo5) and six proteins in kidney (Ago1, Ago2, Ago3, Ago4, Dgcr8, Ran-GTP) were downregulated, indicating an opposite trend. Overall, the data show that miRNA biosynthesis is altered during freezing and differentially regulated across tissues. We suggest that miRNAs are central for the freeze tolerance strategy developed by D. versicolor, and future research will expound upon specific miRNAs and their roles in mediating responses to freezing stress.

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The Amazonia and the Atlantic Forest, separated by the diagonal of open formations, are two ecoregions that comprise the most diverse tropical forests in the world. The Sphaenorhynchini tribe is among the few tribes of anurans that occur in both rainforests, and their historical biogeographic have never been proposed. In this study, we infer a dated phylogeny for the species of the Sphaenorhynchini and we reconstructed the biogeographic history describing the diversification chronology, and possible patterns of dispersion and vicariance, providing information about how orogeny, forest dynamics and allopatric speciation affected their evolution in South America. We provided a dated phylogeny and biogeography study for the Sphaenorhynchini tribe using mitochondrial and nuclear genes. We analyzed 41 samples to estimate the ancestral areas using biogeographical analysis based on the estimated divergence times and the current geographical ranges of the species of Sphaenorhynchini. We recovered three characteristic clades that we recognize as groups of species (S. lacteus, S. planicola, and S. platycephalus groups), with S. carneus and G. pauloalvini being the sister taxa of all other species from the tribe. We found that the diversification of the tribe lineages coincided with the main climatic and geological factors that shaped the Neotropical landscape during the Cenozoic. The most recent common ancestor of the Sphaenorhynchini species emerged in the North of the Atlantic Forest and migrated to the Amazonia in different dispersion events that occurred during the connections between these ecoregions. This is the first large-scale study to include an almost complete calibrated phylogeny of Sphaenorhynchini, presenting important information about the evolution and diversification of the tribe. Overall, we suggest that biogeographic historical of Sphaenorhynchini have resulted from a combination of repeated range expansion and contraction cycles concurrent with climate fluctuations and dispersal events between the Atlantic Forest and Amazonia.

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The roles of bactericidal cathelicidins against bacterial infection have been extensively studied. However, the antibacterial property and mechanism of action of non-bactericidal cathelicidins are rarely known. Herein, a novel naturally occurring cathelicidin (PopuCATH) from tree frog (Polypedates puerensis) did not't show any direct anti-bacterial activity in vitro. Intriguingly, intraperitoneal injection of PopuCATH before bacterial inoculation significantly reduced the bacterial load in tree frogs and mice, and reduced the inflammatory response induced by bacterial inoculation in mice. PopuCATH pretreatment also increased the survival rates of septic mice induced by a lethal dose of bacterial inoculation or cecal ligation and puncture (CLP). Intraperitoneal injection of PopuCATH significantly drove the leukocyte influx in both frogs and mice. In mice, PopuCATH rapidly drove neutrophil, monocyte/macrophage influx in mouse abdominal cavity and peripheral blood with a negligible impact on T and B lymphocytes, and neutrophils, monocytes/macrophages, but not T and B lymphocytes, were required for the preventive efficacy of PopuCATH. PopuCATH did not directly act as chemoattractant for phagocytes, but PopuCATH obviously drove phagocyte migration when it was cultured with macrophages. PopuCATH significantly elicited chemokine/cytokine production in macrophages through activating p38/ERK mitogen-activated protein kinases (MAPKs) and NF-κB p65. PopuCATH markedly enhanced neutrophil phagocytosis via promoting the release of neutrophil extracellular traps (NETs). Additionally, PopuCATH showed low side effects both in vitro and in vivo. Collectively, PopuCATH acts as a host-based immune defense regulator that provides prophylactic efficacy against bacterial infection without direct antimicrobial effects. Our findings reveal a non-bactericidal cathelicidin which possesses unique anti-bacterial action, and highlight the potential of PopuCATH to prevent bacterial infection.

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With the great biodiversity existing on our planet, different organisms have developed defensive strategies based on the use of toxins. Among tetrapods, the Amphibia class are highlighted in this sense, since all its representatives have poison glands in their skin. A very interesting group from the toxinological point of view are the frogs, especially the casque-headed tree frogs. They form a group belonging to the Lophiohylini tribe, in which many species have a skull with high bone coverage, bone projections in the form of spines, dermal coossification and phragmotic behavior, using the head to seal the entrance of their shelters. Aspects of the natural history of representative species of Lophiohylini (Nyctimantis spp, Trachycephalus spp, Corythomantis greeningi and Itapotihyla langsdorffii) were elucidated through fieldwork. These data were associated with cranial anatomical and histological analyzes of the head of each species. The present work verified a correlation between the increase of the cranial bone coverage and its spinous projections related to venom glands with the execution of the phragmosis. It was also evidenced in the skin secretions of these species, significant differences in chemical composition, with the presence of molecules with enzymatic activities widely found in animal venoms, such as hyaluronidases. Furthermore, except for N. galeata and I. langsdorffii, the other species showed evident cytotoxic potential. Through the information gathered, it was possible to list different species of casque-headed tree frogs endowed with a “Toxin Inoculating System” (SIT), defined by the presence of spines related to cutaneous glands containing toxic secretions, which can, therefore, be considered venomous (instead of poisonous) species.

Com a grande biodiversidade existente em nosso planeta, diferentes organismos desenvolveram estratégias defensivas baseadas na utilização toxinas. Entre os tetrápodes, a classe Amphibia merece destaque nesse sentido, visto que todos seus representantes possuem glândulas de veneno em sua pele. Um grupo bastante interessante do ponto de vista toxinológico, são os anuros, em especial as “pererecasde-capacete”. São espécies pertencentes a tribo Lophiohylini, na qual muitas possuem o crânio com alta abrangência óssea, projeções ósseas em formato de espinhos, coossificação dérmica e comportamento fragmótico, utilizando a cabeça para tapar a entrada dos abrigos que ocupam. Através de trabalhos de campo foram elucidados aspectos da história natural de espécies representativas de Lophiohylini (Nyctimantis spp, Trachycephalus spp, Corythomantis greeningi e Itapotihyla langsdorffii). Esses dados foram associados a análises cranianas anatômicas e histológicas da cabeça de cada espécie. O presente trabalho verificou uma correlação entre o aumento da abrangência óssea craniana e suas projeções espinhosas, essas relacionadas a glândulas de veneno com a execução da fragmose. Foi também evidenciado nas secreções cutâneas dessas espécies, diferenças significativas de composição química e a presença de moléculas com atividades enzimáticas amplamente encontradas em peçonhas animais, como hialuronidases. Além disso, com exceção de N. galeata e I. langsdorffii, as demais espécies apresentaram evidente potencial citotóxico. Assim, por meio das informações levantadas, foi possível elencar diferentes espécies de pererecas-de-capacete dotadas de um “Sistema Inoculador de Toxinas” (SIT), definido pela presença de espinhos relacionados a glândulas cutâneas contendo secreções tóxicas, e que podem, por isso, ser consideradas como espécies peçonhentas ao invés de venenosas.

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Anuran advertisement calls play a fundamental role in social interactions related to reproduction, like territory defense and mate attraction. As such, advertisement calls are often shaped by natural and sexual selection. Therefore, to understand the evolution, function, and mechanisms of signal production and perception, it is important to characterize sources and patterns of variation in call properties. We recorded and analyzed the two types of advertisement calls produced by Pacific tree frogs, Hyliola regilla: diphasic and monophasic calls. Specifically, we examined (i) the relationship between call properties and both body size and physical condition, (ii) patterns of within- and among-individual variation in call properties, and (iii) acoustic differences between diphasic and monophasic calls. While only one property of diphasic calls correlates with physical condition, spectral properties of both types of calls correlate with body size. Patterns of within- and among-individual variation were similar between call types and, overall, consistent with patterns reported for closely related species. Monophasic calls have lower frequencies, are delivered faster, and have higher call effort and duty cycle than diphasic calls. We discuss our results in relation to sexual selection and formulate hypotheses about the evolution, function, and mechanisms of acoustic communication in Pacific tree frogs.

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Advertisement calls potentially represent honest signals for delimiting species and sexual selection. Quantitative statistics of individual variation in advertisement call properties can be used to predict female preferences for particular signal properties. In this study, advertisement call properties and their individual variation was analyzed in two endemic treefrog species, Liuixalus hainanus and L. ocellatus. Together with the description of the advertisement calls, our goals included determining whether call properties can be used to distinguish between the two species on the field, which acoustic properties are likely play a role in species or individual recognition, and whether they could predict the signaler's body size. We found that the dominant frequency, call duration, inter-call interval, and note number of monosyllabic calls in L. hainanus were significantly higher or longer than those in L. ocellatus. The dominant frequency was classified as a static property; the call duration, inter-call interval, inter-note interval, and note number were classified as dynamic properties in both species. The inter-note interval of multisyllabic calls was correlated with body mass, and the pulse number of monosyllabic calls was correlated with snout-vent length in L. ocellatus. These results indicate that the dominant frequency strongly contributed to species recognition of L. hainanus and L. ocellatus males. Acoustic properties reflected the signaler's body size in L. ocellatus but did not in L. hainanus. The difference in advertisement call characteristics between the two species may be due to the different reproductive strategies associated with different selection pressures and may promote the sharing of similar habitats by the two species.

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BACKGROUND: 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.

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Background: 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.

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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)

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