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Among vertebrates, the yolk is commonly the only form of nutritional investment offered by the female to the embryo. Some species, however, have developed parental care behaviors associated with specialized food provisioning essential for offspring survival, such as the production of lipidic-rich parental milk in mammals. Here, we show that females of the egg-laying caecilian amphibian Siphonops annulatus provide similarly lipid-rich milk to altricial hatchlings during parental care. We observed that for 2 months, S. annulatus babies ingested milk released through the maternal vent seemingly in response to tactile and acoustic stimulation by the babies. The milk, composed mainly of lipids and carbohydrates, originates from the maternal oviduct epithelium's hypertrophied glands. Our data suggest lactation in this oviparous nonmammalian species and expand the knowledge of parental care and communication in caecilians.

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Sequestration of chemical defenses from dietary sources is dependent on the availability of compounds in the environment and the mechanism of sequestration. Previous experiments have shown that sequestration efficiency varies among alkaloids in poison frogs, but little is known about the underlying mechanism. The aim of this study was to quantify the extent to which alkaloid sequestration and modification are dependent on alkaloid availability and/or sequestration mechanism. To do this, we administered different doses of histrionicotoxin (HTX) 235A and decahydroquinoline (DHQ) to captive-bred Adelphobates galactonotus and measured alkaloid quantity in muscle, kidney, liver, and feces. HTX 235A and DHQ were detected in all organs, whereas only DHQ was present in trace amounts in feces. For both liver and skin, the quantity of alkaloid accumulated increased at higher doses for both alkaloids. Accumulation efficiency in the skin increased at higher doses for HTX 235A but remained constant for DHQ. In contrast, the efficiency of HTX 235A accumulation in the liver was inversely related to dose and a similar, albeit statistically nonsignificant, pattern was observed for DHQ. We identified and quantified the N-methylation of DHQ in A. galactonotus, which represents a previously unknown example of alkaloid modification in poison frogs. Our study suggests that variation in alkaloid composition among individuals and species can result from differences in sequestration efficiency related to the type and amount of alkaloids available in the environment.

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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.

RESUMO

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)

Assuntos

RESUMO

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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The phenomenon of fluorescence can be used by animals to change effective colouration or patterning, potentially to serve functions including intra- and interspecific signalling. Initially believed to be restricted to marine animals, fluorescent colours are now being described in an increasing number of terrestrial species. Here, we describe unique, highly fluorescent patterns in two species of pumpkin toadlets (Brachycephalus ephippium and B. pitanga). We establish that the origin of the fluorescence lies in the dermal bone of the head and back, visible through a particularly thin skin. By comparing them to those of the closely related species Ischnocnema parva, we demonstrate that pumpkin toadlets' bones are exceptionally fluorescent. We characterize the luminescence properties of the toadlets' bones and discuss the potential function of fluorescent patterns in natural lighting conditions.

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Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians' skin, and no antibody recognition on fish and mammals' skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a 'paleo-protein'. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding-as indicated by the molecular models and immunohistochemistry analyses-a consequence of the different cysteine pattern but with conserved biological function.

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Bufotenine is an alkaloid derived from serotonin, structurally similar to LSD and psilocin. This molecule is able to inhibit the rabies virus infection in in vitro and in vivo models, increasing the survival rate of infected animals. Being a very promising molecule for an incurable disease and because of the fact that there is no consensus regarding its neurological effects, this study aimed to evaluate chronic treatment of bufotenine on behavior, pathophysiology, and pharmacokinetics of mice. Animals were daily treated for 21 consecutive days with 0.63, 1.05, and 2.1 mg/animal/day bufotenine and evaluated by open field test and physiological parameters during all the experiment. After this period, organs were collected for histopathological and biodistribution analysis. Animals treated with bufotenine had mild behavioral alterations compared to the control group, being dose-response relationship. On the other hand, animals showed normal physiological functions and no histological alterations in the organs. With high doses, an inflammatory reaction was observed in the site of injection, but with no cellular damage. The alkaloid could be found in the heart and kidney with all doses and in the lungs and brain with higher doses. These results show that the effective dose, 0.63 mg/day, is safe to be administered in mice, since it did not cause significant effects on the animals' physiology and on the CNS. Higher doses were well tolerated, causing only mild behavioral effects. Thus, bufotenine might be a drug prototype for rabies treatment, an incurable disease.

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The emergence and maintenance of animal communication systems requires the co-evolution of signal and receiver. Frogs and toads rely heavily on acoustic communication for coordinating reproduction and typically have ears tuned to the dominant frequency of their vocalizations, allowing discrimination from background noise and heterospecific calls. However, we present here evidence that two anurans, Brachycephalus ephippium and B. pitanga, are insensitive to the sound of their own calls. Both species produce advertisement calls outside their hearing sensitivity range and their inner ears are partly undeveloped, which accounts for their lack of high-frequency sensitivity. If unheard by the intended receivers, calls are not beneficial to the emitter and should be selected against because of the costs associated with signal production. We suggest that protection against predators conferred by their high toxicity might help to explain why calling has not yet disappeared, and that visual communication may have replaced auditory in these colourful, diurnal frogs.

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Toads have a pair of glandular accumulations on each side of the dorsal region of the head known as parotoid macroglands. These macroglands consist of secretory units (granular glands), each one capped with an epithelial plug. When threatened, toads point one of the parotoids toward the aggressor, and if the aggressor squeezes the parotoid with its teeth, jets of poison will come out of the secretory units and hit the predator's oral mucosa, thereby causing poisoning. Our study focused on the mechanism of parotoid function by comparing parotoids from toads naturally attacked by dogs with those manually compressed. We verified that the process of glandular emptying in response to dog bites is very similar to that following manual compression. We observed that the structure of the plug plays an essential role in the release of the poison jets. Our results suggest that the parotoids may act as "bulletproof vests," reducing the impact of the force exerted by predator attacks, and thus may function as a passive antipredator mechanism.

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