Milk provisioning in oviparous caecilian amphibians.

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.

Milk provisioning in oviparous caecilian amphibians

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.

Peering into the unknown world of amphisbaenians (Squamata, Amphisbaenia): A summary of the life history of Amphisbaena alba

Capturing data on the life of fossorial vertebrates is difficult since access to the subterranean environment is made unfeasible by its density and opacity. Collecting specimens is only possible through excavation work, causing damage or even death to the specimens. Due to the obstacles of in situ studies, the scarce information comes from reports obtained indirectly, mainly through specimens preserved in museums. Considering the adaptations to fossoriality, investments in studying these groups could be very enlightening since they would contribute enormously to the knowledge of the evolutionary strategies developed throughout the colonisation of the subterranean world. Amphisbaena alba is the species of Amphisbaenia with the broadest geographic distribution in the world. It occupies virtually all countries in South America except for Chile and southern Argentina. This study, carried out over the last 36 years, aims to provide data on the biology and behaviour of A. alba in captivity and in the field. Our main objective is to provide subsidies to expand the knowledge of the life history of this species and, by extension, of amphisbaenians in general.

Reproductive behaviour, cutaneous morphology, and skin secretion analysis in the anuran Dermatonotus muelleri.

Despite the common poison and mucous glands, some amphibian groups have differentiated glands associated with reproduction and usually present on the male ventral surface. Known as breeding glands or sexually dimorphic skin glands (SDSGs), they are related to intraspecific chemical communication during mating. Until recently, reproduction associated with skin glands was recognized only in salamanders and caecilians and remained unexplored among anurans. The Brazilian microhylid Dermatonotus muelleri (Muller's termite frog) is known for its very toxic skin secretion. Despite the slippery body, the male adheres to the female back during reproduction, as they have differentiated ventral glands. In this paper, we have gathered data proposing an integrative approach correlated with the species' biology and biochemical properties of their skin secretions. Furthermore, we suggest that the adhesion phenomenon is related to arm shortening and rounded body that make amplexus inefficient, although constituting important adaptive factors to life underground.

Dose-dependent alkaloid sequestration and N-methylation of decahydroquinoline in poison frogs.

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.

Skin and poison glands in toads (Rhinella) and their role in defence and water balance

Toads are considered poisonous animals since they have a passive mode of defence relying on cutaneous poison glands, differently from venomous animals who can inject venom in predators/aggressors or prey. Toads of Rhinella marina group are generally large and have a broad distribution in South America, inhabiting a wide range of environments. In this paper, we studied the toads Rhinella icterica from the Atlantic rainforest, and Rhinella jimi from the Brazilian Semiarid Caatinga, analysing aspects of natural history and comparing their skin morphology, the presence of macroglands, their resistance to water loss and rates of water uptake. In periods of extreme drought, R. jimi uses rock cracks as refuges, exposing only the head and regions of accumulation of poison glands. The skin of R. jimi showed higher number of poison glands with hydrophilic content than R. icterica. R. jimi also had a thicker skin, which can be related to its superior resistance to water loss. It also showed high rates of rehydration in association with a peculiar behaviour using the limbs to spread water onto highly glandular skin areas, suggesting that poison glands may also act in water balance in addition to chemical defence.

Reproductive behaviour, cutaneous morphology, and skin secretion analysis in the anuran Dermatonotus muelleri

Despite the common poison and mucous glands, some amphibian groups have differentiated glands associated with reproduction and usually present on the male ventral surface. Known as breeding glands or sexually dimorphic skin glands (SDSGs), they are related to intraspecific chemical communication during mating. Until recently, reproduction associated with skin glands was recognized only in salamanders and caecilians and remained unexplored among anurans. The Brazilian microhylid Dermatonotus muelleri (Muller's termite frog) is known for its very toxic skin secretion. Despite the slippery body, the male adheres to the female back during reproduction, as they have differentiated ventral glands. In this paper, we have gathered data proposing an integrative approach correlated with the species' biology and biochemical properties of their skin secretions. Furthermore, we suggest that the adhesion phenomenon is related to arm shortening and rounded body that make amplexus inefficient, although constituting important adaptive factors to life underground.

Dose-dependent alkaloid sequestration and N-methylation of decahydroquinoline in poison frogs

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.

Morphology of the Cutaneous Poison and Mucous Glands in Amphibians with Particular Emphasis on Caecilians (Siphonops annulatus).

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

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

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.

Morphology of the cutaneous poison and mucous glands in amphibians with particular emphasis on caecilians (Siphonops annulatus)

Caecilians (order Gymnophiona) are apodan, snake-like amphibians, usually with fossorial habits, constituting one of the most unknown groups of terrestrial vertebrates. As in orders Anura (frogs, tree frogs and toads) and Caudata (salamanders and newts), the caecilian skin is rich in mucous glands, responsible for body lubrication, and poison glands, producing varied toxins used in defence against predators and microorganisms. Whereas in anurans and caudatans skin gland morphology has been well studied, caecilian poison glands remain poorly elucidated. Here we characterised the skin gland morphology of the caecilian Siphonops annulatus, emphasising the poison glands in comparison to those of anurans and salamanders. We showed that S. annulatus glands are similar to those of salamanders, consisting of several syncytial compartments full of granules composed of protein material but showing some differentiated apical compartments containing mucus. An unusual structure resembling a mucous gland is frequently observed in lateral/apical position, apparently connected to the main duct. We conclude that the morphology of skin poison glands in caecilians is more similar to salamander glands when compared to anuran glands that show a much-simplified structure.

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

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.

Differences between poison and venom: an attempt at an integrative biological approach

We discuss the use of the terms venom and poison, in the context of integrative biology, with particular emphasis on behaviour and natural history. Our purpose is to reach a broad scientific audience, especially that dedicated to zoology. The meaning of the two terms is reviewed from the secretory perspective, mainly focussed on the reptiles and amphibians. We justify the use of the two words, based on biological and behavioural differences.

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)

Morphological Evidence for an Oral Venom System in Caecilian Amphibians.

Amphibians are known for their skin rich in glands containing toxins employed in passive chemical defense against predators, different from, for example, snakes that have active chemical defense, injecting their venom into the prey. Caecilians (Amphibia, Gymnophiona) are snake-shaped animals with fossorial habits, considered one of the least known vertebrate groups. We show here that amphibian caecilians, including species from the basal groups, besides having cutaneous poisonous glands as other amphibians do, possess specific glands at the base of the teeth that produce enzymes commonly found in venoms. Our analysis of the origin of these glands shows that they originate from the same tissue that gives rise to teeth, similar to the venom glands in reptiles. We speculate that caecilians might have independently developed mechanisms of production and injection of toxins early in their evolutionary history.

Distribution of major toxins in Rhinella marina parotoid macroglands using Desorption-Electrospray-Ionization mass spectrometry imaging (DESI-MSI).

Amphibian cutaneous glands secrete toxins used in different vital functions including passive defense. Through Desorption Electrospray Ionization-Imaging we analyzed the distribution of the major toxins of the toad Rhinella marina parotoid macroglands. Alkaloids and steroids showed characteristic distribution and intensity within the glands and were also present at lower levels on the skin surface. A comprehensive overview of toxins distribution in toads' skin might help to understand their full biological role within the amphibians.

Toads prey upon scorpions and are resistant to their venom: A biological and ecological approach to scorpionism.

In recent years, SE Brazil, the most populous region in the country with an estimated population of 88 million, has been experiencing an alarming increase in scorpions accidents (scorpionism), mainly caused by the yellow scorpion (Tityus serrulatus), or "escorpião amarelo" in Portuguese. This species is considered particularly dangerous to humans and can reproduce by parthenogenesis favouring rapid dispersal and colonization of new environments. Since the 1940s, owing to the growing danger represented by scorpionism, public control policies have been developed, including active search for scorpions, together with the use of toxic substances applied in places most likely to serve as their refuges. Even so, the number of accidents is increasing year by year, presently at an alarming rate. It seems evident that the increase in accidents is directly (or primarily) related to the lack of predators that in healthy environmental conditions would naturally control scorpion populations. However, due to environmental changes, leading to a lack of predators, scorpions have been gradually invading the urban environment. Arachnids and insects in general, as well as some other invertebrates, are preyed upon by anuran amphibians (toads, frogs and tree frogs). Toads (family Bufonidae) are nocturnal, large, and highly voracious animals, capable of actively exploring extensive areas and consuming large numbers of insects and arachnids daily. One of the most common toad species in southeastern Brazil is Rhinella icterica. Both R. icterica and T. serrulatus inhabit the same nocturnal environment. The predatory action of toads, specifically on scorpions, is practically unknown from behavioural and toxinological points of view. Thus, we studied the predatory behaviour of this toad against the yellow scorpion and evaluated the resistance of the amphibian to scorpion venom. Our results show that R. icterica is a voracious predator of T. serrulatus and is extremely resistant to its venom. Human/toad relationship throughout western history has always been very conflicted and possibly one of the factors that most has contributed to human ignorance of the role of these amphibians in maintaining ecological balance. Presently, the control of scorpionism is being performed through active search and/or the use of chemical agents, although showing little efficacy in reducing human accidents. In the medium or long term, more effective actions taking into account the biology of scorpions and their predators have never been taken to reduce these accidents.

Toads prey upon scorpions and are resistant to their venom: a biological and ecological approach to scorpionism: a biological and ecological approach to scorpionism

In recent years, SE Brazil, the most populous region in the country with an estimated population of 88 million, has been experiencing an alarming increase in scorpions accidents (scorpionism), mainly caused by the yellow scorpion (Tityus serrulatus), or "escorpião amarelo" in Portuguese. This species is considered particularly dangerous to humans and can reproduce by parthenogenesis favouring rapid dispersal and colonization of new environments. Since the 1940s, owing to the growing danger represented by scorpionism, public control policies have been developed, including active search for scorpions, together with the use of toxic substances applied in places most likely to serve as their refuges. Even so, the number of accidents is increasing year by year, presently at an alarming rate. It seems evident that the increase in accidents is directly (or primarily) related to the lack of predators that in healthy environmental conditions would naturally control scorpion populations. However, due to environmental changes, leading to a lack of predators, scorpions have been gradually invading the urban environment. Arachnids and insects in general, as well as some other invertebrates, are preyed upon by anuran amphibians (toads, frogs and tree frogs). Toads (family Bufonidae) are nocturnal, large, and highly voracious animals, capable of actively exploring extensive areas and consuming large numbers of insects and arachnids daily. One of the most common toad species in southeastern Brazil is Rhinella icterica. Both R. icterica and T. serrulatus inhabit the same nocturnal environment. The predatory action of toads, specifically on scorpions, is practically unknown from behavioural and toxinological points of view. Thus, we studied the predatory behaviour of this toad against the yellow scorpion and evaluated the resistance of the amphibian to scorpion venom. Our results show that R. icterica is a voracious predator of T. serrulatus and is extremely resistant to its venom. Human/toad relationship throughout western history has always been very conflicted and possibly one of the factors that most has contributed to human ignorance of the role of these amphibians in maintaining ecological balance. Presently, the control of scorpionism is being performed through active search and/or the use of chemical agents, although showing little efficacy in reducing human accidents. In the medium or long term, more effective actions taking into account the biology of scorpions and their predators have never been taken to reduce these accidents.

Life history of frogs of the Brazilian semi-arid (Caatinga), with emphasis in aestivation

The semi-arid region (caatinga), that corresponds to 18.26% (1,540,000 km2) of Brazil, occupies most the northeast region. Rainfall is irregular and concentrated within the first 3 months of the year. Prolonged periods of drought, with low total rainfall, may extend for two or more years. Most of the rivers of this biome are temporary, remaining totally dry during periods of drought. We collected and observed the natural history and biology of the species Proceratophrys cristiceps, Pleurodema diplolister and Physalaemus spp, both in the rainy and dry seasons, in ten field expeditions to the State of Rio Grande do Norte (Brazil). We focused on the morphology of the skin and cutaneous glands, specifically in their defence against desiccation. During the dry season, they form concentrations in the bed of temporary rivers, burrowing themselves in favourable locations within the sand, at depths that may exceed 1.50 m. No morphological evidence was found that there are specific cutaneous adaptations against water loss. We suggest that the cutaneous fragility per se is a cutaneous adaptation to water balance. The behaviour of such anuran species, together with their physiological characteristics, should be the main tools to face the challenge of living in xeric conditions.

Morphological evidence for an oral venom system in caecilian amphibians

Amphibians are known for their skin rich in glands containing toxins employed in passive chemical defense against predators, different from, for example, snakes that have active chemical defense, injecting their venom into the prey. Caecilians (Amphibia, Gymnophiona) are snake-shaped animals with fossorial habits, considered one of the least known vertebrate groups. We show here that amphibian caecilians, including species from the basal groups, besides having cutaneous poisonous glands as other amphibians do, possess specific glands at the base of the teeth that produce enzymes commonly found in venoms. Our analysis of the origin of these glands shows that they originate from the same tissue that gives rise to teeth, similar to the venom glands in reptiles. We speculate that caecilians might have independently developed mechanisms of production and injection of toxins early in their evolutionary history.