Transcriptomic Analysis Reveals Potential Candidate Pathways and Genes Involved in Toxin Biosynthesis in True Toads.

Synthesized chemical defenses have broadly evolved across countless taxa and are important in shaping evolutionary and ecological interactions within ecosystems. However, the underlying genomic mechanisms by which these organisms synthesize and utilize their toxins are relatively unknown. Herein, we use comparative transcriptomics to uncover potential toxin synthesizing genes and pathways, as well as interspecific patterns of toxin synthesizing genes across 10 species of North American true toads (Bufonidae). Upon assembly and annotation of the 10 transcriptomes, we explored patterns of relative gene expression and possible protein-protein interactions across the species to determine what genes and/or pathways may be responsible for toxin synthesis. We also tested our transcriptome dataset for signatures of positive selection to reveal how selection may be acting upon potential toxin producing genes. We assembled high-quality transcriptomes of the bufonid parotoid gland, a tissue not often investigated in other bufonid-related RNAseq studies. We found several genes involved in metabolic and biosynthetic pathways (e.g., steroid biosynthesis, terpenoid backbone biosynthesis, isoquinoline biosynthesis, and glucosinolate biosynthesis) that were functionally enriched and/or relatively expressed across the 10 focal species that may be involved in the synthesis of alkaloid and steroid toxins, as well as other small metabolic compounds that cause distastefulness in bufonids. We hope that our study lays a foundation for future studies to explore the genomic underpinnings and specific pathways of toxin synthesis in toads, as well as at the macroevolutionary scale across numerous taxa that produce their own defensive toxins.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria.

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 µg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001-100 µg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 µg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

Antimicrobial Secretions of Toads (Anura, Bufonidae): Bioactive Extracts and Isolated Compounds against Human Pathogens.

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo, Bufotes, Duttaphrynus, and Rhinella. Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells.

Variation in Bufadienolide Composition of Parotoid Gland Secretion From Three Taxa of Japanese Toads.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

Individual variation in cardiotoxicity of parotoid secretion of the common toad, Bufo bufo, depends on body size - first results.

Anurans secrete a wide diversity of toxins from skin glands to defend themselves against predators and pathogens. Bufonids produce potent poison in parotoid macroglands located in the postorbital region. Parotoid secretion is a rich source of bioactive compounds with cardiotoxic, cytotoxic and hemolytic activity. Poison content and toxicity may vary between species, populations, and among conspecifics inhabiting the same area. In the present paper, we pre-analyzed the individual variation in cardiotoxicity of parotoid extract of common toads (Bufo bufo Linnaeus, 1758) and impact of body mass (BM), snout to vent length (SVL), and body condition (BC) of toad on the poison toxicity. We hypothesized that large toads produce poison with higher cardiotoxicity than smaller ones. Parotoid extract was fractionated by reverse phase chromatography, and then in vitro physiological bioassays were carried out on the semi-isolated hearts of the mealworm beetle (Tenebrio molitor Linnaeus, 1758) to determine cardiotoxicity of the whole poison and separated fractions. Generalized linear mixed models were used to determine effects of BM, SVL, and BC on the poison toxicity. We recorded significant changes in the insect heart contractility after treatment with the whole poison and separated fractions. We found an individual variation in cardiotoxicity of the parotoid extract which was explained by the body size of toad. Poison of smaller toads displayed a negative, whereas poison of larger toads positive, chronotropic effect on the heart contractility. Thus, we conclude that the effectiveness of parotoid secretion in repelling predators may vary depending on the toad individual size.

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.

19-Hydroxy-bufalin, a major bufadienolide isolated from the parotoid gland secretions of the Panamanian endemic toad Rhinella centralis (Bufonidae), inhibits the growth of Trypanosoma cruzi.

American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.

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.

The cost of chemical defence: the impact of toxin depletion on growth and behaviour of cane toads ( Rhinella marina).

Many animals capable of deploying chemical defences are reluctant to use them, suggesting that synthesis of toxins imposes a substantial cost. Typically, such costs have been quantified by measuring the elevation in metabolic rate induced by toxin depletion (i.e. during replenishment of toxin stores). More generally, we might expect that toxin depletion will induce shifts in a broad suite of fitness-relevant traits. In cane toads ( Rhinella marina), toxic compounds that protect against predators and pathogens are stored in large parotoid (shoulder) glands. We used correlational and experimental approaches in field and laboratory settings to investigate impacts of toxin depletion on growth rate and behaviour in cane toads. In free-ranging toads, larger toxin stores were associated with smaller gonads and livers, suggesting energetic trade-offs between toxin production and both reproduction and energy metabolism. Experimental removal of toxin (by manually squeezing parotoid glands) reduced rates of growth in body mass in both captive and free-ranging toads. Radio tracking demonstrated that de-toxined toads dispersed more slowly than did control toads. Given that toxin stores in cane toads take several months to fully replenish, deploying toxin to repel a predator may impose a substantial cost, explaining why toads use toxin only as a final line of defence.

Chondroitin sulfate isolated from the secretion of the venom-producing parotoid gland of Brazilian bufonid.

The parotoid gland of bufonids is characterized as a specialized integument region, formed by different gland types. The secretion elaborated by the largest glandular alveoli has been related to animal chemical defense and is constituted by granular protein content, associated with a basophilic and alcianophilic material with features of glycoconjugates. This study aimed to identify and characterize the glycoconjugates in the secretion of the largest granular gland of the parotoid gland of Rinella icterica by histochemical and immunohistochemical techniques at light microscopy, biochemical methods, and nuclear magnetic resonance spectroscopy. Our results showed that the glycoconjugate content contains a mixture of chondroitin­6­sulfate (C6S) and chondroitin-non-sulfate (C0S). Thus, chondroitin sulfate probably plays an important role in gland physiology, probably protecting the protein content while inside the secretory portion.

MALDI-MS argininyl bufadienolide esters fingerprint from parotoid gland secretions of Rhinella arenarum: Age, gender, and seasonal variation.

In many amphibians, the granular glands can be grouped in special regions forming macroglands. This is the case of toads, characterized by the presence of a pair of parotoid macroglands, strategically located to give protection by poison release in case of attacks. The product secreted consists of a wide variety of chemical compounds including proteins, peptides, biogenic amines, toxic steroidal bufadienolides, and various alkaloids, depending on the species. In this work, using Rhinella arenarum, we have performed, for the first time, the matrix assisted-ultraviolet laser desorption/ionization mass spectrometry and tandem mass spectrometry characterization of the components of the secretion used as crude material, just suspended in MeOH (or MeCN). The crude sample as a whole (whole suspension) was spotted on the matrix assisted-ultraviolet laser desorption plate for analysis. Electrospray ionization-Orbitrap was used for cross-checking experiments. The pattern of signals obtained at m/z ranges 600 to 800 and 1200 to 1600 could be assigned as the argininyl bufadienolide esters fingerprint characteristic of female and male. Variation patterns for gender (female, male), age (non-reproductive, reproductive), and season (non-reproductive, reproductive) are described.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Structural cutaneous adaptations for defense in toad (Rhinella icterica) parotoid macroglands.

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.

Antibacterial, modulatory activity of antibiotics and toxicity from Rhinella jimi (Stevaux, 2002) (Anura: Bufonidae) glandular secretions.

The increase in microorganisms with resistance to medications has caused a strong preoccupation within the medical and scientific community. Animal toxins studies, such as parotoid glandular secretions from amphibians, possesses a great potential in the development of drugs, such as antimicrobials, as these possess bioactive compounds. It was evaluated Rhinella jimi (Stevaux, 2002) glandular secretions against standard and multi-resistant bacterial strains; the effect of secretions combined with drugs; and determined the toxicity using two biologic in vivo models, and a in vitro model with mice livers. Standard strains were used for the determination of the Minimum Inhibitory Concentration (MIC), while for the modulatory activity of antibiotics, the clinical isolates Escherichia coli 06, Pseudomonas aeruginosa 03 and Staphylococcus aureus 10 were used. Modulatory activity was evaluated by the broth microdilution method with aminoglycosides and ß-lactams as target antibiotics. The secretions in association with the antibiotics have a significant reduction in MIC, both the aminoglycosides and ß-lactams. The toxicity and cytotoxicity results were lower than the values used in the modulation. R. jimi glandular secretions demonstrated clinically relevant results regarding the modulation of the tested antimicrobials.

Morphological Changes in Skin Glands During Development in Rhinella Arenarum (Anura: Bufonidae).

Avoiding predation is critical to survival of animals; chemical defenses represent a common strategy among amphibians. In this study, we examined histologically the morphology of skin glands and types of secretions related to chemical skin defense during ontogeny of Rhinella arenarum. Prior to metamorphic climax the epidermis contains typical bufonid giant cells producing a mucous substance supposedly involved in triggering a flight reaction of the tadpole school. An apical layer of alcianophilic mucus covers the epidermis, which could produce the unpleasant taste of bufonid tadpoles. Giant cells disappear by onset of metamorphic climax, when multicellular glands start developing, but the apical mucous layer remains. By the end of climax, neither the granular glands of the dorsum nor the parotoid regions are completely developed. Conversely, by the end of metamorphosis the mucous glands are partially developed and secrete mucus. Adults have at least three types of granular glands, which we designate type A (acidophilic), type B (basophilic) and ventral (mucous). Polymorphic granular glands distribute differently in the body: dorsal granular glands between warts and in the periphery of parotoids contain protein; granular glands of big warts and in the central region of parotoids contain catecholamines, lipids, and glycoconjugates, whereas ventral granular glands produce acidic glycoconjugates. Mucous glands produce both mucus and proteins. Results suggest that in early juveniles the chemical skin defense mechanisms are not functional. Topographical differences in adult skin secretions suggest that granular glands from the big warts in the skin produce similar toxins to the parotoid glands.

Occurrence of parotoid glands in tadpoles of the tropical frog, Clinotarsus curtipes and their role in predator deterrence.

Tadpoles of the tropical bicolored frog, Clinotarsus curtipes are unique in having parotoid glands secreting a white viscous fluid and are structurally similar to granular glands from other amphibians. To ascertain the involvement of these glands and their secretion in predator deterrence, it was tested against a predatory fish, Clarias gariepinus, using a paired choice behavioral assay. The results showed that the fish avoid eating C. curtipes tadpoles when paired with tadpoles of a sympatric species, Sylvirana temporalis. While the fish fed on C. curtipes tadpoles whose parotoid glands were surgically removed, did not touch those with intact glands, suggesting a role for the parotoid gland secretion in predator deterrence. Histochemical and biochemical analyses of the gland secretion revealed the presence of high concentrations of proteins, lipids, and alkaloids. SDS-PAGE showed the presence of proteins with prominent bands at 17 and 50kDa. The presence of other small molecules (950-2000amu) as detected by LC-MS showed the presence of five major peaks. Peaks 1 and 2 are probably tetrodotoxin and/or its analogs. Peaks 3 and 5 are possibly bufalin and argininosuccinic acid, respectively while peak 4 remains unidentified. Thus, secretion of parotoid glands of larval C. curtipes contains chemicals which, either alone or in combination, might be responsible for deterring predators.

Estudo morfológico comparativo do mecanismo de defesa química cutânea em duas espécies de sapos amazônicos (Rhinella marina e Rhaebo guttatus) / Comparative study of the cutaneous chemical defensive system in two species of amazonian toads (Rhinella marina and Rhaebo guttatus)

The skin of amphibians is characterized by the presence of a large number of granular glands (or poison glands). These glands can be distributed throughout the whole body, or can accumulate in certain areas, strategically placed against predator action. This is the case of the parotoid macroglands of toads (Family Bufonidae), which are located dorso-laterally in the post-orbital region. When disturbed, the toads inflate the body and position their parotoids toward the aggressor. Poisoning, however, only occurs if the predator bites the toad and if the gland secretion makes contact mainly with the oral mucosa, configuring the typical amphibian passive defense. Contrary to such passivity, the Amazon toad Rhaebo guttatus is able to voluntarily eject its poison at great distances, through its parotoids. Based on these characteristics, the main objective of this work was the study of the morphology of R. guttatus parotoid through histology and ultrastructure, comparing it with that of Rhinella marina, a sympatric species with typical passive defense. We sought to characterize the structural differences that make possible the voluntary ejection of poison. Additionally, we studied and compared these two species in relation to the defensive behavior, the skin gland morphology, the biochemistry of parotoid secretion (by SDS-PAGE, HPLC and mass spectrometry) and the toxic activity (lethality, edema, nociception, hemorrhage and necrosis) induced by the parotoid secretion in animal models.The histology shows that the parotoid general morphology and the histochemistry of the parotoid contents are very similar in both species. The dermis that frames the secretory units and forms the alveoli is mainly composed of dense collagen fibers, giving to the macrogland a honeycomb appearance. Each alveolus is basically a large syncytial gland, which is connected to the exterior by a duct obstructed by an epithelial plug. Contrary to what occurs with R. marina, the parotoid of R. guttatus is attached to the scapula. This characteristic behavior associated with the behavior of lowering the girdle and inflating the lungs, confers to the animal the possibility of voluntary ejection of the poison. The poison in both R. marina and R. guttatus is composed mainly of proteins, biogenic amines and steroids (bufadienolids), but several molecules are exclusive to each species. When compared to R. marina, the venom of R. guttatus is less lethal and capable of inducing intense edema and nociception. Neither poisons, however, were able to cause hemorrhage or necrosis. R. guttatus was described more than 200 years and there is virtually no information about its biology and natural history. In view of his unusual defense mechanism, it is expected that the chemical arsenal used in its defense is different from other toads with passive defense. Because the behavior of voluntary ejection of poison is only described in R. guttatus, in order to complete this work, it would be important to study the defensive mechanism in other species of this genus. The voluntary ejection of poison would be placed then in a broader context, opening new perspectives of study.

A pele dos anfíbios é caracterizada pela presença de inúmeras glândulas granulosas (ou de veneno). Tais glândulas podem se distribuir por todo o corpo, ou ainda, acumular-se em determinadas regiões, estrategicamente posicionadas contra a ação de predadores. Esse é o caso das macroglândulas parotóides dos sapos (Família Bufonidae), localizadas dorso-lateralmente na região pós-orbital. Quando importunados, os sapos inflam o corpo e posicionam as suas parotóides em direção ao agressor. O envenenamento, entretanto, só ocorre, se o predador morder o sapo e a secreção das glândulas entrar em contato principalmente com a sua mucosa oral, configurando a defesa passiva típica dos anfíbios. Contrariando tal passividade, o sapo amazônico Rhaebo guttatus é capaz de ejetar seu veneno voluntariamente a grandes distâncias, através de suas parotóides. Com base nessas características, o principal objetivo desse trabalho foi estudar a morfologia das parotóides de R. guttatus através de histologia e ultraestrutura, comparando-a com a de Rhinella marina, espécie simpátrica e de defesa passiva típica. Procurou-se caracterizar as diferenças estruturais que possibilitam ejeção voluntária de veneno. Adicionalmente, foram estudados e comparados entre essas duas espécies o comportamento defensivo, a morfologia das glândulas cutâneas da pele, a bioquímica da secreção das parotóides (através de SDS-PAGE, HPLC e espectrometria de massas), bem como as atividades tóxicas (letalidade, edema, nocicepção, hemorragia e necrose) induzidas por essas secreções em modelos animais. A histologia demonstra que a morfologia geral das parotóides e o seu conteúdo histoquímico são muito semelhantes entre as duas espécies. A derme que sustenta as unidades secretoras formando os alvéolos é composta principalmente por fibras colágenas densas, o que confere à macroglândula uma aparência de favo de mel. Cada alvéolo é, basicamente, uma grande glândula sincicial, que se conecta ao exterior por um duto obstruído por um plug epitelial. Ao contrário do que ocorre em R. marina, a parotóide de R. guttatus é aderida à escápula. Essa característica anatômica associada ao comportamento de baixar a cintura escapular e de inflar os pulmões, deve conferir ao animal a possibilidade de ejetar voluntariamente o veneno. Esse veneno, tanto em R. marina como em R. guttatus é composto basicamente por proteínas, aminas biogênicas e esteróides (bufadienolídeos), apresentando, porém, diversas moléculas exclusivas de cada espécie. Quando comparado com R. marina, o veneno de R. guttatus é menos letal e capaz de induzir edema e intensa nocicepção. Nenhum dos dois venenos, no entanto, foi capaz de causar hemorragia ou necrose. R. guttatus foi descrito há mais de 200 anos e praticamente não existe informações sobre a sua biologia e história natural. Diante do seu inusitado mecanismo de defesa, é de se esperar que o arsenal químico utilizado na sua defesa seja diferente dos outros sapos, com defesa passiva. Como o comportamento de ejeção voluntária de veneno só está descrito em R. guttatus, seria importante para a complementação desse trabalho, estudar o mecanismo defensivo de outras espécies do gênero. A ejeção voluntária de veneno seria, assim, inserida em um contexto mais amplo, abrindo novas perspectivas de estudo.