Alkaloid avoidance in poison frog tadpoles

Animals show a spectrum of avoidance-tolerance to foods containing toxic secondary metabolites. However, this spectrum has not been evaluated in animals that may actively seek out these compounds as a chemical defense. Poison frogs sequester toxic and unpalatable alkaloids from their diet, and in some species, tadpoles are exposed to these toxins before the development of their skin granular glands, which are used for toxin compartmentalization. Here, we examined the effects of the alkaloid decahydroquinoline (DHQ) in tadpoles of the Mimetic poison frog, Ranitomeya imitator, using alkaloid supplemented food. We found that although their survival is lowered by the alkaloid, their development is only mildly affected, with no evident effects on their growth. Furthermore, locomotor activity and feeding behavior was altered in the first week of DHQ treatment, probably in part through nicotinic blockade. However, after two weeks, tadpoles learned to avoid the alkaloid by visiting the food area only when necessary to eat. Our results suggest that poison frogs navigate the avoidance-tolerance spectrum during the development of their sequestration machinery.

Who cares for the eggs? Analysis of egg attendance behaviour in Ranitomeya imitator, a poison frog with biparental care

Dendrobatid poison frogs are known for their diverse parental care behaviours, including terrestrial egg attendance. While usually this behaviour is conducted by males, this study compared the pre-hatching investment of males and females in Ranitomeya imitator, a species with biparental care. Although males tended to spend more time with their eggs overall, there was no difference between sexes when comparing different types of care behaviour. Furthermore, both sexes increased general care behaviour when caring for more than one clutch. The finding that the sexes are relatively equal in their contribution to basic parental care forms provides a basis to understand why biparental care is stable in this species.

Aposematism facilitates the diversification of parental care strategies in poison frogs

Many organisms have evolved adaptations to increase the odds of survival of their offspring. Parental care has evolved several times in animals including ectotherms. In amphibians, ~ 10% of species exhibit parental care. Among these, poison frogs (Dendrobatidae) are well-known for their extensive care, which includes egg guarding, larval transport, and specialized tadpole provisioning with trophic eggs. At least one third of dendrobatids displaying aposematism by exhibiting warning coloration that informs potential predators about the presence of defensive skin toxins. Aposematism has a central role in poison frog diversification, including diet specialization, and visual and acoustic communication; and it is thought to have impacted their reproductive biology as well. We tested the latter association using multivariate phylogenetic methods at the family level. Our results show complex relationships between aposematism and certain aspects of the reproductive biology in dendrobatids. In particular, aposematic species tend to use more specialized tadpole-deposition sites, such as phytotelmata, and ferry fewer tadpoles than non-aposematic species. We propose that aposematism may have facilitated the diversification of microhabitat use in dendrobatids in the context of reproduction. Furthermore, the use of resource-limited tadpole-deposition environments may have evolved in tandem with an optimal reproductive strategy characterized by few offspring, biparental care, and female provisioning of food in the form of unfertilized eggs. We also found that in phytotelm-breeders, the rate of transition from cryptic to aposematic phenotype is 17 to 19 times higher than vice versa. Therefore, we infer that the aposematism in dendrobatids might serve as an umbrella trait for the evolution and maintenance of their complex offspring-caring activities.

First characterization of toxic alkaloids and volatile organic compounds (VOCs) in the cryptic dendrobatid Silverstoneia punctiventris

Background Poison frogs are known for the outstanding diversity of alkaloid-based chemical defences with promising therapeutic applications. However, current knowledge about chemical defences in Dendrobatoidea superfamily has two sources of bias. First, cryptic, brown-colored species have been neglected in comparison to those conspicuously colored, and second, there has been little interest in characterizing metabolites other than alkaloids mediating defensive functions. In an effort to contribute to fill the gap of knowledge about cryptic species and broadening the spectrum of compounds analyzed we have applied head-space solid phase microextraction coupled to gas chromatography and mass spectrometry (HS-SPME/GC-MS) for extracting amphibian alkaloids and volatile organic compounds (VOCs) from Silverstoneia punctiventris. Results Using the skin from 8 specimens in 4 biological replicates we have found 33 different compounds. Twenty of them were classified as VOCs into 15 chemical classes including alkanes, alcohols, carbonyl compounds, methylpyridines, benzothiazoles, N-alkylpyrrolidines, pyrazines, and sesquiterpenoids, some of which were previously reported as repellents, defence compounds or defence pheromones in other organisms, and as sex pheromones in a treefrog. Interestingly, six of the remaining compounds were identified as alkaloids previously reported in other toxic/unpalatable dendrobatid frogs. Conclusions This is the first report of alkaloids and VOCs found in the Silverstoneia genus, which has been assumed for decades as non-chemically defended. This study establishes HS-SPME/GC-MS as a new application for a simultaneous approach to amphibian alkaloids and VOCs in poison frogs while opens up new research questions to assess the co-occurrence of both type of compounds and to investigate the evolutionary significance of a defence gradient that includes olfactory avoidance, unpalatability, and toxicity in dendrobatids. In addition, our results show that amphibian alkaloids could have a dual function (olfactory at distance, taste by contact) never explored before neither in Silverstonaeia nor in any other dendrobatid species.

How Phylogenetics Can Elucidate the Chemical Ecology of Poison Frogs and Their Arthropod Prey

The authors have requested that this preprint be removed from Research Square.

Evidence that toxin resistance in poison birds and frogs is not rooted in sodium channel mutations and may rely on “toxin sponge” proteins

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (NaV) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on NaV function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a NaV DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog NaVs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog NaVs. We also show that captivity-raised poison frogs are resistant to two NaV-directed toxins, BTX and saxitoxin (STX), even though they bear NaVs sensitive to both. Moreover, we demonstrate that the amphibian STX “toxin sponge” protein saxiphilin is able to protect and rescue NaVs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.

Rapid toxin sequestration modifies poison frog physiology

Poison frogs sequester chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about the physiological adaptations that confer this unusual bioaccumulation ability. We conducted an alkaloid-feeding experiment with the Diablito poison frog (Oophaga sylvatica) to determine how quickly alkaloids are accumulated and how toxins modify frog physiology using quantitative proteomics. Diablito frogs rapidly accumulated the alkaloid decahydroquinoline within four days, and dietary alkaloid exposure altered protein abundance in the intestines, liver, and skin. Many proteins that increased in abundance with decahydroquinoline accumulation are plasma glycoproteins, including the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with decahydroquinoline accumulation are membrane proteins involved in small molecule transport and metabolism. Overall, this work shows poison frogs can rapidly accumulate alkaloids, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues.