Linking Predator Responses to Alkaloid Variability in Poison Frogs.

Many chemically-defended/aposematic species rely on diet for sequestering the toxins with which they defend themselves. This dietary acquisition can lead to variable chemical defenses across space, as the community composition of chemical sources is likely to vary across the range of (an aposematic) species. We characterized the alkaloid content of two populations of the Dyeing Poison Frog (Dendrobates tinctorius) in northeastern French Guiana. Additionally, we conducted unpalatability experiments with naive predators, Blue Tits (Cyanistes caeruleus), using whole-skin secretion cocktails to assess how a model predator would respond to the defense of individuals from each population. While there was some overlap between the two D. tinctorius populations in terms of alkaloid content, our analysis revealed that these two populations are markedly distinct in terms of overall alkaloid profiles. Predator responses to skin secretions differed between the populations. We identified 15 candidate alkaloids (including three previously undescribed) in seven classes that are correlated with predator response in one frog population. We describe alkaloid profile differences between populations for D. tinctorius and provide a novel method for assessing unpalatability of skin secretions and identifying which toxins may contribute to the predator response. In one population, our results suggest 15 alkaloids that are implicated in predator aversive response. This method is the first step in identifying the causal link between alkaloids and behavioral responses of predators, and thus makes sense of how varying alkaloid combinations are capable of eliciting consistent behavioral responses, and eventually driving evolutionary change in aposematic characters (or characteristics).

Maternal chemical defenses predict offspring defenses in a dendrobatid poison frog.

Within and among populations, alkaloid defenses of the strawberry poison frog (Oophaga pumilio) vary spatially, temporally, and with life history stage. Natural variation in defense has been implicated as a critical factor in determining the level of protection afforded against predators and pathogens. Oophaga pumilio tadpoles sequester alkaloids from nutritive eggs and are, thus, entirely dependent on their mothers for their defense. However, it remains unclear how tadpole alkaloid composition relates to that of its mother and how variation in maternally provisioned defenses might result in varying levels of protection against predators. Here, we demonstrate that natural variation in the alkaloid composition of a mother frog is reflected as variation in her tadpole's alkaloid composition. Tadpoles, like mother frogs, varied in their alkaloid composition but always contained the identical alkaloids found in their mother. Alkaloid quantity in tadpoles was highly correlated with alkaloid quantity in their mothers. Additionally, alkaloid quantity was the best predictor of tadpole palatability, wherein tadpoles with higher alkaloid quantities were less palatable. Mother frogs with greater quantities of alkaloids are, thus, providing better protection for their offspring by provisioning chemical defenses during one of the most vulnerable periods of life.

Maternal Provisioning of Alkaloid Defenses are Present in Obligate but not Facultative Egg Feeding Dendrobatids.

Poison frogs sequester alkaloid defenses from a diet of largely mites and ants. As a result, frogs are defended against certain predators and microbial infections. Frogs in the genus Oophaga exhibit complex maternal care, wherein mothers transport recently hatched tadpoles to nursery pools and return regularly to supply developing tadpoles with unfertilized (nutritive) eggs. Developing tadpoles are obligate egg feeders. Further, female O. pumilio and O. sylvatica maternally provision their nutritive eggs with alkaloid defenses, providing protection to their developing tadpoles at a vulnerable life-stage. In another genus of poison frog, Ranitomeya, tadpoles only receive and consume eggs facultatively, and it is currently unknown if mothers also provision these eggs (and thus their tadpoles) with alkaloid defenses. Here, we provide evidence that mother frogs of another species in the genus Oophaga (Oophaga granulifera) also provision alkaloid defenses to their tadpoles. We also provide evidence that Ranitomeya imitator and R. variabilis eggs and tadpoles do not contain alkaloids, suggesting that mother frogs in this genus do not provision alkaloid defenses to their offspring. Our findings suggest that among dendrobatid poison frogs, maternal provisioning of alkaloids may be restricted to the obligate egg-feeding members of Oophaga.

Deoxybuzonamine Isomers from the Millipede Brachycybe lecontii (Platydesmida: Andrognathidae).

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here, we describe (6aR,10aS,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-anti-trans-deoxybuzonamine (1a)] and (rel-6aR,10aR,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-syn-cis-deoxybuzonamine (1b)], two isomers of deoxybuzonamine found in the chemical defense secretions of the millipede Brachycybe lecontii Wood (Colobognatha, Platydesmida, Andrognathidae). The carbon-nitrogen skeleton of these compounds was determined from their MS and GC-FTIR spectra obtained from the MeOH extract of whole millipedes, along with a subsequent selective synthesis. Their structures were established from their 1D (1H, 13C) and 2D NMR (COSY, NOESY, multiplicity-edited HSQC, HSQC-TOCSY, HMBC) spectra. Additionally, computational chemistry (DFT and DP4) was used to identify the relative configurations of 1a and 1b by comparing predicted 13C data to their experimental values, and the absolute configuration of 1a was determined by comparing its experimental specific rotation with that of the computationally calculated value. This is the first report of dodecahydropyrrolo[2,1-a]isoquinoline alkaloids from a platydesmidan millipede.

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.

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.

Total Synthesis of Decahydroquinoline Poison Frog Alkaloids ent-cis-195A and cis-211A.

The total synthesis of two decahydroquinoline poison frog alkaloids ent-cis-195A and cis-211A were achieved in 16 steps (38% overall yield) and 19 steps (31% overall yield), respectively, starting from known compound 1. Both alkaloids were synthesized from the common key intermediate 11 in a divergent fashion, and the absolute stereochemistry of natural cis-211A was determined to be 2R, 4aR, 5R, 6S, and 8aS. Interestingly, the absolute configuration of the parent decahydroquinoline nuclei of cis-211A was the mirror image of that of cis-195A, although both alkaloids were isolated from the same poison frog species, Oophaga (Dendrobates) pumilio, from Panama.

Gosodesmine, a 7-Substituted Hexahydroindolizine from the Millipede Gosodesmus claremontus.

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here we describe gosodesmine (1), 7-(4-methylpent-3-en-1-yl)-1,2,3,5,8,8a-hexahydroindolizine, a unique alkaloid with some terpene character found in the chemical defense secretions of the millipede Gosodesmus claremontus Chamberlin (Colobognatha, Platydesmida, Andrognathidae). The structure of 1 was suggested by its mass spectra and GC-FTIR spectra and established from its 1H, 13C, and 2D NMR spectra and 1D NOE studies. The 7-substituted indolizidine carbon skeleton of 1 was confirmed by unambiguous synthesis. This is the first report of an alkaloid from a platydesmid millipede and the first report of a 7-substituted indolizidine from an arthropod.

Use of whole-body cryosectioning and desorption electrospray ionization mass spectrometry imaging to visualize alkaloid distribution in poison frogs.

Ambient mass spectrometry is useful for analyzing compounds that would be affected by other chemical procedures. Poison frogs are known to sequester alkaloids from their diet, but the sequestration pathway is unknown. Here, we describe methods for whole-body cryosectioning of frogs and use desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to map the orally administered alkaloid histrionicotoxin 235A in a whole-body section of the poison frog Dendrobates tinctorius. Our results show that whole-body cryosectioning coupled with histochemical staining and DESI-MSI is an effective technique to visualize alkaloid distribution and help elucidate the mechanisms involved in alkaloid sequestration in poison frogs.

Transcriptomic Signatures of Experimental Alkaloid Consumption in a Poison Frog.

In the anuran family Dendrobatidae, aposematic species obtain their toxic or unpalatable alkaloids from dietary sources, a process known as sequestering. To understand how toxicity evolved in this family, it is paramount to elucidate the pathways of alkaloid processing (absorption, metabolism, and sequestering). Here, we used an exploratory skin gene expression experiment in which captive-bred dendrobatids were fed alkaloids. Most of these experiments were performed with Dendrobates tinctorius, but some trials were performed with D. auratus, D. leucomelas and Allobates femoralis to explore whether other dendrobatids would show similar patterns of gene expression. We found a consistent pattern of up-regulation of genes related to muscle and mitochondrial processes, probably due to the lack of mutations related to alkaloid resistance in these species. Considering conserved pathways of drug metabolism in vertebrates, we hypothesize alkaloid degradation is a physiological mechanism of resistance, which was evidenced by a strong upregulation of the immune system in D. tinctorius, and of complement C2 across the four species sampled. Probably related to this strong immune response, we found several skin keratins downregulated, which might be linked to a reduction of the cornified layer of the epidermis. Although not conclusive, our results offer candidate genes and testable hypotheses to elucidate alkaloid processing in poison frogs.

Weak warning signals can persist in the absence of gene flow.

Aposematic organisms couple conspicuous warning signals with a secondary defense to deter predators from attacking. Novel signals of aposematic prey are expected to be selected against due to positive frequency-dependent selection. How, then, can novel phenotypes persist after they arise, and why do so many aposematic species exhibit intrapopulation signal variability? Using a polytypic poison frog (Dendrobates tinctorius), we explored the forces of selection on variable aposematic signals using 2 phenotypically distinct (white, yellow) populations. Contrary to expectations, local phenotype was not always better protected compared to novel phenotypes in either population; in the white population, the novel phenotype evoked greater avoidance in natural predators. Despite having a lower quantity of alkaloids, the skin extracts from yellow frogs provoked higher aversive reactions by birds than white frogs in the laboratory, although both populations differed from controls. Similarly, predators learned to avoid the yellow signal faster than the white signal, and generalized their learned avoidance of yellow but not white. We propose that signals that are easily learned and broadly generalized can protect rare, novel signals, and weak warning signals (i.e., signals with poor efficacy and/or poor defense) can persist when gene flow among populations, as in this case, is limited. This provides a mechanism for the persistence of intrapopulation aposematic variation, a likely precursor to polytypism and driver of speciation.

Experimental evidence for maternal provisioning of alkaloid defenses in a dendrobatid frog.

Dendrobatid frogs sequester alkaloid defenses from dietary arthropods. Here, we provide experimental evidence that mother strawberry poison frogs (Oophaga pumilio) provision alkaloids to tadpoles. Captive-raised females were fed the synthetic alkaloid decahydroquinoline (DHQ), which we subsequently quantified in their skin, eggs, and developing tadpoles. DHQ quantity was positively associated with tadpole mass/development, suggesting high sequestration rates by tadpoles. These data confirm that tadpoles obtain nutrition and alkaloids by feeding exclusively on maternally provisioned eggs.

Comment on Amézquita et al. (2017) "Conspicuousness, color resemblance, and toxicity in geographically diverging mimicry: The pan-Amazonian frog Allobates femoralis".

Amézquita et al. (2017) recently concluded that species of the Allobates femoralis group are toxic to mice at levels equivalent to syntopic alkaloid-containing poison frogs, which they attributed to the presence of alkaloids in skin secretions. However, the chemical composition of skin secretions was not analyzed, and here we present additional data supporting the absence of alkaloids in skin secretions of the Allobates femoralis group. Instead, we suggest the observed toxicity was caused by the anesthetic benzocaine, which was applied to the buccal cavity to euthanize frogs prior to skin removal. We show that orally administered benzocaine is rapidly incorporated into the skin of species that sequester and do not sequester alkaloids, which casts doubt on the conclusion that Allobates femoralis group skin secretions are toxic and makes the results of experiments with alkaloid-containing species of Adelphobates and Ameerega uninterpretable. To prevent experimental errors and misinterpretations in studies of amphibian chemical defense, we encourage researchers to test the chemical composition of samples prior to experimentation, include all necessary controls to detect false positives, conduct small pilot studies for new methods, and consider the limitations of particular methods and their ability to address the intended research questions.

Sequestered Alkaloid Defenses in the Dendrobatid Poison Frog Oophaga pumilio Provide Variable Protection from Microbial Pathogens.

Most amphibians produce their own defensive chemicals; however, poison frogs sequester their alkaloid-based defenses from dietary arthropods. Alkaloids function as a defense against predators, and certain types appear to inhibit microbial growth. Alkaloid defenses vary considerably among populations of poison frogs, reflecting geographic differences in availability of dietary arthropods. Consequently, environmentally driven differences in frog defenses may have significant implications regarding their protection against pathogens. While natural alkaloid mixtures in dendrobatid poison frogs have recently been shown to inhibit growth of non-pathogenic microbes, no studies have examined the effectiveness of alkaloids against microbes that infect these frogs. Herein, we examined how alkaloid defenses in the dendrobatid poison frog, Oophaga pumilio, affect growth of the known anuran pathogens Aeromonas hydrophila and Klebsiella pneumoniae. Frogs were collected from five locations throughout Costa Rica that are known to vary in their alkaloid profiles. Alkaloids were isolated from individual skins, and extracts were assayed against both pathogens. Microbe subcultures were inoculated with extracted alkaloids to create dose-response curves. Subsequent spectrophotometry and cell counting assays were used to assess growth inhibition. GC-MS was used to characterize and quantify alkaloids in frog extracts, and our results suggest that variation in alkaloid defenses lead to differences in inhibition of these pathogens. The present study provides the first evidence that alkaloid variation in a dendrobatid poison frog is associated with differences in inhibition of anuran pathogens, and offers further support that alkaloid defenses in poison frogs confer protection against both pathogens and predators.

The Chemistry of Some Dalodesmidean Millipedes from Tasmania (Diplopoda, Polydesmida).

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. As part of a larger investigation, we describe the chemical constituents of 14 species of Tasmanian millipedes in seven genera. Six species in the genus Gasterogramma were found to produce acyclic ketones, including the pungent unsaturated ketones 1, 2, and 6, and the novel (rel-3R,5S,7S)-3,5,7-trimethyl-2,8-decanedione (7b), for which the stereoconfiguration was established by stereoselective syntheses of pairs of isomers. These compounds have not been detected before in millipede defensive secretions. This report is the first on species of the suborder Dalodesmidea (Polydesmida), a dominant component of the soil and litter fauna of the temperate regions of the Southern Hemisphere.

Chemical characterization of the adhesive secretions of the salamander Plethodon shermani (Caudata, Plethodontidae).

Salamanders have developed a wide variety of antipredator mechanisms, including tail autotomy, colour patterns, and noxious skin secretions. As an addition to these tactics, the red-legged salamander (Plethodon shermani) uses adhesive secretions as part of its defensive strategy. The high bonding strength, the fast-curing nature, and the composition of the biobased materials makes salamander adhesives interesting for practical applications in the medical sector. To understand the adhesive secretions of P. shermani, its components were chemically analysed by energy dispersive X-ray spectroscopy (EDX), inductively coupled plasma mass spectrometry (ICP-MS), amino acid analysis, and spectroscopy (ATR-IR, Raman). In addition, proteins were separated by gel-electrophoresis and selected spots were characterised by peptide mass fingerprinting. The salamander secretion contains a high amount of water and predominantly proteins (around 77% in the dry stage). The gel-electrophoresis and peptide mass fingerprint analyses revealed a de novo set of peptides/proteins, largely with a pI between 5.0 and 8.0 and a molecular mass distribution between 10 and 170 kDa. Only low homologies with other proteins present in known databases could be identified. The results indicate that the secretions of the salamander Plethodon clearly differ chemically from those shown for other glue-producing terrestrial or marine species and thus represent a unique glue system.

Variable Alkaloid Defenses in the Dendrobatid Poison Frog Oophaga pumilio are Perceived as Differences in Palatability to Arthropods.

Conspicuously colored dendrobatid frogs sequester alkaloid defenses from dietary arthropods, resulting in considerable alkaloid variation among populations; however, little is known about how variation is perceived as a defense against predators. Previous studies have found variable alkaloids in the dendrobatid Oophaga pumilio to be associated with differences in toxicity to laboratory mice, suggesting variable defenses are important. Arthropods are natural predators that use chemoreception to detect prey, including frogs, and may therefore perceive variation in alkaloid profiles as differences in palatability. The goal of the present study is to determine how arthropods respond to variable alkaloid defenses in O. pumilio. Frog alkaloids were sampled from individual O. pumilio from ten geographic locations throughout the Bocas del Toro region of Panama and the Caribbean coast of Costa Rica. Alkaloid extracts were used in feeding bioassays with the vinegar fly Drosophila melanogaster and the ant Ectatomma ruidum. Both species of arthropods fed significantly less on frog alkaloid extracts when compared to controls, and differences in alkaloid palatability were observed among frog populations, as well as between sexes and life stages within a population. Differences in alkaloid quantity, richness, and type were the main predictors of arthropod palatability. Our findings also represent the first direct evidence of a palatability spectrum in a vertebrate that sequesters chemical defenses from dietary sources. Further, the presence of a palatability spectrum suggests that variable alkaloid defenses in O. pumilio are ecologically relevant and play an important role in natural predator-prey interactions, particularly with respect to arthropod predators.

Arthropod predation in a dendrobatid poison frog: does frog life stage matter?

Frogs in the family Dendrobatidae are well known for their conspicuous colors and variable alkaloid-based chemical defenses. The aposematic coloration in dendrobatid frogs appears to deter predators with color vision, but relatively little is known about how these frogs are protected and their defenses are perceived by non-color vision dominated predators. The neotropical bullet ant Paraponera clavata and the red-legged banana spider Cupiennius coccineus are predators that avoid adults of the dendrobatid Oophaga pumilio, but readily consume non-toxic frogs. Juvenile O. pumilio possess the same warning coloration as adult O. pumilio, but may be more palatable given that they have lower quantities of defensive chemicals. This may provide juvenile O. pumilio protection from color-sighted predators, while leaving them susceptible to predators that use chemoreception. To test this hypothesis, we presented juveniles and adults of both O. pumilio and the non-chemically defended frog Craugastor bransfordii to bullet ants and banana spiders. Both bullet ants and banana spiders preyed upon C. bransfordii significantly more than on O. pumilio. Adult and juvenile C. bransfordii experienced similar predation rates by both predators. The life stage of O. pumilio significantly predicted predation by bullet ants, with juveniles being consumed significantly more often than adults. However, the life stage of O. pumilio did not predict predation by banana spiders, as no adults or juveniles were consumed. Our study provides evidence that bullet ants can detect differences in chemical defenses between juvenile and adult O. pumilio, resulting in differential predation on the more palatable juvenile frogs. The avoidance of both adults and juveniles by C. coccineus suggests the alkaloids in O. pumilio act as an effective chemical deterrent to banana spiders, regardless of quantity. Overall, our results suggest that differences in alkaloid defenses among life stages in O. pumilio correspond to differences in relative palatability to at least one arthropod predator.

The relationship between poison frog chemical defenses and age, body size, and sex.

INTRODUCTION: Amphibians secrete a wide diversity of chemicals from skin glands as defense against predators, parasites, and pathogens. Most defensive chemicals are produced endogenously through biosynthesis, but poison frogs sequester lipophilic alkaloids from dietary arthropods. Alkaloid composition varies greatly, even among conspecific individuals collected at the same time and place, with some individuals having only a few micrograms of one or a few alkaloids and others possessing >1 mg of >30 alkaloids. The paucity of alkaloids in juveniles and their abundance in adults suggests that alkaloids accumulate over time; however, alkaloid diversity is highly variable among adult poison frogs and has never been studied in relation to individual age. Using skeletochronology to infer individual ages and gas chromatography-mass spectrometry and vapor phase Fourier-transform infrared spectral analysis to identify the defensive chemicals of 63 individuals, we tested the relationship between defensive chemicals and age, size, and sex in the Brazilian red-belly toad, Melanophryniscus moreirae, a poison frog that possesses both sequestered alkaloids and the biosynthesized indolealkylamine bufotenine. RESULTS: Adult females were, on average, older and larger than adult males. Juveniles were smaller but not necessarily younger than adults and possessed bufotenine and 18 of the 37 alkaloids found in adults. Alkaloid richness was positively related to age, but not size, whereas the quantities of sequestered alkaloids and bufotenine were positively related to size, but not age. Defensive chemicals were unrelated to sex, independent of size. CONCLUSIONS: The relationship between alkaloid richness and age appears to result from the gradual accumulation of alkaloids over a frog's lifetime, whereas the relationship between the quantity of defensive chemicals and size appears to be due to the greater storage capacity of larger individuals. The decoupling of age and size effects increases the amount of individual variation that can occur within a population, thereby possibly enhancing anti-predator efficacy. Further, given that both richness and quantity contribute to the overall chemical defense of individual frogs, our results suggest that older, larger individuals are better defended than younger, smaller ones. These considerations underscore the importance of including age in studies of the causes and consequences of variation in poison frog chemical defenses.

Individual and Geographic Variation of Skin Alkaloids in Three Swamp-Forest Species of Madagascan Poison Frogs (Mantella).

Seventy skins of three mantellid frog species from Madagascan swamp-forest habitats, Mantella aurantiaca, M. crocea, and M. milotympanum, were individually examined for skin alkaloids using GC/MS. These poison frogs were found to differ significantly in their alkaloid composition from species of Mantella originating from non-flooded rainforest in eastern Madagascar, which were examined in earlier work. Only 16 of the previously detected 106 alkaloids were represented among the 60 alkaloids from the swamp-forest frogs of the present study. We hypothesize this difference is related mainly to habitat but cannot exclude a phylogenetic component as the three swamp-forest species are a closely related monophyletic group. The paucity of alkaloids with unbranched-carbon skeletons (ant-derived) and the commonness of alkaloids with branched-carbon skeletons (mite-derived) indicate that oribatid mites are a major source of alkaloids in these species of mantellids. Furthermore, most of the alkaloids have an oxygen atom in their formulae. Differences in alkaloids were observed among species, populations of the same species, and habitats. In M. aurantiaca, small geographic distances among populations were associated with differences in alkaloid profiles, with a remote third site illustrating even greater differences. The present study and an earlier study of three other mantellid species suggest that oribatid mites, and not ants, are the major source of alkaloids in the species of mantellids examined thus far.