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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)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022 Wiley Periodicals LLC.)

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here, we describe (6a R ,10a S ,10b R )-8,8-dimethyldodecahydropyrrolo[2,1- a ]isoquinoline [ trans-anti-trans- deoxybuzonamine ( 1a )] and ( rel -6a R ,10a R ,10b R )-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 ( 1 H, 13 C) 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 13 C 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.

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 2 R , 4a R , 5 R , 6 S , and 8a S . 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.

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 1 H, 13 C, 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.

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., (© 2020 John Wiley & Sons, Ltd.)

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.

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.)

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.

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.

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.

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.

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., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The opisthonotal (oil) glands of oribatid mites are the source of a wide diversity of taxon-specific defensive chemicals, and are likely the location for the more than 90 alkaloids recently identified in oribatids. Although originally recognized in temperate oribatid species, alkaloids have also been detected in related lineages of tropical oribatids. Many of these alkaloids are also present in a worldwide radiation of poison frogs, which are known to sequester these defensive chemicals from dietary arthropods, including oribatid mites. To date, most alkaloid records involve members of the superfamily Oripodoidea (Brachypylina), although few species have been examined and sampling of other taxonomic groups has been highly limited. Herein, we examined adults of more than 60 species of Nearctic oribatid mites, representing 46 genera and 33 families, for the presence of alkaloids. GC-MS analyses of whole body extracts led to the detection of 15 alkaloids, but collectively they occur only in members of the genera Scheloribates (Scheloribatidae) and Protokalumma (Parakalummidae). Most of these alkaloids have also been detected previously in the skin of poison frogs. All examined members of the oripodoid families Haplozetidae and Oribatulidae were alkaloid-free, and no mites outside the Oripodoidea contained alkaloids. Including previous studies, all sampled species of the cosmopolitan oripodoid families Scheloribatidae and Parakalummidae, and the related, mostly tropical families Mochlozetidae and Drymobatidae contain alkaloids. Our findings are consistent with a generalization that alkaloid presence is widespread, but not universal in Oripodoidea. Alkaloid presence in tropical, but not temperate members of some non-oripodoid taxa (in particular Galumnidae) deserves further study.

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.

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.

Bufonid poison frogs of the genus Melanophryniscus contain alkaloid-based chemical defenses that are derived from a diet of alkaloid-containing arthropods. In addition to dietary alkaloids, bufadienolide-like compounds and indolealkylamines have been identified in certain species of Melanophryniscus. Our study reports, for the first time, the co-occurrence of large quantities of both alkaloids sequestered from the diet and an endogenously biosynthesized indolalkylamine in skin secretions from individual specimens of Melanophryniscus moreirae from Brazil. GC/MS analysis of 55 individuals of M. moreirae revealed 37 dietary alkaloids and the biosynthesized indolealkylamine bufotenine. On average, pumiliotoxin 267C, bufotenine, and allopumilitoxin 323B collectively represent ca. 90 % of the defensive chemicals present in an individual. The quantity of defensive chemicals differed between sexes, with males possessing significantly less dietary alkaloid and bufotenine than females. Most of the dietary alkaloids have structures with branched-chains, indicating they are likely derived from oribatid mites. The ratio of bufotenine:alkaloid quantity decreased with increasing quantities of dietary alkaloids, suggesting that M. moreirae might regulate bufotenine synthesis in relation to sequestration of dietary alkaloids.

Background: Polytypism in aposematic species is unlikely according to theory, but commonly seen in nature. Ranitomeya imitator is a poison frog species exhibiting polytypic mimicry of three congeneric model species (R. fantastica, R. summersi, and two morphs of R. variabilis) across four allopatric populations (a "mimetic radiation"). In order to investigate chemical defenses in this system, a key prediction of Müllerian mimicry, we analyzed the alkaloids of both models and mimics from four allopatric populations., Results: In this study we demonstrate distinct differences in alkaloid profiles between co-mimetic species within allopatric populations. We further demonstrate that R. imitator has a greater number of distinct alkaloid types than the model species and more total alkaloids in all but one population., Conclusions: Given that R. imitator is the more abundant species in these populations, R. imitator is likely driving the majority of predator-learned avoidance in these complexes. The success of Ranitomeya imitator as a putative advergent mimic may be a direct result of differences in alkaloid sequestration. Furthermore, we propose that automimicry within co-mimetic species is an important avenue of research.

Many organisms use chemical defenses to reduce predation risk. Aposematic dendrobatid frogs sequester alkaloid-based chemical defenses from a diet of arthropods, but research on these defenses has been limited to adults. Herein, we investigate chemical defense across development in a dendrobatid frog, Oophaga pumilio. This species displays complex parental care: at hatching, mothers transport tadpoles to phytotelmata, and then return to supply them with an obligate diet of nutritive eggs for about six weeks. We collected eggs, tadpoles, juveniles, and adults of O. pumilio, and detected alkaloids in all life stages. The quantity and number of alkaloids increased with frog and tadpole size. We did not detect alkaloids in the earliest stage of tadpoles, but alkaloids were detected as trace quantities in nutritive eggs and as small quantities in ovarian eggs. Tadpoles hand-reared with eggs of an alkaloid-free heterospecific frog did not contain alkaloids. Alkaloids that are sequestered from terrestrial arthropods were detected in both adults and phytotelm-dwelling tadpoles that feed solely on nutritive eggs, suggesting that this frog may be the first animal known to actively provision post-hatch offspring with chemical defenses. Finally, we provide experimental evidence that maternally derived alkaloids deter predation of tadpoles by a predatory arthropod.

Several lineages of brightly colored anurans independently evolved the ability to secrete alkaloid-containing defensive chemicals from granular glands in the skin. These species, collectively referred to as 'poison frogs,' form a polyphyletic assemblage that includes some species of Dendrobatidae, Mantellidae, Myobatrachidae, Bufonidae, and Eleutherodactylidae. The ability to sequester alkaloids from dietary arthropods has been demonstrated experimentally in most poison frog lineages but not in bufonid or eleutherodactylid poison frogs. As with other poison frogs, species of the genus Melanophryniscus (Bufonidae) consume large numbers of mites and ants, suggesting they might also sequester defensive alkaloids from dietary sources. To test this hypothesis, fruit flies dusted with alkaloid/nutritional supplement powder were fed to individual Melanophryniscus stelzneri in two experiments. In the first experiment, the alkaloids 5,8-disubstituted indolizidine 235B' and decahydroquinoline were administered to three individuals for 104 days. In the second experiment, the alkaloids 3,5-disubstituted indolizidine 239Q and decahydroquinoline were given to three frogs for 153 days. Control frogs were fed fruit flies dusted only with nutritional supplement. Gas chromatography/mass spectrometry analyses revealed that skin secretions of all experimental frogs contained alkaloids, whereas those of all control frogs lacked alkaloids. Uptake of decahydroquinoline was greater than uptake of 5,8-disubstituted indolizidine, and uptake of 3,5-disubstituted indolizidine was greater than uptake of decahydroquinoline, suggesting greater uptake efficiency of certain alkaloids. Frogs in the second experiment accumulated a greater amount of alkaloid, which corresponds to the longer duration and greater number of alkaloid-dusted fruit flies that were consumed. These findings provide the first experimental evidence that bufonid poison frogs sequester alkaloid-based defenses from dietary sources.

Chemical defenses are widespread among animals, and the compounds involved may be either synthesized from nontoxic precursors or sequestered from an environmental source. Defensive sequestration has been studied extensively among invertebrates, but relatively few examples have been documented among vertebrates. Nonetheless, the number of described cases of defensive sequestration in tetrapod vertebrates has increased recently and includes diverse lineages of amphibians and reptiles (including birds). The best-known examples involve poison frogs, but other examples include natricine snakes that sequester toxins from amphibians and two genera of insectivorous birds. Commonalities among these diverse taxa include the combination of consuming toxic prey and exhibiting some form of passive defense, such as aposematism, mimicry, or presumptive death-feigning. Some species exhibit passive sequestration, in which dietary toxins simply require an extended period of time to clear from the tissues, whereas other taxa exhibit morphological or physiological specializations that enhance the uptake, storage, and/or delivery of exogenous toxins. It remains uncertain whether any sequestered toxins of tetrapods bioaccumulate across multiple trophic levels, but multitrophic accumulation seems especially likely in cases involving consumption of phytophagous or mycophagous invertebrates and perhaps consumption of poison frogs by snakes. We predict that additional examples of defensive toxin sequestration in amphibians and reptiles will be revealed by collaborations between field biologists and natural product chemists. Candidates for future investigation include specialized predators on mites, social insects, slugs, and toxic amphibians. Comprehensive studies of the ecological, evolutionary, behavioral, and regulatory aspects of sequestration will require teams of ecologists, systematists, ethologists, physiologists, molecular biologists, and chemists. The widespread occurrence of sequestered defenses has important implications for the ecology, evolution, and conservation of amphibians and reptiles.

Poison frogs are chemically defended from predators by diverse alkaloids, almost all of which are sequestered unchanged from alkaloid-containing arthropods in the frog diet. Oribatid mites recently have been proposed as a major dietary source of poison frog alkaloids. Here, we report on alkaloids common to an oribatid mite and poison frogs. Gas chromatographic-mass spectrometric analysis of methanol extracts of adult Scheloribates laevigatus (Oribatida: Scheloribatidae) revealed nine alkaloids. Five of these have been detected previously in the skin glands of poison frogs: two isomers of the pumiliotoxin 291G, two isomers of the 5,6,8-trisubstituted indolizidine 209C, and the 5,6,8-trisubstituted indolizidine 195G. The other four alkaloids, a pumiliotoxin, a tricyclic (coccinelline-like), and two isomers of an izidine, were not previously known, but are similar in structure to alkaloids found in poison frogs. Alkaloids were not detected in immature S. laevigatus, suggesting that they are adult-specific and possibly the result of mite biosynthesis. Although most of the alkaloids detected in S. laevigatus are common to poison frogs, the geographic distributions of these organisms are not sympatric. The findings of this study indicate that oribatid mites, and in particular, members of the genus Scheloribates, represent a relatively unexplored arthropod repository for alkaloids and a significant dietary source of alkaloids in poison frogs.

Four five-skin alkaloid extracts of the Madagascan poison frog Mantella baroni from three disturbed collection sites were compared with four five-skin extracts from three undisturbed sites. The number of alkaloids (diversity) was significantly different in M. baroni between undisturbed and disturbed collection sites, with more alkaloids generally being found in frogs from disturbed sites. Two undisturbed sites did not differ from two disturbed sites, but the third disturbed site (coded 6) had more than twice the alkaloid diversity found in frogs from the third undisturbed site (coded 5a/5b). There was no difference in the quantity of alkaloids in M. baroni between undisturbed and disturbed collection sites. The hypothesis that an undisturbed habitat confers a benefit to poison frogs dwelling therein, in allowing for the sequestration of greater alkaloid diversity and amounts, is challenged by our results. In the course of our study, we found that collections of frogs separated by an interval of three months at an undisturbed site differed by only 4% in alkaloid composition over this period, whereas frogs collected at a disturbed site and collected approximately three months later already had a 26% difference in alkaloid composition between the two collections. This constancy of skin alkaloid composition likely reflects a constancy of dietary prey items consumed by frogs at undisturbed sites.

Poison frogs contain an alkaloid-based chemical defense that is sequestered directly from a diet of alkaloid-containing arthropods. Geographic and temporal variation in alkaloid defense is common in poison frogs and is generally attributed to differences in the availability of alkaloid-containing arthropods. Variable chemical defense in poison frogs may have important consequences for predator-prey interactions, requiring a full understanding of the factors involved in explaining such variation. In the present study, we examine alkaloid variation in the dendrobatid poison frog Oophaga pumilio between males and females on Cayo Nancy (Isla Solarte), located in the Bocas del Toro archipelago of Panama. On average, females contained a significantly larger number and quantity of alkaloids when compared to males. Alkaloid composition varied significantly between males and females, illustrating that chemical defense in this population of O. pumilio is sex-dependent. The variation in alkaloids between sexes is attributed to differences in feeding and behavior between males and females. The majority of alkaloids present in the skin of O. pumilio appear to be of oribatid mite origin, supporting the importance of these dietary arthropods in the chemical defense of poison frogs.

The dominant alkaloids previously identified in skin extracts of Amazonian dendrobatid frogs of the genus Ameerega are histrionicotoxins and 2,5-disubstituted decahydroquinolines. Analysis of alkaloids in skin extracts of Ameerega picta from Bolivia revealed that the alkaloid 257A, previously reported as a 2,5-disubstituted decahydroquinoline, is an N-methyl-2,5-disubstituted decahydroquinoline. We characterized alkaloids of another 12 of the more than 25 species recently assigned to the genus Ameerega, and five additional N-methyldecahydroquinolines were identified. In some cases, the relative configuration of the N-methyldecahydroquinolines was determined by comparison with the N-methylated products prepared from the corresponding 2,5-disubstituted decahydroquinolines of known relative configuration. A dietary source for N-methyldecahydroquinolines is unknown; however, myrmicine ants are the likely source for the 2,5-disubstituted decahydroquinolines. The alkaloids in skin extracts of three species of another genus of Amazonian poison frog, Adelphobates, were also characterized, but N-methyldecahydroquinolines were not detected.

Alkaloid profiles for 81 individual mantellid frogs, Mantella baroni (Boulenger 1988) (N = 19), M. bernhardi (N = 51), and M. madagascariensis (Grandidier 1877) (N = 11), from six different populations from Madagascar were examined. Marked individual differences in alkaloid composition (number, type, and amount) were observed between different species and between populations of the same species. Disjunct populations of each of the three species differed significantly in alkaloid composition. Sympatric populations of M. baroni and M. madagascariensis also differed significantly in alkaloid composition. In M. bernhardi, differences in alkaloid composition were marginally associated with different sexes. A total of 111 alkaloids, including isomers, were detected in analysis of the individuals from the three species. The majority (47%) appear likely to be obtained from dietary mites, whereas many of the others (18%) are presumed to be from ants, and a few (4%) are from millipedes. Putative dietary sources for the remaining alkaloids are generally unknown, but beetles are probably the source of at least some of the tricyclic alkaloids (6%). In addition, alkaloid compositions from extracts of groups of individuals from five additional populations of M. baroni and from one population of M. bernhardi (Vences et al. 1994) and one population of M. cowanii (Boulenger 1882) were examined. An additional 50 alkaloids, including isomers, were detected in the combined samples, bringing the total number of alkaloids identified from these four species of mantellid frogs to 161. Alkaloid compositions in mantellid poison frogs are diverse and highly dependent on geographic location that appear to be largely determined by the nature and availability of alkaloid-containing prey items.

A total of 232 alkaloids, representing 21 structural classes were detected in skin extracts from the dendrobatid poison frog Oophaga pumilio, collected from 53 different populations from over 30 years of research. The highly toxic pumiliotoxins and allopumiliotoxins, along with 5,8-disubstitiuted and 5,6,8-trisubstituted indolizidines, all of which are proposed to be of dietary mite origin, were common constituents in most extracts. One decahydroquinoline (DHQ), previously shown be of ant origin, occurred in many extracts often as a major alkaloid, while other DHQs occurred rather infrequently. Histrionicotoxins, thought to be of ant origin, did not appear to possess a specific pattern of occurrence among the populations, but when present, were usually found as major components. Certain 3,5-disubstituted pyrrolizidines and indolizidines, known to be of ant origin, did occur in extracts, but infrequently. Alkaloid composition differed with regard to geographic location of frog populations, and for populations that were sampled two or more times during the 30-year period significant changes in alkaloid profiles sometimes occurred. The results of this study indicate that chemical defense in a dendrobatid poison frog is dependent on geographic location and habitat type, which presumably controls the abundance and nature of alkaloid-containing arthropods.

Alkaloids in the skin glands of poison frogs serve as a chemical defense against predation, and almost all of these alkaloids appear to be sequestered from dietary arthropods. Certain alkaloid-containing ants have been considered the primary dietary source, but dietary sources for the majority of alkaloids remain unknown. Herein we report the presence of approximately 80 alkaloids from extracts of oribatid mites collected throughout Costa Rica and Panama, which represent 11 of the approximately 24 structural classes of alkaloids known in poison frogs. Forty-one of these alkaloids also occur in the dendrobatid poison frog, Oophaga pumilio, which co-occurs with the collected mites. These shared alkaloids include twenty-five 5,8-disubstituted or 5,6,8-trisubstituted indolizidines; one 1,4-disubstituted quinolizidine; three pumiliotoxins; and one homopumiliotoxin. All but the last of these alkaloid classes occur widely in poison frogs. In addition, nearly 40 alkaloids of unknown structure were detected in mites; none of these alkaloids have been identified in frog extracts. Two of these alkaloids are homopumiliotoxins, five appear to be izidines, four appear to be tricyclics, and six are related in structure to poison frog alkaloids that are currently unclassified as to structure. Mites are common in the diet of O. pumilio, as well as in the diets of other poison frogs. The results of this study indicate that mites are a significant arthropod repository of a variety of alkaloids and represent a major dietary source of alkaloids in poison frogs.

Poison frogs contain an alkaloid-based chemical defense that is derived from a diet of certain alkaloid-containing arthropods, which include mites, ants, beetles, and millipedes. Variation in population-level alkaloid profiles among species has been documented, and more than 800 different alkaloids have been identified. In the present study, we examine individual alkaloid variation in the dendrobatid poison frog Dendrobates pumilio among seven populations and between two seasons on Isla Bastimentos, located in the Bocas del Toro archipelago of Panama. Alkaloid profiles vary among populations and between seasons, illustrating that chemical defense in this species can vary on a small spatial and temporal scale. Alkaloid variation among populations is marginally correlated with geographic distance, and close populations have profiles more similar to each other than to distant populations. Individuals within populations also vary in alkaloid profiles. Differences are attributed to both spatial and temporal variations in the availability of alkaloid-containing arthropods. Many of the alkaloids present in the skin of D. pumilio appear likely to be of ant origin, supporting the importance of myrmecophagy in chemical defense among poison frogs. However, a variety of frog skin alkaloids was recently detected in mites, suggesting that mites may also play an important role in chemical defense.

A remarkable diversity of bioactive lipophilic alkaloids is present in the skin of poison frogs and toads worldwide. Originally discovered in neotropical dendrobatid frogs, these alkaloids are now known from mantellid frogs of Madagascar, certain myobatrachid frogs of Australia, and certain bufonid toads of South America. Presumably serving as a passive chemical defense, these alkaloids appear to be sequestered from a variety of alkaloid-containing arthropods. The pumiliotoxins represent a major, widespread, group of alkaloids that are found in virtually all anurans that are chemically defended by the presence of lipophilic alkaloids. Identifying an arthropod source for these alkaloids has been a considerable challenge for chemical ecologists. However, an extensive collection of neotropical forest arthropods has now revealed a putative arthropod source of the pumiliotoxins. Here we report on the presence of pumiliotoxins in formicine ants of the genera Brachymyrmex and Paratrechina, as well as the presence of these ants in the stomach contents of the microsympatric pumiliotoxin-containing dendrobatid frog, Dendrobates pumilio. These pumiliotoxins are major alkaloids in D. pumilio, and Brachymyrmex and Paratrechina ants now represent the only known dietary sources of these toxic alkaloids. These findings further support the significance of ant-specialization and alkaloid sequestration in the evolution of bright warning coloration in poison frogs and toads.

Poison frogs of the neotropical family Dendrobatidae contain a wide variety of lipophilic alkaloids, which are accumulated from alkaloid-containing arthropods. A small millipede, Rhinotus purpureus (Siphonotidae), occurs microsympatrically with the dendrobatid frog Dendrobates pumilio on Isla Bastimentos, Bocas del Toro Province, Panamá. Methanol extracts of this millipede contain the spiropyrrolizidine O-methyloxime 236, an alkaloid previously known only from skin extracts of poison frogs, including populations of D. pumilio. Thus, R. purpureus represents a likely dietary source of such alkaloids in dendrobatid frogs.

Purpose: To provide a head-to-head comparison of the anticaries efficacy associated with two commercially-available and American Dental Association-accepted dentifrices: Crest Cavity Fighting Toothpaste with Fluoristat, containing 0.243% sodium fluoride in a silica base, and Colgate Great Regular Flavor Fluoride Toothpaste, containing 0.76% sodium monofluorophosphate in a dicalcium phosphate dihydrate base. The study was conducted in harmony with the published 1988 American Dental Association guidelines for studies geared toward this purpose., Materials and Methods: The study employed a double-blind, parallel-groups, multi-center two-treatment design, and involved third, fourth, and fifth grade schoolchildren from Newark, New Jersey, and from the Cidra and Lares areas of Puerto Rico. Qualifying subjects were stratified according to age and sex, and were randomly assigned to the two treatment groups, with multiple subjects in the same household all assigned to the dentifrice randomly allocated to the first among them. Caries examinations were conducted in accordance with U.S. Food and Drug Administration guidelines for the clinical evaluation of drugs to prevent dental caries. After treatment assignment, study participants were instructed to brush their teeth at home with their assigned dentifrice at least twice daily. Brushing instructions were reinforced by the presentation of educational films and lectures at school, by semi-annual mailings to parents, and through the periodic distribution of small novelty gifts along with the dentifrice deliveries, in order to enhance the interest and enthusiasm of study participants. Post-baseline examinations were performed after 1 and after 2 yrs of product use. Two thousand four hundred seventy-nine (2,479) subjects completed this 2-yr study. For these subjects, the mean (S.D.) DFS scores at baseline were 2.77 (3.35) for the Crest group, and 2.66 (3.18) for the Colgate group. For caries increment after 1 yr, the respective means were 1.68 (2.53) and 1.70 (2.57). After 2 yrs, the mean caries increments were 3.56 (4.11) for the Crest group, and 3.56 (4.05) for the Colgate group., Results: The analysis of the 2-yr caries increment scores support the conclusion that the anticaries efficacy associated with Colgate Great Regular Flavor Fluoride Toothpaste is equivalent to that associated with Crest Cavity Fighting Toothpaste with Fluoristat, in accordance with the procedures and standards provided by the published guidelines of the American Dental Association. Further, consistent with those same standards, the results of this study serve to lend additional support to the conclusion that dentifrices formulated with sodium monofluorophosphate provide an equivalent level of anticaries efficacy as to those formulated with sodium fluoride.

Participation in a self-administered quality assessment (SAQA) program led to changes in New Jersey dentists' perceptions of practice quality. Ninety-four percent indicated they discovered practice deficiencies. This study suggests that using a self-administered quality assessment program, such as the SAQA program, can lead to a better understanding of a practice's strengths and weaknesses.

Age-related changes in renal function have been attributed to alterations in the chemical composition of the kidney tissues. Hence, the glycosaminoglycan composition of the renal cortex and medulla at varying age intervals was investigated. Glycosaminoglycans were isolated from the tissues by means of digestion with collagenase and pronase and purified by ethanol precipitation. Subsequent separation of various polyanions was accomplished by ion exchange chromatography on a Dowex 1-X2 column, using sodium chloride buffers of increasing ionic strengths. The glycosaminoglycans in each fraction were identified and quantitated by digestion with specific enzymes, including hyaluronidase, chondroitinase AC and ABC. The enzyme resistant material was separated and further digested with nitrous acid to quantitate the proportion of heparon sulfate. The results indicate that the glycosaminoglycan content of the renal medulla was much higher than the cortex at all the age intervals studied, and age-induced reduction was mainly cortical. There was a significant reduction in the heparan sulfate content of the cortex in aging. Interestingly, the major glycosaminoglycan content of the medulla was hyaluronic acid, which showed a sharp increase during aging, whereas heparan sulfate declined. Chondroitin sulfate was not altered due to age in either tissue. The molecular weight of hyaluronic acid was determined by column chromatography. Results indicate that the size of hyaluronate in the cortex was small and did not vary with age. In the medulla of the younger age group, a considerable amount of large size hyaluronate was observed. As age increased, the size decreased. The results strongly suggest that alteration in the renal glycosaminoglycans may be partly responsible for the age related protinuria and ionic imbalance.

In an attempt to better prepare students for dental practice in a litigious environment with cost-containment pressures, quality assurance requirements, and increased patient expectations, the New Jersey Dental School has begun planning and implementing various programs to teach students and faculty ethics, jurisprudence, and risk management. In addition to formal course work, the New Jersey Dental School is implementing a quality assurance and risk management clinical program.