Introduction

Frogs of the family Dendrobatidae are commonly known as Poison Frogs or Dart-Poison Frogs. These frogs occur in Central and South America. Their common name comes from the potent lipophilic alkaloids contained in the granular glands of their skin, which are used by natives of the Choco region of Columbia to poison blowgun darts. Up to fifty darts, which last for up to six months, may be poisoned with the excretions from one frog. These darts are used for hunting birds and mammals. Many of the toxins that have been found in these frogs have been found no where else in nature (Daly et al. 1994). Some of these compounds have been found in arthropods (Daly 1998). This finding, along with experimental evidence that frogs raised in captivity do not produce alkaloids, has lead to the belief that many of the so-called "Dendrobatid" toxins are sequestered from a dietary source (Daly 1998). Seven genera of Anurans have been discovered to contain alkaloid toxins. Four of these genera are in the family Dendrobatidae: Dendrobates, Epipedobates, Minyobates, and Phyllobates. The remaining three genera are from Madagascar (Mantella), South America (Melanophryniscus), and Australia (Pseudophryne). All of these taxa share similar morphological, behavioral, and ecological adaptations (Vences et al. 1997/98). These traits seem to have evolved independently in each group (Daly et al. 1992). The convergent evolution of alkaloid sequestering in correlation with several other traits has powerful implications on the evolution of these groups (Caldwell, 1996, Vences et al., 1997/98). "Dendrobatid" toxins may be used for research in several areas including taxonomy, phylogeny, pharmesuticals, and cell membrane receptor sites and ion channels.

Alkaloid Distribution in Anurans

Lipophilic Alkaloids are limited to seven genera of Anurans cotained in four families: Dendrobatidae, Bufonidae, Myobatrachidae, and Ranidae. The family Dendrobatidae contains six genera: Aromobates, Colostethus, Dendrobates, Epipedobates, Minyobates, and Phyllobates. Dendrobates, Epipedobates, Minyobates, and Phyllobates contain potent lipophilic alkaloid toxins belonging to 20 major structural classes (Daly 1998). Species contained in these genera are small, diurnal, microphagous, and aposematically colored. Aromobates and Colostethus contain little or no toxins (Daly 1994). Colostethus species are cryptically colored and Aromobates is noctrunal. One stream dwelling species, Colostethus inguinalis, has been found to contain tetrodotoxins, which are potent water-soluble toxins (Daly et al. 1994). The tetrodotoxins are widely distributed in nature, especially in marine organisms such as bacteria, snails, a flatworm, a starfish, xanthid crabs, an octopus, puffer fish, gobies, parrot fish, and angel fish. The only terrestrial organisms found to contain tetrodotoxins are amphibians: salamanders and anurans. The most notable example is the genus Atelopus whose species are diurnal, microphagous, and aposematically colored, traits similar to the toxic genera of Dendrobatids (Daly et al., 1994). The Ranid genus Mantella contains pumiliotoxins, allopumiliotoxins, homopumiliotoxins, 5,8-disubstituted indolizidines, 1,4-disubstituted quinolizidines, and decahydroquinolines. Representatives of all these structural classes are found in Dendrobatids (Daly, 1998). Species in the Bufonid genus Melanophryniscus have been found to contain pumiliotoxins, allopumiliotoxins, homopumiliotoxins, several izidine compounds, and several decahydroquinolines. These are all compounds found in the Dendrobatids (Daly, 1998). Both Mantella and Melanophryniscus species occupy the same ecological niche types as Dendrobatids. Species in the genus Pseudophryne are nocturnal but otherwise very similar to other alkaloid containing taxa. They have been found to contain pumiliotoxins, allopumiliotoxins, and a novel class of indolic compounds which have been named the pseudophrynamines (Daly, 1998).

"Dendrobatid" Alkaloid Properties

At least 20 major structural classes of alkaloids have been detected in the skin of Dendrobatid frogs. These classes include the batrachotoxins, histrionicotoxins, pumiliotoxins, allopumiliotoxins, homopumiliotoxins, epibatidine, pyrrolidines, piperidines, decahydroquinolines, pyrrolizidines, pyrrolizidine oximes, indolizidines, quinolizidines, tricyclic gephyrotoxins, pseudophrynamines, cyclopentaquinolizidines, spiropyrrolizidine oximes, coccinellines, and other tricyclic alkaloids. Nearly 500 distinct alkaloids have been isolated from the skins of frogs. Due to this great number only a few have been given a common name. Instead a numbering system is used consisting of a bold faced number, corresponding to the nominal mass of the compound, plus a bold faced letter to distinguish compounds of the same mass. Prefixes such as cis-, trans-, epi, and iso- are used to distinguish stereoisomers of the same compound (Daly, 1998). One of the most potent classes of animal toxins known are the batrachotoxins from species in the genus Phyllobates. These toxins have a steroidal nucleus with additional structures seen in no other classes of compounds (Daly, 1998). These compounds are selective activators of sodium channels. Their action is specifically directed towards voltage-dependent sodium channels of nerve and muscle tissue. Batrachotoxins prevent closing of these channels causing an influx of sodium ions and a depolarization of the cell membrane. This action destroys neve and muscle function in the affected tissue (Myers and Daly,1983). This affect is irreversible causing symptoms such as heart arrhythmias, fibrilation and failure. These toxins have proven to be useful tools in research on sodium channel function (Myers and Daly, 1983). Another potent class of toxins, the pumiliotoxins, is more widespread across taxa. This class of over thirty toxins occurs in all seven of the alkaloid containing genera. These compounds have an indole nucleus plus a side chain and several other substituents. Three hydroxyl groups must be present for full activity. In pumiliotoxins, two of these hydroxyls occur on the side chain and one on the indole ring. In the allopumiliotoxin subclass, one hydroxyl group occurs on the side chain with two on the indole ring, one specifically at the 7’ position (Daly, 1998). Another subclass of pumiliotoxins, the homopumiliotoxins, have a quinolizidine ring in place of the indole ring. Pumiliotoxins also act on voltage-dependent sodium channels causing the release of calcium ions from storage sites in the cell. This results in muscle contraction and then prevents re-accumulation of calcium ions lengthening the time of muscle contraction. These actions occur in heart and skeletal muscle. These myotonic and cardiotonic properties of the pumiliotoxin structural class have potential clinical applications (Myers and Daly, 1983). Another widespread structural class is the histrionicotoxins. These compounds have a nucleus of two six membered rings in perpendicular planes connected at a single carbon, these structures are commonly known as spiropiperidine alkaloids. These toxins act on acetylcholine and end-plate receptors. Their action blocks transmission of action potentials from nerve to muscle cells. Histrionicotoxins also block the action of potassium channels keeping them in the open or closed state. This action lengthens action potentials from nerve cells prolonging muscle contraction (Myers and Daly, 1983). At least sixteen different histrionicotoxins have been isolated from Dendrobatid frogs. They usually have fifteen, seventeen or nineteen carbons (Daly, 1998). An alkaloid that is potentially of high pharmaceutical value was isolated from Epipedobates tricolor. This alkaloid was named Epibatidine. It’s strructure is very similar to a Solanaceae alkaloid, anabasine, and is also very similar to that of nicotine (Fisher et al., 1994). It has been suggested, based on behavioral bioassays, that epibatidine has anagesic properties that are up to three orders of magnitude greater than morphine. In vivo, epibatidine has a potency comparable to compounds such as LSD, clonidine, and fentanyl which are effective in micrograms per kilogram doses (Fisher et al., 1994). The action of epibatidine is as a depolarizing agent of ganglionic nicotinic receptors producing a depolarizing blockade at high enough doses. Epibatidine is found in conjunction with histrionicotoxins. The action of epibatidine on ganglionic nicotinic receptors should allow greatly enhanced activity of the histrionicotoxins (Fisher et al., 1994). Some minor alkaloid components such as decahydroquinolines, pyrrolidines, various izidines, and gephyrotoxins are all noncompetitive blockers of nicotinic channels similar to phencyclidine, quinacrine, chlorpromazine, various local anesthetics and other drugs (Daly, 1998). These similarities to drugs that are known to be useful offer many potential pharmacological uses for Dendrobatid toxins.

Uptake of "Dendrobatid" Alkaloids

It has been seen that offspring from toxic parents do not contain alkaloids when raised in captivity. This is a phenomenon that has been witnessed in Phyllobates terribilis, Phyllobates bicolor, Dendrobates auratus, Dendrobates azureus, Dendrobates leucomelas, Epipedobates tricolor, and Mantella aurantiaca (Daly et al., 1992). Mantella aurantiaca was bred in captivity and the offspring were raised on a diet of cultured crickets and springtails for over a year. These offspring contained no detectable alkaloids upon extraction (Daly et al., 1992). Wild caught Mantella viridis were fed a mixture of allopumiliotoxin 267A, a csi-2-methyl-6-undecanylpiperidine, a 2,5-dimethyl-cis-decahydroquinoline, pumiliotoxins 307A and 323A, isodihydrohistrionicotoxin 285A, and 5,8-disubstituted indolizidines 205A and 207A in the vitamin dust which was added to food insects. Ananysis of these frogs, after 1.5 years on this diet, revealed the same alkaloid profiles as freshly caught specimens but in lower quantities. These alkaloids were found along with several alkaloids from the synthetic mixture added to their diet, namely allopumiliotoxin 267A, the 2,5-dimethyl-cis-decahydroquinoline, 5,8-disubstituted indolizidines 205A and 207A, and trace amounts of the isodihydrohistrionicotoxin 285A. Pumiliotoxins 307A and 323A were also present but could have been retained from their original compliment of alkaloids in the wild. These studies show the ability of species in the genus Mantella to sequester alkaloids that occur in their diet. These studies also show an environmental element in determining the presence alkaloids in their skin and in determining the types of alkaloids that occur when present (Daly et al. 1997). This situation has been observed in several types of Dendrobatids. Captive raised Phyllobates bicolor, fed on wild caught termites and wild caught fruit flies for three years, contained none of the potent batrachotoxins, histrionicotoxins or decahydroquinolines found in wild caught frogs. Indolizidine 195B was detected in the captive frog. This alkaloid is detectable in trace amounts in wild Phyllobates bicolor (Daly et al., 1992)

A situation that has unintentionally allowed much study of this system is the introduction of Dendrobats auratus onto the island of Oahu, Hawaii. These frogs were introduced from Isla Taboga, Panama. Several populations from Panama and Costa Rica were analysed for alkaloid content. These alkaloids varied from one population to another. A population from the mountains in central Panama contained histrionicotoxins, a pumiliotoxin, an allopumiliotoxin, and a 5,8-disubstituted indolizidine as major alkaloids. A population from Costa Rica contained histrionicotoxins, decahydroquinolines, disubstituted indolizidines, disubstituted quinolizidines, disubstiuted pyrrolizidines, and homopumiliotoxins. Pyrrolizidine 223H in this population is identical to an alkaloid found in the thief ant, Solenopsis xenovenenum. . Frogs from Isla Taboga contained histrionicotoxins, pumiliotoxins, allopumiliotoxins, and decahydroquinolines as their major alkaloids. The frogs in Hawaii contained some of the same compounds as the founding population on Isla Taboga: pumiliotoxins 251D, 307A, and 323A, allopumiliotoxin 267A, and decahydroquinoline cis-195A. The Hawaiian population does not contain any histrionicotoxins, which are major alkaloids in the original population from Panama. The Hawaiian population did have a tricyclic alkaloid, 193, which is identical to the beetle alkaloid precoccinelline. This compound was not found in the original population (Daly et al., 1992). Offspring from the Hawaiian population were raised in outdoor terrariums on wild caught termites and wild caught fruit flies. These frogs were found to contain pumiliotoxin 251D, allopumiliotoxin 267A, decahydroquinoline 195A and tricyclic 193, which were also found in the parents. Offspring raised in indoor terrariums on cultured crickets and fruit flies did not contain any detectable alkaloids (Daly et al., 1992). Offspring from the two mainland populations of Dendrobates auratus previously mentioned were raised in indoor terrariums. These offspring were subjected to several different types of environmental stresses: frequent changes in terrariums, bath treatments, visual threats, swabbing, increased fluorescent lighting, and diet modifications. These frogs contained no detectable alkaloids upon analysis (Daly et al., 1992). In feeding experiments with offspring from mainland populations of Dendrobates auratus, alkaloids were accumulated similarly to the accumulation in the Hawaiian offspring (Daly et al., 1994). The alkaloids were retained for several months. It has been demonstrated, in wild caught specimens of Dendrobates auratus, D. azureus, D. lehmanni, D. tinctorius, Epipedobates trivittatus, and Phyllobates terribilis, that alkaloids are retained for several years in captivity (Daly et al., 1994). The alkaloids are retained exclusively in the skin, with no detectable alkaloids in muscle or organ tissue. It is possible that the retention of alkaloids is aided by frogs eating their own shed skin (Daly et al., 1994).

Arthropod Sources of Alkaloids

Many of the alkaloids sequestered in the skins of frogs appear to be obtained in their diet from arthropod sources. Six of the twenty classes of alkaloids detected in Dendrobatid frogs have been found in Myrmicine ants, with the highest number occurring in the genus Solenopsis (Diplorhoptrum) (Jones et al., 1999). The classes thus far detected in Myrmicine ants and Dendrobatid frogs are 2,5-dialkylpyrrolidines, 2,6-dialkylpiperidines, 3,5-dialkylpyrrolizidines, 3,5-dialkylindolizidines, 4,6-dialkylquinolizidines, and 2,5-dialkyldecahydroquinolines (Daly et al., 2000). A total of 18 individual compounds have been detected in both groups from these six classes (Daly et al., 2000). Alkaloid 195C and two decahydroquinolines have been found in a Brazilian Solenopsis (Diplorhoptrum) species and in the same proportions in Mantella betsileo. The discovery of three alkaloids in the same proportions in an ant and in frogs strongly suggests a dietary source for these and related compound in frog skin(Jones et al., 1999). Decahydroquinoline cis-195A has been found in Solenopsis (Diplorhoptrum) species and also in Dendrobatid and Mantella species (Jones et al.,1999). The 4,6-disubstituted quinolizidine, 195C, occurs in Dendrobatids from Panama, Costa Rica, Venezuela, Colombia, Peru, Brazil, and Ecuador and in the Madagascan Mantellas. This compound is identical to a quinolizidine found in a Solenopsis (Diplorhoptrum) species from Brazil. The only other occurrence of 4,6-disubstituted quinolizidines in nature is the plant alkaloid porantherilidine (Jones et al., 1999).

The Myrmicine ant species Monomorium pharaonis produces significant amounts of two alkaloids: a 3,5-disubstituted indolizidine, named monomorine-I, and trans-2-heptyl-5-(5-hexenyl)pyrrolidine. The pyrrolidine occurs in much higher concentrations than the indolizidine. A 3,5-disubstituted indolizidine, 195B, has been detected in the skin of some frogs but is usually a different isomer than monomorine-I. When Monomorium pharaonis was fed to frogs for seven weeks, monomorine-I was accumulated in the skin but none of the pyrrolidine was detected (Daly et al., 1994).

A study was done in Panama on Dendrobates auratus raised in outdoor cages and fed with arthropods contained in leaf litter collected from the same site as the parents. Fourteen of the forty alkaloids that were found in the parents were found in the terrarium raised offspring. Pumiliotoxins, allopumiliotoxins and histrionicotoxins were absent from the terrarium raised frogs but were major alkaloids in the parents (Daly et al., 2000). Ants collected from the site were found to contain several of the alkaloids that were found in the frogs. This important study was the first to find the same alkaloids in sympatric species of frogs and arthropods (Daly et al., 2000).

Several alkaloids from arthropods other than ants have been detected in Dendrobatids. A spiropyrrolizidine and a spiropyrrolidine have been found in millipedes and frogs. Two of the fifteen tricyclic coccinelline alkaloids that have been detected in frogs have also been found in beetles (Daly et al., 2000).

Evolutionary Significance of Alkaloids in the Dendrobatidae and Convergently Evolved Taxa

Specialization for a diet consisting mainly of myrmicine ants and other minute prey is an adaptation shared by the seven genera of alkaloid containing frogs (Caldwell, 1996). Frogs in the genus Dendrobates, whose species contain alkaloids, contained from 50% to 73% ants upon stomach analyses whereas the nontoxic genus Colostethus was found to contain from 12% to 16% ants (Caldwell, 1996). This difference seems to be due to prey preference not merely small body size (Caldwell, 1996). It has been suggested that genetic disposal toward a certain type of prey might determine in which assemblages these frogs could occur rather than interactions within assemblages determining the type of prey taken (Caldwell, 1996). Some costs are involved in microphagous and myrmecophagous specialization including rejection of suitable prey, digestion of heavily chitinous and toxic prey, and constant foraging (Toft, 1995). Several derived traits seem to be shared by alkaloid containing frogs. These traits include microphagous specialization, myrmecophagous specialization, small body size, aposematic coloration, diurnal activity patterns, narrowing of the head and tongue, and decrease in number of maxillary and vomerine teeth (Vences et al., 1997/98). These traits were found to be correlated in comparisons with sister groups, with Mantidactylus as the sister group for Mantella and Colostethus as the sister group for the four alkaloid containing genera of Dendrobatids. The number of traits correlated with diet specialization suggest that foraging ecology could be the storngest driving force in the adaptive radiation of Dendrobatid and other alkaloid containing genera (Toft, 1995). Caldwell (1996) suggested a sequence of events leading to the radiation of toxic species. Diet specialization on toxic ants necessitates the detoxification of prey, especially by excretion through the skin. This would be strengthened by natural selection, if the excretions are toxic to predators, leading to the sequestering of alkaloid toxins in the skin. This would lead to selection for aposematic coloration and then diurnal activity. The end result being the radiation of species. This trend seems to have occurred separately in four groups of anurans: Dendrobatidae, Mantellinae, Pseudophryne, and Melanophryniscus (Vences et al., 1997/98).

References

Clarke, BT(1997) The Natural History of Amphibian Skin Secretions, Their Normal Functioning and Potential Medical Applications. Biological Review, 72, 365-379.

Caldwell, JP(1996) The Evolution of Myrmecophagy and It’s Correlates in Poison Frogs (Family Dendrobatidae). Journal of the Zoological Society of London, 240, 75-101.

Daly, JW(1998) Thirty Years of Discovering Arthropod Alkaloids in Amphibian Skin. Journal of Natural Products, 61:1, 162-172.

Daly, JW, Garraffo, HM, Hall, GSE, Cover, JF Jr.(1997) Absence of Skin Alkaloids in Captive-Raised Madagascan Mantelline Frogs (Mantella) and Sequestration of Dietary Alkaloids. Toxicon, 35:7, 1131-1135.

Daly, JW, Gusovsky, F, Myers, CW, Yotsu-Yamashita, M, Yasumoto, T(1994) First Occurrence of Tetrodotoxin in a Dendrobatid Frog (Colosethus inguinalis), with Further Reports for the Bufonid Genus Atelopus.Toxicon, 32:3, 279-285.

Daly, JW, Garraffo, HM, Jain, P, Spande, TF, Snelling, RR, Jaramillo, C, Rand, AS(2000) Arthropod-Frog Connection: Decahydroquinoline and Pyrrolizidine Alkaloids Common to Microsympatric Myrmicine Ants and Dendrobatid Frogs. Journal of Chemical Ecology, 26:1, 73-85.

Daly, JW, Secunda, SI, Garraffo, HM, Spande, TF, Wiskienski, A, Cover, JF Jr.(1994) An Uptake System for Dietary Alkaloids in Poison Frogs (Dendrobatidae). Toxicon, 32:6, 657-663.

Daly, JW, Secunda, SI, Garraffo, HM, Spande, TF, Wisnieski, A, Nishihira, C, Cover, JF Jr.(1992) Variability in Alkaloid Profiles in Neotropical Poison Frogs (Dendrobatidae): Genetic Versus Environmental Determinants. Toxicon, 30:8, 887-898.

Fisher, M, Huangfu, D, Shen, TY, Guyenet, PG(1994) Epibatidine, An Alkaloid From the Poison Frog Epipedobates tricolor, Is a Powerful Ganglionic Depolarizing Agent. The Journal of Pharmacological and Experimental Therapeutics, 270:2, 702-707.

Jones, TH, Gorman, JST, Snelling, RR, Delabie, JHC, Blum, MS, Garraffo, HM, Jain, P, Daly, JW, Spande, TF(1999) Further Alkaloids Common to Ants and Frogs: Decahydroquinolines and a Quinolizidine. Journal of Chemical Ecology, 25:5, 1179-1193.

Myers, CW, Daly, JW(1983) Dart-Poison Frogs. Scientific American, 248:2, 120-133.

Toft, CA(1995) Evolution of Diet Specialization in Poison-Dart Frogs (Dendrobatidae). Herpetologica, 51:2, 202-216.

Toledo, RC, Jared, C(1995) Cutaneous Granular Glands and Amphibian Venoms. Comparitive Biochemistry and Physiology, 111A:1, 1-29.

Vences, M, Glaw, F, Bohme, W(1997/98) Evolutionary Correlates of Microphagy in Alkaloid-Containing Frogs (Amphibia: Anura). Zoologischer Anzeiger, 236, 217-230.