Arthropod–Frog Connection: Decahydroquinoline and Pyrrolizidine Alkaloids Common to Microsympatric Myrmicine Ants and Dendrobatid Frogs

Neotropical poison frogs (Dendrobatidae) contain a wide variety of lipophilic alkaloids, apparently accumulated unchanged into skin glands from dietary sources. Panamanian poison frogs (Dendrobates auratus) raised in a large, screened, outdoor cage and provided for six months with leaf-litter from the frog's natural habitat, accumulated a variety of alkaloids into the skin. These included two isomers of the ant pyrrolizidine 251K; two isomers of the 3,5-disubstituted indolizidine 195B; an alkaloid known to occur in myrmicine ants; another such indolizidine, 211E; two pyrrolidines, 197B and 223N, the former known to occur in myrmicine ants; two tricyclics, 193C and 219I, the former known to occur as precoccinelline in coccinellid beetles; and three spiropyrrolizidines, 222, 236, and 252A, representatives of an alkaloid class known to occur in millipedes. The alkaloids 211E, 197B, and 223N appear likely to derive in part from ants that entered the screened cage. In addition, the frog skin extracts contained trace amounts of four alkaloids, 205D, 207H, 219H, and 231H, of unknown structures and source. Wild-caught frogs from the leaf-litter site contained nearly 40 alkaloids, including most of the above alkaloids. Pumiliotoxins and histrionicotoxins were major alkaloids in wild-caught frogs, but were absent in captive-raised frogs. Ants microsympatric with the poison frog at the leaf-litter site and at an island site nearby in the Bay of Panamá were examined for alkaloids. The decahydroquinoline (–)-cis-195A and two isomers of the pyrrolizidine 251K were found to be shared by microsympatric myrmicine ants and poison frogs. The proportions of the two isomers of 251K were the same in ant and frog.     

Chemistry of venom alkaloids in someSolenopsis (Diplorhoptrum) species from Puerto Rico

A number of 15-carbon alkaloids have been identified in venom extracts of four Puerto Rican species of ants in the genusSolenopsis (Diplorhoptrum). Workers of a species from El Verde produced thecis andtrans isomers of 2-methyl-6-nonylpiperidine with the latter isomer predominating. The same compounds were identified in queens of a species from Río Grande, but in this species no alkaloids were detected in worker extracts. Workers of aDiplorhoptrum species collected on Mona Island produced primarily atrans-2-methyl-6-(Z-4-nonenyl)piperidine,3, with smaller amounts of thecis isomer, whereas the major compound found in the queens of the same species on Mona Island was (5Z,9Z)-3-hexyl-5-methylindolizidine, identical with the alkaloid produced by queens of a species collected on Cabo Rojo. Surprisingly, workers of the Cabo Rojo species produced (5Z,9Z)- and (5E,9E)-3-butyl-5-propylindolizidine (4 and5, respectively) reported earlier as the 223AB indolizidines from skins of dendrobatid frogs. The possible significance of the qualitative and quantitative differences in the venom alkaloids synthesized by queens and workers is discussed as is the possibility that ants containing such alkaloids may serve as a dietary source for the skin alkaloids used by certain frogs in chemical defense.     

3-Hexyl-5-Methylindolizidine Isomers from Thief Ants, Solenopsis (Diplorhoptrum) Species

The venom alkaloids from the workers of nine collections of Solenopsis (Diplorhoptrum) from California contain either (5E,9E)-3-hexyl-5-methylindolizidine (1c) or (5Z,9E)-3-hexyl-5-methylindolizidine (1d) along with cis-2-methyl-6-nonylpiperidine. The structures of these compounds were determined from their mass spectra and by comparison of their GC-FTIR spectra with those of a synthetic mixture. In view of the facts that a third diastereomer of 3-hexyl-5-methylindolizidine had been reported in previous collections of Solenopsis (Diplorhoptrum) queens from Puerto Rico, and that indolizidines along with other ant venom alkaloids are sequestered by amphibians, the determination of species in this difficult group of ants is significant. In particular, the chemotaxonomic value of the stereochemistry of these venom alkaloids is discussed.     

Enantioselective Hydrogenation of a Methoxypyrone with Cinchona-Modified Palladium

Hydrogenation of 4-methoxy-6-methyl-2-pyrone 1 has been investigated over cinchona-modified Pd/TiO₂. The appropriate start-up procedure including a catalyst reduction—oxidation—reduction cycle and short pretreatment with the modifier in the absence of reactant can remarkably enhance the enantiomeric excess (ee) to the dihydropyrone 2. Another key parameter is the alkaloid/Pd_s molar ratio; the alkaloid concentration in the slurry or the alkaloid/reactant ratio is not crucial. Under the best conditions 94% ee and 95% chemoselectivity to 2 were achieved at 80% conversion of 1, in only 30 min reaction time under ambient conditions. The ee can be further increased by kinetic resolution of 2. In the second reaction step the diasteroselectivity to the cis-tetrahydropyrone 3 is about 99%.     

Alkaloids in the Mite <Emphasis Type="Italic">Scheloribates laevigatus</Emphasis>: Further Alkaloids Common to Oribatid Mites and Poison Frogs

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.     

Chemistry of venom alkaloids in the ant genusMegalomyrmex

Chemical analyses of three species in the Neotropical ant genusMegalomyrmex have identified this taxon as the third myrmicine genus to produce alkaloids as major venom products. Workers ofM. leoninus and workers and ergatoids ofM. goeldii produce one or more of fourtrans-2,5-dialkylpyrrolidines previously identified in other myrmicine genera.M. modestus, on the other hand, is distinctive in producing the novel alkaloid (5E,8E)-3-butyl-5-hexylpyrrolizidine (5d), whose structure was established using a micro-Hofmann degradation sequence. The relationship ofMegalomyrmex to other alkaloid-producing ant genera is discussed along with the possible chemotaxonomic significance of the analyzed species when viewed in terms of the recognized species groups in this genus.     

Biparental Endowment of Endogenous Defensive Alkaloids in <Emphasis Type="Italic">Epilachna paenulata</Emphasis>

Coccinellid beetles contain a variety of defensive alkaloids that render them unpalatable to predators. Epilachna paenulata (Coleoptera: Coccinellidae) is a South American ladybird beetle that feeds on plants of the Cucurbitaceae family. The defensive chemistry of E. paenulata has been characterized as a mixture of systemic piperidine, homotropane, and pyrrolidine alkaloids. Whole body extracts of adult beetles contain four major alkaloids: 2-(2′-oxopropyl)-6-methylpiperidine (1); 1-(6-methyl-2,3,4,5-tetrahydro-pyridin-2-yl)-propan-2-one (2); 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3); and 1-(2″-hydroxyethyl)-2-(12′-aminotridecyl)-pyrrolidine (4). Comparative studies of the defensive chemistry of eggs, larvae, pupae, and adults showed differences in alkaloid composition and concentration among life stages. While adults contained mainly the homotropane 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3), eggs showed the highest concentration of the piperidine 2-(2′-oxopropyl)-6-methylpiperidine (1). We studied the origin of this alkaloid in the eggs by feeding newly emerged, virgin adult beetles with [2-¹³C]-labeled acetate, and by performing crosses between ¹³C-fed and unlabeled males and females. GC-MS analysis of alkaloids from ¹³C-fed males and females showed high incorporation of ¹³C into the alkaloids, as evidenced from a 20–30% increase of isotopic peaks in diagnostic fragment ions, confirming the expected endogenous origin of these alkaloids. In addition, analyses of eggs from different crosses showed that labeled alkaloids from both parents are incorporated into eggs, indicating that E. paenulata males transfer alkaloids to the females at mating. Biparental endowment of chemical defenses into eggs has been shown previously in insects that acquire defensive compounds from dietary sources. To our knowledge, this is the first report of biparental egg endowment of endogenous defenses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Scheloribatid Mites as the Source of Pumiliotoxins in Dendrobatid Frogs<Superscript>†</Superscript>

The strawberry poison frog Dendrobates pumilio (Anura: Dendrobatidae) and related poison frogs contain a variety of dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods microsympatrically distributed in the habitat. In addition to ants, beetles, and millipedes, we found that adults of two species of oribatid mites belonging to the cohort Brachypylina, trophically a lower level of animal than ants and beetles, contain dendrobatid alkaloids. Gas chromatography/mass spectrometry (GC/MS) of hexane extracts of adult Scheloribates azumaensis (Oribatida: Acari) revealed the presence of not only pumiliotoxin 251D (8-hydroxy-8-methyl-6-(2′-methylhexylidene)-1-azabicyclo[4.3.0]nonane), but also precoccinelline 193C and another coccinelline-type alkaloid. From the corresponding extracts of an unidentified Scheloribates sp., pumiliotoxin 237A (8-hydroxy-8-methyl-6-(2′-methylpentylidene)-1-azabicyclo[4.3.0]nonane) was detected as a minor component, and identified by synthesis. The presence of related alkaloids, namely deoxypumiliotoxin 193H, a 6,8-diethyl-5-propenylindolizidine, and tentatively, a 1-ethyl-4-pentenynylquinolizidine, were indicated by the GC/MS fragmentation patterns, along with at least another six unidentified alkaloid components. Thus, one possible origin of pumiliotoxins, coccinellid alkaloids, and certain izidines found in poison frogs may be mites of the genus Scheloribates and perhaps related genera in the suborder Oribatida. Chemical Ecology of Oribatid Mites IV.     

Coadsorption of Cinchona Alkaloids on Supported Palladium: Nonlinear Effects in Asymmetric Hydrogenation and Resistance of Alkaloids Against Hydrogenation

The transient behavior of the adsorption of cinchona alkaloid modifiers on Pd/TiO₂ has been investigated in situ during the enantioselective hydrogenation of 4-methoxy-6-methyl-2-pyrone (1). Modifier mixtures consisting of pairs of alkaloids that alone afford the opposite enantiomers in comparable excess were applied to probe the adsorption behavior and possible nonlinear phenomena. Complementary information has been gathered from an indirect UV-vis study of the adsorption and hydrogenation of cinchonidine and quinidine on Pd/TiO₂. The striking nonlinear behavior of cinchonidine-quinidine and cinchonine-quinine pairs in the hydrogenation of 1, and in the competitive saturation of the quinoline rings of the alkaloids, is attributed to differences in the adsorption strength and geometry of the alkaloids. The results are in good agreement with our former mechanistic model assuming that the quinoline ring of cinchona alkaloid and 1 adsorb parallel to the Pd surface during the enantiodifferentiating step.     

Mandibular gland allomones ofDasymutilla occidentalis and other mutillid wasps

The mandibular gland secretion of the mutillid wasp,Dasymutilla occidentalis, possesses three short-chained ketones-4-methyl-3-heptanone (4MH), 4,6-dimethyl-3-nonanone (4,6DMN), 4,6-dimethyl-3-octanone (4,6DMO)—and several unidentified compounds. This is the first report of 4,6DMN as a natural product and its synthesis is described. These ketones, which are either known to be ant alarm pheromones or are structurally very similar to ant alarm pheromones, appear to function as allomones against ants, major potential predators of mutillid wasps. The major secretory component, 4-methyl-3-heptanone, which was identified in females and/ or males of the species analyzed within the generaDasymutilla, Timulla, Traumatomutilla, andPappognatha, appears to constitute a chemical character of the defensive secretions of these genera.     

Geographic and Seasonal Variation in Alkaloid-Based Chemical Defenses of Dendrobates pumilio from Bocas del Toro, Panama

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 Siphonotid Millipede (Rhinotus) as the Source of Spiropyrrolizidine Oximes of Dendrobatid Frogs

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.     

Individual Variation in Alkaloid Content of Poison Frogs of Madagascar (Mantella; Mantellidae)

Brightly colored Malagasy poison frogs, Mantella spp., sequester lipophilic, basic alkaloids from arthropod prey for their own chemical defense. Consequently, microsympatric prey diversity is expected to influence alkaloid diversity observed in poison frogs. Twenty-two specimens of three Mantella species from four localities in moist forests of southeastern Madagascar were analyzed individually via gas chromatography-mass spectrometry, revealing that they contain over 80 known alkaloids. Frogs within a locality possessed significantly similar alkaloid content and diversity, while frogs from areas that varied in disturbance, elevation, and/or species showed greater differences. Based on dietary data, the larger frog species Mantella baroni consumed more and larger prey, and showed greater diversity in skin alkaloids than significantly smaller Mantella bernhardi. Additionally, frogs from the most pristine locality had the greatest number of alkaloids, whereas individuals from the most disturbed localities had the least. In a comparison of frog alkaloid profiles over a 10- to 14-yr period, alkaloid turnover, and thus presumably alkaloid-source arthropod turnover, was high in a disturbed locality and low in the pristine primary forest locality. We demonstrate that the nonlethal transcutaneous amphibian stimulator (TAS) is effective for harvesting alkaloids from poison frogs; future studies using this device could obtain larger sample sizes without harming local frog populations.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Neotropical ant gardens

In ant gardens of lowland Amazonia, parabiotic ant speciesCamponotus femoratus andCrematogaster cf.limata parabiotica cultivate a taxonomically diverse group of epiphytic plants, whose establishment is restricted to arboreal carton ant nests. Epiphyte seeds are collected by workers ofCa. femoratus, the larger of the two ants, and stored unharmed in brood chambers where they subsequently germinate. Although seeds of some ant-garden epiphytes bear nutritional rewards, previous studies have shown that these rewards are not sufficient to explain the pattern of ant attraction to seeds. Five aromatic compounds occur frequently in and on the seeds of most ant-garden epiphytes and may be chemical cues by which ants recognize propagules of their symbiotic plants. The most widely distributed of these is methyl 6-methylsalicylate [6-MMS]1, previously reported as a major mandibular gland product in relatedCamponotus species and present in trace quantities inCa. femoratus males. (–)-Citronellol6 (previously unreported inCamponotus) was the principal volatile constituent in extracts of male heads, and (–)-mellein7 was present in small quantities. Discovery of 6-MMS inside the mandibular glands of maleCa. femoratus (and its presence in analogous glands of related ants) offers preliminary support for Ule's (1906) hypothesis that seeds attract ants by mimicking ant brood. In addition, the likely fungistatic activity of seed compounds suggests that they could retard microbial pathogens of ants and plants in the organic detritus of nest gardens. While the presence of identical seed compounds in so many unrelated plant lineages might represent a remarkable case of convergent evolution, other interpretations are possible.     

(5Z,9Z)-3-alkyl-5-methylindolizidines fromSolenopsis (Diplorhoptrum) species

The alkaloidal venom components of two species of thief ants,Solenopsis (Diplorhoptrum) species AA andS. (Diplorhoptrum)conjurata have been found to contain (5Z,9Z)-3-hexyl-5-methylindolizidine and a mixture of (5Z,9Z)-3-ethyl-5-methylindolizidine andcis-2-methyl-6-nonyl-piperidine,trans-2-methyl-6-nonylpiperidine,cis-2-methyl-6-undecylpiperidine, and hexadecanoic acid.Monomorium pharaonis was similarly investigated and found to contain the indolizidine and pyrrolidines previously described (Ritter et al., 1977b). Both indolizidines were synthesized along with their stereoisomers and separated by preparative gas chromatography. Spectral studies revealed the stereochemistry to be 5Z,9Z in both cases. The stereochemistry of 2-butyl-5-pentylpyrrolidine inM. phaeronis has also been established. Biosynthetic relationships are discussed.     

Components of male aggregation pheromone of strawberry blossom weevil, Anthonomus rubi herbst. (Coleoptera:Curculionidae)

The strawberry blossom weevil, Anthonomus rubi, is a major pest of strawberries in the United Kingdom and continental Europe. As part of a project to develop noninsecticidal control methods, the pheromone system of this species was investigated. Comparison of volatiles produced by field-collected, overwintering individuals of each sex led to identification of three male-specific compounds—(Z)-2-(3,3-dimethylcyclohexylidene)ethanol, (cis)-1-methyl-2-(1-methylethenyl)cyclobutaneethanol, and 2-(1-methylethenyl)5-methyl-4-hexen-1-ol (lavandulol)—in amounts of 6.1, 1.2, and 0.82 g/day/male. The first two compounds are components of the aggregation pheromone of the boll weevil, Anthonomus grandis, grandlure II and grandlure I, respectively. Grandlure I was the (1R,2S)-(+) enantiomer and lavandulol was a single enantiomer, although the absolute configuration was not determined. Trace amounts of the other two grandlure components (Z)-(3,3-dimethylcyclohexylidene)acetaldehyde (grandlure III) and (E)-(3,3-dimethylcyclohexylidene)acetaldehyde (grandlure IV) were also detected. (E,E)-1-(1-Methylethyl)-4-methylene8-methyl-2,7-cyclo-decadiene (germacrene-D), a known volatile from strawberry plants, Fragaria ananassa, was collected in increased amounts in the presence of pheromone-producing weevils. Male weevils only produced pheromone on F. ananassaand not on scented mayweed, Matracaria recutita, or cowparsley, Anthriscus sylvestris, although these are known food sources. In field trials using various combinations of synthetic grandlures I, II, III, and IV and lavandulol, significantly more weevils were caught in traps baited with blends containing grandlure I and II and lavandulol than in those baited with blends without lavandulol or unbaited controls. Addition of grandlure III and IV had no significant effect on attractiveness. Horizontal sticky traps were found to be more effective than vertical sticky traps or standard boll weevil traps. In mid-season females predominated in the catches, but later more males than females were trapped.     

Isolation and structure of glucosylceramides from the starfish Cosmasterias lurida

From the water-insoluble lipid fraction of the methylene chloride/methanol extract of the starfish Cosmasterias lurida, two new glucosylceramides together with a known glucosylceramide, ophidiacerebroside E, were isolated by chromatographic procedures and characterized by spectroscopic (¹H and ¹³C nuclear magnetic resonance, mass spectrometry) methods. The new compounds were identified as (2S, 3R, 4E, 8E, 10E)-1-(β-d-glucopyranosyloxy)-3-hydroxy-2-[(R)-2-hydroxyheptadecanoyl)amino]-9-methyl-4,8,10-octadecatriene (3) and (2S,3R,4E,8E,10E)-1-(β-d-glucopyranosyloxy)-3-hydroxy-2-[(R)-2-hydroxyoctadecanoyl)amino]-9-methyl-4,8,10-octadecatriene (4).     

Bulk anionic ring opening polymerization of silacyclopent-3-enes

Summary Poly[1,1-diphenyl-1-sila-cis-pent-3-ene](I), poly[1,1-dimethyl-1-sila-cis-pent-3-ene](II), poly[1-methyl-1-phenyl-1-sila-cis-pent-3-ene](III), poly[1-methyl-sila-cis-pent-3-ene](IV), and poly[1-phenyl-1-sila-cis-pent-3-ene](V) were prepared by bulk anionic ring opening polymerization of the corresponding 1-silacyclopent-3-enes, at room temperature by use n-butyllithium or t-butyllithium and HMPA as co-catalysts. No solvent (THF or diethyl ether) was utilized.     

New Sphingolipids with a Previously Unreported 9-Methyl-C<Subscript>20</Subscript>-sphingosine Moiety from a Marine Algous Endophytic Fungus <Emphasis Type="Italic">Aspergillus niger</Emphasis> EN-13

Asperamides A (1) and B (2), a sphingolipid and their corresponding glycosphingolipid possessing a hitherto unreported 9-methyl-C₂₀-sphingosine moiety, were characterized from the culture extract of Aspergillus niger EN-13, an endophytic fungus isolated from marine brown alga Colpomenia sinuosa. The structures were elucidated by spectroscopic and chemical methods as (2S,2′R,3R,3′E,4E,8E)-N-(2′-hydroxy-3′-hexadecenoyl)-9-methyl-4,8-icosadien-1,3-diol (1) and 1-O-β-d-glucopyranosyl-(2S,2′R,3R,3′E,4E,8E)-N-(2′-hydroxy-3′-hexadecenoyl)-9-methyl-4,8-icosadien-1,3-diol (2). In the antifungal assay, asperamide A (1) displayed moderate activity against Candida albicans.     

Dihydropyrrolizine attractants for arctiid moths that visit plants containing pyrrolizidine alkaloids

Adults of three species of arctiid moths (Cisseps fulvicollis, Ctenucha virginia, andHalysidota tessellaris) are attracted to plants that contain pyrrolizidine alkaloids (PAs). The moths use olfactory cues to locate these plants, then feed on leaves, flowers, and roots with the proboscis. To investigate the chemical basis of attraction, sticky traps were baited with roots of a PA-containing plant,Eupatorium maculatum, alkaloids ofE. maculatum, and several derivatives of these alkaloids. Volatile derivatives of the bicyclic pyrrolizidine skeleton attracted all three arctiid species. The dihydropyrrolizines, (S)-(+)-hydroxydanaidal and (R)-(–)-hydroxydanaidal, proved to be the most attractive compounds tested, accounting for over 70% of the moths captured. Different alkaloid derivatives attracted different proportions of male and femaleCisseps. Both (S)-(+)-hydroxydanaidal (52% male) and (R)-(–)-hydroxydanaidal (71% male) attracted a significantly lower percentage ofCisseps males thanE. maculatum roots (87% male).Cisseps males possess eversible scent organs (coremata) that are displayed during courtship. Analysis of corematal extracts revealed the presence of hydroxydanaidal.Cisseps moths thus resemble danaine and ithomiine butterflies, both in their attraction to PA sources and in the presence of PA derivatives in the male scent organs.