Mass Spectrometry of Aliphatic Macrolides, Important Semiochemicals or Pheromones.

Macrolides are a relatively common structural motif prevalent in Nature. However, the structures of these large ring lactones have been relatively difficult to elucidate via NMR spectroscopy due to the minute amounts of compounds that are sometimes obtainable from natural sources. Thus, GC-MS analysis of individual macrolactones has become the method of choice for the structural identification of these compounds. Here we discuss the mass spectrometric behavior of aliphatic macrolides, evaluating spectra from numerous compounds of various ring size, including derivatives containing methyl branches as well as double bonds. The specific fragmentation of these macrolactones under electron impact conditions allows for the development of a general rule set aimed at the identification of similar compounds by mass spectrometry. In addition, the mass spectra of dimethyl disulfide adducts of unsaturated macrolides are discussed. The mass spectra of almost 50 macrolides are presented.

Biosynthesis of polybrominated aromatic organic compounds by marine bacteria.

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

Take time to smell the frogs: vocal sac glands of reed frogs (Anura: Hyperoliidae) contain species-specific chemical cocktails.

Males of all reed frog species (Anura: Hyperoliidae) have a prominent, often colourful, gular patch on their vocal sac, which is particularly conspicuous once the vocal sac is inflated. Although the presence, shape, and form of the gular patch are well-known diagnostic characters for these frogs, its function remains unknown. By integrating biochemical and histological methods, we found strong evidence that the gular patch is a gland producing volatile compounds, which might be emitted while calling. Volatile compounds were confirmed by gas chromatography-mass spectrometry in the gular glands in 11 species of the hyperoliid genera Afrixalus, Heterixalus, Hyperolius, and Phlyctimantis. Comparing the gular gland contents of 17 specimens of four sympatric Hyperolius species yielded a large variety of 65 compounds in species-specific combinations. We suggest that reed frogs might use a complex combination of at least acoustic and chemical signals in species recognition and mate choice.

Macrolides and alcohols as scent gland constituents of the Madagascan frog Mantidactylus femoralis and their intraspecific diversity.

Acoustic and, to a lesser degree, visual signals are the predominant means of signaling in frogs. Nevertheless, certain lineages such as the mantelline frogs from Madagascar use the chemical communication channel as well. Males possess femoral glands on the hind legs, which recently have been shown to contain volatile compounds used in communication as pheromones. Many mantelline species occur in sympatry, and so far species recognition is regarded to occur mainly by acoustic signals. The analysis of the gland constituents of Mantidactylus femoralis by GC/MS revealed the presence of volatile macrolides and secondary alcohols. The new natural products mantidactolides A (4) and B (6), as well as several methyl carbinols, were identified, and their structures were confirmed by synthesis. The analysis of individuals from different locations of Madagascar revealed the presence of two groups characterized by specific patterns of compounds. While one group contained the alcohols and mantidactolide B, the other showed specific presence of the macrolides phoracantholide I (1) and mantidactolide A (4). Genetic analysis of some individuals showed no congruence between genetic relatedness and gland constituents. Several other individuals from related species had different gland compositions. This suggests that a basic set of biosynthetic machinery might be available to a broader group of related species.

Genetic diversity, phylogeny and evolution of alkaloid sequestering in Cuban miniaturized frogs of the Eleutherodactylus limbatus group.

The miniaturized frogs of the Eleutherodactylus limbatus group, endemic to Cuba, have recently been shown to contain alkaloids in their skin, representing the fifth independent evolutionary origin of alkaloid sequestering in anurans. Based on a comprehensive sampling of all known species of the group we here assess their range-wide phylogeography using DNA sequences of the mitochondrial cytochrome b and the nuclear Rag-1 gene. We found E. etheridgei, E. cubanus, and E. orientalis to be genetically highly distinct, with uncorrected pair-wise distances >12% in the cytochrome b gene and >5% in the 16S rRNA gene, and without haplotype sharing in the Rag-1 gene. On the contrary, E. iberia, E. jaumei, E. limbatus, and one unnamed lineage from Gran Piedra mountains had lower mitochondrial divergences to each other (<6% in cytochrome b; <3% in 16S) and extensive Rag-1 haplotype sharing, suggesting that their species status requires careful revision. All species and lineages of the group occur in the mountain areas of eastern Cuba and only one of them, E. limbatus, colonized central and western Cuba. Populations of this latter lineage are only weakly genetically differentiated, and its range expansion to western Cuba is probably Pleistocenic, demonstrating that also miniaturized frog species are able of considerable range expansions (over hundreds of kilometers) in short time spans. A phylogeny based on about 5 kb of mitochondrial and nuclear genes places, with maximum support, E. etheridgei basalmost in the group, followed by E. cubanus, E. orientalis, and the E. iberia-jaumei-limbatus clade. The screening of skin alkaloids revealed the presence of alkaloids in all lineages of the group, but alkaloids known to be sequestered by other anurans were not found in the basalmost E. etheridgei. Furthermore, individuals of the derived lineages of the E. iberia-jaumei-limbatus clade had a much larger variety of different alkaloid compounds (3-18) than the other three species (0-4). Traces of alkaloids were found in other species only remotely related to the E. limbatus group, i.e., E. caspari, E. goini, E. ricordii, and E. tetajulia. According to these findings, it can be hypothesized that the capacity to sequester dietary alkaloids into the skin evolved first in an ancestral, litter-dwelling and small-sized Cuban Eleutherodactylus, followed by evolution of miniaturization and diurnality in the early representatives of the E. limbatus group, and eventually by increased brightness of color with probable aposematic function and a highly efficient mechanism of alkaloid sequestering.

Discovery of skin alkaloids in a miniaturized eleutherodactylid frog from Cuba.

Four phylogenetically independent lineages of frogs are currently known to sequester lipid-soluble skin alkaloids for which a dietary source has been demonstrated. We report here a remarkable fifth such instance, in Eleutherodactylus iberia and Eleutherodactylus orientalis, two species of miniaturized frogs of the family Eleutherodactylidae from Cuba. Six pumiliotoxins and two indolizidines were found in E. iberia, one of the smallest frogs in the world and characterized by a contrasting colour pattern for which we hypothesize an aposematic function. Analyses of stomach content indicated a numerical prevalence of mites with an important proportion of oribatids-a group of arthropods known to contain one of the pumiliotoxins detected in E. iberia. This suggests that miniaturization and specialization to small prey may have favoured the acquisition of dietary skin alkaloids in these amphibians.