1. Are insect-synthesized retronecine esters (creatonotines) the precursors of the male courtship pheromone in the arctiid moth Estigmene acrea?
T Hartmann, C Theuring, E A Bernays J Chem Ecol. 2003 Nov;29(11):2603-8. doi: 10.1023/a:1026361628676.
The pyrrolizidine alkaloid (PA) profiles were determined for adults of the polyphagous arctiid Estigmene acrea, which as larvae had fed on artificial diet supplemented with Crotalaria-pumila powder with known concentrations of PAs. The larvae always had a free choice between alkaloid-containing and plain diets. The alkaloid profiles of adults revealed a striking sexual dimorphism. Both sexes contained macrocyclic PAs of the monocrotaline type sequestered from the diet and, in addition, a substantial proportion of supinidine and retronecine esters synthesized by the insects from necine bases derived from the dietary alkaloids and necic acids of insect origin. These insect alkaloids accounted for 35% and 55% of total PAs in males and females, respectively. The difference was that in females the retronecine esters (creatonotines) made up 58 microg (43% of total PAs), while males contained a fivefold lower proportion, 12 microg (13%). Four of the ten male individuals analyzed were found devoid of creatonotines. Based on the experimental data in combination with evidence from the literature, it is suggested that the creatonotines are direct pheromone precursors in E. acrea. It is hypothesized that this may represent a general mechanism of hydroxydanaidal formation from diverse macrocyclic PAs in arctiids.
2. Acquired and partially de novo synthesized pyrrolizidine alkaloids in two polyphagous arctiids and the alkaloid profiles of their larval food-plants
T Hartmann, C Theuring, T Beuerle, L Ernst, M S Singer, E A Bernays J Chem Ecol. 2004 Feb;30(2):229-54. doi: 10.1023/b:joec.0000017975.16399.c3.
The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A-C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1beta,2beta-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40-70% (E. acrea) and 17-37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.
3. Acquisition, transformation and maintenance of plant pyrrolizidine alkaloids by the polyphagous arctiid Grammia geneura
T Hartmann, C Theuring, T Beuerle, E A Bernays, M S Singer Insect Biochem Mol Biol. 2005 Oct;35(10):1083-99. doi: 10.1016/j.ibmb.2005.05.011.
The polyphagous arctiid Grammia geneura appears well adapted to utilize for its protection plant pyrrolizidine alkaloids of almost all known structural types. Plant-acquired alkaloids that are maintained through all life-stages include various classes of macrocyclic diesters (typically occurring in the Asteraceae tribe Senecioneae and Fabaceae), macrocyclic triesters (Apocynaceae) and open-chain esters of the lycopsamine type (Asteraceae tribe Eupatorieae, Boraginaceae and Apocynaceae). As in other arctiids, all sequestered and processed pyrrolizidine alkaloids are maintained as non-toxic N-oxides. The only type of pyrrolizidine alkaloids that is neither sequestered nor metabolized are the pro-toxic otonecine-derivatives, e.g. the senecionine analog senkirkine that cannot be detoxified by N-oxidation. In its sequestration behavior, G. geneura resembles the previously studied highly polyphagous Estigmene acrea. Both arctiids are adapted to exploit pyrrolizidine alkaloid-containing plants as "drug sources". However, unlike E. acrea, G. geneura is not known to synthesize the pyrrolizidine-derived male courtship pheromone, hydroxydanaidal, and differs distinctly in its metabolic processing of the plant-acquired alkaloids. Necine bases obtained from plant acquired pyrrolizidine alkaloids are re-esterified yielding two distinct classes of insect-specific ester alkaloids, the creatonotines, also present in E. acrea, and the callimorphines, missing in E. acrea. The creatonotines are preferentially found in pupae; in adults they are largely replaced by the callimorphines. Before eclosion the creatonotines are apparently converted into the callimorphines by trans-esterification. Open-chain ester alkaloids such as the platynecine ester sarracine and the orchid alkaloid phalaenopsine, that do not possess the unique necic acid moiety of the lycopsamine type, are sequestered by larvae but they need to be converted into the respective creatonotines and callimorphines by trans-esterification in order to be transferred to the adult stage. In the case of the orchid alkaloids, evidence is presented that during this processing the necine base (trachelanthamidine) is converted into its 7-(R)-hydroxy derivative (turneforcidine), indicating the ability of G. geneura to introduce a hydroxyl group at C-7 of a necine base. The creatonotines and callimorphines display a striking similarity to plant necine monoesters of the lycopsamine type to which G. geneura is well adapted. The possible function of insect-specific trans-esterification in the acquisition of necine bases derived from plant acquired alkaloids, especially from those that cannot be maintained through all life-stages, is discussed.
