Acquisition, transformation and maintenance of plant pyrrolizidine alkaloids by the polyphagous arctiid Grammia geneura.

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.

Specific recognition, detoxification and metabolism of pyrrolizidine alkaloids by the polyphagous arctiid Estigmene acrea.

Evidence is presented that the polyphagous arctiid Estigmene acrea is well adapted to sequester and specifically handle pyrrolizidine alkaloids of almost all known structural types representative of the major plant families with pyrrolizidine alkaloid-containing species, i.e. Asteraceae with the tribes Senecioneae and Eupatorieae, Boraginaceae, Fabaceae, Apocynaceae and Orchidaceae. The adaptation of E. acrea to pyrrolizidine alkaloids includes a number of specialized characters: (i) highly sensitive recognition of alkaloid sources by pyrrolizidine alkaloid-specific taste receptors; (ii) detoxification of pyrrolizidine alkaloids by N-oxidation catalyzed by a specific flavin-dependent monooxygenase; (iii) transfer and maintenance of all types of pyrrolizidine N-oxides through all developmental stages; (iv) conversion of the various structures into the male courtship pheromone hydroxydanaidal most probably through retronecine and insect specific retronecine esters (creatonotines) as common intermediates; (v) specific integration into mating behavior and defense strategies. Toxic otonecine derivatives, e.g. the senecionine analogue senkirkine, which often accompany the common retronecine derivatives and which cannot be detoxified by N-oxidation do not affect the development of E. acrea larvae. Senkirkine is not sequestered at all. Non-toxic 1,2-saturated platynecine derivatives that frequently occur together with toxic retronecine esters are sequestered and metabolized to hydroxydanaidal, indicating the ability of E. acrea to aromatize saturated pyrrolizidines. Although pyrrolizidine alkaloids, even if they are offered continuously at a high level (2%) in the larval diet, are non-toxic, E. acrea larvae are not able to develop exclusively on a pyrrolizidine alkaloid-containing plant like Crotalaria. Therefore, E. acrea appears to be specifically adapted to exploit pyrrolizidine alkaloid-containing plants as "drug source" but not as a food source.

Acquired and partially de novo synthesized pyrrolizidine alkaloids in two polyphagous arctiids and the alkaloid profiles of their larval food-plants.

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.

Are insect-synthesized retronecine esters (creatonotines) the precursors of the male courtship pheromone in the arctiid moth Estigmene acrea?

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.

Uptake and metabolism of pyrrolizidine alkaloids in Longitarsus flea beetles (Coleoptera: Chrysomelidae) adapted and non-adapted to alkaloid-containing host plants.

Several Longitarsus flea beetle species sequester pyrrolizidine alkaloids acquired from their Asteraceae and Boraginaceae host plants. We carried out feeding and injection experiments using radioactively labeled pyrrolizidine alkaloids to investigate the physiological mechanisms of uptake, metabolism and storage of alkaloids in adult beetles. We examined six Longitarsus species belonging to different phylogenetic clades in a comparative approach. All species that accepted pyrrolizidine alkaloids in a preceding food choice study showed the ability both to store pyrrolizidine alkaloid N-oxides and to metabolize tertiary pyrrolizidine alkaloids into their N-oxides. Regardless of whether the beetles' natural host plants contain pyrrolizidine alkaloids or not, these species were found to possess an oxidizing enzyme. This oxygenase appears to be specific to pyrrolizidine alkaloids: [3H]Atropine and [14C]nicotine, two alkaloids not related to pyrrolizidine alkaloids, were neither stored nor N-oxidized by any of the tested species. One species, L. australis, that strictly avoids pyrrolizidine alkaloids behaviorally, exhibited a lack of adaptations to pyrrolizidine alkaloids on a physiological level as well. After injection of tertiary [14C]senecionine, beetles of this species neither N-oxidized nor stored the compounds, in contrast to L. jacobaeae, an adapted species that underwent the same treatment. L. jacobaeae demonstrated the same efficiency in N-oxidation and storage when fed or injected with tertiary [14C]senecionine.

Biochemical processing of plant acquired pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi.

Leaf beetles of the genus Platyphora, feeding on plant species containing pyrrolizidine alkaloids of the lycopsamine type, not only sequester these alkaloids and concentrate them in their exocrine defensive secretions, but also specifically process the plant acquired alkaloids. Using P. boucardi as subject, three mechanisms were studied: (i). utilization of host plant alkaloids that are not sequestered per se; (ii). elucidation of the mechanism of the already documented C-7 epimerization of heliotridine O(9)-monoesters; (iii). the specificity of insect catalyzed necine base esterification. P. boucardi does not sequester the triester parsonsine, the principal alkaloid of its host plant Prestonia portobellensis (Apocynaceae). Beetles fed with a purified mixture of nor-derivatives of parsonsine, obtained from Parsonsia laevigata, did not sequester the triesters but transformed them by partial degradation into monoesters that are accumulated in the defensive secretions. The mechanism of the previously described transformation of rinderine into intermedine by C-7 epimerization was elucidated by feeding C-7 deuterated heliotrine (3'-methylrinderine). The transformation of heliotrine into epiheliotrine (3'-methylintermedine) catalyzed by P. boucardi is accompanied by complete loss of deuterium, indicating the same mechanism of an oxidation-reduction process via a ketone intermediate as recently demonstrated in a pyrrolizidine alkaloid sequestering lepidopteran. P. boucardi is able to form ester alkaloids from five different necine bases fed as radioactively labeled substrates. However, besides C-7 epimerization the beetles are not able to convert simple necine bases into retronecine. The functional importance of the various alkaloid transformations is discussed in comparison to striking parallels of analogous reactions known from pyrrolizidine alkaloid sequestering Lepidoptera.

Sequestration, metabolism and partial synthesis of tertiary pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi.

Platyphora boucardi leaf-beetles sequester tertiary pyrrolizidine alkaloids of the lycopsamine type acquired from their host-plant Prestonia portobellensis (Apocynaceae) and synthesize their own alkaloids from exogenous retronecine and aliphatic 2-hydroxy acids. Tracer studies with [14C]rinderine and its N-oxide revealed that P. boucardi sequesters both alkaloidal forms with the same efficiency, but accumulates exclusively tertiary alkaloids. There is no substantial alkaloid accumulation in the body outside the defensive glands. Feeding studies with [2H][14C]rinderine confirmed that P. boucardi specifically epimerizes rinderine to its stereoisomers intermedine and lycopsamine. Feeding studies with [2H][14C]retronecine proved the ability of P. boucardi to synthesize O7- and O9-(2-hydroxyisovaleryl)-retronecine and O7-lactyl-O9-(2-hydroxyisovaleryl)-retronecine. Both, alkaloids of the lycopsamine type and self-synthesized retronecine esters accumulate in the defensive secretions at concentrations up to 38 mM and 33 mM, respectively. The different biochemical strategies to maintain pro-toxic pyrrolizidine alkaloids and to prevent self-poisoning, developed by specialized insects, are compared. There are two major findings: (1) the chemical defense mediated by plant acquired pyrrolizidine alkaloids in the taxonomically related palaearctic Oreina and neotropical Platyphora leaf beetles have been evolved independently, since the biochemical mechanisms of storing and maintaining the alkaloids is completely different in the two genera; (2) unexpected parallels exist between taxonomically unrelated Coleoptera and Lepidoptera in their ability to synthesize the same retronecine esters and to catalyze the same site-specific epimerizations of the lycopsamine stereoisomers.

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles.

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.

Sites of synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L.

(14)C-Labelled alkaloid precursors (arginine, putrescine, spermidine) fed to Senecio vulgaris plants via the root system were rapidly taken up and efficiently incorporated into the pyrrolizidine alkaloid senecionine N-oxide (sen-Nox) with total incorporations of 3-6%. Considerable amounts of labelled sen-Nox were translocated into the shoot and were directed mainly into the inflorescences, the major sites of pyrrolizidine-alkaloid accumulation. Detached shoots of S. vulgaris were unable to synthesize pyrrolizidine alkaloids, indicating that the roots are the site of their biosynthesis. Further evidence was obtained from studies with in-vitro systems established from S. vulgaris: root cultures were found to synthesize pyrrolizidine alkaloids but not cell-suspension cultures, tumor cultures or shoot-like teratomas obtained by transformation with Agrobacterium tumefaciens. Studies on transport of [(14)C]sen-Nox, which was fed either to detached shoots or to the root system of intact plants, indicate that the alkaloid N-oxide does not simply follow the transpiration stream but is specifically channelled to the target tissues such as epidermal stem tissue and flower heads. Exogenously applied [(14)C]senecionine is rapidly N-oxidized. If the phloem path along the stem is blocked by a "steam girdle" translocation of labelled sen-Nox is blocked as well. Root-derived sen-Nox accumulated below the girdle and only trace amounts were found in the tissues above. It is most likely that the root-to-shoot transport of sen-Nox occurs mainly if not exclusively via the phloem. In accordance with previous studies the polar, salt-like N-oxides, which are often considered to be artifacts, were found to be the real products of pyrrolizidine-alkaloid biosynthesis as well as the physiological forms for long-distance transport, tissue-specific distribution and cellular accumulation.

Accumulation of quinolizidine alkaloids in plants and cell suspension cultures: genera lupinus, cytisus, baptisia, genista, laburnum, and sophora.

The patterns of quinolizidine alkaloids in cell cultures of 10 species of Fabaceae were analyzed by high-resolution GLC and GLC-MS and compared with the alkaloids present in the leaves of the respective plants. Lupanine was produced in all 10 cell suspension cultures as the main alkaloid. It was accompanied by sparteine, tetrahydrorhombifoline, 17-oxosparteine, 13-hydroxylupanine, 4-hydroxylupanine, 17-oxolupanine, and 13-hydroxylupanine esters as minor alkaloids in some species. The alkaloid patterns of the plants differed markedly in that alpha-pyridone alkaloids were the major alkaloids in the genera Cytisus, Genista, Laburnum and Sophora but were not accumulated in the cell cultures. These data further support the assumption that the pathway leading to lupanine is the basic pathway of quinolizidine alkaloids biosynthesis and that the other alkaloids are derived from lupanine.