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

Abstract

Tracer feeding experiments with ¹⁴C-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.

Introduction

Plant pyrrolizidine alkaloids with certain structural features such as the presence of a 1–2 double bond, esterification of the allylic hydroxyl group and a free or esterified second hydroxyl group (Fig. 1) are potentially toxic (Mattocks, 1986, Cheeke, 1989). If these alkaloids are ingested by a herbivore they are passively absorbed as tertiary alkaloids and are converted into reactive pyrrolic metabolites by multisubstrate cytochrome P450 oxidases. These pyrrolic intermediates easily react with biological nuclophiles, with the consequence of detrimental cytotoxic and genotoxic effects. In vertebrates this bioactivation is catalyzed by microsomal cytochrome P450 enzymes (EC 1.14.14.1) (Winter and Segall, 1989). These enzymes are mainly localized in liver and lung tissues. They are components of the xenobiotic metabolism and transform absorbed foreign compounds (xenobiotics) into excretable metabolites. In the case of pyrrolizidine alkaloids per se non-toxic compounds are converted into toxic metabolites which are responsible for the hepatotoxicity and pneumotoxicity of pyrrolizidine alkaloids in vertebrates (Mattocks, 1986, Cheeke, 1989). Insects which have a similar xenobiotic metabolism with microsomal cytochrome P450 enzymes (Hodgson, 1985, Brattsten, 1992) should be affected by pyrrolizidine alkaloids similarly as vertebrates. In fact, pyrrolizidine alkaloids were shown to be genotoxic in the Drosophila wing spot test (Frei et al., 1992).

A number of insect species from unrelated taxa have developed adaptations to sequester pyrrolizidine alkaloids from their host plants and utilize them against their own predators. Pyrrolizidine alkaloid sequestering species are found among Lepidoptera (many moths or butterflies of the Arctiidae, Danainae and Ithomiinae) (Boppré, 1986, Schneider, 1987, Hartmann, 1999, Hartmann and Witte, 1995) and Coleoptera such as leaf beetles (Chrysomelidae) of the genus Oreina (Pasteels et al., 1988b, Pasteels et al., 1994, Pasteels et al., 1996, Hartmann et al., 1997). In addition isolated occurrences of alkaloid sequestering species are known from the Orthoptera, i.e. the African grasshopper Zonocerus (Bernays et al., 1977, Fischer and Boppré, 1997) and Homoptera, i.e. Aphis jacobaeae, a specialist phloem-feeder on Senecio species (Witte et al., 1990).

The question is, how pyrrolizidine alkaloid sequestering insects protect themselves against alkaloid toxicity. Arctiids (Ehmke et al., 1990, Hartmann et al., 1990) and Zonocerus (Biller et al., 1994) store plant-derived pyrrolizidine alkaloids exclusively in the N-oxide state. N-oxidation converts the tertiary alkaloid into a derivative which cannot be bioactivated. Some vertebrates detoxify potentially toxic tertiary alkaloids by N-oxidation (Cheeke, 1994). For example, guinea pigs possess a microsomal multisubstrate flavin monooxygenase (EC 1.14.13.8) which efficiently converts ingested pyrrolizidine alkaloids into their N-oxides. In guinea pig liver N-oxigenation by far exceeds cytochrome P450 mediated bioactivation (Miranda et al., 1991). This explains the high resistance of guinea pigs to the toxic effects of pyrrolizidine alkaloids. Similarly, Arctiids store sequestered pyrrolizidine alkaloids in the safe N-oxide state (Lindigkeit et al., 1997).

Pyrrolizidine alkaloid sequestration in leaf beetles differs from sequestration in lepidopterans. Pyrrolizidine alkaloid sequestering leaf beetles have two alkaloid storage sites: (i) exocrine defensive glands located in the elytre and the pronotum of adult beetles from which the defensive secretion is actively released upon attack (Pasteels et al., 1988a, Pasteels et al., 1989, Pasteels et al., 1994); (ii) the whole body in which adults (Ehmke et al., 1991, Rowell-Rahier et al., 1991, Pasteels et al., 1992) and larvae (Dobler and Rowell-Rahier, 1994, Ehmke et al., 1999) store alkaloids acquired from the host plant. The major storage site in the body is the hemolymph. Like lepidopterans, Oreina larvae and adults store the alkaloids as N-oxides. However, in contrast to lepidopterans they are not able to efficiently convert the tertiary alkaloids into the respective N-oxides (Ehmke et al., 1991).

The purpose of this study was to elucidate the mechanisms by which pyrrolizidine alkaloid sequestering leaf beetles, e.g. O. cacaliae, handle tertiary alkaloids and protect themselves against pyrrolizidine alkaloid toxicity. Chrysolina coerulans was chosen as a non-adapted control. The results provide some general insight into the mechanisms and strategies adopted by leaf beetles during their evolutionary adaptation to host plant-derived chemical defense. These mechanisms will be compared with the those adopted by lepidopteran species utilizing host-derived pyrrolizidine alkaloids as defensive compounds.