Selective Transport Systems Mediate Sequestration of Plant Glucosides in Leaf Beetles: a Molecular Basis for Adaptation and Evolution

Selective transport systems mediate sequestration of plant glucosides in leaf beetles: A molecular basis for adaptation and evolution Ju¨ rgen Kuhn†, Eva M. Pettersson†, Birte K. Feld†, Antje Burse†, Arnaud Termonia‡, Jacques M. Pasteels‡, and Wilhelm Boland†§ †Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-Strasse 8, D-07745 Jena, Germany; and ‡Behavioral and Evolutionary Biology, Universite´Libre de Bruxelles, P.O. Box 160͞12, 50 Avenue F. D. Roosevelt, 1050 Brussels, Belgium Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved August 12, 2004 (received for review April 10, 2004) Chrysomeline larvae respond to disturbance and attack by everting they may constitute a rather complex blend, covering different dorsal glandular reservoirs, which release defensive secretions. types of compound classes from different biosynthetic pathways, The ancestral defense is based on the de novo synthesis of mono- as shown in Scheme 1. terpene iridoids. The catabolization of the host-plant O-glucoside A basal group of chrysomelid larvae that specialized on plant salicin into salicylaldehyde is a character state that evolved later in families, such as Salicaceae, Brassicaceae, or Ranunculaceae, two distinct lineages, which specialized on Salicaceae. By using produces iridoid monoterpenes de novo (7, 8). The early bio- two species producing monoterpenes (Hydrothassa marginella and synthetic steps toward, e.g., chrysomelidial, lead from mevalonic Phratora laticollis) and two sequestering species (Chrysomela po- acid to the O-glucoside of 8-hydroxygeraniol (9), whereas the puli and Phratora vitellinae), we studied the molecular basis of final transformations into the active compounds are carried out sequestration by feeding the larvae structurally different thioglu- in the reservoir (Fig. 1B) (10). Further advanced species use cosides resembling natural O-glucosides. Their accumulation in the defensive products whose synthesis depends partly or exclusively defensive systems demonstrated that the larvae possess transport on secondary metabolites from the plant host. Interestingly, a systems, which are evolutionarily adapted to the glycosides of subset of the enzymes (e.g., glucosidase and oxidase) needed for their host plants. Minor structural modifications in the aglycon iridoid biosynthesis is also required in Chrysomela species that result in drastically reduced transport rates of the test compounds. secrete salicylaldehyde, derived from phenol glycosides of the Moreover, the ancestral iridoid-producing leaf beetles already food plants (Fig. 1C) (11). The most evolved species (e.g., possess a fully functional import system for an early precursor of Chrysomela interna) sequester several plant-derived precursors the iridoid defenses. Our data confirm an evolutionary scenario in simultaneously; e.g., glucosides of (3Z)-hex-3-en-1-ol and phe- which, after a host-plant change, the transport system of the leaf nylethanol. After cleavage in the reservoir, the free aglycons are beetles may play a pivotal role in the adaptation on new hosts by esterified with isobutyric acid and 2-methylbutyric acid derived selecting plant-derived glucosides that can be channeled to the from the insects’ internal pools of amino acids (12, 13). defensive system. This shift from autogenous biosynthesis in the basal group to a combinatorial biosynthesis modifying sequestered compounds lant-derived secondary metabolites play an important role in with autogenous components in the highest evolved species is Pthe coevolution of host plants and herbivorous insects. believed to originate from host-plant changes and by subsequent Several insects have overcome the chemical barrier of noxious adaptation of the enzymes. According to phylogenetic analyses, plants and, thereby, have successfully colonized a niche that is this shift from de novo synthesis to the host-derived defense has not readily available to nonadapted competitors. Further ad- evolved independently at least twice (14, 15). However, the vanced strategies additionally exploit the plant-derived toxins for molecular mechanisms facilitating the transitions among these the insects’ own antipredator defense. This use of plant-derived different types of chemical defenses are only partially known. secondary metabolites requires a complex scenario of adapta- Because the basal species and the more advanced sequestering tion, which is unlikely to occur frequently. The insects have to species share not only the principal architecture of their defen- develop (i) a selective import system that selects for the appro- sive system but also several enzymatic activities (see Fig. 1), the priate compound(s) from the plethora of chemicals present in shift from de novo to host-derived defense may require only small the food and (ii) a mode of hemolymph transport and storage changes in the specificity of enzymes present in the glands and capabilities for the toxic compounds that excludes negative reservoir (11, 16). Certainly, also the ability to acquire ‘‘unfa- interferences with the insects own physiological processes (Fig. miliar’’ plant metabolites from food plays a pivotal role because 1A). The successful uptake and use of highly toxic pyrrolizidines the import system is the first molecular mechanism that selects (1), cardenolides (2), iridoid glycosides (3), cyanogenic glyco- for appropriate molecules that are delivered to the glandular sides (4), and glucosinolates (5) by Lepidopterans provide system. several examples for such successful adaptations. Although these We investigated possible mechanisms for this adaptation by examples clearly document the flow of the compounds from comparing the glycoside transport in two iridoid-producing plants to the insect, almost nothing is known about the molecular species and in two salicin-sequestering leaf beetles, in particular basis of the import by and transport within the insect. Chry- the genus Chrysomela, the major taxon in which plant-derived somelid larvae provide a well suited system for study of the defense has evolved. We first established a molecular phylogeny, mechanistic basis of sequestration at different levels of evolu- which included the genera known to secrete iridoids and those tionary adaptation. Leaf beetles (Coleoptera: Chrysomelidae) constitute a very large group of species (6), each of which feeds on a restricted number of plant taxa. In many species, the adults This paper was submitted directly (Track II) to the PNAS office. and͞or the larvae (especially in the subtribe Chrysomelina) Data deposition: The sequences reported in this paper have been deposited in the GenBank protect themselves against predation by releasing defensive database (accession nos. AY027591–AY027628 and AY027695–AY027760). secretions from serial dorsal reservoirs (7). The defensive se- §To whom correspondence should be addressed. E-mail: [email protected]. cretions may contain a single compound or a few compounds, or © 2004 by The National Academy of Sciences of the USA 13808–13813 ͉ PNAS ͉ September 21, 2004 ͉ vol. 101 ͉ no. 38 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402576101 Downloaded by guest on September 27, 2021 Fig. 1. Principal route for plant-derived metabolites into the defensive Scheme 1. gland. (A) At least two transport systems, (i) from the gut to hemolymph and (ii) from hemolymph to the reservoir, are necessary for sequestration. (B) De novo production of the iridoid monoterpene chrysomelidial in the basal group Chemicals. The thioglucosides 1 and 10 (Scheme 2) represent (H. marginella and P. laticollis). (C) Sequestration of salicin and subsequent hydrolysis-resistant, synthetic S-analogs of natural O-glucosides conversion into salicylaldehyde (C. populi and P. vitellinae). Although the involved in the biosynthesis of chrysomelidial (Fig. 1B) and different toxins vary in both structure and biosynthetic origin, the reservoir 1 enzymes involved (a, glucosidase; b, oxidase) are the same (7). Double arrows salicin (Fig. 1C). The synthesis of has been described (19). indicate multistep enzymatic reactions. The thioglucosides of (3Z)-hex-3-en-1-ol 2 and phenylethanol 3 were obtained by Mitsunobu-coupling of the free alcohols with 2,3,4,6-tetra-O-acetyl-␤-D-thioglucopyranose and deprotection sequestering salicin. From this phylogenetic analysis, we selected with methoxide in methanol (20). 2 and 3 served as hydrolysis- for transport experiments species that branched at the basis of resistant analogs of O-glucosides found in Salicaceae and Betu- each of the two groups. As representatives of iridoid-producing laceae (12). Aromatic thioglucosides 437 were obtained by leaf beetles, we chose Hydrothassa marginella and Phratora means of a Ko¨nigs–Knorr protocol using the thiocresols and laticollis, and we chose Chrysomela populi and Phratora vitellinae 2,3,4,6-tetra-O-acetyl-␣-D-glucopyranosyl bromide (19). An for the salicylaldehyde-secreting species. The latter indepen- analogous procedure afforded the isomeric o-, m-, and p- dently evolved the ability to sequester salicin within the group of mercaptobenzoates 8, 9, 10, starting from the isomers of methyl ␣ iridoid-producing species. By providing a rigorous structural mercaptobenzoates and 2,3,4,6-tetra-O-acetyl- -D-glucopyrano- proof for the molecular basis of transport in combination with a syl bromide. The thiogalactoside 11 was obtained from methyl selection of evolutionary relevant species, we offer mechanistic o-mercaptobenzoate and galactopyranosyl bromide.

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