Selective transport systems mediate sequestration of plant glucosides in leaf beetles: a molecular basis for adaptation and evolution

Abstract

Chrysomeline larvae respond to disturbance and attack by everting dorsal glandular reservoirs, which release defensive secretions. The ancestral defense is based on the de novo synthesis of monoterpene iridoids. The catabolization of the host-plant O-glucoside salicin into salicylaldehyde is a character state that evolved later in two distinct lineages, which specialized on Salicaceae. By using two species producing monoterpenes (Hydrothassa marginella and Phratora laticollis) and two sequestering species (Chrysomela populi and Phratora vitellinae), we studied the molecular basis of sequestration by feeding the larvae structurally different thioglucosides resembling natural O-glucosides. Their accumulation in the defensive systems demonstrated that the larvae possess transport systems, which are evolutionarily adapted to the glycosides of their host plants. Minor structural modifications in the aglycon result in drastically reduced transport rates of the test compounds. Moreover, the ancestral iridoid-producing leaf beetles already possess a fully functional import system for an early precursor of the iridoid defenses. Our data confirm an evolutionary scenario in which, after a host-plant change, the transport system of the leaf beetles may play a pivotal role in the adaptation on new hosts by selecting plant-derived glucosides that can be channeled to the defensive system.

Fig. 1.

Principal route for plant-derived metabolites into the defensive 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 (H. marginella and P. laticollis). (C) Sequestration of salicin and subsequent conversion into salicylaldehyde (C. populi and P. vitellinae). Although the different toxins vary in both structure and biosynthetic origin, the reservoir enzymes involved (a, glucosidase; b, oxidase) are the same (7). Double arrows indicate multistep enzymatic reactions.

Scheme 1.

Scheme 2.

Fig. 6.

Mean abundance and standard error of concentration in secretion and hemolymph of C. populi after feeding on compound 10 for various time intervals. The concentration of 10 was found to be time-dependent in secretion samples. Differences are indicated by different letters (P < 0.05).

Fig. 4.

RP-HPLC separation of components from the secretion of P. vitellinae after 48 h of feeding on leaves of P. canadensis impregnated with thiosalicin 10.

Fig. 5.

Mass spectrum of thiosalicin 10. Analyses were carried out in the atmospheric pressure chemical ionization (APCI) mode. All thioglucosides displayed comparable spectra with strong quasimolecular ions [M + H₂O]⁺ and [M + H]⁺.

Fig. 2.

Maximum-parsimony reconstruction of the chemical defensive strategies on the maximum-parsimony strict consensus tree. Bootstrap values are given above the branches. Red, autogenous monoterpene iridoids; green, host-derived defense based on salicylaldehyde derived from salicin; blue, mixed metabolism (esterification of de novo carboxylic acids by plant alcohols). Asterisks indicate taxa that have a dual defense, combining the host-derived metabolism and the mixed metabolism. O, Oreina; Ch, Chrysolina; H, Hydrothassa; P, Phratora; Ph, Phaedon; G, Gonioctena; P, Plagiodera; and C, Chrysomela. Boxes indicate the species chosen to study the transport mechanisms. A, feeding on Asteraceae; B feeding on Betulaceae; Br, feeding on Brassicaceae; L, feeding on Lamiaceae; P, feeding on Polygonaceae; R, feeding on Ranunculaceae; and S, feeding on Salicaceae.

Fig. 3.

Superposition of the molecular structures of salicin and thiosalicin. Data were obtained by x-ray crystallography (22). The C—S bonds are longer than the C—O analogs. The angle of the glycosidic bond in salicin (C—O—C, 117.7°) is significantly larger than in thiosalicin 10 (C—S—C, 102.4°).

Fig. 7.

Survey of transport efficacies as determined for the two leaf-beetle groups of iridoid-producing (H. marginella and P. laticollis) and sequestering (C. populi and P. vitellinae) species fed with different thioglucosides 1–11.