Seed treatment with clothianidin induces changes in plant metabolism and alters pollinator foraging preferences.

Neonicotinoids, systemic insecticides that are distributed into all plant tissues and protect against pests, have become a common part of crop production, but can unintentionally also affect non-target organisms, including pollinators. Such effects can be direct effects from insecticide exposure, but neonicotinoids can affect plant physiology, and effects could therefore also be indirectly mediated by changes in plant phenology, attractiveness and nutritional value. Under controlled greenhouse conditions, we tested if seed treatment with the neonicotinoid clothianidin affected oilseed rape's production of flower resources for bees and the content of the secondary plant products glucosinolates that provide defense against herbivores. Additionally, we tested if seed treatment affected the attractiveness of oilseed rape to flower visiting bumblebees, using outdoor mesocosms. Flowers and leaves of clothianidin-treated plants had different profiles of glucosinolates compared with untreated plants. Bumblebees in mesocosms foraged slightly more on untreated plants. Neither flower timing, flower size nor the production of pollen and nectar differed between treatments, and therefore cannot explain any preference for untreated oilseed rape. We instead propose that this small but significant preference for untreated plants was related to the altered glucosinolate profile caused by clothianidin. Thereby, this study contributes to the understanding of the complex relationships between neonicotinoid-treated crops and pollinator foraging choices, by suggesting a potential mechanistic link by which insecticide treatment can affect insect behavior.

Proposal for field sampling of plants and processing in the lab for environmental metabolic fingerprinting.

BACKGROUND: Samples for plant metabolic fingerprinting are prepared generally by metabolism quenching, grinding of plant material and extraction of metabolites in solvents. Further concentration and derivatisation steps follow in dependence of the sample nature and the available analytical platform. For plant material sampled in the field, several methods are not applicable, such as, e.g., collection in liquid nitrogen. Therefore, a protocol was established for sample pre-treatment, grinding, extraction and storage, which can be used for analysis of field-collected plant material, which is further processed in the laboratory. Ribwort plantain (Plantago lanceolata L., Plantaginaceae) was used as model plant. The quality criteria for method suitability were high reproducibility, extraction efficiency and handling comfort of each subsequent processing step. RESULTS: Highest reproducibility of results was achieved by sampling fresh plant material in a solvent mixture of methanol:dichloromethane (2:1), crushing the tissue with a hand-held disperser and storing the material until further processing. In the laboratory the material was extracted threefold at different pH. The gained extracts were separated with water (2:1:1 methanol:dichloromethane:water) and the aqueous phases used for analysis by LC-MS, because the polar metabolites were in focus. Chromatograms were compared by calculating a value Xi for similarities. Advantages and disadvantages of different sample pre-treatment methods, use of solvents and solvent mixtures, influence of pH, extraction frequency and duration, and storing temperature are discussed with regard to the quality criteria. CONCLUSIONS: The proposed extraction protocol leads to highly reproducible metabolic fingerprints and allows optimal handling of field-collected plant material and further processing in the laboratory, which is demonstrated for an exemplary field data-set. Calculation of Xi values is a useful tool to judge similarities between chromatograms.

Iridoid biosynthesis in Chrysomelina larvae: Fat body produces early terpenoid precursors.

Larvae of the Chrysomelina species Phaedon cochleariae and Gastrophysa viridula produce monoterpenoids (iridoids) to defend themselves against predatory attacks by presenting the toxins upon attack as droplets on the top of nine pairs of dorsal glands. Although the conversion of 8-hydroxygeraniol-8-O-beta-d-glucoside into the iridoids in the glandular reservoir has been studied in detail, the synthesis of the glucosidically bound precursor received only limited attention. We compared larvae of the two iridoid producing species with those of Chrysomela populi, a sequestering species producing salicylaldehyde, in terms of the key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and isoprenyl diphosphate synthases involved in the biosynthesis of the iridoid precursor. Increased HMGR transcript abundance, high HMGR activity and accumulation of geraniol indicating geranyl diphosphate synthase activity was observed only in the fat body of the iridoid producing larvae in comparison to other larval tissues and to the tested tissues of C. populi. These results correlate with the identification of glucosidically bound 8-hydroxygeraniol in the fat body of the iridoid producers. We suggest that in P. cochleariae and G. viridula glucosidically bound 8-hydroxygeraniol is produced by the fat body and transferred via the hemolymph into the glandular reservoir for further conversion into iridoids.

Sequestration of plant-derived phenolglucosides by larvae of the leaf beetle Chrysomela lapponica: thioglucosides as mechanistic probes.

Feeding larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) acquire characteristic O-glucosides from the leaves of their food plants. The glucosides are selectively channeled from the gut to the defensive gland. Subsequent enzymatic transformations generate a blend of different defensive compounds, e.g., salicylaldehyde and two series of 2-methylbutyl and isobutyryl esters. By using systematically modified and hydrolysis-resistant thioglucosides as structural mimics of the plant-derived glucosides, e.g., salicin and its o-, m-, and p-isomers 1, 2, and 3; o-, m-, and p-cresols 5, 6, 7; along with thioglucosides of 2-phenylethanol 9 and (3Z)-hexenol 10, we demonstrated that the larvae of C. lapponica are able to sequester a broad range of structurally different thioglucosides with comparable efficiency. This sharply contrasts with the sequestration habitus previously observed in Chrysomela populi and Phratora vitellinae, which secrete almost pure salicylaldehyde and posses a highly specific transport mechanism for salicin (Kuhn et al., Proc. Natl. Acad. Sci. USA 101:13808-13813, 2004). Also, neither C. lapponica nor C. populi sequester in their gland the thioglucoside of 8-hydroxygeraniol, the mimic of the glucoside specifically transported by larvae secreting iridoid monoterpenes (Phaedon cochleariae, Gastrophysa viridula). Accordingly, leaf beetle larvae possess selective membrane carriers in their gut and their defensive systems that match the orientation of the functional groups of glucosides from their food plants probably by embedding the substrate in a network of hydrogen bonds inside the membrane carriers. The synthesis and the spectroscopic properties of the test compounds along with a comparative evaluation of the transport capabilities of larvae of C. populi and C. lapponica are described.

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

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.