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Complex metabolism of aromatic glucosinolates in Pieris rapae caterpillars involving nitrile formation, hydroxylation, demethylation, sulfation, and host plant dependent carboxylic acid formation

Agerbirk, Niels, Olsen, Carl Erik, Poulsen, Eva, Jacobsen, Niels, Hansen, Paul Robert
Insect biochemistry and molecular biology 2010 v.40 no.2 pp. 126-137
natural toxicants, hydrolases, metabolites, Pieris rapae, plant pests, frass, leaves, glucosinolates, insect pests, hydroxylation, larvae, enzyme activity, chemical structure, excretion, carboxylic acids, nitriles, laboratory techniques, metabolic detoxification, chemical constituents of plants, Arabis hirsuta, phenolic compounds
We investigated the metabolism of two chain elongated phenolic glucosinolates and the corresponding O-methyl derivatives upon ingestion by caterpillars of the butterfly Pieris rapae (L.). The glucosinolates (GSLs) were 4-hydroxyphenethylGSL, (R)-2-hydroxy-2-(4-hydroxyphenyl)ethylGSL, 4-methoxyphenethylGSL, and (R)-2-hydroxy-2-(4-methoxyphenyl)ethylGSL, variously occurring in foliage of two Arabis species: Arabis hirsuta (L.) Scop. and Arabis soyeri Reut. & Huet subsp. subcoriacea (Gren. ex Nyman) Breitstr. (Brassicaceae). Frass from caterpillars reared on each Arabis species contained two sulfated nitriles (4-sulfates of 3-(4-hydroxyphenyl)propanenitrile and 3-hydroxy-3-(4-hydroxyphenyl)propanenitrile) as apparent GSL metabolites. Comparison of glucosinolate levels in foliage and levels of sulfated nitriles in frass, and experiments with isolated GSLs spiked to crucifer foliage and ingested by P. rapae, demonstrated that phenolic GSLs and the corresponding O-methyl derivatives were metabolised to sulfated nitriles, and that metabolites lacking a β-hydroxy group were partially hydroxylated in this position during metabolism in P. rapae. In contrast, an induction experiment did not show increased levels of β-hydroxylated GSLs in A. soyeri plants upon caterpillar feeding. Frass contents of other putative GSL metabolites from the interaction with the two Arabis species differed significantly; caterpillars reared on A. hirsuta excreted significant amounts of four carboxylic acids (3-(4-hydroxyphenyl)propanoic acid, 3-hydroxy-3-(4-hydroxyphenyl)propanoic acid, and the corresponding 4-sulfates), which were low or absent when the caterpillars were reared on A. soyeri. The excreted carboxylic acids could be formed by hydrolysis of nitriles to carboxylic acids in caterpillar guts by an ingested nitrilase enzyme from A. hirsuta foliage; this hypothesis was supported by demonstration of 3-(4-hydroxyphenyl)propanenitrile hydrolysing nitrilase activity (E.C. 3.5.5.x) in a crude A. hirsuta extract. Some hypothetic metabolites, glycine conjugates of phenolic carboxylic acids, were not detected. Conditions for group separation and HPLC isolation of intact GSLs and sulfated metabolites were optimised, NMR spectroscopic data of the compounds are reported, and evolutionary and ecological implications are discussed.