Antifeedants and Feeding Stimulants in Bark Extracts of Ten Woody Non-host Species of the Pine Weevil, Hylobius abietis

Bark of ten woody species, known to be rejected as a food source by the pine weevil, Hylobius abietis, were sequentially extracted by a Soxhlet apparatus with pentane followed by methanol. Species were alder (Alnus glutinosa), aspen (Populus tremula), beech (Fagus sylvatica), guelder rose (Viburnum opulus), holly (Ilex aquifolium), horse chestnut (Aesculus hippocastanum), lilac (Syringa vulgaris), spindle tree (Evonymus europaeus), walnut (Juglans regia), and yew (Taxus baccata). Bark of each species was collected in southern Scandinavia during the summer. Resulting extracts were tested for antifeedant activity against the pine weevil by a micro-feeding choice assay. At a dose corresponding to that in the bark, methanol extracts from Aesculus, Taxus, Ilex, and Populus were antifeedant active, while pentane extracts of Aesculus, Fagus, Syringa, and Viburnum were stimulatory. Four known antifeedants against H. abietis, the straight-chained carboxylic acids, hexanoic and nonanoic acid (C6 and C9), carvone, and carvacrol were identified by gas chromatography (GC)-mass spectrometry (MS) in several extracts. The major constituents were identified and tested for feeding deterrence. The aromatic compounds benzyl alcohol and 2-phenylethanol are new non-host plant-derived feeding deterrents for the pine weevil. Additionally, two feeding stimulants, beta-sitosterol and 5-(hydroxymethyl)-2-furaldehyde, were identified. One active methanol extract of Aesculus bark was sequentially fractionated by liquid chromatography, and major compounds were tentatively identified as branched alcohols and esters of hexanoic acid. Five commercially available hexanoate esters and two commercially available branched alcohols were identified as new active antifeedants. Both stimulatory and inhibiting compounds were found in the same extracts and co-eluted in the same or adjacent fractions. The mix of semiochemicals of opposite activity in each extract or fraction could explain the stimulatory-, inhibitory-, or sometimes neutral activity. Generally, such co-occurrence confounds the isolation of antifeedants.