A precision apparatus, with solid phase micro-extraction monitoring capability, for incorporation studies of gaseous precursors into insect-derived metabolites

Abstract

An apparatus is described that facilitates the determination of incorporation levels of isotopelabelled, gaseous precursors into volatile insect-derived metabolites. Atmospheres of varying gas compositions can be generated by evacuation of a working chamber followed by admission of the required levels of component gases, using a precision, digitised pressure read-out system. Insects such as fruit-flies are located initially in a small introduction chamber, from which migration can occur downwards into the working chamber. The level of incorporation of labelled precursors is continuously assayed by the Solid Phase Micro Extraction (SPME) technique and GC-MS analyses. Experiments with both Bactrocera species (fruit-flies) and a parasitoid wasp, Megarhyssa nortoni nortoni (Cresson) and 18 O-labelled dioxygen illustrate the utility of this system. The isotope effects of 18 O on the carbon-13 NMR spectra of 1,7-dioxaspiro[5,5]undecane are also described.

Biosynthesis of insect spiroacetals

“…For studies of 18 O incorporation from dioxygen, a more elaborate system was developed and its components and operation, again using SPME-GCMS methods, have been illustrated and described elsewhere 38 and readers are directed there for detailed descriptions of its operation.…”

“…22,50 The major components of the volatile emissions were found to be N-(3-methylbutyl)propanamide (37) and (E,E)-2,8-dimethyl-1,7dioxaspiro [5.5]undecane (4). Another amide, N-(3-methylbutyl)acetamide (38), the even carbon-numbered spiroacetal (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane (6), and (E,E)-2-methyl-8-n-propyl-1,7-dioxaspiro [5.5]undecane (25) were present at low levels, with the relative intensities of amides to spiroacetals being dependent on the sampling conditions. Enantioselective GCMS analysis of a pentane extract of whole, crushed female abdomens, revealed a suite of spiroacetals incorporating from nine to thirteen carbons and including certain unusual, even carbon numbered spiroacetals (Fig.…”

“…S. noctilo infests and causes damage to commercial pine forests and so M. nortoni has been used as a biological control agent. 94 A coloured photograph of this spectacular wasp is contained in the work of Fletcher et al 38 Davies and Madden 76 reported in 1985 that the mandibular gland secretion of M. nortoni contained a number of stereoisomeric spiroacetals of the 2,8-dimethyl-1,7-dioxaspiro-[5.5]undecane (4) and 2-ethyl-7-methyl-1,6-dioxaspiro [4.5]decane (23) systems as well as the corresponding dihydropyran ((2S)-36), undecan-2-one (74) and 2-undecanol (75) (Fig. 23).…”

“…Very careful further examination revealed traces of the isomeric dihydropyran 131, and 2,6-undecanediol (81). 75 Determination of oxygen atom incorporation from [ 18 O 2 ]dioxygen into this compound suite was conducted with the apparatus already described, 38 and a single wasp provided sufficient volatiles for successful SPME-coupled GCMS analyses. 50 The size of M. nortoni meant that a partial evacuation/ refill strategy was adopted that minimised trauma to the wasp, and generated an atmosphere that was ca.…”

Spiroacetal biosynthesis in fruit flies is complex: distinguishable origins of the same major spiroacetal released by different Bactrocera spp.

“…7 Recently, an essentially complete biosynthetic scheme was proposed for a (Hymenoteran) Giant Ichneumon wasp Megarhyssa nortoni, which also produces (E,E)-1 as a major volatile, on the basis of incorporation studies with labelled precursors (including oxygen mapping) and intermediate identification. 8 In M. nortoni, b-oxidation and subsequent decarboxylation of a b-keto fatty acid led to 2-undecanone (9), which yields 4 via P450 mediated hydroxylation at an unactivated C-H bond. Following oxidation/reduction, the resulting alkyltetrahydropyranol (cyclised 2), characteristic of Bactrocera spiroacetal biosynthesis, undergoes hydroxylation, with oxygen incorporation from [ 18 O 2 ]-dioxygen, and cyclisation to yield (E,E)-1.…”