Solid Phase Micro-extraction (SPME) with In Situ Transesterification: An Easy Method for the Detection of Non-volatile Fatty Acid Derivatives on the Insect Cuticle

Abstract

Triacylglycerides (TAGs) and other non-volatile fatty acid derivatives (NFADs) occur in large amounts in the internal tissues of insects, but their presence on the insect cuticle is controversially discussed. Most studies investigating cuticular lipids of insects involve solvent extraction, which implies the risk of extracting lipids from internal tissues. Here, we present a new method that overcomes this problem. The method employs solid phase micro-extraction (SPME) to sample NFADs by rubbing the SPME fiber over the insect cuticle. Subsequently, the sampled NFADs are transesterified in situ with trimethyl sulfonium hydroxide (TMSH) into more volatile fatty acid methyl esters (FAMEs), which can be analyzed by standard GC/MS. We performed two types of control experiments to enable significant conclusions: (1) to rule out contamination of the GC/MS system with NFADs, and (2) to exclude the presence of free fatty acids on the insect cuticle, which would also furnish FAMEs after TMSH treatment, and thus might simulate the presence of NFADs. In combination with these two essential control experiments, the described SPME technique can be used to detect TAGs and/or other NFADs on the insect cuticle. We analyzed six insect species from four insect orders with our method and compared the results with conventional solvent extraction followed by ex situ transesterification. Several fatty acids typically found as constituents of TAGs were detected by the SPME method on the cuticle of all species analyzed. A comparison of the two methods revealed differences in the fatty acid compositions of the samples. Saturated fatty acids showed by trend higher relative abundances when sampled with the SPME method, while several minor FAMEs were detected only in the solvent extracts. Our study suggests that TAGs and maybe other NFADs are far more common on the insect cuticle than usually thought.

The chemical basis of mate recognition in two parasitoid wasp species of the genus Nasonia

“…TAGs have been found to play an essential role in mate recognition in the parasitoid wasp Lariophagus distinguendus (Förster) (Kühbandner et al., ), and act as a brood pheromone in drone brood of the honey bee Apis mellifera L. (Koeniger & Veith, ). As they are not detected by standard GC‐MS methods without transesterification into fatty acid methyl esters (Kühbandner & Ruther, ), NFADs were not detectable in the chemical analysis of cuticular lipids in this study.…”

“…Previous studies on the chemical basis of mate recognition in N. giraulti did not differentiate clearly between complete cuticular lipid extracts and fractions of extracts gained from purification procedures (Buellesbach et al., ; Giesbers et al., ). Irrespective of the solvent used for extractions, complete cuticular lipid extracts may contain more polar lipids, such as free fatty acids, alcohols, glycerides, sterols, aldehydes, or ketones, in addition to the non‐polar lipids, i.e., CHCs (Lockey, ; Buckner, ; Kühbandner & Ruther, ). Furthermore, polar cuticular lipids have been shown to function as pheromones in various insects (Buckner, ; Yew et al., ; Kühbandner et al., ).…”

Application of headspace solid‐phase microextraction followed by gas chromatography coupled with mass spectrometry to determine esters of carboxylic acids and other volatile compounds inDermestes maculatusandDermestes aterlipids

“…An equally good technique for analyzing the surface compounds is SPME. For example, SPME‐GC/MS was used to identify Bagrada hilaris cuticular hydrocarbons (De Pasquale, Guarino, Peri, Alonzo, & Colazza, ), Neoclytus acuminatus aggregation pheromones (Lacey et al, ) and Lariophagus distinguendus nonvolatile fatty acids (Kühbandner & Ruther, ). The influence of our extraction method on the extractability of insect lipids can seen by comparing the results obtained in our work with the results presented, for example, in Buszewska‐Forajta et al ().…”

Chemical Ecology of the Parasitoid Wasp Genus Nasonia (Hymenoptera, Pteromalidae)

“…CHCs are the most abundant CLs found on the insects' cuticular surface and are easily detectable and identifiable by gas chromatography and mass spectrometry (GC/MS). Other CLs are the more polar lipids such as aldehydes, alcohols, ketones, wax esters, or non-volatile fatty acid derivatives (NFADs) some of which are not detected by standard GC/MS methods without prior derivatization (Buckner, 1993;Kühbandner and Ruther, 2015). As a result, they are often neglected in studies investigating mate recognition in insects and the term CHCs is often erroneously used interchangeably with CLs.…”