Neglected very-long-chain hydrocarbons and the incorporation of body surface areas reveal novel perspectives for chemical profile analysis in insects

Most of what we know about insect cuticular hydrocarbons (CHCs) has been informed by analyses with gas-chromatography coupled with mass spectrometry (GC-MS). However, this analysis technique has its limit, particularly in detecting compounds in a chain length range beyond around C40. Here, we attempt to differentiate the range of CHCs detectable by GC-MS with the largely neglected very-long-chain CHC range up to C58 analyzed by silver-assisted laser desorption/ionization mass spectrometry (Ag-LDI-MS), in seven species of the order Blattodea. For all tested species we unveiled a considerable range of very-long-chain CHCs not detectable by traditional GC-MS technology. This strongly suggests that general studies on insect CHCs routinely miss compounds in that range, and we encourage future studies to implement technologies capable of detecting very-long-chain CHCs. Furthermore, we implement 3D scanned insect body surface areas when quantifying CHC amounts per GC-MS, as Ag-LDI-MS is less suited to quantify total CHC contents. Proportional CHC distributions differed considerably when adjusted for body surface areas, suggesting the importance of their consideration. Moreover, correlations between body surface areas and GC-MS assessed CHC amounts were not significant, further hinting the inaccessibility of whole CHC profile ranges by this technique, potentially by missing the clearly present very-long-chain CHCs