Spatial and chemical heterogeneity of insect cuticular hydrocarbon layers: insights from extraction methods and acclimatization regimes

Abstract Insect cuticles are covered by a complex mixture of cuticular hydrocarbons (CHCs) that are essential for desiccation resistance and chemical communication, yet the physical structure of CHC layers remains poorly understood. We extracted CHCs from five insect species (desert locusts, house crickets, American cockroaches and two ant species) using solvents of differing polarity and extraction durations, and from insects reared under different temperature regimes. Extractions with n-hexane were enriched in relatively solid CHCs (n-alkanes and terminally branched monomethylalkanes), while extractions with dichloromethane and dichloromethane–methanol yielded proportionally more fluid compounds (internally branched monomethyl and dimethylalkanes, unsaturated hydrocarbons). Solid-phase CHCs were proportionally more abundant in shorter extractions, whereas fluid-phase compounds were proportionally more abundant in prolonged or sequential extractions. These patterns are consistent with a model of cohesive solid domains surrounded by more accessible fluid regions, though this interpretation remains indirect and alternative explanations (e.g. differential solubility and diffusion kinetics) cannot be excluded. CHCs of similar phase behaviour covaried across treatments, indicating that they aggregate within shared domains. Warm acclimatization increased the proportion of solid compounds, whereas fluctuating temperatures enhanced CHCs with intermediate phase behaviour. Our results suggest spatial and chemical heterogeneity in the CHC layer, with patterns of differential extractability consistent with clustering into phase-distinct domains, though direct imaging is required to confirm spatial topology. This potential heterogeneity may facilitate CHC multi-functionality, with the phase behaviour and domain membership of each CHC depending on its biophysical properties relative to the individual's overall CHC profile.