Cuticular Lipid Topology on Insect Body Surfaces Studied by Synchrotron Radiation FTIR ATR Microspectroscopy

The cuticular lipid covering the integument of insects is exposed to the environment and involved in a variety of functions offered by insect body surfaces, ranging from protection against the environment, such as the control of water transpiration, the reduction of abrasive damage, and the prevention of pathogen intrusion, to the communication between insects from intraspecific to interspecific interactions. In comparison with the importance of their physiological functions, there is remarkably little information on the structure and physical property of cuticular lipids on insect body surfaces. The lipid layer on the outer exoskeleton is very thin, estimated on the order of 0.01-1 μm or less, and this has led to a lack of practical methodologies for detailed structural analyses. To fill this devoid, we have exploited the characteristics of Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy, which allows us to conduct a chemical analysis on insect body surfaces and also to investigate depth-dependent structural changes. We have applied a combination of FTIR ATR microspectroscopy with IR radiation provided by a synchrotron facility to obtain in situ two-dimensional (2D) information of the cuticular lipid layer on the surface of the integument. The 2D FTIR spectra measured on the two-spotted cricket and the American cockroach show that the IR bands due to the cuticular lipid, such as CH₂ symmetric and antisymmetric stretch, ν_a(CH₂) and ν_s(CH₂), change in intensity significantly, depending on the location of measurements. As if to keep pace with this, the bands of the amide group for the underlying cuticular layer also change in intensity significantly, although the changes are in the opposite direction; as the lipid bands increase in intensity, the amide band decreases, and vice versa. The ATR spectral analysis, which takes into account the characteristics of the evanescent wave, points out that the lipid layer would vary tens of times in the range of 0.01-1 μm significantly. The ν_a(CH₂) and ν_s(CH₂) bands show frequency shifts, which correlate to some extent with their intensity changes, suggesting that the drastic uneven distribution of the cuticular lipid would be related to the solid-liquid phase separation and also the coarsening of the solid phase domains. The formation of such topological features, significant heterogeneity in the lipid layer thickness, and solid-liquid phase ratios would be accompanied by the partitioning of lipid components according to molecular structures and physicochemical properties. Considering that each lipid component in insect body surface lipids is involved in various physiological roles, the segregation of lipid components during the formation of such heterogeneous structures is thought to have a significant impact on the functionality of the insect body surface.