Flat does not mean 2D: Using X‐ray microtomography to study insect wings in 3D as a model for comparative studies

Abstract

Abstract While not generally used for relatively flat structures, X‐ray microtomography can provide decisive insights for comparative anatomy and evolutionary studies of flat structures, thereby solving long‐standing ecological and evolutionary issues. This is particularly true for the study of insect wings, organs that have played a central role in the evolution and ecological success of insects, the first organisms to develop powered flight. We present the challenges inherent in studying insect wings by microtomography, as well as solutions to overcome them. We highlight the impact of insect preparation (i.e. dry or ethanol‐preserved specimens, complete specimens or only part of the wings, wings stained with iodine or with a 0.3% solution of phosphotungtic acid—PTA) and of the acquisition mode (phase contrast or not), on scan quality and ease of interpretation. We compare our results with those obtained by more ‘traditional’ techniques, based on examination of the wings with a binocular or SEM microscope. We show that X‐ray microtomography is effective in establishing vein homologies—a crucial step in many evolutionary studies in insects—by accessing the internal structures of the wing base itself, where veins originate. It can also be used for difficult tasks such as distinguishing between crossveins and main veins, establishing vein polarity (i.e. concave vs. convex veins) or vein fusions, situations that complicate the establishment of vein homology hypotheses and the completion of subsequent evolutionary studies. Unstained, ethanol‐preserved specimens give the least conclusive results, while dry specimens and ethanol‐preserved wings stained with PTA give the best results. We show that 3D tools provide crucial insights for the study of insect wings, illustrating how CT‐scan tools may contribute to a change in dimension, introducing quantification and functional approaches and hopefully stimulating new studies for other seemingly 2D organs in several other fields, including plant studies. We highlight, nonetheless, that this tool must be used in coordination with more traditional observational techniques, at least for insect wings, to provide a more complete picture of these fundamental organs.