Digital imaging system

To analyze the content of MHC I, MHC IIa, IIb, IId/x isoforms in the extracts, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed [19 (link)]. Samples were prepared within a dilution buffer (5% β-MEtOH, 5% SDS, 20% glycerol, 125 mM Tris, pH 6.8, and 0.1% bromophenol blue) and 2 μg of myofibrillar protein was loaded. Gels run at constant voltage (70 V) for 24 h at 4 °C. All gels were stained with Coomassie R250 and scanned using a Canon digital imaging system. The density of bands was estimated using the UN-Scan-IT software and the percentages of the MHC isoforms were determined by the software from the height (peak intensity) and area of the curve, with area boundaries determined manually.

The right forewing of every specimen was dissected and mounted in a water droplet between microscopic slides. In order to obtain repeatable pictures of wings that were as coplanar as possible, we removed the thick part at the base of the wings beforehand. Pictures were taken with a Visionary Digital imaging system, using Infinity lenses and a Canon 60D camera with a constant magnification.
Forewing size and shapes were assessed using 19 2D landmarks recorded with TPSDig2 software [30 ] (Fig 1). Landmarks of all specimens were superimposed once, using a generalized Procrustes analysis (GPA; [31 ]). This analysis extracts information of location, orientation and size from the raw landmark coordinates measured on the pictures in order to retain only the geometric shapes. Aligned landmark coordinates were then projected into the linear tangent space [32 ]. Shape variables were assessed as the scores of the 34 Principal Components (PC) with eigenvalues greater than zero from a Principal Component Analysis (PCA) on these tangent coordinates. In this multidimensional space, each point represented a potential wing shape and each vector depicted a shape change.
The forewing size was estimated by the log-transformed centroid size from the Procrustes analysis [33 ].