Elemental and compositional analyses

Using an EDS apparatus mounted in an SEM (EDAX Inc., Mahwah, NJ, USA), the chemical elements in the SLP cuticles were assessed in the same six samples used for the SEM observations. In the compositional analysis, six fresh SLP samples were resected from the locust femur under a biological dissection microscope (XTL-165-VT, Phenix Ltd., Jiangxi, China) and prepared by drying to a powder and mixing with KBr. Then, the SLP samples were tested in a vacuum (vacuum pressure: ~100 Pa) using FTIR (VERTEX 70V, Bruker Inc., Karlsruhe, Germany). In addition, six cuticle specimens from the distal-dorsal part of the same six locust femurs were tested to determine differences between the compositions of the SLP cuticles and the distal-dorsal cuticles of the femur.

All the preparation and measurement methods of CLSM were the same as those in the literature^20,21 and are briefly described as follows. Before the CLSM measurement, six fresh SLP samples were resected along the middle cross-section (indicated by the red line in Fig. 1b) using a razor blade and were immersed in glycerine (≥99.0%; Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). Then, the glycerine-coated SLP cross-sections were placed on a cover slip (thickness = 0.16 ± 0.01 mm; Citotest Labware Manufacturing Co., Ltd, Jiangsu, China) with additional glycerine around them. A CLSM instrument (Zeiss LSM 710META, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) was used for observing the autofluorescences of the specimens under the excitations of 405 nm, 488 nm, 555 nm, and 639 nm laser lines. The transmitting light wavelengths of the emission filter were 420–480 nm (bandpass), ≥490 nm (longpass), ≥560 nm (longpass), and ≥640 nm (longpass), respectively. All the different fluorescences were excited and detected sequentially for avoiding possible interference between the different fluorescences. Laser power values between 3% and 50% were selected, depending on the intensity of the respective autofluorescence. The digital gain and offset values were defined as the default values of the CLSM software ZEN (i.e., 1 and 0, respectively). The pinhole size for each autofluorescence was adjusted slightly by approximately one Airy value to achieve identical thickness of the respective optical sections. The line average of all image stacks was set to 2. According to the rough cross-section of the specimens, the entire thickness of the specimens for the CLSM visualization was set between 100 μm and 200 μm to obtain the whole cross-sectional view of both the SLP and the cover plate. Both the slice thickness and interval values were determined automatically by the ZEN software. Maximum intensity projections were performed for all image stacks using the software. Pseudo-colors of the four different autofluorescences were chosen for the final micrographs as follows: blue (excitation = 405 nm and emission = 420–480 nm), green (excitation = 488 nm and emission ≥490 nm), and red (for both: excitation = 555 nm and emission ≥560 nm, excitation = 639 nm and emission ≥640 nm). To rule out the effect of bleaching on the autofluorescence results, all the micrographs were obtained from the first CLSM visualization procedure of the specimens.