Cuticle thickness evaluation

On the third day post‐injection, six randomly selected M. mediator for each injected group were dissected and evaluated using histological analysis. In order to prepare samples for cryosectioning M. mediator larvae were rinsed with PBS and fixed with 4% paraformaldehyde overnight at 4 °C. Fixed samples were soaked with 30% sucrose as a cryoprotector and then frozen in tissue‐freezing medium. The whole body of M. mediator larvae was used for preparing sagittal cross‐sections with 7 µm thickness using cryomicrotome Leica CM1950 (Leica, Wetzlar, Germany). To estimate cuticle thickness, a series of sections were stained with chitin‐binding dye Calcofluor White M2R (Sigma‐Aldrich, St. Louis, MO, USA) and visualized using a LSM 780 Confocal Microscope (Zeiss, Dublin, CA, USA) (440 nm excitation wavelength and 500 nm wavelength emission parameters). We did not observe a statistically significant difference between the dorsal and ventral locations of the cuticle in M. mediator larvae. So, for further analysis, we randomly selected cuticle regions with the exceptions of the head and the last posterior abdominal segment. Digital images of six selected areas for every sample were recorded and used for manual morphometric measurements with ImageJ. All measurement sets were tested for normality using D'Agostino‐Pearson omnibus normality test and then compared with the Mann–Whitney test.