Axiocam icc5

We fixed and stored the specimens in absolute ethanol and mounted the dissected appendages in a neutral balsam mounting medium. All studied specimens are deposited in the Insect Museum, Jiangxi Agricultural University, Nanchang, China (JXAUM).
Photographs were taken with a digital camera Zeiss AxioCam Icc 5 attached to a digital microscope Zeiss Stereo Discovery V12. Line drawings were made by the GNU Image Manipulation Program [36 ]. Species identifications were based on traditional morphological criteria. Terminology of morphology follows Schmidt [37 ].
For morphometric analysis, we selected 42 specimens representing seven sky-island Ligidium species of southwest China that were revealed by molecular delimitation. To evaluate variation in body shape, 16 landmarks were placed on pereonites 1–7 (Fig. 1a). The images were converted into TPS format using tpsUtil 1.56 [38 ]. Landmarks for all images were aligned, rotated and scaled using Procrustes superimposition [39 (link)]. Afterwards, we recorded the landmarks using TPSdig 2.17 [40 ] and analysed the data with MorphoJ [41 (link)]. To visualize shape variations across morphospace, we conducted principal component analyses (PCAs) and canonical variate analyses (CVAs). Mahalanobis and Procrustes distances were applied to evaluate the morphological variations among species.

After 5mC immunodetection, slides were analyzed using a Carl Zeiss Imager Z2 fluorescence microscope with fluorescent lighting HSP, 120 W. The images were recorded with an AxioCam ICc5 digital camera and immersion lens with a ×100 magnification. The specific location of the cell nuclei with the micronuclei was also recorded. After subsequent FISH, the same slides were analyzed using the same equipment. The specific locations for each previously recorded cell nucleus and micronucleus were found after FISH and hybridization signals were captured. The frequencies of MN with specific signals and without signals were calculated. For the group treated with MH, a total of 350 nuclei with MN were evaluated. In order to analyze the presence of DNA methylation signals within the rDNA foci, 50 MN with rDNA signals were evaluated.

Myoblast migration was assessed by a wound healing assay, which was performed after reaching 80–100% confluency (day 4) in 6-well plates using duplicates from three independent cell cultures. Initially, the cell monolayers were mechanically scratched (“wound”) in the shape of a cross by a 200 µL sterile tip in the center of each well. The cell debris was removed by two washes with PBS, and the myoblasts were incubated in DMEM media with 1% antibiotics and 2% fetal bovine serum (Sigma-Aldrich, USA) to reduce the cell proliferation rate. The wound areas were evaluated at 0, 6, 12, 18, 24, 30 and 48 hours, and images were captured under an inverted microscope coupled to the digital camera AxioCam ICc5 (Zeiss, Germany). The wound areas corresponding to the area mean of the four lines in the cross-shaped wound were measured using ImageJ® software^{63 (link)}. The wound areas were used to calculate the reduction in the wound area size over time (wound closure) and the variation in wound closure over the time interval (migration rate or migration speed).