Xradia micro xct 200

One female and one male adult specimen of H. subterraneus were anesthetized and fixed in Bouin’s solution overnight. Preparations were washed in 70% ethanol, dehydrated in a graded ethanol series, and incubated in a 1% iodine solution (iodine resublimated in 99% ethanol; Carl Roth #X864.1) for 12 h. Preparations were washed several times in pure ethanol and critical-point-dried. Finally, samples were fixed on insect pins with super glue. Scans were performed with a Zeiss Xradia MicroXCT-200 (Imaging Center of the Department of Biology, University of Greifswald) at 30 kV, 6 W, and 4 s exposure time resulting in a pixel size of ca. 0.96 μm (male specimen) and 40 kV, 8 W, and 1 s exposure time resulting in a pixel size of ca. 0.95 μm (female specimen). Tomography projections were reconstructed using the XMReconstructor software (Zeiss Microscopy) resulting in images stacks (TIFF format). All scans were performed by using Binning 2 (resulting in noise reduction) and subsequently reconstructed by using Binning 1 (full resolution) to avoid information loss. Image stacks were further processed using Amira 6.4 (Thermo Fischer) and Drishti 2.4 [81 ].

After anesthetization, two adult specimens were fixed in Bouin’s solution overnight. The subsequent preparation followed the protocol by Sombke et al. [33 (link)]. Preparations were rinsed in several changes of PBS (phosphate buffered saline, 0.1 M, pH 7.4), dehydrated in a graded ethanol series and incubated in a 1% iodine solution (iodine resublimated in 99% ethanol; Carl Roth #X864.1) for 12 h. Preparations were rinsed several times in pure ethanol and critical-point-dried. Finally, samples were fixed on insect pins with super glue. Scans were performed with a Zeiss Xradia MicroXCT-200. Dissected walking legs (pair 10) were scanned with a 10× objective lens resulting in a 2.19 μm pixel size; ultimate legs were scanned with a 4× objective lens unit resulting in 5.05 μm pixel size. Dissected ventral nerve cord ganglia associated with walking legs were scanned with a 20x objective lens resulting in a 0.93 μm pixel size (compare also Schendel et al. [24 ]). The ultimate leg associated ganglion 15 was scanned using a 20× objective lens resulting in a 1.09 μm pixel size. Tomography projections were reconstructed using the XMReconstructor software (Zeiss Microscopy) resulting in image stacks (TIFF format). All scans were performed using binning 2 (resulting in noise reduction) and subsequently reconstructed using binning 1 (full resolution) to avoid information loss.

Micro-CT scans were performed using an X-ray microscope (Xradia MicroXCT-200; Carl Zeiss Microscopy GmbH, Jena, Germany) that uses a 90-kV/8 W tungsten X-ray source and switchable scintillator-objective lens units as described by Sombke et al. (2015) (link). The heads of fixed animals (Bouin; two specimens) were contrasted in iodine solution (2% iodine resublimated (Cat. #X864.1; Carl Roth GmbH, Karlsruhe, Germany) in 99.5% ethanol), critical point-dried using a fully automatic critical point dryer Leica EM CPD300 (Leica Microsystems, Wetzlar, Germany) and scanned dry (scan medium air). Tomography projections were reconstructed using the reconstruction software XMReconstructor (Carl Zeiss Microscopy GmbH, Jena, Germany), resulting in image stacks (DICOM format) with a pixel size of about 5.8 µm for the 4 × objective and 1.9 µm for the 10 × objective.