Xradia xct 200

After fixation (see protocol above), a female opisthosoma with plugged genital openings and three male pedipalps were prepared for an X-ray microscopy scan (Xradia XCT-200, Carl Zeiss Microscopy GmbH) in the following way: after dehydration, samples were stained overnight using 1% iodine solution (in pure ethanol). After washing in pure ethanol, samples were critical point dried (BalTec 30) and subsequently mounted on insect pins using super glue. Scans were then obtained using the 40x objective lens unit with the following scan parameters: 40 kV, 8 W, 200 μA, exposure time 30 sec/frame. Reconstructed image stacks were created using XMReconstructor software (Carl Zeiss Microscopy GmbH) and the subsequent segmentation (delineation) of the structures of interest in the male and female genitalia was performed with Amira 5.4.5 (Visualization Science Group, FEI). Measurements were obtained using the 3D length and volume measurement tool in Amira 5.4.5 (Visualization Science Group, FEI) and transformed to actual sizes based on the pixel to μm ratio.

To illustrate and compare the spermathecal morphology between the female developmental stages in situ, opisthosomata of one early-subadult female, five late-subadult females and four adult females were fixed in Duboscq-Brasil [43 ] and kept in 80% ethanol. After dehydration, samples were contrasted overnight using a 1% iodine solution (in pure ethanol). After washing in 80, 90 and 100% ethanol, the samples were critical point dried with a BAL-TEC CPD 030 and mounted on insect pins using super glue. Scans were performed in an Xradia XCT-200 (Carl Zeiss Microscopy GmbH) using the 4x and 10x objective lens unit with the following scan parameters: 40 kV, 8 W, 200 μA, exposure time 7 s/frame. Reconstructed image stacks were created using XMReconstructor software (Carl Zeiss Microscopy GmbH). Data were visualized and processed using the 3D analysis software AMIRA 5.6 (Visualization Science Group, FEI).

X-ray micro-computed tomography (µCT) was performed at the Imaging Center of the Department of Biology, University of Greifswald, using a XRadia XCT-200 (Carl Zeiss Microscopy GmbH, Jena, Germany). X-ray source settings were 40 kV and 8 W with a source-to-sample distance of 30 mm, sample-to-detector distance of 120 mm. The XRadia XCT-200 is equipped with switchable scintillator-objective lens units, in this case the 0.39×objective was used. 1600 projections with 1.25 s exposure time were recorded with binning 2, resulting in images of 1024 × 1024 px, with a system-based calculated pixel size of 13.49 µm. Tomographic reconstruction was performed (binning 1) using XMReconstructor (Carl Zeiss Microscopy GmbH, Jena, Germany), resulting in image stacks (TIFF format). Tiff image stacks were further post-processed in Fiji (Schindelin et al., 2012 (link)).