Iodine

Fixed specimens were dehydrated via an ascending ethanol series, incubated in solution of 2% iodine (resublimated; Carl RothGmbH&Co. KG, Karlsruhe, Germany; cat. #X864.1) in 99.5% ethanol for ∼48 h at ambient temperature, briefly rinsed in 99.5% ethanol (3–4× 10 min) and either transferred into a vial in ethanol (wet scan) or critical point-dried with a Leica EM CPD300 and glued with the posterior body pole on plastic welding rods (dry scan). Scans were performed with an Xradia MicroXCT-200 (Carl Zeiss Microscopy GmbH). Scan settings were individually optimized for each specimen, including objective choice (0.39×, 4×, 10×) according to size. Scans were performed under 40 kV/200 µA/8W, exposure times ranged from 0.4 to 1.5 s. Tomography projections were reconstructed using the XMReconstructor software (Carl Zeiss Microscopy GmbH) with TIFF format image stacks as output. All scans were performed with Binning 2 to reduce noise and subsequently reconstructed with Binning 1 (=full resolution) to avoid information loss. Processing and 3D visualization of the image stacks (including highlighting of cephalic appendages and removal of nontarget structures) were performed with Imaris (version 7.0.0., Bitplane AG, Switzerland) as described previously (Scholtz and Brenneis 2016 ).