Material and Methods
We investigated a single slab from lithographic limestones of Southern Germany (Solnhofen area, Upper Jurassic, Tithonian, southern Germany) found in the hobby quarry near Eichstätt with a single small fossil specimen. The fossil was formerly part of the private collection of Michael Fecke, Langenberg, now transferred to the State Museum of Natural History Stuttgart (SMNS 70409).
For comparison three modern larvae were documented: a thoracican lepadomorph nauplius of the group Lepadidae from the Museum National d’Histoire Naturelle, Paris (MNHN IU-2014-5478), a thoracican balanomorph nauplius (teaching collection LMU Munich), and a rhizocephalan nauplius, Peltogaster paguri, from the private collection of Jens T. Høeg in the Zoological Museum Copenhagen.
The fossil specimen was documented with macro-photography, stereo-photography and fluorescence micro-photography to extract as much information as possible from it. The lepadomorph nauplius was documented with macro-photography. The balanomorph nauplius was documented with fluorescence micro-photography. The rhizocephalan nauplius was documented with scanning electron microscopy.
Macro-photography and stereo-photography combined with composite imaging were performed (following e.g. Haug et al., 2012; 2013a), both under cross-polarized light. We used a Canon EOS Rebel T3i camera with Canon MP-E (65 mm) macro lens. Illumination was provided by the Canon Macro Twin Lite MT-24EX flash from two opposing sides. Fluorescence microscopy of the fossil was performed on an inverse fluorescence microscope BZ-9000 (BIOREVO, Keyence) with about 40 times magnification recording autofluorescence under blue light (GFP, 488 nm; for details on autofluorescence imaging, see Haug et al., 2011b). Fluorescence microscopy on the balanomorph nauplius was performed on a Zeiss AxioScope 2 with about 200 times magnification recording autofluorescence under UV light (DAPI, 358 nm). For macro-photography and micro-photography stacks of images (of different focal planes) were recorded to overcome limited depth of field. Adjacent stacks were recorded to overcome limitations in field of view. Scanning electron microscopy of the rhizocephalan nauplius was performed on a JEOL 6335-F scanning electron microscope at the Zoological Museum in Copenhagen.
Stacks of images were fused to sharp images using the freeware CombineZP. Resulting sharp images were stitched to panorama images using Adobe Photoshop CS3 or Elements 11. All images were optimized (sharpness, histogram, saturation) and dirt particles or background was removed manually using a lasso tool in Adobe Photoshop CS3.
Interpretations of structures are presented by color-marked versions of the images. Structures apparent in the fluorescence and stereo-photography were marked with the lasso tool in Adobe Photoshop CS3 on a desaturated half image of the stereo image (Haug et al., 2012).
A simplified reconstruction of the fossil was assembled by mirroring missing structures (Haug et al., 2015d). For explaining structures, a virtual 3D model of a modern cirripede nauplius was reconstructed in Blender 2.49 (Blender Foundation), based on drawings from Miller and Roughgarden (1994).