Die Embryonalentwicklung der Paradiesschnecke ... - TOBIAS-lib

Kapitel 4
72 or 96 hours at 6 ◦ C. The second incubation as well as all following steps
were performed in the dark. After incubation, the embryos were washed
4 times à 30 minutes in PBS and then transferred to ScaleB4 (8 M urea,
0.1% (wt/vol) Triton X and 10% (wt/vol) glycerol in double destilled water
(Hama et al., 2011) for at least a 5 weeks clearing of the samples at room
temperature. Before data analysis, the clearing solution was exchanged by
a mounting solution (ScaleB4 with 2.4 ml glycerol). The embryos were then
analyzed with a confocal laser scanning microscope (Leica TCS SPE, Leica
Microsystems, with Leica Application Suite - Advanced Fluorescence (LAS-
AF, Version 2.6.0.7266)) and evaluated with FIJI-ImageJ (fiji.sc). Image
editing was done in Gimp Image Editor and in Inkscape. Additionally, the
stained nervous structures were three-dimensionally modeled by using Amira
5.2.1.
Results
Histology and 3D-reconstruction of the anatomy of adults
Both the histological sections and the 3D models clearly showed how the lack
of an external mantle and shell influences the whole anatomy of a snail. In
Fig. 1A a sagittal section of the control, C1, is shown. The corresponding
“sluggish” snail, P1, is displayed in Fig. 1B. The older snail, P2, is shown
in Fig. 1C,D. The histological sections were the basis of the 3D models
and provided additional information that facilitated the interpretation of
the models. The anatomy of the specimens will be described using the 3Dreconstructions.
Fig. 2 shows complete 3D reconstructions of the three investigated snails.
The control individual, C1, is depicted in the upper row viewed from the left
(Fig. 2A) and from the right side (Fig. 2B). This individual represents a
“normally” developed snail, it will not be described here in detail but mainly
be used for comparisons. It is obvious that a large part of the snail’s body is
shaped by the shell (not shown in the reconstructions); especially the coiling
of the digestive gland is conspicuous. In contrast, the two sluggish snails in
113