Structural and biochemical analysis of the Leptinotarsa decemlineata (Coleoptera; Chrysomeloidea) crystalline chorionic layer.

The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.