Heterodera glycines: eggshell ultrastructure and histochemical localization of chitinous components.

The eggshell in most nematodes consists of an outer vitelline layer, a middle chitinous and an inner lipid layer. Earlier work with eggs of Heterodera glycines suggests the presence of two chitinous layers but the vitelline layer was not observed. From our observation the outer chitin layer described in past literature is actually a vitelline layer. Histochemical analysis has demonstrated that chitin is absent from the outer envelope. Electron microscope observations of the eggshell show a waxy appearance and osmium staining consistent with that of the proteinaceous vitelline layer found in other nematodes. Lectin localization also shows that the eggshell continues to develop past fertilization with the delivery and integration of eggshell precursors. Contrary to previous reports, we propose that the ultrastructure of the eggshell H. glycines follows the common three-layer structure observed in other nematodes.

Heterodera glycines: density-based gradient separation of eggs.

An efficient technique was developed for separating early and late stages of embryonic development in eggs of Heterodera glycines. This technique takes advantage of density changes that occur during embryogenesis in the developing embryo and egg to partition the egg within a sucrose step gradient. Sorted samples of eggs separated with 82% enrichment for pre-gastrula early embryos and 93% enrichment for first and second stage unhatched juveniles as late embryos. Subpopulations enriched for either developmental stage are available for use in generating stage-specific cDNA libraries, normalization of subpopulations to synchronize development, biochemical characterization, and many other uses.