Three-dimensional visualization of insect morphology using confocal laser scanning microscopy.

Cuticular structures of insects are often microscopic and intricately complex; among the most complex structures are male genitalia. Genitalic structures are essential in taxonomic and phylogenetic studies of insects. Using well-described species from two disparate dipteran genera, we demonstrate the utility of confocal laser scanning microscopy for studying the morphological characters of fly genitalia by taking advantage of the autofluorescent properties of cuticle material. Reconstructions of confocal data sets obtained from genitalic structures embedded in two commonly used entomological mounting media (euparal and glycerin jelly) are presented. Aberration artefacts often observed in confocal data obtained from thick specimens were analysed and strategies for their minimization are discussed. Our results indicate that confocal laser scanning microscopy and 3D reconstruction are excellent techniques for visualizing small, complex, autofluorescent structures in flies. These techniques could have a profound impact on the quality of information provided by 3D representations of insect structures over more traditional methods of visualization.

Changes in DNA loop domain structure during spermatogenesis and embryogenesis in the Syrian golden hamster.

The DNA in eukaryotic cells is organized into loop domains that are 25 to 100 kilobases long and attached at their bases to the nuclear matrix. This organization plays major roles in DNA replication and transcription. We examined changes in DNA loop structure of the 5S rDNA gene cluster in the Syrian golden hamster as a function of cellular differentiation by direct visualization with fluorescent in situ hybridization. The 5S rDNA cluster is large enough to encompass more than one loop domain but small enough that individual loop domains can still be resolved. We found that the sizes of the 5S rDNA loops are much smaller, and that the numbers of loops per locus are larger, in all pluripotent cell types than they are in adult somatic tissue. Within the pluripotent spermatogenic cell lineage, the loop domain organization was cell specific. The loop size decreased during the early stages of spermatogenesis but did not change during spermiogenesis, suggesting that DNA loop structure is independent of the chromatin condensation that occurs when protamines replace histones. In early embryonic cells, the loop structure remained small, but in differentiated somatic cells, it became much larger. We suggest that these changes in the 5S rDNA loop domain structure may be related to the maintenance or loss of developmental potential.