Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes. is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy. The copulatory and haptoral sclerites of monogenoids are microscopic and intricately complex. They vary between closely related species more than many other anatomical structures and thus provide important characters for species identification. diagnosis of higher taxa, and for phylogenetic and coevolutionary studies (Gusev, 1978; Boeger and Kritsky, 1989, 19977 2001: Gerasev, 1989, 1998; Desdevises et al., 2001; Kritsky and Boeger, 2003). Traditional methods for visualizing without destruction of the specimen. LSCFM, however, is not without disadvantage and may introduce some artifacts. For example, Klaus et al. (2003), who examined insect genitalia, indicated that axial distortion, particularly along the z-axis, may be associated with LSCFM when observing thick specimens. However, comparison of LSCFM images of monogenoidean sclerites with respective sclerites of other specimens oriented in the same plane as those of the LSCFM images suggests that axial distortion is minimal, probably owing to their comparatively smaller size and thickness.