Development of a light microscopy stain for the sclerites of Gyrodactylus von Nordmann, 1832 (Monogenea) and related genera.

Recent developments in semi-automated identification techniques and the increasing ability to rapidly access digital images and taxonomic descriptions offer to increase the range of individuals capable of performing taxonomic identifications. The present study details methodological approaches undertaken in developing a dedicated stain for the visualisation of monogenean haptoral skeletal elements and reproductive sclerites. The histochemical protocols centre around the use of fluorescent dyes and standard light and laser scanning confocal microscopy to support studies of the functional morphology of these hard structures in small, relatively uncompressed specimens, making these structures more amenable to semi-automated analysis and identification techniques. Staining of the sclerites was achieved using a tissue digestion step to remove the tegument and tissues enclosing the sclerites and then staining them in situ with 40 mM chromothrope 2R (C2R) containing 3 mM phosphotungstic acid (PTA) and 0.5% acetic acid (AA) at room temperature for up to 2 days. Visualisation of the armature of the male copulatory organ of warm water Gyrodactylus species was achieved using 40 mM C2R containing 3 mM PTA for 3 days, whilst cold water species were best stained in 6.4 mM C2R for 1 day without an NaOH pre-treatment. The developed techniques allow for good visualisation of the skeletal elements in a number of monogenean groups and promise to assist the preparation and identification/description of specimens. The 2D/3D digital images of specimens prepared in this manner should provide a useful resource for taxonomists and others needing material to assist specimen identification.