Use of confocal laser scanning microscopy in systematics of insects with a comparison of fluorescence from different stains

Confocal laser scanning microscopy has become a valuable tool for a wide range of investigations in the biological sciences, but its use in insect systematics has been neglected. Confocal microscopy depends on the degree of fluorescence of the examined specimens, which is aided either by fluorescent dyes or autofluorescence of the specimen. This study provides methods for using a combination of fluorescent dyes and autofluorescence to provide images that document the value of confocal microscopy for systematic research with insects. Fluorescence was compared from Lepidoptera genitalia dissections that were unstained or stained with merbromin (mercurochrome), safranine, chlorazol black E, eosin Y, eosin Y + chlorazol black E, and orange-G. The unstained specimen showed that chitin autofluorescences to a small degree. The comparison of stains showed that use of eosin Y provides the best images, followed by safranine and mercurochrome. Orange-G and chlorazol black are the least fluorescent and provide poor images, even when chlorazol black is combined with eosin.