Detection of sperm within Gyrodactylus (Platyhelminthes, Monogenea) tissues using fluorescence and transmission...

A rapid fluorescent staining method demonstrating spermatozoa within gyrodactylid monogeneans is described. Gyrodactylids fixed and stained in 2% acetic acid containing 1 microgram ml-1 bisBenzimide (Hoechst 33258) were viewed using epifluorescence microscopy. In addition to staining sperm in the testis, seminal vesicle and seminal receptacle, the technique highlighted sperm in the gut and cytoplasmic lining of the uterus, locations from which they had not previously been recorded. The technique was more rapid than transmission electron microscopy (TEM), which was used to confirm the observations. Fluorescence microscopy provided an overview of sperm orientation and distribution that could otherwise be obtained only from serial ultrathin sections. Using the fluorescence technique and/or TEM we have located migrating sperm in the tissues of Gyrodactylus turnbulli, G. bullatraudis and G. gasterostei, although these sperm do not appear to enter the tissues of embryos in utero. The technique can be used to study insemination patterns in gyrodactylid populations and in experimental studies of gyrodactylid reproduction.