Three-dimensional images of rigid by Lasser Scanning Confocal Fluoreccence microscopy as a tool for taxonomic studies on Dactylogyridae (Monogenea)

M. A. ROSSIN; M. M. IRIGOITIA; TIMI JUAN TOMÁS; P. N. DE FRANCESCO

Brno

Simposio; 8th International Symposium on Monogenea; 2017

Dactylogyridae is overwhelmingly the most abundant and diverse taxon among monogeneans in continental waters of South America. Their small body size requires considerable sampling effort and training for collecting and identifying the worms from the gills, skin, nasal cavities, and other microhabitats. Indeed diagnostic characteristic as sclerites and male copulatory complex are generally less than 100 µm long and are essential for taxonomic description and identification of species. Here, a combination of simple and routine methods for three dimensional morphological studies on hard structures is proposed for dactylogirids: SDS treatment for clarification of specimens and enzymatic digestion with Proteinase K for freeing sclerotized structures, followed by laser confocal microscopy. This method is applicable to fresh or fixed specimens and does not require staining or dehydration. Indeed, stable autofluorescence emission is detectable at 500-530 nm for bars, anchors, and male copulatory complex when excited by argon laser. Advantages of this protocol over previous methodologies for taking laser confocal images are discussed. Open access software for image processing was used for three-dimensional reconstruction of sclerotized structures generating models and full 360-degrees rotation videos.