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

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

Brno

Simposio; 8th International Symposium on Monogenea; 2017

One of the main problems in the study of the structural and functional morphology of small organisms, such as dactylogyrid monogeneans, is the lack of a comprehensive threedimensional description of the diagnostic structures, since bi-dimensional line drawings and microphotographs provide partial pictures of their structural complexity. Dactylogyridae is overwhelmingly the most abundant and diverse taxon among monogeneans in continental waters of South America. Their small body size requires a considerable sampling effort and training to collect and identify worms from gills, skin, nasal cavities and other microhabitats. In fact, diagnostic features such as sclerites and the male copulatory complex are generally less than 100 μm in length and are essential for the taxonomic description and identification of species. The objective of this study is to propose a simple and routine method for morphological studies by Laser Scanning Confocal Fluorescence Microscopy, combining 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. In fact, stable autofluorescence emission was detectable at 500-530 nm for bars, anchors and male copulatory complex when excited with argon laser. Readily available software for image processing was used for three-dimensional reconstruction of sclerotized structures generating 3D models and 360-degree full-rotation views, as well as orthogonal projections and morphometric analysis. The advantages of this protocol over previous methodologies for confocal laser imaging are discussed.