Localization of EPS components secreted by freshwater diatoms using differential staining with fluorophore-conjugated lectins and other fluorochromes

Abstract

Diatoms produce extracellular polymeric substances (EPS), which mainly consist of carbohydrates and may form different morphological structures. We studied the localization and structure of EPS secreted by 17 diatom species that were isolated from epilithic biofilms from the littoral zone of Lake Constance (Germany). We used six different lectins labelled with FITC (fluorescein isothiocynate) and DAPI (diamidino phenylindole) to localize and visualize the structure of secreted EPS and/or cell-wall-associated EPS, while DTAF (dichlorotriazinyl aminofluorescein) was used to label cell-wall-associated EPS only. The diatoms were categorized according to the structure of the secreted EPS i.e. pads, capsules, tubes or stalks. Among eight pad-producing diatoms, three Fragilaria species showed variable lectin binding indicating the presence of different carbohydrate components. Other pad-forming diatoms like Synedra, Diatoma, Asterionella, and Melosira generally showed binding to at least two different lectins. On the other hand, we did not observe any lectin binding to the capsules of Staurosira and one Achnanthes isolate. We further detected differences in the carbohydrate composition of tube-like EPS structures in two Cymbella species and were able to demonstrate developmental stages of tube formation. The stalk secreting species Gomphonema and Cymbella showed labelling by only one lectin, while EPS of Caloneis alpestris were stained by all lectins and fluorochromes used indicating a highly complex composition.