A fluorescence-based assay for in vitro screening of Saprolegnia inhibitors

The incidence of fish pathogenic oomycetes, Saprolegnia, has increased significantly in aquaculture since the ban of malachite green. For the efficient characterization of anti-Saprolegnia therapeutics, simple accurate methods are required. However, the current screening methods are limited by time, and none of them are confirming the viability of treated spores or hyphae. In this study, a modified fluorescence-based assay for the in vitro screening of Saprolegnia inhibitors has been developed. This method involves the use of FUN-1 viability dye combined with calcofluor white M2R, and is based on the formation of orange-red cylindrical intravacuolar structures (CIVS) in metabolically active spores, hyphae and biofilms. Heat-killed and bronopol-treated Saprolegnia spores, hyphae and biofilms exhibited diffuse bright green fluorescence which confirms complete loss of viability. For boric acid-treated spores, no germination was observed. However, tiny CIVS were observed in 50% of treated spores which indicated reduction in their viability. Our results proved that FUN-1 dye is an efficient tool to distinguish between live and dead Saprolegnia spores, hyphae and biofilms and to monitor the change in Saprolegnia viability during qualitative evaluation of potential anti-Saprolegnia compounds.