CARD-FISH samples were fixed with paraformaldehyde (1% final concentration) for 1 h (4 °C in the dark) before filtration on a 0.8 µm, polycarbonate filter with a vacuum pump (< 200 mm Hg). Filters were then dehydrated using successive 50%, 80%, and 100% ethanol solutions, dried and stored in the dark at −20 °C. FISH staining was then performed according to [8 (link)]. The prevalence was estimated from microscope observations with an Olympus BX-51 epifluorescence microscope (Olympus Optical) equipped with a mercury light source, a Wide Blue filter set (Chroma Technology, VT, USA) and fluorescence filter sets for PI (excitation: 536 nm; emission: 617 nm) and FITC (excitation: 495 nm; emission: 520 nm).
Prevalence was determined by averaging infection counts on a minimum of 80 cells per replicate. Prevalence was characterized in: non-infected host cells, early stage (one or more dinospores of Amoebophrya sp. in the cytoplasm), and advanced stages (intermediate and beehive stages) as described in [42 (link)]. The progeny count (i.e., the number of dinospores per infected host) was estimated by dividing the maximal concentration of dinospores by the concentration of infected hosts in advanced stages.
Prevalence was determined by averaging infection counts on a minimum of 80 cells per replicate. Prevalence was characterized in: non-infected host cells, early stage (one or more dinospores of Amoebophrya sp. in the cytoplasm), and advanced stages (intermediate and beehive stages) as described in [42 (link)]. The progeny count (i.e., the number of dinospores per infected host) was estimated by dividing the maximal concentration of dinospores by the concentration of infected hosts in advanced stages.
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