FISH Probes for the Detection of the Parasitic Dinoflagellate Amoebophrya sp. Infecting the Dinoflagellate Akashiwo sanguinea in Chesapeake Bay

Abstract

A comparison of the small subunit rRNA sequences of a Chesapeake Bay strain of the dinoflagellate Akashiwo sanguinea and the dinoflagellate Arnoebophrya sp. parasitizing it revealed several potential target sites that could be used to detect the parasite through in situ hybridization. The fluorescence of probed cells under various conditions of hybridization was measured by using a spot meter on a Nikon UFX-II camera attachment so that the effect of various hybridization parameters on probe binding could be determined. Probes directed against both the junction between helices 8 and 11 and helix 46 could detect the parasite, although the helix 811 1 probe produced a stronger signal under the conditions tested. The fluorescence of the probed cells increased with increasing hybridization time up to approximately twelve hours. The background fluorescence was lower at the wavelengths used to detect Texas Red than at * those used to detect fluorescein, so probled cells were more distinct when Texas Red was used as the label. Cells stored in cold 4 paraformaldehyde for a year still bound the probes. Young stages of the parasite could be seen more readily after in situ hybridization 1 than after protargol impregnation.