FISH probes for the detection of the parasitic dinoflagellate Amoebophrya sp. infecting the dinoflagellate Akashiwo sanguinea in Chesapeake Bay

A comparison of the small subunit rRNA sequences of a Chesapeake Bay strain of the dinoflagellate Akashiwo sanguinea and the dinoflagellate Amoebophrya 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 8/11 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 probed cells were more distinct when Texas Red was used as the label. Cells stored in cold paraformaldehyde for a year still bound the probes. Young stages of the parasite could be seen more readily after in situ hybridization than after protargol impregnation.     

Multiple strains of the parasitic dinoflagellate Amoebophrya exist in Chesapeake Bay

Small subunit rRNA sequences were amplified from Amoebophrya strains infecting Karlodinium micrum, Gymnodinium instriatum and an unidentified Scrippsiella species in Chesapeake Bay. The alignable parts of the sequences differed from each other and from the previously reported rRNA sequence of the Amoebophrya strain infecting Akashiwo sanguinea in Chesapeake Bay by 4 to 10%. This is a greater degree of difference than sometimes found between sequences from separate genera of free-living dinoflagellates. These sequence differences indicate that the Amoebophrya strains parasitizing dinoflagellates in Chesapeake Bay do not all belong to the same species. In spite of their relative dissimilarity, the sequences do group together into a single clade with high bootstrap support in phylogenetic trees constructed from the sequences.     

Dinoflagellate Evolution: Speculation and Evidence*†

SYNOPSIS. Nuclear features of dinoflagellates that were used originally to support the Mesocaryota concept are reviewed. The fibrillar diameter of dinoflagellate chromatin, low level of chromosomal basic proteins, membrane attachment of chromosomes and swirl pattern observed in sectioned chromosomes are features that support a prokaryotic affinity. The presence of repeated and highly complex DNA, a S-phase of DNA synthesis in the cell cycle, presence of basic proteins, and the reinterpretation of extranuclear microtubules as a spindle support the contention that dinoflagellates are eukaryotes. This combination of prokaryotic and eukaryotic features suggests that dinoflagellates are a geologically old group and that perhaps they diverged from the higher eukaryotic lineage before evolution of eukaryotic chromatin but after the evolution of repeated DNA. The 2 patterns of carotenoid composition exemplified by the presence of peridinin or fucoxanthin suggest separate origins of dinoflagellate plastids, perhaps by prokaryotic and eukaryotic capture. It is suggested that the species possessing fucoxanthin obtained their plastids by capture of photosynthetic eukaryotes. A new class and order, Syndiniophyceae and Syndiniales, are proposed for the dinoflagellates with low chromosome numbers, V-shaped chromosomes, chromosomes containing a sufficient quantity of basic proteins detectable histochemically, possession of centrioles associated with mitosis, intracellular parasitism as a mode of nutrition, and lack of a cellular covering containing plates. Ultrastructural and paleontologic evidence indicates that the thecate is more primitive than the nonthecate condition. The Prorocentrales are considered to be primitive and their thecal construction is reinterpreted as having epithecal and hypothecal regions surrounding a flagellar pore region containing 7 plates. Acritarchs resemble cysts of modern dinoflagellates in size, structure, and chemical composition except for the absence of a polygonal excystment aperture and lack of any indication of transverse and longitudinal flagellar grooves on the acritarchs. The suggestion that some acritarchs may have dinoflagellate affinities is supported by the occurrence of modern dinoflagellates (Prorocentrales) which lack a theca of numerous polygonal plates and lack transverse and longitudinal flagellar arrangement. The Prorocentrales, as opposed to the more typical Dinophyceae, perhaps represent the type of organism that produced some acritarchs.     

The Phylogenetic Position of Amoebophrya sp. Infecting Gymnodinium sanguineum

The small-subunit rRNA sequence of a species of Amoebophrya infecting Gymnodinium sanguineum in Chesapeake Bay was obtained and compared to the small subunit rRNA sequences of other protists. Phylogenetic trees constructed with the new sequence place Amoebophrya between the remaining dinoflagellates and other protists.