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Molecular Quantification of Symbiotic Dinoflagellate Algae of the Genus Symbiodinium

Jeannette E. Loram^1,*, Neil Boonham², Peter O'Toole¹, Henry G. Trapido-Rosenthal^3, and Angela E. Douglas^1,

¹ Department of Biology, University of York, York, YO10 5YW, UK
² Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK
³ Bermuda Institute of Ocean Sciences, Ferry Reach, St. George's, GE 01, Bermuda

To whom correspondence should be addressed, at Department of Biology (Area 2), University of York, PO Box 373, York, YO10 5YW, UK. E-mail: aed2{at}york.ac.uk

The dinoflagellate microalga Symbiodinium is the dominant algal symbiont in corals and related marine animals. To explore the incidence of mixed infections, methods employing real-time quantitative polymerase chain reaction (QPCR) and fluorescence in situ hybridization (FISH) were developed. In experiments focusing on Symbiodinium clades A and B, QPCR and FISH results were well correlated and generally more precise and sensitive than those from the endpoint PCR–restriction fragment length polymorphism analysis (PCR-RFLP) traditionally used for this application, thus increasing the detected incidence of mixed infections. For example, the prevalence of mixed infections in the sea anemone Condylactis gigantea was 40% by PCR-RFLP and 80%–90% by QPCR and FISH. However, the use of QPCR and FISH was limited by inter-host variation in the rRNA gene copy number per Symbiodinium cell, precluding any single conversion factor between QPCR signal and Symbiodinium cell number; and one FISH probe that gave excellent hybridization efficiency with cultured Symbiodinium yielded variable results with Symbiodinium from symbioses. After controlling for these caveats, QPCR studies revealed that field-collected hosts previously described as universally unialgal bore up to 1.6% of the alternative clade. Further research is required to establish the contribution that algal cells at low density in symbiosis and external to the symbiosis make to the minor clade.

Abbreviations: DAPI, 4'–6'-diamidino-2-phenylindole • DGGE, denaturing gradient gel electrophoresis • FISH, fluorescence in situ hybridization • FITC, fluorescein isothiocyanate • QPCR, quantitative polymerase chain reaction • RFLP, restriction fragment length polymorphism

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