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Use of Quantitative Real-Time PCR to Investigate the Dynamics of the Red Tide Dinoflagellate Lingulodinium polyedrum

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Abstract

A new method based on quantitative real-time polymerase chain reaction (qPCR) was developed and applied to quantify the red tide dinoflagellate Lingulodinium polyedrum in natural seawater samples and in laboratory cultures. The method uses a Molecular Beacon™ approach to target a species-specific region of the small subunit ribosomal RNA gene. The accuracy of the method was verified by microscopical counts using cultures of the dinoflagellate isolated from coastal waters near Los Angeles, CA, and with natural water samples spiked with cultured L. polyedrum. The method was applied to document the pattern and timing of vertical migration by the dinoflagellate in a 2-m water column on an 11:13 h light/dark photoperiod established in the laboratory. Positive phototaxis of L. polyedrum resulted in dense aggregations of the dinoflagellate within the top few centimeters of the water column during the light period. This pattern of distribution was readily established by both methods, although abundances of L. polyedrum determined using qPCR were higher than abundances determined by microscopy in the morning and lower in the afternoon and evening. These differences may have been a consequence of variability in the DNA content per cell because of synchrony of cell division. Counts using both methods to analyze natural samples collected from coastal waters in the Long Beach–Los Angeles area and adjacent San Pedro Channel were in close agreement. However, the qPCR method exhibited greater sensitivity than the microscopical method when L. polyedrum was present at low abundances, and qPCR had a much higher rate of sample throughput than microscopy. The development of this new approach for enumerating L. polyedrum provides a useful tool for studying the ecology of this important red tide species.

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Acknowledgments

We thank R. A. Schaffner, J. M. Rose, and A. Schnetzer (USC, Los Angeles) for helpful discussions and help with graphics and statistics, the Centre for Embedded Network Sensing (CENS) for providing the glass column for our laboratory work, and the Deutscher Akademischer Austauschdienst (DAAD) for their financial support. This project was additionally supported by National Science Foundation grants MCB-0084231, OPP-0125437, The Centre for Embedded Networked Sensing (CENS) under the NSF Cooperative Agreement CCR-0120778, and Environmental Protection Agency grant RD-83170501.

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  1. Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, AHF 301, Los Angeles, CA, 90089-0371, USA

    Stefanie D. Moorthi, Peter D. Countway, Beth A. Stauffer & David A. Caron

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Moorthi, S.D., Countway, P.D., Stauffer, B.A. et al. Use of Quantitative Real-Time PCR to Investigate the Dynamics of the Red Tide Dinoflagellate Lingulodinium polyedrum . Microb Ecol 52, 136–150 (2006). https://doi.org/10.1007/s00248-006-9030-3

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