RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS

Authors

  • Holly A. Bowers,

    1. Institute of Human Virology, University of Maryland, 725 West Lombard Street, Baltimore, Maryland 21201, USA
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  • Carmelo Tomas,

    1. University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane, Wilmington, North Carolina 28409, USA
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  • Torstein Tengs,

    1. Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA
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  • Jason W. Kempton,

    1. South Carolina Department of Natural Resources, Marine Resources Research Institute, 331 Fort Johnson Road, Charleston, South Carolina 29422, USA
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  • Alan J. Lewitus,

    1. Belle W. Baruch Institute for Marine and Coastal Sciences, University of South Carolina, Georgetown, South Carolina 29442, USA
    2. South Carolina Department of Natural Resources, Marine Resources Research Institute, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, South Carolina 29412, USA
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  • David W. Oldach

    1. Institute of Human Virology, University of Maryland, 725 West Lombard Street, Baltimore, Maryland 21201, USA
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  • 1Received: 19 August 2005. Accepted 24 August 2006.

Abstract

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.

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