ORIGINAL ARTICLE Assessment of Microzooplankton Grazing on Heterosigma akashiwo Using a Species- Specific Approach Combining Quantitative Real-Time PCR (QPCR) and Dilution Methods Elif Demir & Kathryn J. Coyne & Martina A. Doblin & Sara M. Handy & David A. Hutchins Received: 23 June 2006 / Accepted: 17 April 2007 / Published online: 4 July 2007 # Springer Science + Business Media, LLC 2007 Abstract Delaware’ s Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top–down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species- specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H. akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific micro- zooplankton grazing pressure on H. akashiwo ranged from 0.88 to 1.88 day −1 at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F. japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H. akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplank- ton species within a mixed community. Introduction Microzooplankton grazing accounts for a significant loss factor of phytoplankton and bacteria in marine systems [45]. Selective grazing by zooplankton that are <200 μm shapes phytoplankton community structure [70] and assists nutrient recycling and regeneration [26], thus establishing a link between lower and higher tropic levels [59]. Micro- zooplankton grazing can remove enough cells to prevent or diminish blooms [60], creating a top–down control (removal of organisms by consumers) on community dynamics that may determine the fate of harmful algal blooms. Several studies have demonstrated the direct negative effects of grazing on potentially harmful algae such as Chattonella antiqua [1, 69], Micromonas spp. [16], and Alexandrium minutum [7, 19]. Heterotrophic dinoflagellates have also been found to graze on laboratory cultures of the raphidophytes Fibrocapsa japonica [66] and Heterosigma akashiwo [9]. Conversely, grazing pressure on a mixed community may also indirectly favor the growth of harmful algal species, as is the case with Aureococcus anophagefferens [20]. Blooms of a mixed raphidophyte consortium composed of C. subsalsa, Heterosigma akashiwo, C. cf. verruculosa Microb Ecol (2008) 55:583–594 DOI 10.1007/s00248-007-9263-9 E. Demir : K. J. Coyne : S. M. Handy : D. A. Hutchins (*) College of Marine and Earth Studies, University of Delaware, Lewes, DE 19958, USA e-mail: dahutch@usc.edu M. A. Doblin Institute of Water and Environmental Resource Management/ Department of Environmental Science, University of Technology, Sydney, Westbourne St, Gore Hill, Sydney, NSW 2065, Australia D. A. Hutchins Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA