Development and Application of a Monoclonal-Antibody Technique for Counting Aureococcus anophagefferens , an Alga Causing Recurrent Brown Tides in the Mid-Atlantic United States

Abstract

The identification and enumeration of microorganismal species in natural aquatic assemblages is an essential prerequisite for ecological studies of these populations. The ability to distinguish between closely related taxa is especially critical when these species pose health and environmental risks. Traditionally, protistan species (microalgae and protozoa) have been identified morphologically and enumerated by using light or electron microscopy. Light microscopy (bright field, phase, and differential interference contrast) has been used to identify many protists that possess distinct morphological features, whereas electron microscopy has been used effectively for many small algae and protozoa (e.g., cell sizes of Յ10 m). Protists typically have been counted by epifluorescence microscopy (25) or by using settling techniques and inverted light microscopy (30).Unfortunately, these approaches have significant disadvantages for ecological studies in which it is necessary to identify and count small protists in large numbers of samples in a timely manner. Morphological features that are relevant for species identification are not always easy to discern by methods that are most commonly used for enumeration. For example, transmitted and epifluorescence microscopy do not allow visualization of morphological features that are pertinent for species identifications of many small protists (e.g., striations on frustules of diatoms or body scales on chrysomonads that can be observed only by electron microscopy). In addition, microscopic analyses generally are time-consuming, and the processing of large numbers of samples that are typical in ecological surveys and experiments may require weeks or months to complete. In order to circumvent these shortcomings, new approaches based in modern immunology and genetics have emerged that are able to provide rapid and accurate identification and enumeration of microbial species.Immunological approaches for identifying and enumerating marine microalgae have become commonplace within the last two decades. These methods and their ecological applications for the identification of phytoplankton have been summarized (19,31). Both polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) have been developed for use by microbial ecologists. Immunological probes have proven useful for identifying species of cyanobacteria (10), raphidophytes (29), dinoflagellates (22), pelagophytes (3, 21), and other minute algal taxa (11,24) and even for distinguishing between toxic and nontoxic strains of harmful algae (6). An added advantage of this approach is that these methods often can be converted to formats that are significantly more rapid than routine microscopical counts (32).Aureococcus anophagefferens is a pelagophyte alga that typifies the difficulties of accurately identifying and enumerating small protistan species in natural water samples. The alga is minute (ϳ2 to 4 m in diameter) and spherical, lacks flagella and body scales, and has few other features that might easily distinguish it ...

Effect of the northern quahog Mercenaria mercenaria on the development of blooms of the brown tide alga Aureococcus anophagefferens

“…The abundance of Aureococcus anophagefferens at the beginning of all 3 experiments in the seawater at our study site was near or below the limit of detection of our method (< 5000 cells ml -1 ) (Caron et al 2003). In order to begin each experiment with low but measurable populations of brown tide cells in the mesocosms, seawater was collected in acid-washed containers for use as an inoculum.…”

“…Seawater samples for counts of Aureococcus anophagefferens were preserved with 10% gluteraldehyde prepared with 0.22 µm filtered natural seawater (Sherr & Sherr 1993) for a final preservative concentration of 1%. A. anophagefferens cells were enumerated using the monoclonal antibody colorimetric technique of Caron et al (2003). Replicate 25 to 72 ml samples were filtered onto Gelman GF/F glass fiber filters for the measurement of total chl a concentration using a Turner Designs fluorometer, Model TD-700 (Arar & Collins 1992).…”

Seasonal and Interannual Trends in Nitrogen and Brown Tide in Maryland's Coastal Bays

“…For the years 1999-2002, the but-fuco pigment record of Trice et al (2004) is used. For the years 1999-2004, direct cell count data are available from application of monoclonal and polyclonal antibody techniques (Anderson et al 1989, Caron et al 2003. In order to convert the direct cell count data …”

Role of zooplankton in the onset and demise of harmful brown tide blooms (Aureococcus anophagefferens) in US mid-Atlantic estuaries

“…Flow cytometry samples were flash frozen in liquid nitrogen and stored at -80°C. A. anophagefferens sampled were kept at 4°C and densities were determined using an ELISA-based monoclonal antibody labeling technique (Caron et al 2003). This method estimates abundances of A. anophagefferens in natural samples to a lower threshold concentration of approximately 5 × 10 3 cells ml -1…”