Search

Dinoflagellates are a diverse group of protists, and approximately half of the ca. 2000 extant species are obligate heterotrophs. Heterotrophic dinoflagellates are widespread and often abundant in aquatic ecosystems, contributing to the transfer of organic matter in aquatic food webs and representing a link between primary production and metazooplankton. Knowledge of heterotrophic dinoflagellate nutrition and feeding behavior is needed to better evaluate the role and impact of these ecologically significant protists in microbial communities. The dinoflagellate genus Pfiesteria contains P. piscicida and P. shumwayae, two potentially toxic, heterotrophic species commonly found in temperate estuarine and coastal waters of the U.S. Atlantic coast. Pfiesteria spp. are among the relatively few heterotrophic dinoflagellates that have been maintained in long-term culture, which has facilitated research on the biology and ecology of these species. Detailed studies on the physiology and biochemistry of Pfiesteria spp., however, have been complicated by the inability to culture these dinoflagellates in the absence of living prey and bacteria. The present research focused on one Pfiesteria species, P. shumwayae. Pfiesteria shumwayae strains cultured bacteria-free on a fish cell line were used to investigate phosphatase enzymes in this dinoflagellate. Cellular localization of enzyme activity was determined with the fluorescent probe ELF-97. Phosphatase activity also was evaluated at three different pH values using traditional colorimetric methods. The location of enzyme activity and supporting colorimetric measurements suggested that acid phosphatases predominate in P. shumwayae and have a general catabolic function. Advanced culturing methods also were developed that permitted additional insight into Pfiesteria shumwayae feeding behavior and nutritional requirements. A complex, bi-phasic culture medium was formulated that supported the axenic growth of two P. shumwayae strains. A major component of the medium was chicken egg yolk, suggesting that this species may have a biochemical requirement for one or more lipids. Further, P. shumwayae cells ingested the yolk particles in the medium, permitting detailed microscopic examination of feeding behavior in this dinoflagellate. Subsequently, a semi-defined medium was formulated that supported the axenic growth of three Pfiesteria shumwayae strains. The medium contained high concentrations of dissolved and particulate organic compounds, including amino acids and lipids, and permitted additional insight into the possible biochemical requirements of this heterotrophic dinoflagellate. Maximum cell yields attained in the semi-defined medium were up to 10 times higher than those obtained when P. shumwayae was cultured with living fish or microalgae in xenic cultures. These investigations provided new information on the digestion, feeding behavior, and nutrition of the heterotrophic dinoflagellate Pfiesteria shumwayae. Information on the occurrence and role(s) of phosphatases in heterotrophic dinoflagellates is limited, and P. shumwayae is only the second heterotrophic dinoflagellate species examined for phosphatase activity. Development of first a complex and then semi-defined axenic culture medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of specific biochemical requirements of P. shumwayae, needed to advance understanding of the nutritional ecology of this species. Further, this culturing method provides a source of Pfiesteria shumwayae cells free from other metabolizing cells, permitting physiological and ecological investigations that would otherwise be complicated by the presence of prey and⁄or bacteria.