AXENIC CULTIVATION OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA SHUMWAYAE AND OBSERVATIONS ON FEEDING BEHAVIOR¹

Abstract

Pfiesteria shumwayae Glasgow et J. M. Burkh. [=Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester] is a heterotrophic dinoflagellate commonly found in temperate, estuarine waters. P. shumwayae can feed on other protists, fish, and invertebrates, but research on the biochemical requirements of this species has been restricted by the lack of axenic cultures. An undefined, biphasic culture medium was formulated that supported the axenic growth of two of three strains of P. shumwayae. The medium contained chicken egg yolk as a major component. Successful growth depended on the method used to sterilize the medium, and maximum cell yields (10(4) · mL(-1) ) were similar to those attained in previous research when P. shumwayae was cultured with living fish or microalgae. Additionally, P. shumwayae flagellate cells ingested particles present in the biphasic medium, allowing detailed observations of feeding behavior. This research is an initial step toward a chemically defined axenic culture medium and determination of P. shumwayae metabolic requirements.

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“…The epicone of attached dinoflagellates sometimes became deformed as the cells attempted to ingest the yolk particles.'' Based on these observations, these pseudopodia likely function in attachment to food items, and they appear to be most similar to pseudopodia of the heterotrophic dinoflagellate A. ocellatum (Brown, 1934;Nigrelli, 1936;Lom and Lawler, 1973;Skelton et al, 2008). This organism is an ectoparasite of fish, and is apparently closely related to Pfiesteria species (e.g.…”

“…The medium contained high levels of certain dissolved and particulate organic substances, including amino acids and lipids. The P. shumwayae flagellate cells were attracted to insoluble lipids present in the medium and appeared to feed on them, suggesting, as did the work of Skelton et al (2008), that phagotrophy may be required for growth under axenic conditions. See Section 9.2.1 for further information about feeding by axenically grown P. shumwayae.…”

“…A novel feeding mechanism Recent research has led to the discovery of a novel feeding mechanism in dinoflagellates, as manifested by P. shumwayae. In the above-described experiments with two strains of P. shumwayae in axenic CYM, Skelton et al (2008) noted that the flagellate cells swam slowly in tight circles at the bottom of the culture flasks, generally oriented so that the cell antapex faced toward the culture flask bottom. The movement of the transverse flagellum created a roughly spherical clearing (easily discerned at 40Â) that was mostly devoid of egg yolk granules.…”

“…Skelton et al (2008) suggested that the yolk particles may have met a biochemical requirement, or, alternatively, that the yolk particles were needed to induce phagotrophy and digestive vacuole formation, while the cells likely also engaged in concurrent uptake of various dissolved substrates from the medium. In this regard, the presence or addition of particulates to culture media has been reported to stimulate phagotrophy in other heterotrophic protists grown axenically (see Skelton et al, 2008, and references therein).…”

“…Starting with the monoxenic cultures of Parrow et al (2005), Skelton et al (2008) first accomplished axenic growth of P. shumwayae on an undefined, biphasic chicken egg yolk medium (CYM-MP Biomedicals #1003749; final concentration 0.25-0.5%) as a major component. Growth was evaluated after sterilization by autoclaving versus by sterile filtration (0.22 mm pore size), and was compared to growth on untreated CYM.…”