PHOTOTAXIS AND AEROTAXIS IN A CALCIFYING ALGA P.Jackie Duke1 ,Wendy Callejas2, Lan Doan1, and Mary Marsh3 1 Dept.

of Orthodontics, Dental Branch, University of Texas Health Science Center at Houston, 2University of Houston- Downtown, 3Dept. of Biochemistry and Molecular Biology, Medical School, University of Texas Health Science Center at Houston, Houston, TX 77225, USA.
INTRODUCTION The coccolithophore, Pleurochrysis carterae, is a unicellular marine alga, with an outer covering of scales known as coccoliths, instead of a cell wall (Figure 1; 1). Coccoliths are composed of calcium carbonate (CaCO3) in the form of calcite, and are formed intracellularly in the Golgi cisternae in a very precise and well defined process before being positioned outside the cell membrane (1). Because this is an excellent model of calcification in a single cell, we proposed P. carterae for flight on the International Space Station, and spent several years in experiment development prior to having the experiment deselected from flight. In addition to being a model for calcification, P. carterae was to have been used to address questions of reproduction, cellular polarity, secretion, and gravitaxis in the microgravity environment (2-4). Requiring only low levels of light and capable of being stored for over 70 days in the cold and dark before use, P. carterae remains an excellent choice for space flight studies (5,6). Additional descriptions of the proposed experiment and various educational links regarding this alga can be found at (7). reached, concomitant with the start of bioconvection (convection due to movement of microorganisms.) The bioconvective process begins with the accumulation of cells at the surface of the medium, which in turn requires that the cells swim up (8). The objective of the current study was to determine if taxes other than gravi- or gyrotaxis, specifically photo- and aerotaxis, were the cause of surface accumulation of cells. Because our deselection was due to lack of videomicroscopy, macroscopic methods were used in these experiments. MATERIALS AND METHODS Cells from a stock cell culture of P.carterae, Plymouth strain 136, a swimming strain, were expanded in F/2 medium (18oC), then divided into 4 t-flasks. After 24 hours, cultures were observed for accumulations of cells in the region of the air bubble. For phototaxis, box covers, with only a small opening at the bottom of one side, were placed over the t-flasks as an obstruction of the light source. Cells in these cultures did not accumulate in any region of the culture, and cultures did not grow due to the lack of light. The t-flasks were then placed horizontally in the incubator with the boxes covering all but the top 25% of the flask. The air bubble was located in the dark region of the flask. Flasks were incubated overnight, and observed for accumulation of cells in particular regions of the flask.

Figure 2: T-flask from phototaxis experiment. Cells are visible as a cloud in the medium, and are located in the left hand side of the flask which was exposed to light during culture in the incubator. Figure 1: Scanning electron micrograph showing coccooiths on the surface of P. carterae. Photo by Dr. Mary Marsh.

RESULTS AND DISCUSSION During the initial culture of the cells to expand cell numbers, a green halo of cells surrounded the air bubble in each flask, demonstrating aerotaxis. The first phototaxis experiment used boxes that completely covered the flask, with only a small opening for light. This arrangement did not support cell growth as after the incubation period, no accumulations of cells were seen. This agrees with previous experiments in which we
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In preparation for the gravitaxis portion of the spaceflight experiment, we spent considerable time assessing gravitaxis in P carterae at 1G, using the Wintrack 2000 software. In these studies, we determined that cells of Pleurochrysis carterae do not start to move in a measurably oriented manner until a certain cell density is

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demonstrated that no increase in cell number is seen in cultures of P carterae kept in the dark. In the partially covered t-flasks, cells accumulated in the portion of the flask that was not covered, demonstrating phototaxis (Figure 2). The bubbles in these flasks were carefully sequestered in the dark region, so that the aerotaxic response would not interfere with the phototaxic response. The lack of accumulations around the air bubble in these cultures demonstrates that the cells response to light is stronger than the response to the presence of air. Similar results were seen in experiments with Euglena gracilis, a fresh water photosynthetic alga (9). These simple experiments demonstrate that macroscopic methods can be used to observe movement of populations of algal cells, thus obviating the need for microscopic observations to see overall cell accumulations. These observations, which can be captured automatically on videotape in several of the current cell culture systems developed for ISS, can be used to determine if bioconvection occurs in space. Although bioconvection, according to theory, should not occur in a microgravity environment, there may be enough g disturbances on ISS to begin the process, which might be then continued by swimming action of the organisms themselves. The results in these studies were definitely density dependentat lower densities the cloud of cells would not be visible to the naked eye. At higher densities, bioconvection would occur. Finally, such results must be taken into account when designing space flight experimentsfor example, the light source should be perpendicular to the gravity vector in the culture system to ensure that the cells are responding to gravity and not to light. Alternatively, during g-response experiments, an infared source of light can be used. CONCLUSIONS From this experiment we conclude that P.carterae is aerotaxic and phototaxic, and that the phototaxic response is stronger than the aerotaxis response. In the ocean, P. carterae is found near the surface of the water, due probably to phototaxis and aerotaxis, not gravitaxis or gyrotaxis. Future experiments will determine the response to air during the nonphotosynthetic period of the light:dark cycle. ACKNOWLEDGEMENTS Supported by UTHSC Summer Research Program for High School Students 2004 (WC) and NASA NAG21498 (MM). Porterfield, D.M. 1997. Orientation of motile unicellular algae to oxygen: oxytaxis in Euglena. Biol. Bull. 193: 229-230. REFERENCES Algae and Dentistry:http://www.db.uth.tmc.edu/ orthodont/Duke%20NASA/index.html Bees, M. A. and Hill, N.A. 1997. Wavelengths of bioconvection patterns. Journal of Experimental Biology 200: 1515-1526. Duke, P.J., and Montufar-Solis, D. 2000. Pleurochrysis carterae: a model to study biologically directed mineralization in space. First International Symposium on Microgravity Research and Applications in Physical Sciences and Biotechnology, ESA Publications Division, ESA SP-454:337-348. Marsh, M. 1999. Biomineralization in Coccolithophores. Gravitational and Space Biol Bulletin. 12: 5-15. Montufar-Solis, D., Duke P.J., and Marsh, M.E. 2003. Bioconvection in cultures of the calcifying unicellular alga Pleurochrysis carterae. J Gravitational Physiology. 10: 270-272. Montufar-Solis D., Marsh M.E., and Duke, P.J. 2002. Cell density affects cell motility in P. carterae cultures. In: Proceedings of the Eight European Symposium on Life Sciences Research in Space, Stockholm, Sweden, June 27, , ESA SP-501, pp293-294. Montufar-Solis, D., Marsh, M.E., and Duke, P.J. 2002. Cell density affects cell motility in P. carterae cultures. Journal of Gravitational Physiology 9:P265-266. Montufar-Solis, D., and Duke, P.J. 2001. Pleurochrysis carterae is an excellent model to study biomineralization and gravitaxis on the ISS. American Institute of Aeronautics and Astronautics Bulletin. AIAA (4989):478483.

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