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ABSTRACT. Chromera velia (Chromerida: Alveolata) is a photosynthetic, unicellular organism closely related to parasitic apicom- plexa. Diurnal rhythmicity of ...
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Eukaryotic Microbiology J. Eukaryot. Microbiol., 57(5), 2010 pp. 444–446 r 2010 The Author(s) Journal compilation r 2010 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2010.00495.x

Effect of Nutrient Concentration and Salinity on Immotile–Motile Transformation of Chromera velia JIN TAO GUO,a KATE WEATHERBY,a,b DEE CARTERb and JAN SˇLAPETAa Faculty of Veterinary Science, University of Sydney, New South Wales 2006, Australia, and bSchool of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia

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ABSTRACT. Chromera velia (Chromerida: Alveolata) is a photosynthetic, unicellular organism closely related to parasitic apicomplexa. Diurnal rhythmicity of an immotile–motile transformation has been observed but its role in the life cycle remains largely unknown. Using a multiwell system, we show that salinity and f-medium concentration significantly affect the percentage of motile C. velia cells. An inverse relationship between salinity and motility in C. velia occurred, and flagellation was also suppressed at high nutrient levels. These results suggest a low salinity environment with relatively low nutrient levels enables flagellate transformation during the diurnal cycle of C. velia. Key Words. Flagellate, f-medium, life cycle, motility.

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photosynthetic organism associated with hard coral and related to medically important apicomplexan parasites has been described and named Chromera velia (Moore et al. 2008). Recently, the ultrastructure of the motile form of C. velia was characterised, where it was found to be biflagellated and similar in appearance and size to predatory colpodellids (Weatherby et al. 2010). Understanding factors behind the transformation of C. velia from the immotile to the motile form may shed light on the physiology, biology, and ecology of C. velia. The immotile stage of C. velia could be the stage living in a coral host or might drift within the ocean, and the degree of transformation to the flagellated form may be a response to the surrounding levels of nutrients and salinity. The aim of this study was to assess the effect of different f-medium and salinity concentrations on the proportion of flagellated stages in C. velia cultures, using a multiwell format.

number of flagellated cells reaches a maximum (data not shown). To determine the percentage of motile cells we used two techniques: (1) the whole well was observed for 1 min and the percentage of motile cells was estimated as 0%, 1%, 5%, and 10% followed by steps of 10% up to 100%; and (2) a representative field of view was photographed and the numbers of immotile and motile cells were enumerated from the photograph. Pearson’s product–moment correlation coefficient between two arrays of data were calculated for the percentage of motile C. velia counted from digital photos and by visual estimations (function ‘‘PEARSON’’, MS Excel 2003, Microsoft Corporation, Redmond, WA). Statistical evaluation of the experimental treatments used the restricted maximum likelihood method in GenStat package (VSN International, rel. 11 January 2008). A model including a coordinate within the 96-well plates was assumed to evaluate if medium (f-medium level; salinity) or time (days in culture) significantly affected the results. The a-level was set at 0.05.

MATERIALS AND METHODS Organism and culture conditions. Chromera velia (Chromerida: Alveolata) was isolated from stony coral Leptastrea purpurea (Moore et al. 2008). Cells were maintained in f/2 culture medium (AlgaBoost f/2 1000  no silicate media, AusAqua Pty. Ltd., Wallaroo, SA, Australia), salinity 40 g/L (sea salt, Sigma S9883, Castle Hill, NSW, Australia); 24–25 1C under a 12:12 h light:dark cycle and 165 mmol/m2/s (PAR sensor, LI-190, Li-Cor Biosciences, Lincoln, NE). Nutrition and salinity experiments. A randomised block design in sterile Falcon MicroTest 96-well plates (BD Australia, North Ryde, NSW, Australia) was set up in quadruplicate. Plates contained four different concentrations of f-medium (f/4, f/2, f, 5f; salinity 40 g/L) and four salinities (20, 40, 60, and 80 g/L; in f/2), and were seeded with C. velia at density of 105 cells/ml. Each 96well plate had five randomly distributed replicates (200 ml) of C. velia cultures for each experimental condition. Sea salt only (no f-medium) and f/2 medium only (no added salt) served as negative controls, respectively. Cultures were observed and counted on days 2, 4, 6, and 8 using an inverted Olympus CKX41 light microscope equipped with phase contrast (Olympus Australia, Mt Waverley, Vic., Australia) and a Moticam 2300 digital camera (Motic, Causeway Bay, Hong Kong). Enumeration and statistical evaluation of the percentage of motile cells. All observations were undertaken after growth in 6–9 h of light following the 12-h dark period, which is when the Corresponding Author: Jan Sˇlapeta, Faculty of Veterinary Science, University of Sydney, McMaster Building B14, New South Wales 2006, Australia—Telephone number: 161 2 9251 2025; FAX number: 161 2 935 17348; e-mail: [email protected]

RESULTS AND DISCUSSION Both nutrient levels and salinity significantly affected the degree of the motile–immotile transition in C. velia (F-probability o0.001). An inverse relationship between salinity and the percentage of motile C. velia was observed. In all cultures, the proportion of motile cells declined over time (F-probability o0.001). The highest proportion of motile C. velia was in lower nutrient concentrations (Fig. 1A) while no nutrients and 5fmedium inhibited motile transformation. Salinity levels from 20 to 60 g/L were suitable for the immotile–motile transformation of C. velia (Fig. 1B), but the percent of motile cells dropped dramatically at 0 and 80 g/L salt. Salinity of 20 g/L promoted the highest level of motile C. velia with a mean percent of flagellates of 39.8  16.4% over days 2–8. Salinity, culture age, and salinity–time interaction significantly affected the percentage of motile cells (F-probability for all treatments o0.001). Apart from the negative controls, all f-medium and salinity concentrations gave a positive growth rate (data not shown). Motility is likely to be important in marine microorganisms for maintaining cells in favourable environments, for moving cells to more favourable environments or for seeking a compatible mate in order to undergo sexual reproduction. Salinity and nutrient levels are key variables in marine ecosystems that can change due to depth, rainfall, proximity to land, and the presence of other organisms. Thus, organisms with the capacity for movement may respond actively to these changes. To date, C. velia has only been isolated from corals and it may form an association with them similar to the dinoflagellate genus Symbiodinium, which lives in an endosymbiotic relationship with corals and other marine invertebrates (Moore et al. 2008; Trench 1993). Like

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C. velia, cultured Symbiodinium alternates between a motile flagellated form and a coccoid non-motile form, with the cells becoming motile during the light phase of a diurnal cycle (Fitt and Trench 1983; Freudenthal 1962). In the endosymbiotic state, Symbiodinium cells are arrested in the immotile form by host factors while motile stages have been speculated to be a dispersal or infectious form involved in establishing symbiosis (Koike et al. 2004; Trench 1993). It is possible that in C. velia, the flagellate is also involved in initiating infection and the symbiotic process with corals or other marine organisms, but further research is required to assess this interaction. It is yet to be demonstrated whether C. velia undergoes sexual reproduction. Laboratory studies with toxin-producing Gymnodinium dinoflagellates have suggested that sexuality is stimulated when the intracellular nutrient concentrations fall to a threshold value (Blackburn, Hallegraeff, and Bolch 1989; Ellegaard, Kulis, and Anderson 1998). For C. velia the level of transformation into the motile form was higher in low nutrient media (i.e. f/4 and f/2) compared with the higher nutrient concentrations, suggesting nutrient limitation might trigger flagellation. It is also likely that other factors in the medium influence the transformation to the flagellate form. Oxygen levels and pH are known to influence the life cycle transformation in the marine stramonopile Chattonella marina and the dinoflagellates Peridinium aciculiferum and Tuberculodinium vancampoa (Liu et al. 2007; Phillips and Fawley 2002; Zonneveld and Susek 2007). This investigation experimentally demonstrates the effect of sea salt and f-medium concentration on the level of immotile– motile transformation in C. velia. The ecological role of the motile stage of C. velia is yet to be resolved. However, the appearance of flagellates in freshly transferred cultures and the fact that the percent of flagellate forms increased in conditions that were not optimal but were not toxic for growth suggest a role in moving C. velia to a more favourable environment. The motile stage was very scarce or absent in the most extreme conditions (i.e. 0 and 80 g/L sea salt; no f-medium and 5f-medium). The experimental system described is a first step in elucidating the significance of the motile stage and its diurnal rhythmicity. ACKNOWLEDGMENTS

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We thank Michelle Robinson, John Yim, and Rene Rowson for technical assistance, Robert Sutak for discussions, Peter Ralph for insights into coral and algal physiology, and Peter Thomson for insights into the statistic analyses. This study was supported by the Australian Research Council, Discovery Project DP0986372.

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Fig. 1. The percentage of motile Chromera velia grown under different f-medium (A) and salinity (B) conditions over 8 days in 96-well plates. Each condition had five replicates on a single plate and each plate was repeated in quadruplicate. The percent of motile C. velia from the photocounting method was calculated (black columns) as a fraction of motile cells from the total number of cells on the photo and also visually estimated by observing each well for 1 min (grey columns); these methods were highly correlated in both experimental treatments: Pearson’s product–moment correlation coefficients were 0.985 and 0.957 for salinity and f-medium concentration, respectively. All observations were made after 6–9 h of light. The average percentage is shown with error bars representing one standard deviation (n 5 4).

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Weatherby, K., Murray, S., Carter, D. & Sˇlapeta, J. 2010. Surface and flagella morphology of the motile form of Chromera velia revealed by field-emission scanning electron microscopy. Protist, doi: 10.1016/ j.protis.2010.02.003. Zonneveld, K. A. F. & Susek, E. 2007. Effects of temperature, light and salinity on cyst production and morphology of Tuberculodinium vancampoae (the resting cyst of Pyrophacus steinii). Rev. Palaeobot. Palynol., 145:77–88. Received: 02/09/10, 02/23/10, 05/28/10, 06/06/10; accepted: 06/08/10