J. Eukaryot. Microbiol., 52(6), 2005 pp. 492–499 r 2005 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2005.00058.x Assessment of the Cell Viability of Cultured Perkinsus marinus (Perkinsea), a Parasitic Protozoan of the Eastern Oyster, Crassostrea virginica, Using SYBRgreen–Propidium Iodide Double Staining and Flow Cytometry PHILIPPE SOUDANT,a,1 FU-LIN E. CHUb and ERIC D. LUNDb aLEMAR, UMR 6539, IUEM-UBO, Technopole Brest-Iroise, Place Nicolas Copernic, 29280 Plouzane´, France, and bVirginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062, USA ABSTRACT. A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. mari- nus meront cells, cultivated at 28 1C, were incubated in triplicate for 30 min at À 80 1C, À 20 1C, 5 1C, and 20 1C for cold-shock treatments and at 32 1C, 36 1C, 40 1C, 44 1C, 48 1C, 52 1C, and 60 1C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at À 20 1C and the lowest PVC was obtained at À 80 1C (54.0%). After 30 min of heat shocks at 40 1C and 44 1C, PVC decreased slightly but significantly compared to cells maintained at 28 1C. When cells were heat shocked at 48 1C, 52 1C, and 60 1C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at 44 1C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11–25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells. Key Words. Cell growth, cell mortality, culture phases, cytomorphology, temperature tolerance. UMEROUS methodologies have been described and em- P. marinus. Four life-cycle stages—trophozoites (meronts, N ployed for assessments of number and viability of in vitro merozoites), prezoosporangium (hypnospores), zoosporangium, cultured cells and microorganisms. While microscopic methods and biflagellated zoospores—have been identified and described are simple and accommodating, they are not effective for studies (Perkins 1966, 1988). Trophozoites are considered to be the pri- that require numeration and cell viability measurement of a large mary agents for disease transmission (Chu 1996; Perkins 1988). In number of samples. In this context, analysis by flow cytometry culture, cell number and morphology of P. marinus vary depend- (FCM) has advantages over microscopic approaches, since it ing on nutrient composition and culture conditions (see review, allows multi-parametric analyses of a single cell and can be Villalba et al. 2004). Different life-cycle stages and different sizes performed on a large numbers of cells in a very short time of the same stage (e.g. schizonts or mother cells and different sizes (100–1,000 cells/s) (Barbesti et al. 2000; Dias and Lima 2002; of trophozoites) can be found in the same culture (Sunila, Ham- Gre´gori et al. 2001; Kato and Bowman 2002). ilton, and Dungan 2001). In aquatic ecology, FCM is routinely employed for determina- Recently, FCM protocols using nucleic acid double staining, tion of abundance, viability, and activity of viruses (Marie et al. SYBRgreen I or II and propidium iodine (PI), have been devel- 1999), bacteria (Bernard et al. 2001; Bunthof et al. 1999; Lebaron, oped to assess abundance and viability of cultivated bacteria (Bar- Catala, and Parthuisot 1998; Lebaron et al. 2001; Mortimer, besti et al. 2000), bacteria in natural environments (Gre´gori et al. Mason, and Gant 2000; Roth et al. 1997; Williams et al. 1998), 2001), and oyster hemocytes (Delaporte 2005). SYBR green I or II phytoplankton (Gre´gori et al. 2002; Marie et al. 1997; Marie et al. stain the nucleic acids of both live and dead cells while PI stains 2000; Olson, Zettler, and Durand 1993), and planktonic protozo- only nuclei of cells that have lost their membrane integrity (dead ans (Lindstro¨m, Weisse, and Stadler 2002; Parrow and Burkholder cells and cells with damaged membrane). 2002; Wong and Whiteley 1996). Rapid and reliable FCM meth- The objectives of the present study were to test the feasibility ods have also been developed to enumerate and to evaluate of using SYBRgreen–PI double staining with FCM to measure growth, viability, infectivity, and metabolic activities of gastro- cell number, viability, size, and complexity of cultured P. marinus enteric protozoan parasites, such as Cryptosporidium spp. and meront cells under different temperature-shock treatments and Giardia spp. (Campbell, Robertson, and Smith 1992; Foster et al. at different growth phases. Additionally, the assay, which uses 2003; Kato and Bowman 2002; Medema et al. 1998; Schupp and 3,(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyphenyl)-2-(4- Erlandsen 1987; Vesey et al. 1994; see also Quintero-Betancourt, sulfophenyl)-2H-tetrazolium and phenazine ethosulfate (MTS/ Peele, and Rose 2002 for review), and of intra-erythrocytic pro- PMS) to assess cell viability and proliferation based on dehydro- tozoans, such as Plasmodium falciparum (Srivastava, Rottenberg, genation of oxidizable substrates by dehydrogenase of live cells and Vaidya 1997), Babesia bovis (Wyatt, Goff, and Davis 1991), coupled with the reduction of the tetrazolium dye, was utilized as Leishmania amazonensis (Bertho, Cysne, and Coutinho 1992; a comparison for the viability measurement by FCM. Due to the Guinet et al. 2000), and Theileria sergenti (Yagi et al. 2000). great importance of temperature on disease progression in the field Perkinsus marinus is one of the two important parasites of the (Burreson and Ragone-Calvo 1996), the effects on P. marinus Eastern oyster, Crassostrea virginica, and presently the disease meronts of in vitro exposure to high and low temperature may caused by this parasite is most prevalent among Eastern oyster provide insight into the epidemiology of this disease. populations along the east and Gulf coasts of the United States. Temperature and salinity are the two important environmental factors affecting the progression and geographic distribution of MATERIALS AND METHODS 1 In vitro cell culture. Meronts of P. marinus were cultivated Philippe Soudant was a collaborator via a fellowship under the as previously described (Chu et al. 2002) in the medium defined OECD Co-operative Research Programme: Biological Resource Man- agement for Sustainable Agriculture Systems. by La Peyre, Faisal, and Burreson (1993). The medium was pre- Corresponding Author: F.-L. E. Chu, Virginia Institute of Marine pared with artificial seawater (ASW) and adjusted to an os- Science, College of William and Mary, Gloucester Point, Virginia molarity of 590 (equivalent to salinity 20 psu, Lund, Chu, and 23062, USA—Telephone number: 1-804-684-7349; FAX number: Harvey 2004), then sterilized by 0.2 mm filtration, and stored 1-804-684-7186; e-mail: [email protected] at 4 1C until use. Meronts were inoculated (1 Â 106 cells/ml) in 492 SOUDANT ET AL.—VIABILITY OF PERKINSUS MARINUS BY FLOW CYTOMETRY 493 10-ml aliquots of medium in T-10 tissue culture flasks and culti- tween 0.2 and 1.0 Â 106 cells/ml were analyzed. Data acquisition vated at 28 1C. was made in logarithmic mode (four decades). Treatment of the FCM data was performed with the software Experiments WinMDI v. 2.8 (Joseph Trotterr). Biparametric representations Cold and heat shocks. Seven-day-old meront cell cultures (density plots) of ethanol-fixed cells (Fig. 1A) and live cells (Fig. grown at 28 1C were sampled and distributed (2 ml) in equivalent 1D) showed that the P. marinus cells are homogeneously distrib- numbers (3.1 Æ 0.2 Â 106 cells/ml for the cold shock and uted in terms of Forward SCatter (FSC) and Side SCatter (SSC) 4.3 Æ 0.3 Â 106 cells/ml for the heat shock) in polypropylene parameters and were easily defined by a circular region. FSC, tubes (capacity 5 15.0 ml). Cells were incubated for 30 min in corresponding to the diffracted light on the small angle (detected triplicate for cold-shock treatments at À 80 1C, À 20 1C, 5 1C, and in line with the incident light source), is proportional to the size. 20 1C and for heat-shock treatments at 32 1C, 36 1C, 40 1C, 44 1C, SSC, corresponding to the diffracted light on the right angle, is 48 1C, 52 1C, and 60 1C. The meront cultivation temperature of proportional to the cell complexity or granular content. Geometric 28 1C was used as the control (reference) temperature. After in- means of these parameters were used to characterize, in a relative cubation (30 min) was terminated and as samples were cooled manner, cell size and complexity of P. marinus, and expressed as down or warmed up to room temperature, all cell suspensions Arbitrary Units (AU). After gating cells on R1 of the SSC-FSC were filtered through an 80-mm mesh to eliminate large pieces of density plot, FL1 fluorescence using a histogram allowed visual- debris. Then, 100 ml of the resulting cell suspension from each ization of the SYBRgreen-stained particles, which are either replicate of each treatment were pipetted into a tube containing live or dead cells (Fig. 1B, E). After secondary gating of the 500 ml of 20 psu artificial sea water (ASW), equivalent to the os- SYBRgreen-stained cells on R2 of the FL1 histogram, the FL3 molarity of the P. marinus culture medium.