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 marinus (), a Parasitic Protozoan of the Eastern , 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 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 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 falciparum (Srivastava, Rottenberg, genation of oxidizable substrates by dehydrogenase of live cells and Vaidya 1997), 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 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 , 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. fluorescence histogram allowed visualization of the SYBR- and Meront cells at different ages. Meront cell cultures 7, 14, PI-stained cells, which were considered dead (Fig. 1C, F). 25, 28, 38, 42, and 47 days old were sampled and processed as The FL1 histogram of R1-gated ethanol-fixed cells showed a aforementioned. large peak of green fluorescence corresponding to DNA contain- Analysis of viability, size, and complexity of P. marinus ing P. marinus cells (Fig. 1B). These cells accounted for 95.4% of meront cells by FCM. Two nucleic acid fluorescent dyes were the events presented in R1. After gating on both R1 and R2, the used simultaneously for this assay: the permeant SYBRgreen I FL3 histogram of the ethanol-fixed cells also showed a large peak (ex 5 497 nm, em 5 520 nm, S-7563; Molecular Probes, Eugene, corresponding to cells that incorporated PI (Fig. 1C). All cells OR) and the impermeant propidium iodide (PI, ex 5 535 nm, with a red fluorescence above 30 were considered as dead cells, em 5 617 nm, P4170; Sigma, St. Louis, MO). The permeant presumably due to the loss of membrane integrity. The M2 marker SYBRgreen I purchased from Molecular Probes was a showed that 99% of the ethanol-fixed cells were dead. The SYBR- 10,000 Â concentrate stock solution; no information on either mass green peak of fresh (non-fixed) cells prepared from the same cul- or molar concentration was provided from the manufacturer. SYBR ture was similar to that obtained with fixed cells (Fig. 1E). stains both live and dead cells and PI stains only cells that have lost However, the PI peak was smaller than for the fixed cells (cf. membrane integrity. Thus, SYBR fluorescence on the yellow-flu- Fig. 1C, E), and was used to measure the percentage of dead cells orescence detector (FL1) differentiates live and dead P. marinus in fresh cell preparations (Fig. 1F). In this sample, cells with a red cells from other particles (e.g. cell debris) present in the medium fluorescence above 30 (AU) accounted for 40.2% of SYBR- and from instrument ‘‘noise,’’ while PI allows the detection of dead stained cells. Therefore, it is estimated that the assayed culture cells of P. marinus on the red-fluorescence detector (FL3). contained 40% dead cells (or 60% of viable cells). To estimate Dead meront cells, prepared by killing the meront cells with cell concentration of the cell suspension analyzed by FCM, the absolute ethanol, were used as negative controls for viability flow rate of the instrument was measured using a known concen- measurement. Briefly, 1 ml of cell suspension was transferred tration of fluorospheres (fluorescent flow-count fluorospheres, slowly to 4 ml of absolute ethanol at À 20 1C while vortexing at Coulter PN6605359). top speed. Cells were incubated at À 20 1C for 15 min, pelleted by MTS/PMS assay. The effects of temperature shocks on the centrifugation, and then resuspended in 1 ml of 0.2-mm filtered viability of P. marinus meronts were also assessed using the MTS/ ASW at room temperature. The cells were allowed to rehydrate PMS assay. The MTS/PMS assay assesses cell viability and pro- for 15 min prior to staining. liferation based on dehydrogenation of oxidizable substrates by Fifteen minutes prior to analysis, 6 ml SYBRgreen I (final con- dehydrogenase of live cells coupled with the reduction of the tet- centration:1/10,000 of the commercial stock solution) and PI (fi- razolium dye. It indirectly measures the number of viable cells nal concentration: 10 mg/ml) were added to the cell suspension. since the amount of mitochondrial dehydrogenase present in the FCM measurements of double-stained meront cells were carried samples is directly proportional to the number of viable cells. This out using a Beckman Coulters EPICSs AltraTM flow cytometer assay has been applied to measure viability and proliferation of (Miami, FL) connected to a computer using PC Expo32 as data other cultured cells, including P. marinus (Dungan and Hamilton acquisition software. Optical alignment and stability were moni- 1995). tored daily using 10-mm diam. fluorescent flow-count fluoro- The 3,(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyphenyl)-2- spheres (Coulter PN6605359). To adjust the FCM settings fixed (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was obtained (dead) and live cell samples stained with SYBRgreen I, PI or from Promega (CellTiter 96 AQ, Cat.#G1111, Madison, WI) and SYBRgreen I1PI were assayed. Cells with single stain (SYBR- phenazine methosulfate (PMS) was obtained from Sigma (P9625, green I or PI) allowed adjusting the electronic compensations be- St. Louis, MO). MTS and PMS reagents were prepared according tween detectors (FL1 versus FL3). Non-stained cell samples were to manufacturer’s instructions (Promega, CellTiter 96 Aqueous also assayed to ensure that cell auto-fluorescence remained below Non-Radioactive Cell Proliferation Assay, Technical Bulletin 10 on both detectors. #169, Promega Corporation, Madison, WI). Briefly, 20 ml of the Meront cell samples from cold- and heat-shock treatments, and MTS/PMS working solution were added to 100 ml of cell suspen- at different ages (see above in the ‘‘experiments’’ section) were sion in a flat-bottomed 96-well microtiter plate. The micro-plate double stained with SYBRgreen I1PI. Generally, acquisition time was then incubated for 3 h at 28 1C in the dark. The absorbance of 30 s was found sufficient to obtain between 2,000 and 10,000 (optical density, OD) of formazan, the reduction product of analyzed cells. In all the assays, meront cell concentrations be- MTS/PMS, was measured at 450 nm using a Tecan GENios 494 J. EUKARYOT. MICROBIOL., VOL. 52, NO. 6, NOVEMBER– DECEMBER 2005

ab104 Gates: R1 256 R2 103 P. marinus M1 R1 M1=95.4% of R1

102 Complexity Events

101

100 0 100 101 102 103 104 100 101 102 103 104 Size Green fluorescence

d 104 c Gates: R1*R2 258 103 P. marinus M2 R1 M2=99.9% of R2*R1

102 Complexity Events

101

0 100 100 101 102 103 104 100 101 102 103 104 Red fluorescence Size

efGates: R1*R2 Gates: R1 512 512 R2

M2 M1 M2=96.7% of R1 M2=40.2% of R2*R1 Events Events

0 0 100 101 102 103 104 100 101 102 103 104 Green fluorescence Red fluorescence Fig. 1. Forward Scatter and Side Scatter density plot (A and D), yellow-green fluorescence histogram (B and E), and red fluorescence histogram (C and F) of ethanol-fixed and live meronts of Perkinsus marinus, respectively, stained with SYBRgreen1PI. SOUDANT ET AL.—VIABILITY OF PERKINSUS MARINUS BY FLOW CYTOMETRY 495 micro-plate reader (Maennedorf, Switzerland). Instrument con- (Po0.001) correlated [R2 5 96.4; PVC(MTS/PMS) 5 6.911 trol and data acquisition by MS-ExcelTM were provided by the 0.956 Â PVC(FCM)]. computer software XFLUOR4 V 4.50 (Tecan, Maennedorf, Swit- Viability, size, and complexity after heat shocks. Cell via- zerland). Samples used for the MTS/PMS assay were from the bility measured by FCM (Fig. 4A). The PVC was slightly higher same stocks as those measured by FCM and assayed at two dilu- than 93% from 28 1Cto361C. Slight but significant decreases in tions: 1 and 1/5. The OD value of the 1/5 dilution was used when PVC occurred at 40 1C and 44 1C: the PVC at 40 1C and 44 1C OD values of non-diluted cell suspensions were above 1.0, and were 91.5% and 84.4%, respectively. PVC was reduced to 33.8% corrected accordingly with the dilution factor. As the number of after heat shock of 48 1C for 30 min. When meronts were heat cells used for the assay was identical at all temperatures, the per- shocked at 52 1C and 60 1C for 30 min, heavy mortality occurred centage of viable cells at different temperature shocks was ex- and PVC decreased to 8.0% and 3.4%, respectively. Cell com- pressed as the percentage of the OD measured at the control/ plexity was relatively stable between 28 1C and 44 1C and ranged reference temperature of 28 1C according to the formula OD at from 60.4 to 64.8 AU. Similarly, meront sizes were constant, different temperatures/OD at 28 1C Â 100%. ranging from 68.7 to 70.9 AU between 28 1C and 40 1C. However, Statistical analysis. Data were analyzed statistically using the both cell complexity and size changed when meronts were heat software STATGRAPHICS v.4.1. One factor analysis of variance shocked at  44 1C. Cell size decreased, but cell complexity in- (ANOVA) was utilized to compare the differences (1) of percent- creased, significantly with increasing incubation temperatures age of viable cells, (2) of morphological characteristics (size and (Table 2). complexity as measured, respectively, by the FSC and SSC pa- Cell viability measured by MTS/PMS assay (Fig. rameters) under different temperature-shock treatments, (3) of the 4B). The percentage of viable cells was  95% between 32 1C cell concentration, (4) of the percentage of viable cells, and (5) of and 40 1C (Fig. 4B), but the PVC decreased significantly when morphological characteristics (FSC and SSC) of meront cultures meronts were heat shocked at 44 1C and further decreased to at different ages. Differences were considered significant when P- 10.1% at 60 1C. Similarly to the cold-shock treatment, percentages value was o0.05. When results were significant at the Po0.05, of viable cells measured by the MTS/PMS assay paralleled those Fisher’s least significant difference (LSD) procedure (multiple range test) was used to discriminate among the means. a 128 Gates: R1 RESULTS FCM analysis of P. marinus cells stained with SYBRgreen or PI and SYBRgreen1PI. When the FL1 fluorescence inten- sity of cells stained with SYBRgreen I1PI was compared to cells stained with only SYBRgreen I (Fig. 2A), FL1 was reduced by 2.5-fold. In contrast, FL3 fluorescence intensity of cells stained with SYBRgreen I1PI was increased by 2.7-fold when compared SYBRgreen+PI SYBRgreen with cells stained with only PI (Fig. 2B). The observed phenom- Events ena are called ‘‘fluorescence resonance energy transfers’’ or more trivially ‘‘quenching.’’ It occurs between two nucleic acid fluoro- chromes when they are both closely bound to the nucleic acid.

Cell viability, size, and complexity after cold shocks. Cell viability measured by FCM (Fig. 3A). Meront cell viability was 0 0 1 2 3 4 expressed as percent of viable cells (PVC). PVC of 7 day-old 10 10 10 10 10 meronts (in suspension) incubated at the control/culture temper- Green fluorescence ature of 28 1C for 30 min was 93.7%. Incubation of meronts at 20 1C and 5 1C for 30 min did not change the cell viability com- b 128 pared to the control. But a slight decrease of PVC (92.7%) was observed after 30 min at À 20 1C and the lowest PVC was ob- tained after 30 min at À 80 1C (54.0%). Thirty-minute tempera- ture shocks at 20 1C, 5 1C, À 20 1C, and À 80 1C did not change the mean P. marinus cell size and complexity as assessed by the PI SYBPgreen+PI FSC and SSC parameters, respectively. Size ranged from 81.5 to 88.7 AU and complexity ranged from 85.7 to 91.0 AU (Table 1).

Cell viability measured by MTS/PMS assay (Fig. Events 3B). With the MTS/PMS assay, incubation of meronts at 20 1C and 5 1C for 30 min did not reduce cell viability compared to those incubated at the control temperature, but PVC decreased signif- icantly, almost by half, after a temperature shock at À 20 1C for 30 min. This is different from the result obtained using FCM: only a slight decrease of PVC was noted after a temperature shock 0 at À 20 1C for 30 min (cf. Fig. 3A, B). With a temperature shock 100 101 102 103 104 of À 80 1C, meront viability was significantly reduced by 5-fold Red fluorescence 1 compared to the control reference temperature of 28 C and Fig. 2. A. Yellow-green fluorescence (FL1) of ethanol-fixed meronts by 3-fold compared to the viability at À 20 1C. Overall, the per- of Perkinsus marinus stained with SYBRgreen1PI (empty histogram) or centage of viable cells as measured by the MTS/PMS assay fol- SYBRgreen alone (solid histogram); B. Red fluorescence of ethanol-fixed lowed the same pattern as the percentage of viable cells (PVC) meronts of P. marinus stained with with SYBRgreen1PI (empty histo- measured by FCM. These two measurements were significantly gram) or PI alone (solid histogram). 496 J. EUKARYOT. MICROBIOL., VOL. 52, NO. 6, NOVEMBER– DECEMBER 2005

a a

a a a b c

d

e f

b b ab a ab b

c

d

e e

Fig. 3. Cell viability of Perkinsus marinus meronts (Mean, SD, n 5 3) Fig. 4. Cell viability of Perkinsus marinus meronts (mean, SD, n 5 3) at different cold-shock temperatures as measured by SYBRgreen/PI dou- at different heat-shock temperatures, as measured by SYBRgreen/PI dou- ble-staining flow cytometry assay (A) and by MTS/PMS micro-plate assay ble-staining flow cytometry assay (A) and by MTS/PMS micro-plate assay (B). The percentage of cell viability is expressed as percentage of the OD (B). The percentage of cell viability is expressed as a percentage of the OD at the control/reference temperature of 28 1C, measured at 450 nm (OD at at the control/reference temperature of 28 1C, measured at 450 nm (OD at different temperatures/OD at 28 1C Â 100). Different letters denote sig- different temperatures/OD at 28 1C Â 100). Different letters denote sig- nificant differences at Po0.05 among treatments. nificant differences at Po0.05 among treatments.

6 2 centration of 5.9 Æ 1.8 Â 10 cells/ml was recorded. The PVC measured by FCM (R 5 93.1; Po0.001; PVC(MTS/PMS) 5 1[(0.099910.0009) Â PVC(FCM)). decreased steadily and significantly from 93.6% at 7 d to 79.0% at Meront viability and morphology at different growth phas- 25 d (Fig. 5), and then reached a plateau varying at 11.2–24.5% es. Cell concentration (Table 3) increased rapidly between day 7 6 6 and day 14 from 2.9 Æ 1.2 Â 10 to 8.1 Æ 0.9 Â 10 cells/ml. Table 2. Cell size and cell complexity of Perkinsus marinus meronts at There was no significant change in cell concentrations from day different heat-shock temperatures, as measured, respectively, by Forward 14 to day 47 with the exception of day 25 at which a cell con- Scatter for cell size and Side Scatter parameters for cell complexity (mean, SD, n 5 3).

Table 1. Cell size and cell complexity of Perkinsus marinus meronts at Temperature ( 1C) Size (AU) Complexity (AU) different cold-shock temperatures, as measured, respectively, by Forward Scatter for cell size and Side Scatter parameters for cell complexity (mean, Mean SD Mean SD SD, n 5 3). 28 69.1a 2.3 64.8a 2.1 32 70.9a 1.1 63.6a 1.7 a a Temperature ( 1C) Size (AU) Complexity (AU) 36 69.3 1.0 61.1 1.2 40 68.7a 0.9 60.4a 1.3 Mean SD Mean SD 44 51.4c 0.5 63.6a 1.1 c b 28 86.1 4.4 90.1 5.5 48 51.0 1.3 81.1 2.7 52 46.8b 1.6 88.3c 2.4 20 88.7 4.8 89.8 2.6 c d 5 85.8 4.5 86.6 5.1 60 50.3 3.0 123.9 4.9 À 20 84.4 3.9 91.0 3.5 À 80 84.6 0.6 85.7 0.5 Different letters denote significantly different at Po0.05 among treat- ments. SOUDANT ET AL.—VIABILITY OF PERKINSUS MARINUS BY FLOW CYTOMETRY 497

protozoan, may reflect the difference in chromosomal structures between prokaryote cells and unicellular cells. Experimental infections revealed that prevalence and intensity a b of P. marinus in are positively correlated with temperature c (Chu 1996; Chu et al. 1993; Chu and La Peyre 1993). Generally, P. marinus proliferates and develops rapidly at high temperatures between 20 1C and 30 1C (Chu and Greene 1989; Chu, Soudant, and Lund 2003; Dungan and Hamilton 1995; Gauthier and Vasta d 1995; Volety and Chu 1997). Dungan and Hamilton (1995) showed that P. marinus could proliferate between 10 1C and f f 40 1C, while no proliferation occurred at 4 1C. On the other hand, Gauthier and Vasta (1995) did not report significant differences in e proliferation between 4 1C and 15 1C. Lower metabolic activity in meronts was also observed at lower temperature. Chu et al. (2003) demonstrated that the uptake and assimilation of exogenous lipids and triacylglycerol lipase activities was drastically reduced in meronts of P. marinus grown for 7 d at 10 1C as compared to those cultivated at 18 1C and 28 1C. Fig 5. Cell viability of Perkinsus marinus meronts at different growth Perkinsus marinus appeared to be more sensitive to heat shock phases (culture days) as measured by SYBRgreen/PI double-staining flow than to cold shock. Cold shock of meront cells at 5 1C did not cytometry assay (mean, SD, n 5 3). Different letters denote significant produce significant cell death compared to their cultivation tem- differences at Po0.05 between culture days. perature of 28 1C. A cold-shock treatment of meront cells at À 20 1C for 30 min only induced 1% increase of cell mortality (percentages of viable cells were 92.6% at À 20 1C versus 93.6% between 38 and 47 d. Morphological parameters (Table 3) were at 28 1C). However, a temperature as low as À 80 1C killed cells also significantly different among culture ages. Cell size and rapidly with a mortality more than 50%. complexity decreased rapidly from 7 to 28 d, and then no signif- Also, cold temperature shocks did not cause a change in cell icant change occurred afterward. size and complexity as compared to those maintained at 28 1C. The observed tolerance to cold temperature in meronts of P. mari- nus was not surprising, since P. marinus can be easily cryo-pre- DISCUSSION served (Dungan and Hamilton 1995; Gauthier and Vasta 1995). Chu and Greene (1989) showed that hyponospores of P. marinus Results from the present study indicate that it is feasible to as- could tolerate temperatures as low as 4 1C for 4–7 d. The tested sess cell viability, size, and complexity of meronts of P. marinus heat-shock temperatures of 32 1C, 36 1C, 40 1C, 44 1C, 48 1C, and using FCM with double nucleic acid stains (i.e. SYBRgreen I and 52 1C are comparable to those encountered by P. marinus within propidium iodide, PI). Treating P. marinus meronts with 100% its host oyster in the field. Oysters are ectothermic with body ethanol apparently killed almost all the cells. Nearly 100% of the temperatures close to the ambient environment. For those living in ethanol-treated cells took up the PI base on FCM analysis. When the intertidal zone, a rapidly fluctuating environment could expe- combined with SYBRgreen, the magnification of PI fluorescence rience temperatures as high as 55 1C during low-tide exposure (2.7-fold) was due to fluorescence resonance energy transfer (also (Willson and Burnett 2000). called quenching) from SYBRgreen to PI. This phenomenon oc- Indeed, temperatures in the intertidal may exceed the thermal curs between two nucleic acid fluorochromes when they are both tolerances of P. marinus (Bushek et al. 2002). Results of our study closely bound to the nucleic acid (Barbesti et al. 2000). However, are in agreement with the findings of previous studies that tem- the quenching from SYBRgreen I to PI obtained with the P. mari- peratures higher than 35 1C inhibited the proliferation and induced nus cells was not as high as those observed when applied to bac- mortality in P. marinus (Dungan and Hamilton 1995; La Peyre teria (Barbesti et al. 2000; Gre´gori et al. 2001). This difference in and Faisal 1996). As heat-shock temperature rose to 36–40 1C, quenching intensity between bacteria and P. marinus, a parasitic mortality began and increased dramatically from 40 1Cto441C. All these results suggest that P. marinus is less thermal tolerant than its host, the Eastern oyster. Galtsoff (1964) reported survival Table 3. Cell size and cell complexity of Perkinsus marinus meronts at 1 different growth phases, as measured, respectively, by Forward Scatter of emersed intertidal oysters at 46–49 C for up to 2–3 h. Ingle for cell size and Side scatter parameters for cell complexity (mean, SD, et al. (1971) also reported survival of intertidal Gulf oysters at n 5 2–4). 49.5 1C. In a recent study, we noted that no mortality occurred in two different geographic oyster stocks after heat shocks at 41 1C 1 Days of Size Complexity Cell concentration and 42 C for 1 h (Encomio and Chu 2003; Encomio 2004). culture (AU) (AU) (cell/ml) The significant increase of cell complexity in P. marinus after heat shock at temperatures of 48 1C and above may be due to Mean SD Mean SD Mean SD protein denaturation as protein denaturation is known to increase 7 86.1a 4.4 90.1a 5.5 2.9 Â 106a 1.2 Â 106 cell complexity and light scattering in mammalian models (Le- 14 60.8b 3.5 50.5b 2.4 8.1 Â 106b 0.9 Â 106 Pock 2005). Moreover, Tirard et al. (1995) showed that induction 25 63.0b 8.6 60.6c 11.5 5.9 Â 106c 1.8 Â 106 c bd 6d 6 of heat shock proteins (Hsp), which are produced to limit protein 28 43.3 2.0 40.9 2.4 10.3 Â 10 1.2 Â 10 denaturation, occurred above 46 1CinP. marinus. These authors 38 43.9c 4.5 43.0bd 1.7 9.6 Â 106bd 0.2 Â 106 c d 6d 6 also observed that Hsp was produced at a higher temperature of 42 43.5 1.4 37.2 1.8 10.4 Â 10 0.7 Â 10 1 47 46.4c 1.4 39.2d 1.0 9.4 Â 106bd 0.4 Â 106 46 CinP. marinus than in C. virginica, which was induced from 39 1Cto411C. Based on these observations, they concluded that Different letters indicate the significant differences between culture P. marinus was more thermo-tolerant than the oyster. However, phases. one could also argue that an inability to mount a rapid Hsp 498 J. EUKARYOT. MICROBIOL., VOL. 52, NO. 6, NOVEMBER– DECEMBER 2005 production in response to temperature shock could result in de- 0131108). Contribution number 2681 from the Virginia Institute creased survival at high temperatures. Additionally, Tirard et al. of Marine Science, College of William and Mary, USA and Con- (1995) did not monitor the viability of the cells on which they tribution number 962 of the IUEM, European Institute for Marine measured their Hsp production. Studies, Brest, France, are acknowledged. The cell cultures of P. marinus at stationary phase exhibited a surprisingly high cell mortality. Thus, the mortality occurring in aging cell cultures may result in bias in studies aiming to examine LITERATURE CITED cellular and metabolic activities of different phases of the growth Barbesti, S., Citterio, S., Labra, M., Baroni, M. D., Neri, M. G. & Sgorbati, cycle of P. marinus cultures. We also observed that the cell mor- S. 2000. Two and three-color fluorescence flow cytometry analysis of tality at stationary phase varied among batches of cell culture immunoidentified viable bacteria. Cytometry, 40:214–218. (unpubl. data). Thus, cell viability measurement is recommended Bernard, L., Courties, C., Duperray, C., Scha¨fer, H., Muyzer, G. & Le- for any future cellular and metabolic study. Baron, P. 2001. A new approach to determine the genetic diversity of In addition to directly counting cells employing FCM and mi- viable and active bacteria in aquatic ecosystems. Cytometry, 43: croscopy, several bioassays have been described for assessment of 314–321. cell viability and/or proliferation in in vitro culture. These assays Bertho, A. L., Cysne, L. & Coutinho, S. G. 1992. Flow cytometry in the include measurements using neutral red uptake, reduction of a study of the interaction between murine macrophages and the protozoan parasite Leishmania amazonensis. J. Parasitol., 78:666–671. tetrazolium dye, and uptake of radioactively labeled thymidine 3 Borenfreund, E. & Puerner, J. A. 1985. Toxicity determined in vitro by ( H-thymidine) (e.g. Borenfreund and Puerner 1985; Buttke, morphological alterations and neutral red absorption. Toxicol Lett., McCubrey, and Owen 1993; Dungan and Hamilton 1995; Gillis 24:119–24. et al. 1978; Green, Reade, and Ware 1984; Volety and Chu 1997). Bunthof, C. J., Van den Braak, S., Breeuwer, P., Rombouts, F. M. & Abee, These assays provide quantitative measurements of live cells T. 1999. Rapid fluorescence assessment of the viability of stressed based on their biological functions: uptake of neutral red via pin- Lactobacillus lactis. Appl. Environ. Microbiol., 65:3681–3689. ocytosis (neutral red assay); dehydrogenation of oxidizable subst- Burreson, E. M. & Ragone-Calvo, L. M. 1996. Epizootiology of Perkinsus rates by dehydrogenase coupled with the reduction of the marinus disease of oysters in , with emphasis on data tetrazolium dye (MTS/PMS assay); and uptake of 3H-thymidine since 1985. J. Shellfish. Res., 15:17–34. Bushek, D., Porter, D., Coen, L. D., Bobo, M. Y. & Richardson, D. L. into cellular DNA. The results of cell viability measurement em- 2002. Status and trends of Dermo and MSX in South Carolina. J. Shell- ploying FCM are in good agreement with those obtained from the fish Res., 21:386. MTS/PMS assay, based on their highly significant correlation. Buttke, T. M., McCubrey, J. A. & Owen, T. C. 1993. Use of an aqueous Nevertheless, these two assays assess two different biological soluble tetrazolium/formazan assay to measure viability and prolifera- functions related to cell viability: mitochondrial dehydrogenase tion of lymphokine-dependent cell lines. J. Immunol. Methods, for the MTS/PMS assay and loss of membrane integrity for the 157:233–240. FCM assay. The MTS/PMS assay does not directly measure cell Campbell, A. T., Robertson, L. J. & Smith, H. V. 1992. Viability of viability, but provides an estimation of cell viability via the meas- Cryptosporidium parvum oocysts: correlation of in vitro excystation urement of mitochondrial NADH- and NADPH-dependent de- with inclusion or exclusion of fluorogenic vital dyes. Appl. Environ. Microbiol., 58:3488–3493. hydrogenase activities in living cells. It can be noted that the Chu, F.-L. E. 1996. Laboratory investigations of susceptibility, infectivity MTS/PMS assay detected a greater effect on parasite viability and transmission of Perkinsus marinus in oysters. J. Shellfish. Res., compared with the FCM assay for cold shocks at À 20 1Cto 15:57–66. À 80 1C and for heat shocks at 44–48 1C. So, it is likely that met- Chu, F.-L. E. & Greene, K. H. 1989. Effect of temperature and salinity on abolic activities such as mitochondrial dehydrogenase are firstly in vitro culture of the oyster , Perkinsus marinus (Apicom- affected by thermal stress and that lost of membrane integrity oc- plexa: Perkinsea). J. Invert. Pathol., 53:260–268. curs with the increased intensity of the stress. Employing the Chu, F.-L. E. & La Peyre, J. F. 1993. Perkinsus marinus susceptibility and SYBRgreen–PI double staining facilitates the direct measure- defense-related activities in Eastern oysters, Crassostrea virginica: tem- ments of living and dead cells, and cells with damaged mem- perature effects. Dis. Aquat. Org., 16:223–234. Chu, F.-L. E., Soudant, P. & Lund, E. D. 2003. Perkinsus marinus, a pro- branes, with additional information concerning cell size and tozoan parasite of Eastern oyster (Crassostrea virginica): effects of complexity changes. The time-consuming microscopic trypan temperature on the uptake and metabolism of fluorescent lipid analogs blue exclusion assay also directly gives measurement of cell vi- and lipase activities. Exp. Parasitol., 105:121–130. ability. However, in addition to the potential problem of ineffi- Chu, F.-L. E., Burreson, C. S., Volety, A. K. & Constantin, G. 1993. Per- cient uptake of the dye by the nucleus of dead meronts, kinsus marinus susceptibility in Eastern (Crassostrea virginica) and considering the morphology (e.g. eccentric , numerous Pacific (Crassostrea gigas) oysters: temperature and salinity effects. lipid droplets, and small, slightly convex centrifugally located J. Shellfish. Res., 12:360. nucleus) and size (approximately 2–5 mm) of meronts (Perkins Chu, F.-L. E., Lund, E. D., Soudant, P. & Harvey, E. 2002. De novo 1996), it may be difficult to obtain unbiased/correct counting be- arachidonic acid synthesis in Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica. Mol. Biochem. Parasitol., tween live and dead cells. 119:179–190. In summary, the present study demonstrated that it is feasible to Delaporte, M. 2005. Modulation des parame`tres he´mocytaires par la nu- employ FCM to assess cell viability, cell size, and cell complexity trition chez l’huıˆtre creuse Crassostrea gigas. Implication dans les mor- of meronts of P. marinus exposed to different temperature shock talite´s estivales. The`se de doctorat, Biologie et Production animale. treatments and at different growth phases. Thus, FCM assay could Universite´ de Rennes. 370 p. be used routinely to monitor cell viability and morphological Dias, N. & Lima, N. 2002. A comparative study using a fluorescence- changes in P. marinus cultures under different culture conditions. based and a direct-count assay to determine cytotoxicity in Tetrahy- mena pyriformis. Res. Microbiol., 153:313–322. Dungan, C. F. & Hamilton, R. M. 1995. Use of tetrazolium-based cell ACKNOWLEDGMENTS proliferation assay to measure effects of in vitro conditions on Perkin- sus marinus () proliferation. J. Eukaryot. Microbiol., The authors thank Georgeta Constantin and Helen Quinby for 42:379–388. technical assistance. This study was supported in part by a grant Encomio, V. G. 2004. 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