Plankton Benthos Res 8(4): 178–185, 2013 Plankton & Benthos Research © The Plankton Society of Japan

Carbon to volume relationship of () during cell divisions

YUKI ISHIWATA, NOBUAKI OHI, MITSUKO OBATA & SATORU TAGUCHI*

Soka University, Faculty of Engineering, Laboratory of Biological Oceanography, 1–236 Tangi-Cho, Hachioji, Tokyo 192–8577, Japan Received 8 February 2013; Accepted 23 August 2013

Abstract: Dependency of carbon cell quota (C) on cell volume (V) was studied for the Prymnesiophyte Isochrysis galbana. Changes in the cell volume and carbon quota during cell divisions were examined at 25°C in a continuous cul- ture with an enriched f/2 medium under a 12/12 h light/dark cycle at an irradiance of 45, 150, and 365 μmol m－2 sec－1. Carbon quota estimates followed the relation log C=b log V+log a, where b is the slope and log a is the intercept of the relationship. Significant relationships between carbon quota and cell volume during cell divisions were obtained at the three irradiances tested. The value of the slope and the intercept ranged from 0.59 to 0.98 and from 0.22 to －0.64, respectively, however, they were not significantly different between the three irradiances. The relationship between carbon quota to cell volume during cell divisions also corresponded well to previously published carbon quota to cell volume relationship values within the size range of nanophytoplankton ranging from 2 to 20 μm, even though the ir- radiances used in the experiments differed from those studied previously. Due to the close similarity in cell size and division manner between the majority of nanophytoplankton communities and I. galbana, the results of the present study can be applied to natural assemblages of nanophytoplankton to estimate their carbon biomass in the euphotic layer regardless of sampling time on a given day.

Key words: allometry, carbon quota, cell division, cell volume, irradiance

toplankton community structure can be quantified from Introduction estimated biomass (Ishizaka et al. 1997, Shalapyonok et al. Phytoplankton is currently responsible for about 45% of 2001, Cermeño et al. 2005). However, analyzing the com- global primary production (Falkowski & Raven 2007). munity structure of phytoplankton depends on the accu- However, the biomass of phytoplankton amounts to less racy of the determination of carbon quota and cell volume than 1% of the total photosynthetic biomass on our planet. relationships. Estimating phytoplankton biomass has been one of the The first detailed study of the relationship between car- most essential components to understand plankton ecol- bon quota and cell volume was made by Mullin et al. ogy. The difficulty in estimating the amount of phyto- (1966). However, the relationships described to date have planktonic carbon is due to the co-occurrence of other mainly been reported for phytoplankton larger than 20 μm. matter (e.g., bacteria and detritus) with significant frac- Since a substantial contribution to biomass by small phyto- tions of non-phytoplanktonic carbon, so that living phyto- plankton such as nanoplankton (2–20 μm) in oceanic areas planktonic carbon cannot be accurately determined from has been recognized (e.g., Malone 1980), the carbon to vol- total particulate carbon. Carbon is the principal structural ume relationship has been studied extensively for nano- component of phytoplankton and is important for commu- phytoplankton under only one irradiance condition (Verity nity biomass and bioenergetics. Estimates of phytoplank- et al. 1992, Montagnes et al. 1994). The slopes of carbon ton biomass in terms of carbon are usually made using em- quota to cell volume for nanophytoplankton have been pirically derived carbon quota to cell volume relationships found to be greater in this size fraction than in earlier work (e.g. Mullin et al. 1966, Strathmann 1967). Therefore phy- on phytoplankton larger than 20 μm (Mullin et al. 1966). Cell volume and carbon quota is known to change dur- * Corresponding author: Satoru Taguchi; E-mail, [email protected] ing cell division. The carbon quota increases during light Carbon to volume relationship of Isochrysis galbana 179 periods and decreases during dark periods under light and scalar quantum sensor (Model QSL 100, Biospherical In- nutrient saturated conditions in laboratory experiments struments, USA) at 45, 150, and 365 μmol m－2 sec－1. The (e.g. Kohata & Watanabe 1989, Varela & Harrison 1999) light cycle was maintained at a 12/12 h light/dark cycle by except for diatom (Berges et al. 1995). Little is cool-white fluorescent tubes (Panasonic, Osaka, Japan). known about the dependency of carbon quota on cell vol- The growth medium was the natural seawater f/2 formula- ume during cell division (DuRand & Olson 1998). The tion (Guillard & Ryther 1962). The base for the medium changes in cell volume and carbon quota during cell divi- was coastal surface seawater (salinity of 35.0) collected off sions have not been considered when interpreting carbon the Manazuru coast (35°09′49″N, 139°10′33″E). Seawater quota to cell volume relationships in natural assemblages was pre-filtered through 0.2 μm membrane filter (Milli- of nanophytoplankton (Verity et al. 1992, Montagnes et al. pore, USA) with added nutrients, trace metals, and vita- 1994), and collection of field samples for carbon estimation mins at f/2 concentrations. Cultures were continuously di- has been conducted without an awareness of the changes in luted with fresh media at a fixed rate with a peristaltic cell volume and carbon quota during cell division. The car- pump to maintain stationary phase (Tokyo Rikakikai Co. bon quota to cell volume relationship should be determined Ltd., Tokyo, Japan). The culture was acclimatized to each during cell division to obtain precise biomass measure- light condition for two weeks. ments. Cell numbers were determined once a day to confirm Allometric modeling i.e. size scaling, is a powerful ap- synchronized and steady growth. Dilution rates were re- proach for ecological research concerning biological corded daily by measuring culture overflow volumes. Once oceanography (e.g. Banse 1976, Taguchi 1976). Cell vol- steady growth was confirmed, the daily growth rate (μ) ume and carbon quota have been used to estimate growth was calculated using the equation: and respiratory rates in phytoplankton (e.g. Banse 1976, ν Tang 1995). Allometric models deal with cell volume and/ µ = V or carbon quota, and those relationships are assumed to be c constant regardless of the environmental conditions (e.g. where v is the volume of fresh media and Vc is the culture Tang 1995). If this constancy indeed holds, volume mea- volume. Daily growth rates were 0.12 d－1 under surements should be useful for ecological studies. 45 μmol m－2 sec－1, 0.45 d－1 under 150 μmol m－2 sec－1, and For the experiments on carbon quota to cell volume rela- 0.53 d－1 under 365 μmol m－2 sec－1 in the present study. tionships regulated by cell division, we chose the Prymne- Samples were collected for the determination of cell densi- siophyte Isochrysis galbana as a model species because it ties, cell volumes, and particulate organic carbon every is classified as an open-ocean eukaryotic nanophytoplank- two hours for 48 hours to repeat the one day cell division ter based on its size and the division pattern of its cells cycle. Aseptic techniques were used to minimize bacterial (Nelson & Brand 1979, Ohi et al. 2002). We measured cell contamination throughout the incubation experiment. volume and cell carbon quota under a 12/12 h light/dark Cell densities and volumes cycle for 48 hours at three light intensities. We discuss how our results can aid in the understanding of the dependency Samples for the determination of cell density and cell of cell quota on the cell volume during cell division and volume were preserved with 2% formaldehyde. Cell den- the interpretation of the carbon quota to cell volume rela- sity of 200–600 cells was measured under a microscope tionship of the nanophytoplankton community in the field with a hemocytometer (Erma Inc., Tokyo, Japan) at a mag- by comparing our results with published data. nification of 200×. Cell size of 50–100 cells was measured under a microscope at a magnification of 1000× with an ocular ruler calibrated with a micrometer. Cell volumes Materials and Methods (V) were calculated assuming I. galbana is an ellipsoid, by the formula: Culture conditions and sampling π Isochrysis galbana Parke (NEPCC633) was obtained V= rr2 6 12 from The North East Pacific Culture Collection (NEPCC) at the University of British Columbia. It is free-living ellip- where π refers to the circular constant, and r1 and r2 are the soid flagellate cell with a mean equivalent spherical diame- long and short radius of ellipsoid, respectively (Hillebrand ter of 5.1 μm, and a mean volume of 68 μm3. The cultures et al. 1999). were grown in a continuous-culture system in a glass, dou- Since we kept cells with formaldehyde, we conducted ble-walled 1 L volume vessel as described by Laws & Ban- time-series experiments to determine when shrinkage oc- nister (1980). Cultures were kept at a temperature of 25°C curred due to preservatives. We corrected the fixed cell by immersion in circulating water baths, stirred continu- volume to the fresh one, by multiplying the fixed volume ously by a magnetic Teflon-coated stir bar at 200 revolu- by a factor of 1.34 (Iwasawa et al. 2009). tions min－1 and bubbling sterile air through the culture. Photosynthetically active radiation was determined by a 180 Y. Ishiwata et al.

Particulate organic carbon cepts, and their significance, were calculated according to Triplicate subsamples were filtered onto Whatman GF/A Sokal & Rohlf (1995). Comparisons of regression lines glass fiber filters pre-combusted at 500°C for 2 h for the among irradiances were conducted by ANCOVA (Snede- analysis of particulate organic carbon (POC). POC was an- cor & Cochran 1989). alyzed on an elemental analyzer (Fison, Model EA-1108) Published data calibrated using acetanilide as the standard (Nagao et al. 2001). Cellular carbon quotas were calculated by dividing Previously published data on maxima and minima in ei- POC by cell density. Cellular carbon densities were calcu- ther cell volume or carbon quota during cell divisions of lated by dividing carbon quota by cell volume. nanophytoplankton were obtained from the following sources: Eppley & Coatsworth (1966), Kohata & Watanabe Mathematical and statistical analyses (1986, 1989), Berdalet et al. (1992), DuRand & Olson The light and dark maximum or minimum deviation (1998), Varela & Harrison (1999), DuRand et al. (2002), from the mean of the entire 48 hours measures the ampli- and Ohi et al. (2002, 2003). From these sources, we se- tude of the change during cell divisions at the experimental lected the data giving both the cell volume and carbon irradiances. Differences in the averages and amplitudes of quota during cell division: Kohata & Watanabe (1989), the variables during cell division among the different irra- DuRand & Olson (1998), Varela & Harrison (1999) and diances were analyzed using ANOVA. Standard errors of DuRand et al. (2002). The cell volumes and carbon averages and amplitudes were calculated by dividing stan- quotas were then transformed to log scale. When the dard deviation by the square root of the number of data change in cell volume and carbon quota during cell divis- points. ion had a slope, the slope was corrected to zero to obtain The periodicity of the rhythm was analyzed by the sine a maximum and minimum. function based on Kieding et al. (1984) and modified as follows: Results (+tC )π Y= AB + sin  Change in cell volume and carbon quota during cell 12  division where Y is the estimate of a variable during cell division Cell volume and carbon quota of Isochrysis galbana in- (e.g. carbon quota), A is the average, B is the amplitude of creased during the light period and decreased during the the sine function, t is time and C is the difference between dark period at the three irradiances (Figs. 1 and 2). Aver- values at t=0 and t=24. A nonlinear least-squares regres- ages of cell volume and carbon quota over light and dark sion analysis was used to test the degree of fitness of the cycles differed significantly among the three irradiance estimated value to the observed value. Since the amplitude levels (ANOVA; p<0.001), and decreased with increasing might be related to the average, in order to standardize the irradiance as a decaying exponential function (p<0.001 for change during cell division in the amplitude of both cell cell volume, p<0.01 for carbon quota; Fig. 3A). Significant volume and carbon quota, the magnitude of change during linear relationships between the amplitudes of cell volume cell division (MCD) was calculated for the first 24 hour- and carbon quota were obtained with irradiance (p<0.05 and the second 24 hour-period as follows: for cell volume, p<0.01 for carbon quota; Fig. 3B). The magnitude of change during cell division (MCD), a syn- Max－ Min onym for amplitude of cell volume and carbon quota, in- MCD = ×100 (%) Min creased linearly when irradiance ranged from 45 to 365 μmol m－2 sec－1 (p<0.001; Fig. 3C). The slopes of the where Max is the maximum value and Min is the minimum MCD of carbon quota and cell volume to irradiance were value of each variable, respectively. The MCD allows di- not significantly different (ANCOVA, p>0.05). rect comparisons between cell volume and carbon quota. Carbon quota to cell volume relationship during cell Log transformation division Apparent linear relationships between cell volume (V) Significant relationships between carbon quota and cell and carbon quota (C) were evaluated with least-squares volume during cell division were obtained at the three light regression model I of the form: intensities (p<0.01 for 45 and 150 μmol m－2 sec－1, p<0.001 for 365 μmol m－2 sec－1; Fig. 4). The slopes of the line with Log C =ba log V + log 95% confidence intervals were 0.59±0.40 pgC μm－3, 0.66± 0.39 pgC μm－3, and 0.98±0.27 pgC μm－3 under 45, 150, where b is the slope and log a is the intercept of the rela- and 365 μmol m－2 sec－1, respectively. The intercepts of the tionship between log V and log C. Confidence intervals line with 95% confidence intervals were 0.22±0.79, and standard errors around regression slopes and inter- －0.0098±0.74, and －0.64±0.48 under 45, 150, and Carbon to volume relationship of Isochrysis galbana 181

Fig. 1. Change in cell volume during cell division of Isochry- Fig. 2. Change in carbon quota during cell division of Isochry- sis galbana (open circles) with the fitted curve (solid line) at an sis galbana (open circles) with the fitted curve (solid line) at an irradiance of 45 (A), 150 (B), and 365 μmol m－2 sec－1 (C). Verti- irradiance of 45 (A), 150 (B), and 365 μmol m－2 sec－1 (C). See cal bars indicated one standard deviation of the mean. Solid bars Fig. 1 for vertical bar and solid bar legend. denote dark periods. lowed us to measure the change of cell volume and carbon 365 μmol m－2 sec－1, respectively. The slope and the inter- quota during cell division at a given growth irradiance. cept were not significantly different among the light treat- Generally, the changes in cell volume and carbon quota ments (ANCOVA, p>0.05). within a one day cycle are characterized by a daytime in- crease due to photosynthesis and a nighttime decrease due to cell division. However, even within the nanophytoplank- Discussion ton size range, some diatoms display a distinctively differ- The changes in the cell volume and carbon quota during ent cell division pattern (Nelson & Brand 1979, Chisholm cell division were consistent with previous studies con- & Costello 1980, Berges et al. 1995, Needoba & Harrison ducted on nanophytoplankton such as Prymnesiophyceae 2004). Berges et al. (1995) showed the change in cell vol- (Varela & Harrison 1999), Prasinophyceae (Kohata & ume over one day in the diatom Thalassiosira pseudonana Watanabe 1989, DuRand et al. 2002), and Chlorophyceae had two apparent peaks due to cell division over both the (DuRand & Olson 1998). The use of steady state cultures light and dark periods, while the carbon quota exhibited a grown in continuous culture compared to batch cultures al- similar change to other phytoplankton taxa. Even if one 182 Y. Ishiwata et al.

Fig. 4. Relationship between cell volume and carbon quota for Isochrysis galbana during cell division at irradiances of 45 (A), Fig. 3. Relationship between irradiance and the average (A), 150 (B), and 365 μmol m－2 sec－1 (C). Broken lines denote the the amplitude (B) and magnitude of change during cell division 95% confidence intervals of the mean. (MCD) (C) for cell volume (μm3 cell－1) (solid circles) and carbon quota (pgC cell－1) (open circles) in Isochrysis galbana. Error bars denote standard deviation around the mean (n=12). 45 μmol m－2 sec－1 on a 12/12 h light/dark cycle with con- tinuous culture. The growth rate in Montagnes et al. (1994) deals with the change of cell volume and carbon quota dur- was 0.32 d－1, whereas it was 0.12 d－1 in the present study. ing cell division in the natural nanophytoplankton commu- The growth stage when cells were harvested in Montagnes nity in the ocean, careful consideration needs to be paid to et al. (1994) and Fujiki & Taguchi (2002) was mid- or late- the community structure. logarithmic, whereas it was in the stationary phase in the A variable range of carbon quota values can be found in present study. Cellular biochemical components such as the literature even for the same species (Falkowski et al. the protein, carbohydrate and lipid contents (main compo- 1985, Montagnes et al. 1994, Fujiki (personal communica- nents in the carbon quota) are relatively higher in the sta- tion; Fig. 5). For example, the carbon quota was 6.97 pgC cell－1 tionary phase than in the logarithmic phase (Brown et al. for I. galbana at 16°C under 20–60 μmol m－2 sec－1 on a 1996). Different culture conditions and difference in the 14/10 h light/dark cycle with batch culture (Montagnes et growth stage when cells were harvested between Mon- al. 1994), whereas the carbon quota in the present study tagnes et al. (1994) or Fujiki & Taguchi (2002) and the was 23.8 pgC cell－1 for the same species at 25°C under present study might be the reason for the relatively higher Carbon to volume relationship of Isochrysis galbana 183

Fig. 5. Comparison of the relationship between carbon quota Fig. 6. Relationship between the magnitude of change during and irradiance for Isochrysis galbana determined using data cell division (MCD) for cell volume (closed circles) and carbon from Falkowski et al. (1985), inverted triangles; Montagnes et al. quota (open circles) and irradiance in nanophytoplankton, deter- (1994), open triangle; Fujiki (personal communication), open cir- mined using data from Eppley & Coatsworth (1966), Kohata & cles; and the present study, closed circles. Watanabe (1986), Kohata & Watanabe (1989), Berdalet et al. (1992), DuRand & Olson (1998), Varela & Harrison (1999), Du- Rand et al. (2002), Ohi et al. (2002), Ohi et al. (2003), and the carbon quota in the present study. Since cells divide faster present study with the fitted curve (solid line). with increasing irradiance, the average cell volume and carbon quota decreased, but their amplitudes increased in the present study (Fig. 3A and B). This pattern was con- Once nitrogen is limited, the cell volume decreases and the sisted with data provided by Fujiki (personal communica- carbon quota or cell carbon density per unit cell volume in- tion). Carbon quotas in Fujiki (personal communication) creases. Similar trends in cell volume and carbon quota changed from 23.6 to 10.6 pgC cell－1 with increasing irra- under nitrogen-starved conditions were also reported in a diance from 25 to 750 μmol m－2 sec－1 under a 12/12 h light/ different strain of Isochrysis sp. even at the high growth dark cycle with the same species at the same temperature rate of 1 day－1 under a day-night light cycle (Lacour et al. as in the present study under semi-continuous batch cul- 2012). A similar nitrogen-limited condition can be found at ture (Fig. 5). The stage of harvesting in Fujuki & Taguchi the sea surface in the euphotic layer where limited nitrogen (2002) was mid- or late-logarithmic phase. Different cul- usually reduces the growth of phytoplankton, and light is ture methods and differences in the growth stage when saturated under the light/dark cycle. Future studies are cells were harvested between Fujiki & Taguchi (2002) and needed to investigate the carbon quota to cell volume rela- the present study might be the cause for the relatively tionship under nitrogen-limited condition and under a higher carbon quotas observed in the present study. Oppo- light/dark cycle to determine if the present relationship be- site patterns have been reported for the relationship be- tween cell volume and carbon quota is held under those tween carbon quota and irradiance with the same species conditions. at the same temperature as in the present study (Falkowski The response of MCD estimated using cell volume and/ et al. 1985). Carbon quotas in Falkowski et al. (1985) in- or carbon quota showed a hyperbolic relationship when the creased from 10.3 to 20.0 pgC cell－1 with increasing irradi- published experimental data were included (Eppley & ance from 30 to 600 μmol m－2 sec－1 under 24 h continuous Coatsworth 1966, Kohata & Watanabe 1986, 1989, Berdalet light with the same species as in the present study under a et al. 1992, DuRand & Olson 1998, Varela & Harrison 1999, turbidostat culture (Fig. 5). The mechanisms responsible DuRand et al. 2002, Ohi et al. 2002, 2003; p<0.001; Fig. for the changes in cell volume and carbon quota with irra- 6). The initial slope that was obtained was almost linear, diance are not well understood. Microalgal cell volume particularly for the irradiance range employed in the pres- may be under the control of carbon metabolism (Claustre ent study. The initial slope was not different from the slope & Gostan 1987), and different responses in cell volume of the relationship between irradiance and cell volume or and carbon quota under different day lengths might be due carbon quota (Fig. 3C). Analysis of the diel observations to the change in carbon metabolism responsible for the published for five different species (Pyramimonas parkeae, light/dark cycle. Nannochloris sp., Emiliania huxleyi, Micromonas pusilla, In nitrogen-limited chemostat culture, the carbon quota Isochrysis galbana) and including our results yields a sig- of I. galbana increased to 18.0 pgC cell－1 under continuous nificant regression line (p<0.001; Fig. 7). light conditions at 175 μmol m－2 sec－1 due to a decrease in the growth rate to 0.18 d－1 (Herzig & Falkowski 1989). Log C = 0.97 log V－ 0.58 184 Y. Ishiwata et al.

can be fitted to a single line within the nanophytoplankton size range. The results of the present study also suggest that light intensity might not affect this carbon quota to cell volume relationship. If such a trend is a general feature for nanophytoplankton in the natural environment, this would suggest that estimating biomass from the carbon quota to cell volume relationship may be justified in some oceanic situations̶regardless of the sampling time and the depth of the euphotic layer. Since the cell size and divi- sion manner of I. galbana is similar to that of the majority of nanophytoplankton, the results of the present study pro- vide important information for the interpretation of the carbon quota to cell volume relationship in other nanophy- toplankton in the ocean.

Acknowledgements Fig. 7. Relationship between cell volume and carbon quota of We thank V. S. Kuwahara, S. C. Y. Leong, and F. phytoplankton during cell division. Solid lines denote regression Rassoulzadegan for valuable comments that improved the lines or simple lines connecting the maximum and minimum val- manuscript. Original data on cell volume and carbon quota ues during diel variation in Micromonas pusilla (DuRand et al. for Nannochloris sp., provided by M. D. DuRand, and 2002; open circles); Nannochloris sp. (DuRand & Olson 1998; Isochrysis galbana, provided by T. Fujiki, are greatly ap- open triangles); Emiliania huxleyi (Varela & Harrison 1999; open squares); Pyramimonas parkeae (Kohata & Watanabe 1989; preciated. Figures were prepared by T. Katayama. This open diamonds); Isochrysis galbana (present study at an irr- research was partially supported by a Sasakawa Scientific adiance of 45 μmol m－2 sec－1; solid circles, at an irradiance Research Grant from the Japan Science Society. of 150 μmol m－2 sec－1; solid triangles, at an irradiance of －2 －1 365 μmol m sec ; solid squares). Broken line denotes the References regression line for the five species. See Materials and Methods section for details. Banse K (1976) Rates of growth, respiration and photosynthesis of unicellular algae as related to cell size̶A review. J Phycol 12: 135–140. This relationship could be characterized as phylogenetic Berdalet E, Latasa M, Estrada M (1992) Variations in biochemi- allometry (Gould 1966). What kind of relationship exists cal parameters of Heterocapsa sp. and Olithodiscus luteus between MCD and the slope of carbon quota to cell vol- grown in 12 : 12 light : dark cycles. Hydrobiologia 238: 139– ume during cell division? Generally, the degree of leaning 147. of the linear function indicates the ratio of an increment of Berges JA, Cochlan WP, Harrison PJ (1995) Laboratory and field y to an increment of x. When applied to the present study, responses of algal nitrate reductase to diel periodicity in irra- an increment of x or y means the amount of change in car- diance, nitrate exhaustion, and the presence of ammonium. bon quota or cell volume within a day, in other words, the Mar Ecol Prog Ser 124: 259–269. MCD of carbon quota or cell volume. Because both carbon Brown MR, Dunstan GA, Norwood SJ, Miller KA (1996) Effects of harvest stage and light on the biochemical composition of quota and cell volume change in a similar manner during the diatom Thalassiosira pseudonana. J Phycol 32: 64–73. cell division, the slope of the carbon quota to cell volume Cermeño P, Marañón E, Rodríguez J, Fernández E (2005) Large- relationship may not vary significantly with irradiance, sized phytoplankton sustain higher carbon-specific photosyn- even though the MCD of carbon quota and cell volume thesis than smaller cells in a coastal eutrophic ecosystem. Mar changes hyperbolically with irradiance. Investigations on Ecol Prog Ser 297: 51–60. the carbon quota to cell volume relationship during cell di- Chisholm SW, Costello JC (1980) Influence of environmental vision of nanophytoplankton, over a wide range of cell vol- factors and population composition on the timing of cell divi- umes, will help in understanding the ecology of nanophy- sion in Thalassiosira fluviatilis (Bacillariophyceae) grown on toplankton communities. Although the carbon quota to cell light/dark cycles. J Phycol 16: 375–383. volume relationship applies for the size range of nanophy- Claustre H, Gostan J (1987) Adaptation of biochemical composi- toplankton occurring in the ocean, the species composition tion and cell size to irradiance in two microalgae: possible should be taken into consideration, particularly diatoms ecological implications. Mar Ecol Prog Ser 40: 167–174. due to their characteristic manner of cell division in the eu- DuRand MD, Green RE, Sosik HM, Olson RJ (2002) Diel varia- photic layer (e.g. Needoba & Harrison 2004). tions in optical properties of Micromonas pusilla (Prasinophy- The present results indicate that the relationship be- ceae). J Phycol 38: 1132–1142. tween cell volume and carbon quota during cell division DuRand MD, Olson RJ (1998) Diel patterns in optical properties Carbon to volume relationship of Isochrysis galbana 185

of the chlorophyte Nannochloris sp.: Relating individual-cell Malone TC (1980) Algal size. In: The physiological ecology of to bulk measurements. Limnol Oceanogr 43: 1107–1118. phytoplankton (ed Morris I). University of California Press, Eppley RW, Coatsworth JL (1966) Culture of the marine phyto- Berkeley and Los Angeles, pp. 433–463. plankter, Dunaliella tertiolecta, with light-dark cycles. Micro- Montagnes DJS, Berges JA, Harrison PJ, Taylor FJR (1994) Esti- biol 55: 66–80. mating carbon, nitrogen, protein, and chlorophyll a from vol- Falkowski PG, Dubinsky Z, Wyman K (1985) Growth-irradiance ume in marine phytoplankton. Limnol Oceanogr 39: 1044– relationships in phytoplankton. Limnol Oceanogr 30: 311–321. 1060. Falkowski PG, Raven JA (2007) Aquatic Photosynthesis, 2nd Mullin MM, Sloan PR, Eppley RW (1966) Relationship between edition. Princeton University Press, Princeton, 484 pp. carbon content, cell volume, and area in phytoplankton. Lim- Fujiki T, Taguchi S (2002) Variability in chlorophyll a specific nol Oceanogr 11: 307–311. absorption coefficient in marine phytoplankton as a function Nagao N, Toda T, Takahashi K, Hamasaki K, Kikuchi T, Taguchi of cell size and irradiance. J Plankt Res 24: 859‒874. S (2001) High ash content in net-plankton samples from shal- Gould SJ (1966) Allometry and size in ontogeny and phylogeny. low coastal water: Possible source of error in dry weight mea- Biol Rev 41: 587–640. surement of zooplankton biomass. J Oceanogr 57: 105–107. Guillard RRL, Ryther JH (1962) Studies of marine planktonic di- Needoba JA, Harrison PJ (2004) Influence of low light and a － － atoms. I. Cyclotella nana Hustedt and Detonula confervacea light:dark cycle on NO3 uptake, intracellular NO3 , and nitro- (Cleve) Gran. Can J Microbiol 8: 229–239. gen isotope fractionation by marine phytoplankton. J Phycol Herzig R, Falkowski PG (1989) Nitrogen limitation in Isochrysis 40: 505–516. galbana (Haptophyceae). I. Photosynthetic energy conversion Nelson DM, Brand LE (1979) Cell division periodicity in 13 spe- and growth efficiencies. J Phycol 25: 462–471. cies of marine phytoplankton on a light : dark cycle. J Phycol Hillebrand H, Dürselen CD, Kirschtel D, Pollingher U, Zohary T 15: 67–75. (1999) Biovolume calculation for pelagic and benthic microal- Ohi N, Ishiwata Y, Taguchi S (2002) Diel patterns in light gae. J Phycol 35: 403–424. absorption and absorption efficiency factors of Isochrysis Ishizaka J, Harada K, Ishikawa K, Kiyosawa H, Furusawa H, galbana (Prymnesiophyceae). J Phycol 38: 730–737. Watanabe Y, Ishida H, Suzuki K, Handa N, Takahashi M Ohi N, Shino M, Ishiwata Y, Taguchi S (2003) Light absorption (1997) Size and taxonomic plankton community structure and of Isochrysis galbana (Prymnesiophyceae) under day-night carbon flow at the equator, 175°E during 1990–1994. Deep-Sea cycle at high-light irradiance. Plankton Biol Ecol 50: 1–9. Res II 44: 1927–1949. Shalapyonok A, Olson RJ, Shalapyonok LS (2001) Arabian Sea Iwasawa K, Murata A, Taguchi S (2009) Cell shrinkage of phytoplankton during Southwest and Northeast Monsoons Isochrysis galbana (Prymneshiophyceae) during storage with 1995: composition, size structure and biomass from individual preservatives. Plankton Benthos Res 4: 120–121. cell properties measured by flow cytometry. Deep-Sea Res II Kieding N, Rudolph N, Møller U (1984) Diurnal variation in in- 48: 1231–1261. flux and transition intensities in the S phase of hamster cheek Snedecor GW, Cochran WG (1989) Statistical methods, 8th edi- pouch epithelium cells. In: Cell cycle clocks (ed Edmunds LJr). tion. Iowa State University Press, Iowa, 374 pp. Marcel Dekker, New York, pp. 135–159. Sokal RR, Rohlf FJ (1995) Biometry, 3rd edition. Freeman, New Kohata K, Watanabe M (1986) Synchronous division and the pat- York, 451 pp. tern of diel vertical migration of Heterosigma akashiwo Strathmann RR (1967) Estimating the organic carbon content of (Hada) Hada (Raphidophyceae) in a laboratory culture tank. phytoplankton from cell volume or plasma volume. Limnol J Exp Mar Biol Ecol 100: 209–224. Oceanogr 12: 411–418. Kohata K, Watanabe M (1989) Diel changes in the composition Taguchi S (1976) Relationship between photosynthesis and cell of photosynthetic pigments and cellular carbon and nitrogen in size of marine diatoms. J Phycol 12: 185–189. Pyramimonas parkeae (Prasinophyceae). J Phycol 25: 377– Tang EPY (1995) The allometry of algal growth rate. J Plankton 385. Res 17: 1325–1335. Lacour T, Sciandra A, Talec A, Mayzaud P (2012) Diel variations Varela DE, Harrison PJ (1999) Effect of ammonium on nitrate of carbohydrates and neutral lipids in nitrogen-sufficient and utilization by Emiliania huxleyi, a from the nitrogen-starved cyclostat cultures of Isochrysis sp. J Phycol oceanic northeastern Pacific. Mar Ecol Prog Ser 186: 67–74. 48: 966–975. Verity PG, Robertson CY, Tronzo CR, Andrews MG, Nelson JR, Laws EA, Bannister TT (1980) Nutrient-limited and light-limited Sieracki ME (1992) Relationships between cell volume and the growth of Thalassiosira-fluviatilis in continuous culture, with carbon and nitrogen content of marine photosynthetic nano- implications for phytoplankton growth in the ocean. Limnol plankton. Limnol Oceanogr 37: 1434–1446. Oceanogr 25: 457–473.