Invest. Mar., Valparaíso, 34(2): 97-108, 2006

Dynamics of oxygen production / consumption in Dunaliella salina, Thalassiosira weissfiogii and Heterocapsa triquetra circulating within a simulated upper mixed layer*

Dinámica de producción / consumo de oxígeno en Dunaliella salina, Thalassiosira weissfiogii and Heterocapsa triquetra circulando dentro de una capa superficial de mezcla simulada*

Elena S. Barbieri¹, Virginia E. Villafañe¹ & E. Walter Helbling¹

¹Estación de Fotobiología Playa Unión & Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Casilla de Correos N° 15. 9103, Rawson, Chubut, Argentina

Dirección para correspondencia

ABSTRACT. Oxygen production / consumption dynamics in three phytoplankton species (20-25 µm in effective diameter), Dunaliella salina (Chlorophyceae), Thalassiosira weissfiogii (Bacillariophyceae) and Heterocapsa triquetra (Dinophyceae), was experimentally determined when cells circulated within a simulated upper mixed layer (UML). Samples were exposed to three radiation treatments receiving: a) full solar radiation (PAB, 280-700 nm), b) PAR+UV-A (PA, 320-700 nm), and c) only PAR (P, 400-700 nm). Two pathways were simulated (as if the cells started to circulate from the surface or from the bottom of the UML): 1) downward circulation (i.e., from 100% to 9% irradiance and back to 100%), and 2) upward circulation (i.e., from 9% to 100% irradiance and back to 9%). There were no significant differences among radiation treatments (p < 0.05) and photosynthetic inhibition was only due to PAR. We found important inter-specific differences in O₂ rates when cells circulated within the simulated UML, D. salina was affected by both high and low irradiances whereas T. weissfiogii was only inhibited by high irradiances. On the other hand, H. triquetra showed the least variability and it benefited by fluctuating radiation regimes. We also determined differences in the depth integrated O₂ production when species performed a complete rotation within the simulated UML, with the highest values in H. triquetra and the lowest in D. salina. Our findings suggest that the different pathways of the cells circulating in the water column should be considered at the time to assess primary productivity in areas exposed to changing meteorological conditions throughout the year, and hence with variable UMLs.

Key words: Dunaliella salina, Heterocapsa triquetra, Thalassiosira weissfiogii, mixing, oxygen rates, photosynthetically active radiation, ultraviolet radiation.

RESUMEN. Se determinó experimentalmente la dinámica de producción/consumo de oxígeno en tres especies fitoplanctónicas (20-25 µm de diámetro efectivo): Dunaliella salina (Chlorophyceae), Thalassiosira weissfiogii (Bacillariophyceae) y Heterocapsa triquetra (Dinophyceae), cuando las células circularon dentro de una capa superficial de mezcla (CSM) simulada. Las muestras fueron expuestas a tres tratamientos de radiación recibiendo: a) toda la radiación solar (PAB, 280-700 nm), b) PAR+RUV-A (PA, 320-700 nm), y c) sólo PAR (P, 400-700 nm). Se simularon dos recorridos (como si las células circularan desde la superficie o desde la base de la CSM): 1) circulación hacia abajo (i.e., desde el 100% al 9% de la irradiancia y nuevamente al 100%), y 2) circulación hacia arriba (i.e., desde el 9% al 100% de la irradiancia y nuevamente al 9%). No se encontraron diferencias significativas entre tratamientos de radiación (p < 0,05) y la inhibición fotosintética se debió sólo a PAR. Se encontraron importantes diferencias inter-específicas en las tasas de oxígeno cuando las células circularon dentro de la CSM simulada, D. salina fue afectada tanto por altas como por bajas irradiancias, mientras que T. weissfiogii solo fue inhibida por altas irradiancias. En cambio, H. triquetra mostró la menor variabilidad y se benefició por los regímenes fluctuantes de irradiancia. También se determinaron diferencias en la producción de O₂ integrada en profundidad cuando las especies realizaron una rotación completa dentro de la CSM simulada, con los mayores valores en H. triquetra y los menores en D. salina. Estos resultados sugieren que los diferentes recorridos de las células circulando en la columna de agua deberían ser considerados al momento de determinar la productividad primaria en áreas expuestas a condiciones meteorológicas cambiantes durante el año, y consecuentemente, con CSMs variables.

Palabras clave: Dunaliella salina, Heterocapsa triquetra, Thalassiosira weissfiogii, mezcla, tasa de oxígeno, radiación fotosintética activa, radiación ultravioleta.

INTRODUCTION

Fluctuations of physical factors are widely known to affect significantly aquatic organisms (Steele, 1985); in particular, variations in the underwater radiation field have profound effects on phytoplankton. These fluctuations in irradiance occur by changes in the depth of the upper mixed layer (UML), a layer of homogeneous characteristics that is generally produced by wind stress and solar heating (Neale et al., 2003). Mixing causes phytoplankton to move in the water column and hence alter their exposure to solar radiation. This has important consequences on physiological processes, such as photosynthesis rates and respiration (Marra, 1978; Yoder & Bishop, 1985; Kroon et al., 1992), growth (Ibelings et al., 1994; Litchman, 2000) and nutrient uptake (Litchman et al., 2004). Moreover, fluctuating light regimes may stimulate the growth of different species, hence potentially changing the phytoplankton taxonomic composition of the community (Litchman, 1998).

Studies have shown variable effects of mixing on phytoplankton photosynthetic rates. For example, Marra (1978) determined that vertical mixing significantly enhanced primary productivity, whereas Kroon et al. (1992) found that photosynthesis rates were depressed under fluctuating radiation regimes; on the other hand, Yoder & Bishop (1985) did not find differences in photosynthetic rates when comparing them with those at fixed irradiances. The combined effects of mixing and solar ultraviolet radiation (UVR, 280-400 nm) are also known to affect phytoplankton primary productivity (Helbling et al., 1994; Neale et al., 1998; Kohler et al., 2001; Barbieri et al., 2002). Furthermore, mixing speed strongly affects photosynthetic rates (Helbling et al., 1994, 2003; Neale et al., 1998) and Helbling et al. (2003) demonstrated that fast mixing favored the utilization of UVR as source of energy for photosynthesis in tropical phytoplankton, whereas slow mixing caused a stronger UVR-induced photoinhibitory effect. On the other hand, Helbling et al. (1994) and Neale et al. (1998) working with Antarctic phytoplankton showed higher inhibition with rapid mixing, thus demonstrating that the interactive effects of UVR and mixing are strongly dependant on the specific sensitivity of phytoplankton assemblages as well as on the exposure response (i.e., irradiance / dose).

Although much progress have been done to understand phytoplankton responses when exposed to fluctuating radiation regimes, we are not aware of any study assessing primary productivity in mixed environments that has specifically considered the pathway that cells perform within the UML, i.e., if cells circulate from low towards high irradiances or vice versa. In this study, we simulated the rotation of phytoplankton cells (Dunaliella salina; Chlorophyceae, Thalassiosira weissfiogii; Bacillariophyceae and Heterocapsa triquetra; Dinophyceae), within the UML under two extreme conditions (or pathways): in one of such pathways, cells that were at the bottom of the UML circulated upward to high irradiances (i.e., towards the surface) and back again ("upward" circulation). In the other type of circulation, cells that were at the surface moved downward to low irradiances (i.e., towards the bottom of the UML) and back again ("downward" circulation). As cells size plays an important role in the acclimation and responses to solar radiation (Helbling et al., 2001a) we are simplifying inter-specific comparisons by using organisms with a similar effective diameter (20-25 µm).

MATERIALS AND METHODS

Cultures

Three marine phytoplankton species (20-25 µm in effective diameter) were used in the experiments: Dunaliella salina (Dunal) Teodoresco (Clorophyceae), Thalassiosira weissfiogii (Grunow) G. Fryxell et Hasle (Bacillariophyceae) and Heterocapsa triquetra (Ehrenberg) Stein (Dinophyceae). The organisms used in these experiments were obtained from the Institut für Botanik und Pharmazeutische Biologie (Friedrich Alexander Universität, Germany) and from the Algae Culture Collection of Estación de Fotobiología Playa Unión. Cells were grown in seawater-enriched medium f/2 (Guillard & Ryther, 1962) in a temperature controlled (20°C) illuminated chamber (12:12, light: dark period) receiving PAR irradiance of 37 W·m^-2, provided by 10 fiuorescent Philips daylight lamps.

Experimentation

When cultures reached the exponential growth phase, they were transferred, during the light period, to an oxymeter (Real Time Computer Inc., model Oxym 5). The oxymeter has five 20 ml quartz tubes inside of an acrylic UV transparent chamber with circulating water as temperature control (Fig. 1). Five oxygen microelectrodes (Yellow Spring Instruments Co., model 5331) were attached to the quartz tubes. Gentle mixing was done with a magnetic stirrer to maintain oxygen concentration homogeneous inside each quartz tube and to prevent the formation of bubbles. To test the effects of the different wavebands, samples were put in the quartz tubes under three treatments: a) PAB treatment (samples exposed to UVR+PAR; 280700 nm) uncovered tubes; b) PA treatment (samples exposed to UV-A+PAR; 320-700 nm) tubes covered with a cut-off filter (Montagefolie, Folex, Dreieich, Germany, 50% transmission at 320 nm) and, c) P treatment (samples exposed only to PAR; 400-700 nm) tubes covered with Ultraphan -395 film (UV Opak, Digefra, Munich, Germany, 50% transmission at 395 nm) (the spectra of filters and materials are published in Figueroa et al. 1997). The oxymeter containing the samples was then exposed to natural radiation, and data from the five oxygen sensors, together with temperature and PAR irradiance were acquired every two seconds and recorded in a laptop computer.

Figure 1. Schematic representation of the experimental set-up. Samples were put in the oxymeter under three radiation treatments (PAB, PA and P). Circulation of cells within the UML was simulated by adding / taking out neutral density screens that were placed in front of the oxymeter. A laptop computer recorded O2 evolution with a frequency of one datum every two seconds. Figura 1. Representación esquemática del diseño experimental. Las muestras se colocaron en un oxímetro bajo tres tratamientos de radiación (PAB, PA y P). La circulación de las células en la CSM se simuló agregando / quitando mallas de densidad neutra que se colocaron en frente al oxímetro. Una computadora portátil registró la evolución de O2 con una frecuencia de un dato cada dos segundos.

Mixing was simulated based on real weather conditions of the Patagonia costal area, with a mean daily wind speed of 88 km·h^-1 during spring and summer (Villafañe et al., 2004). We used different combinations of neutral density screens (Helbling et al., 1994; Barbieri et al., 2002) (Fig. 1) so that the simulated depth of the UML was 10 m (z_UML=10 m, Barbieri et al., 2002). Our imposed mixing conditions, together with the irradiance at which the cells were grown before used in experimentation -37 W·m^-2 -were chosen to establish the worst case scenario, as this irradiance value represents roughly the bottom of the simulated UML using an attenuation coefficient (k_PAR) = 0.3 m-1 (as previously determined for the study area) and the surface irradiance (Barbieri et al., 2002). For each species, we performed two types of experiments (as if cells circulated in two different pathways) and each experiment was repeated twice. In one set of experiments ("downward" circulation) cells moved from 100% to 9% irradiance and back to 100%. In the other set of experiments ("upward" circulation), cells moved from 9% to 100% irradiance and back to 9%. We simulated four discrete steps of irradiance (i.e., 100%, 85%, 47% and 9%) in the UML by adding / taking out neutral density screens every 20 min, so that the total duration of each experiment (i.e., one rotation within the UML, 20 min at each irradiance step) was 160 min. Although the simulated "upward" and "downward" circulations were done during different days (to keep the incubations centered on local noon) they were done under clear sky conditions (without cloud cover infiuence), with relatively similar PAR irradiance conditions during each step. The mean incident PAR irradiances registered were of 313, 348 and 364 W·m^-2for experiments carried out with D. salina, T. weissfiogii and H. triquetra, respectively. All experiments were carried out at Estación de Fotobiología Playa Unión (EFPU, 43.3°S, 65°W) during the austral summer, during the period december 2003 - january 2004.

Assessment of oxygen rates and integrated oxygen production

Oxygen evolution (in mg O₂·L^-1) was plotted as a function of time. The data from each 20-min interval were adjusted to a lineal function (best fit) so that the slope represented O₂ production rates (in µg O₂·L^-1·min^-1). Oxygen production rates were integrated over the turnover time (i.e., from 0 to 160 min) as well as from 0 to 10 m (i.e., the depth of the simulated UML). Then, and to compare the integrated production among species, O₂ production rates were normalized by cell concentration. The integrated O₂ production over the simulated UML was thus calculated as:

Analyses and measurements

Chlorophyll-a (chl-a) and UV-absorbing compounds: samples for chl-a were taken at the beginning and at the end of each experiment whereas those for spectral absorption characteristics were only taken at the beginning. An aliquot of 15-50 mL of sample was filtered onto Whatman GF/F filters (25 mm) and the pigments extracted in 7 mL of absolute methanol overnight at 4°C (Holm-Hansen & Riemann, 1978). After the extraction period, the sample was centrifuged and the extract scanned between 250 and 750 nm using a Hewlett Packard spectrophotometer (model HP-8453E). The peak height at 334 nm was used as an estimator of the concentration of UV-ab-sorbing compounds (Dunlap et al., 1995). The same sample was used to determine chl-a concentration from readings taken before and after acidification using a Turner Designs fiuorometer (model TD 700) (Holm-Hansen et al., 1965).

Enumeration of cells: phytoplankton samples were taken at the beginning and at the end of each experiment; the samples were fixed with buffered formalin (final concentration in the sample 0.4%). Quantitative analyses of cells were done using a Sedwick-Rafter chamber following the technique described in Villafañe & Reid (1995).

Radiation measurements: irradiance levels during experiments were monitored using a broad-band filter radiometer (ELDONET, Real Time Computer Inc.) which is permanently installed on the roof of the EFPU. This sensor collects data of incident solar radiation once per min for PAR (400-700 nm), UV-A (315-400 nm) and UV-B (280-315 nm).

Statistics: in the three species studied, no significant differences (p > 0.05) were established between radiation treatments at any irradiance level (see below). Therefore, we combined and used the data from the five oxymeter channels (i.e., as quintuplicate samples) to calculate mean O₂ rates and to perform all statistics. The Kruskal-Wallis non-parametric test (Zar, 1984) was used to establish differences between irradiance levels (confidence level = 0.05) as well as in the integrated O₂ production.

RESULTS

The absorption characteristics (i.e., optical density (O.D.) normalized by chl-a concentration) of D. salina, T. weissfiogii and H. triquetra are shown in Fig. 2. The three species showed the chl-a peaks at 440 and 665 nm; carotenoids (l _max = 470 nm) were noticeable in D. salina and T. weissfiogii whereas in H. triquetra only a small shoulder of these compounds was observed. On the other hand, this dinofiagellate had a clear peak of UV-absorbing compounds (l _max = 334 nm) whereas in T. weissfiogii their amount were very small; they were virtually absent in D. salina. In all experiments, the concentration of cells was comparable and it varied between 1,800 and 2,200 cells·mL^-1.

Figure 2. Initial absorption characteristics (Optical density per unit chl-a) as a function of wavelength for Dunaliella salina (thin line), Thalassiosira weissfiogii (broken line) and Heterocapsa triquetra (thick line). Figura 2. Características de absorción iniciales (Densidad óptica por clor-a) en función de la longitud de onda de Dunaliella salina (línea fina), Thalassiosira weissfiogii (línea entrecortada) y Heterocapsa triquetra (línea gruesa).

In D. salina (Fig. 3) O₂ evolution rates at the beginning of experiments were negative (i.e., O₂ consumption) regardless if samples were exposed to a "downward" or an "upward" circulation. When the "upward" circulation was imposed to the cells, O₂ rates increased significantly (p < 0.001) as soon as the cells moved towards high irradiances, with positive O₂ rates measured all the way up to the maximum experimental irradiance (i.e., 374 W·m^-2) as well as during the way back to low irradiances -32 W·m^-2. There was a slight negative value of -2.5 µg O₂·L^-1·min^-1 when cells were exposed to irradiances of 172 W·m^-2; this value, however, was not significantly different (p > 0.05) from that of 0.93 µg O₂·L^-1·min^-1 measured at the lowest irradiance at the end of the "upward" circulation. When cells circulated "downward" towards low irradiances, O₂ consumption was measured in the first two steps of the simulated UML (i.e., at 343 and 201 W·m^-2) as well as at the lowest experimental irradiance (i.e., 20 W·m^-2); during the rest of the experiment however, there was a positive O₂ balance. The maximum O₂ rate was determined during the "upward" circulation -14.7 µg O₂·L^-1·min^-1 at 319 W·m^-2. In the "downward" circulation though, maximum O₂ production values were significantly lower (p < 0.05) (9.3 µg O₂·L^-1·min^-1 at 233 W·m^-2), than that determined during the "upward" circulation.

Figure 3. Mean O2 production rates (µg O2 L-1·min-1) for Dunaliella salina as a function of PAR irradiance during the "upward" (open squares) and "downward" (black circles) circulations. The horizontal lines indicate the standard deviation, and the arrows represent the starting point of each circulation. Figura 3. Tasa media de producción de O2 (µg O2 L-1·min-1) para Dunaliella salina en función de la irradiancia PAR durante el movimiento "hacia arriba" (cuadrados blancos) y "hacia abajo" (círculos negros). Las líneas horizontales indican la desviación estándar y las fiechas el comienzo de cada circulación.

In T. weissfiogii (Fig. 4) O₂ rates had a relatively high value of 15.95 µg O₂·L^-1·min^-1 at the beginning of the "upward" circulation (i.e., 23 W·m^-2) but they decreased with increasing irradiances. At the maximum irradiance (i.e., 357 W·m^-2) O₂ rates at the end of the first half of the experiment (6.68 µg O₂·L^-1·min^-1) were not significantly different (p > 0.05) from that at the beginning of the second part (0.55 µg O₂·L^-1·min^-1). When samples returned to low irradiances, however, O₂ rates were negative (~ -8.40 - -7.03 µg O₂·L^-1·min^-1). Samples of T. weissflogii during the "downward" circulation initially displayed very high O₂ consumption rates of 40.57 µg O₂·L^-1·min^-1 at 317 W·m^-2. As soon as irradiance decreased, we measured O₂ production, as high as 20.23 µg O₂·L^-1·min^-1 at 253 W·m^-2. Relatively high O₂ production rates (i.e., 16-19 µg O₂·L^-1·min^-1) were determined at intermediate irradiances; however, slight O₂ consumption was determined at the lowest irradiance (i.e., 31 W·m^-2) but O₂ rates increased again towards the end of this experiment.

Figure 4. Mean O2 production rates (µg O2 L-1·min-1) for Thalassiosira weissfiogii as a function of PAR irradiance during the "upward" (open squares) and "downward" (black circles) circulations. The horizontal lines indicate the standard deviation, and the arrows represent the starting point of each circulation. One datum is missing during the "upward" circulation. Figura 4. Tasa media de producción de O2 (µg O2 L-1·min-1) para Thalassiosira weissfiogii en función de la irradiancia PAR durante el movimiento "hacia arriba" (cuadrados blancos) y "hacia abajo" (círculos negros). Las líneas horizontales indican la desviación estándar y las fiechas el comienzo de cada circulación. Se perdió un dato durante la circulación "hacia arriba".

Finally, the samples of H. triquetra (Fig. 5) moving from low towards high irradiances had positive O₂ rates throughout almost all the experiment, with maximum production values of 12.70 µg O₂·L^-1·min^-1 at 160 W·m^-2. A minimum value of -10.20 µg O₂·L^-1·min^-1 was found at the end of the "upward" circulation (i.e., at 30 W·m^-2). During the "downward" circulation, samples of H. triquetra had positive O₂ rates values during the first half of the experiment (i.e., when circulating towards low irradiances). However, O₂ was slightly consumed at intermediate irradiances (i.e., 189 W·m^-2), recovering to 15.36 µg O₂·L^-1·min^-1 at 350 W·m^-2.

Figure 5. Mean O2 production rates (µg O2 L-1·min-1) for Heterocapsa triquetra as a function of PAR irradiance during the "upward" (open squares) and "downward" (black circles) circulations. The horizontal lines indicate the standard deviation, and the arrows represent the starting point of each circulation. One datum is missing during the "downward" circulation. Figura 5. Tasa media de producción de O2 (µg O2 L-1·min-1) para Heterocapsa triquetra en función de la irradiancia PAR durante el movimiento "hacia arriba" (cuadrados blancos) y "hacia abajo" (círculos negros). Las líneas horizontales indican la desviación estándar y las fiechas el comienzo de cada circulación. Se perdió un dato durante la circulación "hacia abajo".

To compare the photosynthetic performance (O₂ consumption / production) of the species studied, we calculated the depth integrated O₂ production during a complete rotation within the simulated UML (Fig. 6). All species produced O₂ during the "downward" circulation. During this circulation, no significant differences in O₂ production rates (p > 0.05) were found between T. weissfiogii and H. triquetra but O₂ production in D. salina was significantly lower (p < 0.05). When mixing started from low towards high irradiances, the three species displayed significant differences in the depth integrated O₂ production (p < 0.05).

Figure 6. Integrated O2 production of D. salina, T. weissfiogii and H. triquetra during the "upward" (light bars) and "downward" (dark bars) circulations within a simulated UML (zUML = 10 m). The lines on top of the bars represent the standard deviation. Full explanation in the text. Figura 6. Producción integrada de O2 para D. salina, T. weissfiogii y H. triquetra durante la circulación "hacia arriba" (barras claras) y "hacia abajo" (barras oscuras) dentro de una CSM simulada (ZCSM = 10 m). Las líneas sobre las barras representan la desviación estándar. La explicación completa se encuentra en el texto.

H. triquetra had the highest integrated production (2.68 µg O₂·m^-2·celL^-1) whereas D. salina the lowest - O₂ consumption (-0.64 µg O₂·m^-2·celL^-1); T. weissfiogii produced O₂ at a rate of 0.29 µg O₂·m^-2·celL^-1. Finally, we determined significant inter-specific differences (p < 0.05) in the integrated O₂ production: D. salina and T. weissfiogii had higher integrated O₂ production in the "downward" as compared to the "upward" circulation; in H. triquetra however, the integrated O₂ production during the "downward" was less than half of that in the "upward" circulation.

DISCUSSION

Lack of UVR-induced photoinhibition

Photosynthetic rates are clearly associated to the radiation quality under which the cells are exposed, with PAR being mostly responsible for photosynthesis, whereas UVR is generally considered a stress factor for this process (see review by Villafañe et al., 2003 and references therein). However, a wide range of responses to UVR is also reported: while some species are very resistant, e.g., from tropical environments (Helbling et al., 1992, 2001b) some others, e.g., from polar areas, are especially sensitive even under low UVR levels (Helbling et al., 1992, 1996). Other studies have also reported the utilization of UVR (mainly UV-A) when PAR levels are relatively low (Barbieri et al., 2002) or when phytoplankton cells mix fast within the UML (Helbling et al., 2003). In our study though, we did not find significant effects of solar UVR when the species were exposed to a variable mixing regime. This could be related to the fact that when cells changed their irradiance exposure, from the relatively low irradiances during the culturing condition at 37 W·m^-2, to natural solar radiation, they were inhibited by PAR and thus, the contribution of UVR-inhibition was not significant. As previously mentioned, the irradiance conditions at which the cultures were maintained was similar to those received by the cells at the bottom of the UML. The relatively high PAR-induced photoinhibition as found in this study (Figs. 3-5) is also in agreement with other studies carried out in Patagonia (Villafañe et al., 2004) with phytoplankton exposed to similar radiation levels as that used in our experiments. In these studies, the authors reported that the UVR contribution to photoinhibition was relatively high only in winter, when PAR levels were low (Villafañe et al., 2004). Thus, the relatively high PAR-induced photoinhibition as compared to that of UVR seems to be a rather general feature for phytoplankton exposed to natural radiation levels as those received in the Patagonia area, with PAR / UVR ratios varying between 6.9-8.0 in summer and winter, respectively.

Mixing effects on O₂ rates: Inter-specific differences in O₂ production

Even under similar experimental conditions, there was an important degree of variability in the responses of the three phytoplankton species circulating within our simulated UMLs. This inter-specific variability is thought to occur by one or several processes (Litchman, 2000 and references therein), including differences in sensitivity among taxa (Richardson et al., 1983), variations in respiration rates which are dependant on irradiance (Falkowski et al., 1994), or variations in allocation of resources to protect the cells (Raven, 1994). For example, O₂ rates in D. salina were strongly inhibited by both high and low irradiances at the beginning of circulation (Fig. 3) but after that, O₂ rates increased again, although without reaching very high production. On the other hand, O₂ rates of T. weissfiogii (Fig. 4) were inhibited only by high irradiances in the "downward" circulation, whereas those in H. triquetra (Fig. 5) were rather similar throughout the irradiances changes simulated in our experiments.

The significant inhibition of O₂ rates observed in D. salina (Fig. 3) and T. weissfiogii (Fig. 4) during the "downward" circulation was rather expected, as cells were transferred from relatively low (i.e., 37 W·m^-2, culture conditions) to high PAR irradiances (> 300 W·m^-2, experimental conditions). Even though this change in irradiances is large, we observed similar changes in the study area which were produced by intense mixing due to wind stress (Villafañe et al., 2004) and by variable cloud cover that result in changes in irradiances of one order of magnitude occurring within minutes (Helbling et al., 2005). These changing conditions obviously imply an energy cost for the cells to acclimate their photosynthetic apparatus to the variable radiation conditions (Kiefer, 1973) and in the case of our experiments this was translated in a reduction of O₂ rates. However, our data do suggest dynamic rather than chronic photosynthetic inhibition in the three species studied. The mechanism of chronic inhibition is the light-dependent photodegradation of the D1 protein in photosystem II (PS II) which leads to a decrease in the photosynthetic electron transport (Osmond, 1994) and so, the photosynthetic apparatus is protected from excessive radiation energy. In contrast, dynamic photoinhibition is based on the xanthophyll cycle identified in higher plants and in many macroalgae (Häder & Figueroa, 1997) so that when cells are exposed to excessive solar radiation, the quantum yield of PS II decreases and the excitation energy is dissipated thermally. In D. salina, photosynthetic inhibition during the "downward" circulation lasted longer (i.e., two steps in the rotation, Fig. 3) than in the other two species, suggesting that although irradiance decreased to ~ 60% of the initial value, PAR still inhibited photosynthesis. On the other hand, the better performance of T. weissfiogii compared to that of D. salina might be related to its absorption characteristics (Fig. 2) and to its siliceous structure that might prevent solar radiation to reach vital targets within the cell (e.g., photosystems, DNA, etc.) by acting as a passive shield. In fact, this species recovered very fast from the change of irradiance from laboratory to experimental conditions under solar radiation. For example, during the "downward" circulation the small change in irradiance from 320 to 290 W·m^-2 that occurred over a 20 min interval, was enough to acclimate and to significantly increase O₂ rates from ~ -40 µg O₂·L^-1·min^-1 to a maximum of ca. 20 µg O₂·L^-1·min^-1.

The responses to mixing of H. triquetra (Fig. 5) were rather different as compared to that in the other two species, as it did not show high variability in O₂ rates in both "upward" and "downward" circulations. One might think that this response is related to the different absorption characteristics of this species (Fig. 2) as compared to that of D. salina and T. weissfiogii that did not have significant amounts of UV-absorbing compounds, as it is normally the case in most Chlorophyceae and many diatom species (Banaszak, 2003). On the other hand, although H. triquetra had high concentrations of UV-absorbing compounds, as seen in many dinofiagellate species (Banaszak, 2003) and that would protect them from UVR stress, there were no significant differences between samples exposed and non-exposed to UVR; in fact, PAR contributed for most of photoinhibition. Thus, it is apparent that these UV-absorbing compounds are not responsible for the relatively low inhibition, unless these compounds are involved in other processes than just preventing UVR to reach vital parts of the cell. In fact, several additional functions of these compounds have been suggested (see review by Bandaranayake, 1998) and at least one of them, mycosporine-glycine, has been found to have antioxidant activity with mild effects as scavenger for free radicals (Dunlap & Yamamoto, 1995). Even though here we are not testing the role of these compounds, we can not rule out the possibility that they might be involved in other metabolic processes within the cell, thus resulting in an overall better and faster acclimation of H. triquetra to the changing irradiance conditions. Overall, it is seen that the differential species' performance is translated in important inter-specific differences in the integrated O₂ production (Fig. 6): the highest production rates were determined in H. triquetra, whereas the lowest values (i.e., O₂ consumption) were registered in D. salina. Additionally, relatively large differences were determined according to the pathway followed by each species: D. salina and T. weissfiogii had higher O₂ rates when circulation started at high irradiances, whereas the opposite occurred in H. triquetra (Fig. 6).

Ecological implications

Our results may have important ecological implications for natural assemblages of phytoplankton in areas exposed to a variable atmospheric and climatologically conditions such as those of the Patagonian coast (Villafañe et al., 2004). In this study, we compared the responses of three microplanktonic species that are typical of summer (D. salina), spring (T. weissfiogii) and harmful algae bloom conditions (H. triquetra). We showed that they displayed a differential behavior in relation to mixing, being H. triquetra the species that obtained more benefits from fluctuating irradiance conditions, whereas D. salina would be more affected in such changing environment (Fig. 6). Changes in irradiance as those used in our experimental set-up would be found, for example, in our study site in the Patagonian region, where daily doses of solar radiation vary from 1 to 14 MJ·m^-2 for PAR, and from 150 to 2000, and 5 to 42 kJ·m^-2, for UV-A and UV-B, respectively (Villafañe et al., 2004). In addition, the study area is characterized by strong winds with a mean daily value of 88 km·h^-1and maximum wind speed frequency in the range of 12-16 Km·h^-1during spring and summer; on the other hand, relatively calm weather is characteristic from winter time (Villafañe et al., 2004). Under these conditions, variable portions of the euphotic zone - Eu (that in general extend down to ca 15 m) are mixed, and the upper mixed layer (UML) can encompass from ca 50% to almost the whole E (Barbieri et al., 2002).

It is important to point out that in the experiments we did not simulate the differential attenuation of solar irradiance within the UML so that our ratios of energy (short versus long wavelengths) were maximal, and thus they represented the worst-case scenario. However, as mentioned above, we did not find any significant differences between radiation treatments, even with these high energies ratios. Whether phytoplankton acclimate in situ to those variable radiation regimes will depend on several factors, not only on the fluctuating irradiance levels received by cells, but also on other variables such as nutrient status, cell size and factors of intrinsic origin (i.e., specific sensitivity). In Patagonia, it has been reported that phytoplankton blooms (dominated by microplankton diatoms) occur during winter (Villafañe et al., 2004). However, and based on our results, if the responses of H. triquetra are indeed representative for the Dynophyceae group, their productivity would be favored under windy conditions. In fact, studies carried out by in Patagonia by Gayoso (2001) and Villafañe et al. (2004) have reported relatively high concentrations of dinofiagellates during the windy season (i.e., spring-summer) including toxic species such as Alexandrium tamarense. However, during this season phytoplankton biomass is relatively low (Villafañe et al., 2004) as availability of nutrients appear to be the most important variable limiting growth (Helbling et al., 2005).

We conclude from our results that at the time to assess phytoplankton acclimation to solar radiation, not only the changes in irradiance are important but also if they occur towards high or low levels. Under mixing conditions in the water column, in which various species co-exist, the inter-specific responses would lead to a dominance of the fittest in terms of adaptation to changes in irradiance that, in our case, it would mean that mixing would favor the development and growth of dinofiagellates.

ACKNOWLEDGMENTS

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (PICT2000 N° 07-08184 and PICT 2003 N° 01-13388) and Fundación Playa Unión. We thank Dr. D.P. Häder who kindly provided some cultures used in the experiments and J.I. Albarracín that helped in cultures' maintenance; C. Barrio helped with experimental set-up. We are grateful with the comments and suggestions of one reviewer that contributed to improve this manuscript. This is contribution Nº 90 of the Estación de Fotobiología Playa Unión.

REFERENCES

Banaszak, A.T. 2003. Photoprotective physiological and biochemical responses of aquatic organisms to UVR. In: E.W. Helbling & H.E. Zagarese (eds.). UV effects in aquatic organisms and ecosystems. The Royal Society of Chemistry, Cambridge, pp. 329-356.

Bandaranayake, W.M. 1998. Mycosporines: are they nature's sunscreensfi Nat. Prod. Rep., 15: 159-172.

Barbieri, E.S., V.E. Villafañe & E.W. Helbling. 2002. Experimental assessment of UV effects on temperate marine phytoplankton when exposed to variable radiation regimes. Limnol. Oceanogr., 47: 1648-1655.

Dunlap, W.C. & Y. Yamamoto. 1995. Small molecular antioxidants in marine organisms: antioxidant activity of mycosporine-glycine. Comp. Biochem. Physiol., 112(B): 105-114.

Dunlap, W.C., G.A. Rae, E.W. Helbling, V.E. Villafañe & O. Holm-Hansen. 1995. UV-absorbing compounds in natural assemblages of Antarctic phytoplankton. Antarct. J. US., 30: 323-326.

Falkowski, P.G., R. Greene & Z. Kolber. 1994. Light utilization and photoinhibition of photosynthesis in marine phytoplankton. In: N. Baker & J. Bowyer (eds.). Photoinhibition of photosynthesis: from molecular mechanisms to the field. Bios Scientific, Oxford, pp. 407-432.

Figueroa, F.L., S. Salles, J. Aguilera, C. Jiménez, J. Mercado, B. Viñegla, A. Flores-Moya & M. Altamirano. 1997. Effects of solar radiation on photoinhibition and pigmentation in the red alga Porphyra leucosticta. Mar. Ecol. Progr. Ser., 151: 81-90.

Gayoso, A.M. 2001. Observations on Alexandrium tamarense (Lebour) Balech and other dinofiagellate populations in golfo Nuevo, Patagonia (Argentina). J. Plankton Res., 23: 463-468.

Guillard, R.R.L. & J.H. Rhyter. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol., 8: 229-239.

Häder, P.D. & F.L. Figueroa. 1997. Photoecophysiology of marine macroalgae. Photochem. Photobiol., 66: 1-14.

Helbling, E.W., H.C. Eilertsen, V.E. Villafañe & O. Holm-Hansen. 1996. Effects of ultraviolet radiation on post-bloom phytoplankton populations in Kvalsund, North Norway. J. Photochem. Photobiol., B-Biol., 33: 255-259.

Helbling, E.W., K. Gao, R.J. Gonçalves, H. Wu & V.E. Villafañe. 2003. Utilization of solar UV radiation by coastal phytoplankton assemblages off SE China when exposed to fast mixing. Mar. Ecol. Progr. Ser., 259: 59-66.

Helbling, E.W., V.E. Villafañe & E.S. Barbieri. 2001a. Sensitivity of winter phytoplankton communities from Andean lakes to artificial ultraviolet-B radiation. Rev. Chil. Hist. Nat., 74: 273-282.

Helbling, E.W., V.E. Villafañe & O. Holm-Hansen. 1994. Effects of ultraviolet radiation on Antarctic marine phytoplankton photosynthesis with particular attention to the infiuence of mixing. In: C.S. Weiler & P.A. Penhale (eds.). Ultraviolet radiation in Antarctica: measurements and biological effects. American Geophysical Union, Washington, DC, pp. 207-227.

Helbling, E.W., V.E. Villafañe, A.G.J. Buma, M.Andrade & F. Zaratti. 2001b. DNA damage and photosynthetic inhibition induced by solar UVR in tropical phytoplankton (Lake Titicaca, Bolivia). Eur. J. Phycol., 36: 157-166.

Helbling, E.W., V.E. Villafañe, M. Ferrario & O. Holm-Hansen. 1992. Impact of natural ultraviolet radiation on rates of photosynthesis and on specific marine phytoplankton species. Mar. Ecol. Progr. Ser., 80: 89-100.

Helbling, E.W., E.S. Barbieri, M.A. Marcoval, R.J. Gonçalves & V.E. Villafañe. 2005. Impact of solar ultraviolet radiation on marine phytoplankton of Patagonia, Argentina. Photochem. Photobiol., 81: 807-818.

Holm-Hansen, O. & B. Riemann. 1978. Chlorophyll a determination: improvements in methodology. Oikos, 30: 438-447.

Holm-Hansen, O., C.J. Lorenzen, R.W. Holmes & J.D.H. Strickland. 1965. Fluorometric determination of chlorophyll. J. Cons. Explor. Mer., 30: 3-15.

Ibelings, B.W., B.M.A. Kroon & L.R. Mur. 1994. Acclimation of photosystem II in a cyanobacterium and a eukaryotic green alga to high and fluctuating photosynthetic photon fiux densities, simulating light regimes induced by mixing in lakes. New Phytol., 128: 407-424.

Kiefer, D.A. 1973. Chlorophyll a fiuorescence in marine centric diatoms: responses of chloroplasts to light and nutrient stress. Mar. Biol., 23: 39-46.

Kohler, J., M. Schmitt, H. Krumbeck, M. Kapfer, E. Litchman & P.J. Neale. 2001. Effects of UV on carbon assimilation of phytoplankton in a mixed water column. Aquat. Sci., 63: 294-309.

Kroon, B.M.A., M. Latasa, B.W. Ibelings & L.R. Mur. 1992. The effect of dynamic light regimes on Chlorella. I. Pigments and cross sections. Hydrobiologia, 238: 71-78.

Litchman, E., C.A. Klausmeier & P. Bossard. 2004. Phytoplankton nutrient competition under dynamic light regimes. Limnol. Oceanogr., 49: 1457-1462.

Litchman, E. 2000. Growth rates of phytoplankton un-der fluctuating light. Freshwat. Biol., 44: 223-235.

Litchman, E. 1998. Population and community responses of phytoplankton to fluctuating light. Oecologia, 117: 247-257.

Marra, J. 1978. Phytoplankton photosynthetic response to vertical movement in a mixed layer. Mar. Biol., 46: 203-208.

Neale, P.J., E.W. Helbling & H.E. Zagarese. 2003. Modulation of UVR exposure and effects by vertical mixing and advection. In: E.W. Helbling & H.E. Zagarese (eds.). UV effects in aquatic organisms and ecosystems. The Royal Society of Chemistry, Cambridge, pp. 107-134.

Neale, P.J., R.F. Davis & J.J. Cullen. 1998. Interactive effects of ozone depletion and vertical mixing on photosynthesis of Antarctic phytoplankton. Nature, 392: 585-589.

Osmond, C.B. 1994. What is photoinhibitionfi Some insights from comparison of shade and sun plants. In: N. Baker & J. Bowyer (eds.). Photoinhibition of photosynthesis: from molecular mechanisms to the field. Bios Scientific, Oxford, pp. 1-24.

Raven, J.A. 1994. The cost of photoinhibition to plant communities. In: N. Baker & J. Bowyer (eds.). Photoinhibition of photosynthesis: from molecular mechanisms to the field. Bios Scientific, Oxford, pp. 449-464.

Richardson, K., J. Beardall & J.A. Raven. 1983. Adaptation of unicellular algae to irradiance: an analysis of strategies. New Phytol., 93: 157-191.

Steele, J.H. 1985. A comparison of terrestrial and marine ecological systems. Nature, 313: 355-358.

Villafañe, V.E. & F.M.H. Reid. 1995. Métodos de microscopía para la cuantificación del fitoplancton. In: K. Alveal, M.E. Ferrario, E.C. Oliveira & E. Sar (eds.). Manual de métodos ficológicos. Universidad de Concepción, Concepción, pp. 169-185.

Villafañe, V.E., E.S. Barbieri & E.W. Helbling. 2004. Annual patterns of ultraviolet radiation effects on temperate marine phytoplankton off Patagonia, Argentina. J. Plankton Res., 26: 167-174.

Villafañe, V.E., K. Sundbäck, F.L. Figueroa & E.W. Helbling. 2003. Photosynthesis in the aquatic environment as affected by UVR. In: E.W. Helbling & H.E. Zagarese (eds.). UV effects in aquatic organisms and ecosystems. The Royal Society of Chemistry, Cambridge, pp. 357-397.

Yoder, J.A. & S.S. Bishop. 1985. Effects of mixinginduced irradiance fluctuations on photosynthesis of natural assemblages of coastal phytoplankton. Mar. Biol., 90: 87-93.

Zar, J.H. 1984. Biostatistical analysis. Prentice Hall, New Jersey, USA, 718 pp.

Recibido: 16 mayo 2006; Aceptado 20 octubre 2006

Corresponding author: Walter Helbling (whelbling@efpu.org.ar)

* Trabajo presentado en el XXV Congreso de Ciencias del Mar de Chile y XI Congreso Latinoamericano de Ciencias del Mar (COLACMAR), realizados en Viña del Mar, entre el 16 y 20 de mayo de 2005.