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Interactive effects of ultraviolet radiation and nutrient addition on growth and photosynthesis performance of four species of marine phytoplankton
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Journal of Photochemistry and Photobiology B: Biology 89 (2007) 78–87 www.elsevier.com/locate/jphotobiol
Interactive effects of ultraviolet radiation and nutrient addition on growth and photosynthesis performance of four species of marine phytoplankton M. Alejandra Marcoval
a,b,c
, Virginia E. Villafan˜e
a,b
, E. Walter Helbling
a,b,*
a
c
Estacio´n de Fotobiologı´a Playa Unio´n, Casilla de Correos No. 15, 9103 Rawson, Chubut, Argentina b Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina Universidad Nacional de Mar del Plata, Estacio´n Costera J. J. Na´gera, Ruta 11 Km 24, 7600 Chapadmalal, Buenos Aires, Argentina Received 6 August 2007; received in revised form 13 September 2007; accepted 13 September 2007 Available online 19 September 2007
Abstract Experiments (6–8 days) were carried out during the austral summer of 2005 in Chubut, Argentina (43° S, 65° W) to determine the interactive effects of solar UVR (280–400 nm) and nutrient addition on growth and chlorophyll fluorescence of four species of marine phytoplankton – the diatoms Thalassiosira fluviatilis Hustedt and Chaetoceros gracilis Schu¨tt, and the dinoflagellates Heterocapsa triquetra (Ehrenberg) Stein and Prorocentrum micans (Ehrenberg). Samples were incubated under three radiation treatments (two sets of each radiation treatment): (a) samples exposed to full solar radiation (PAR + UVR, PAB treatment, 280–700 nm); (b) samples exposed to PAR and UV-A (PA treatment, 320–700 nm) and (c) samples exposed only to PAR (P treatment, 400–700 nm). At the beginning of the experiments, nutrients (i.e., NaPO4H2 and NaNO3) were added to one set of samples from each radiation treatment (‘‘N’’ cultures) whereas in the other, the nutrients concentration was that of the culture medium. At all times, the lowest growth rates (l) were determined in the PAB treatments, where enriched cultures had significantly higher l (P < 0.05) than non-enriched cultures. Daily cycles of photochemical quantum yield (Y) displayed a pattern of relatively high values early in the morning with a sharp decrease at noon; recovery was observed late in the afternoon. In general, higher Y values were determined in enriched cultures than in non-enriched cultures. As the experiments progressed, acclimation (estimated as the difference between Y at noon and that at time zero) was observed in all species although in variable degree. All species displayed some degree of UVR-induced decrease in the photochemical quantum yield, although it was variable among treatments and species. However, this effect decreased with time, and this pattern was more evident in the dinoflagellates, as the concentration of UV-absorbing compounds increased. Thus, under conditions of nutrient enrichment as may occur by river input or by re-suspension by mixing, dinoflagellates outcompete with diatoms because they may have a higher fitness under UVR stress. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Chaetoceros gracilis; Heterocapsa triquetra; Nutrients; Photosynthesis; Prorocentrum micans; Phytoplankton; Thalassiosira fluviatilis; Ultraviolet radiation; UV-absorbing compounds
1. Introduction Solar ultraviolet radiation (UVR, 280–400 nm) is a natural stress factor that has the potential to affect negatively * Corresponding author. Address: Estacio´n de Fotobiologı´a Playa Unio´n, Casilla de Correos No. 15, 9103 Rawson, Chubut, Argentina. Tel.: +54 2965 498019; fax: +54 2965 496269. E-mail address: [email protected] (E.W. Helbling).
1011-1344/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2007.09.004
phytoplankton organisms by reducing both growth and photosynthetic rates [1] and by damaging the DNA molecule [2]. Studies have shown that in combination with other abiotic factors (e.g., nutrient status/availability, temperature, mixing) the responses of phytoplankton to UVR can be potentiated. Particularly, when considering the interactive effects of UVR and nutrient status, a study [3] demonstrated the increased sensitivity to UVR when dinoflagellates grew in nutrient-limited conditions. Shelly
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et al. [4] showed for a chlorophyte species that the decline in growth rates and fluorescence parameters (i.e., effective quantum yield) after phosphorus starvation was significantly faster in the presence of UVR. Frost and Xenopoulos [5] working in boreal lakes of Canada, also showed that phytoplankton growth was co-regulated by phosphorus limitation and UVR suppression, with highest growth rates found in high-phosphorous, low-UVR treatments. Also, Aubriot et al. [6] showed that the phosphorous nutritional status modulated the sensitivity to UVR of natural assemblages from a southern Atlantic Ocean coastal lagoon. Even the negative effects caused by UVR, exposure of phytoplankton to short wavelengths of the solar spectrum has been frequently associated with the synthesis/accumulation of UV-absorbing compounds such as mycosporine like amino acids – MAAs [7]. The presence of these nitrogenous compounds (with absorption maxima between 310 and 360 nm) represents an acclimation mechanism against UVR stress, as seen in studies evaluating the photosynthetic performance of diatoms [8] and dinoflagellates [9]. These compounds are commonly found in relatively large species of dinoflagellates and diatoms [7]; on the other hand, for small cells the useful concentration would be too high and osmotically disadvantageous [10]. It is known that the synthesis and posterior accumulation of MAAs depends not only on the radiation climate (i.e., quality and quantity) but also on other environmental variables, such as salinity, temperature, or nutrient availability [11,12]. For example, studies carried out by Korbee Peinado et al. [13] revealed that not only the quality and quantity of the energy received induced the MAAs synthesis in the rhodophyte Porphyra columbina, but also high concentration of ammonium significantly increased the content of MAAs. However, for Grateloupia lanceola it was found [14] that although the addition of ammonium benefited the recovery of photosynthetic activity, the content of MAAs did not follow the same pattern hence, it could not be concluded that this was the cause of enhanced recovery. In any case, it is clear that the overall impact of UVR upon phytoplankton – expressed as the balance between the net (negative) effect and the acclimation capacity (e.g., through the presence of potentially protective compounds) – depends not only on solar radiation exposure but also on its interaction with other environmental variables. The aim of our study is to evaluate the interactive effects of solar UVR and nutrient addition on growth and photochemical performance of four species of marine phytoplankton – the diatoms Thalassiosira fluviatilis Hustedt and Chaetoceros gracilis Schu¨tt, and the dinoflagellates Heterocapsa triquetra (Ehrenberg) Stein and Prorocentrum micans (Ehrenberg). We further explored the ability of these organisms to potentially acclimate to the radiation conditions imposed throughout the experiment by the synthesis of UV-absorbing compounds. The species chosen are of ecological relevance as they are
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either an important component of marine phytoplankton communities and/or they are potentially toxic in coastal and oceanic waters. 2. Materials and methods 2.1. Organisms and culture conditions The phytoplankton species used in this study were the diatoms T. fluviatilis Hustedt (mean cell size 10 lm) and C. gracilis Schu¨tt (mean cell size 5 lm) and the dinoflagellates H. triquetra (Ehrenberg) Stein (mean cell size 20 lm) and P. micans (Ehrenberg) (mean cell size 50 lm) which were obtained from the Algae Culture Collection at Estacio´n de Fotobiologı´a Playa Unio´n (EFPU). C. gracilis, T. fluviatilis and P. micans were originally isolated from the Argentinean Sea whereas H. triquetra was obtained from the North Sea. Cells were kept in seawater-enriched medium f/2 [15] in an illuminated chamber (Sanyo model ML 350) at 20° C using a 12L:12D photoperiod with PAR (Photosynthetically Active Radiation, 400–700 nm) irradiance of 70 W m2 until experimentation (see below). The experiments were carried out at EFPU (43° S, 65° W), Chubut, Argentina, during the austral summer of 2005 during the periods: February 2–8, February 11–16, February 28– March 7 and March 14–20 for experiments performed with C. gracilis, T. fluviatilis, P. micans and H. triquetra, respectively. 2.2. Experimentation/sampling protocol For each experiment (i.e., with each species) aliquots of the monospecific culture were put in twelve 4-l UVtransparent containers (Plexiglas UVT, GS 2458, Ro¨hm and Haas, Darmstadt, Germany). Six of these containers received supplemental nutrients (i.e., 0.6 and 0.7 mM of NaPO4H2 and NaNO3, respectively) – (‘‘N’’) cultures, whereas in the other six the nutrient concentration was that of the original culture medium. Three radiation treatments were imposed on the samples, with two containers from each nutrient treatment exposed either to: (1) full solar radiation (UVR, 280–400 nm, and PAR, 400–700 nm) – uncovered containers – PAB treatment; (2) UV-A (320–400 nm) and PAR – containers covered with UV cut-off filter foil (Montagefolie, No. 10155099, Folex) (50% transmission at 320 nm) – PA treatment; and (3) only PAR – containers covered with Ultraphan film (UV Opak, Digefra) (50% transmission at 395 nm) – P treatment (the spectra of these materials are published in Figueroa et al. [16]). The containers were placed in a bath with running water as temperature control (17– 20 °C) and exposed to natural radiation for 6–8 days. At the beginning of each experiment and then, on a daily basis, samples were taken to determine chlorophyll a (chl a) concentration, absorption spectral characteristics and cell concentration (see below). Additionally, samples were collected three times during the day (i.e., 8 am, 1 pm
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and 5 pm) to determine fluorescence parameters (see below). 2.3. Analyses and measurements The analyses and measurements were conducted as described below: 2.3.1. Chlorophyll a (chl a) Chl a concentration was measured by filtering 50–100 ml of sample onto a Whatman GF/F filter (25 mm) and extracting the photosynthetic pigments in absolute methanol [17]. The fluorescence of the methanolic extract was measured using a Turner Designs fluorometer (model TD700) before and after acidification; chl a concentration was calculated from these readings [18] (it should be noted that the fluorometer is routinely calibrated against spectrophotometric readings). 2.3.2. UV-absorbing compounds UV-absorbing compounds were determined by filtering 50–100 ml of sample onto a Whatman GF/F filter (25 mm) and extracting these compounds in absolute methanol overnight. The estimation of concentration of UVabsorbing compounds was done by peak analysis of the scans (250–750 nm) using a Hewlett Packard spectrophotometer (model HP 8453E). The peak height at 334 nm was considered as an estimate of UV-absorbing compounds concentration [19]. We are aware that other studies (e.g., [20]) established the use of 20% methanol as the best extraction solvent for these compounds. Nevertheless, and since we were limited by the volume of samples, we considered that this procedure was appropriate for the purposes and aim of our investigation, even it might slightly underestimate the amount of UV-absorbing compounds. Once scanned, the same sample was used to determine the chl a concentration fluorometrically.
2.3.5. Radiation measurements and other atmospheric parameters Incident solar radiation was continuously measured using a broad band ELDONET radiometer (Real Time Computers Inc., Germany) that measures UV-B (280– 315 nm), UV-A (315–400 nm) and PAR (400–700 nm) and stores the data with a frequency of one reading per minute. In addition, continuous monitoring of other atmospheric parameters (e.g., temperature, humidity and wind speed and direction) was carried out using a meteorological station (Oregon Scientific model WMR-918). 2.3.6. Statistics/treatment of data The duration of each experiment (i.e., 6–8 days) together with the rapid changes in radiation levels that normally occur in the study area did not allow for repetition of experiments under similar radiation conditions. However, and within each experiment, treatments were done in duplicate, and duplicate samples were collected for each analysis/measurement. In the case of photochemical yield measurements, however, samples were measured 10 times with the PAM fluorometer. The data are reported as the mean and half range between duplicates. The non-parametric Kruskal Wallis test [23] was used to assess for significant differences between the samples exposed to different treatments using a 95% confidence limit. The decrease of the photochemical quantum yield at each wavelength interval (i.e., photochemical quantum yield in the PAB and PA treatments relative to that in the P control) over the incubation period was calculated as Decrease by UV-B ¼ ½ðY P Y PAB Þ ðY P Y PA Þ=ðY P Þ 100 Decrease by UV-A ¼ ðY P Y PA Þ=ðY P Þ 100 where YP, YPA, and YPAB are the photochemical quantum yield in the P, PA, and PAB treatments, respectively. 3. Results
2.3.3. Cell counts Aliquots of cell cultures (10 ml) were fixed with buffered formalin (final concentration in the sample = 0.4% of formaldehyde) and counted using an inverted microscope (Leica model DM IL) with a 1-ml Sedwick–Rafter chamber. 2.3.4. Photochemical quantum yield A portable pulse amplitude modulated fluorometer (Water-ED PAM, Walz, Germany) was used to determine in vivo chlorophyll fluorescence of the photosystem II. The photochemical effective quantum yield (Y) was calculated using the equations of Genty et al. [21] and Weis and Berry [22] as Y ¼ ðF 0m F t Þ=F 0m ¼ F 0v =F 0m where F 0m is the maximal fluorescence induced by a saturating light pulse (ca. 5300 lmol photons m2 s1 in 0.8 s), and Ft the current steady state fluorescence induced by weak actinic light in light-adapted cells.
Total ozone column concentrations and daily doses of solar radiation during the study period – February 2– March 20, 2005 (Julian days 33–79) are shown in Fig. 1. Ozone column concentration over Playa Unio´n (data obtained from http://toms.gsfc.nasa.gov) was variable but relatively high throughout the study period, with maximum and minimum ozone concentrations of 298 and 241 Dobson Units (D.U.) on Julian days 79 and 57, respectively (Fig. 1a). Daily doses of PAR, UV-A and UV-B (Fig. 1b–d) were variable due to differences in cloud cover but nevertheless, there was a clear decreasing trend towards the end of the study period. UV-B daily doses varied between 11 and 42 kJ m2 (Fig. 1b) whereas UV-A between 400 and 1500 kJ m2 (Fig. 1c). PAR followed the same trend as UVR wavebands, with daily doses ranging between 2100 and 10,500 kJ m2 (Fig. 1d). The ratio of UV-B to PAR during the study period ranged between 0.0033 and 0.0051, on Julian days 68 and 50, respectively.
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Fig. 1. (a) Total ozone column concentration (in Dobson Units, D.U.) during the study period (Julian days 33–79) and (b–d) Daily doses of solar radiation (in kJ m2) (Julian days 33–79). (b) UV-B, 280–315 nm; (c) UVA, 315–400 nm; (d) PAR, 400–700 nm. The experiments with Chaetoceros gracilis, Thalassiosira fluviatilis, Prorocentrum micans and Heterocapsa triquetra were carried out from February 2 to February 8, 2005, February 11 to February 16, 2005, February 28 to March 6, 2005 and March 14 to March 20 for, respectively.
The daily variations of PAR irradiance during the four experiments are presented in Fig. 2. Maximum PAR irradiance levels were higher (350 W m2) during experiments carried out with C. gracilis (Fig. 2a) and T. fluviatilis (Fig. 2b) than during those carried out with dinoflagellates (300 W m2) (Fig. 2c and d). In general, PAR irradiance conditions (and also that of UV-A and UV-B, data not shown) were rather similar within each experiment, except for the last 2 days of the C. gracilis experiment (Fig. 2a) where cloud cover resulted in ca. 50% reduction in daily doses. Growth (expressed as cells and chl a concentration) of C. gracilis, T. fluviatilis, P. micans and H. triquetra exposed to different radiation/nutrient treatments is presented in Fig. 3. The most evident feature was the lack of a lag phase in the diatoms (Fig. 3a–d), with the exception of C. gracilis samples exposed to full radiation. In the other treatments, however, the concentration of C. gracilis cells and chl a
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Fig. 2. Daily variations of PAR irradiance (in W m2) during the experiments carried out with (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra.
increased as soon as the experiment started (Fig. 3a and b). In samples under the P and PA treatments, the cultures that received additional nutrients had higher cells and chl a concentrations than those that did not receive them. Regardless of the nutrient treatment, the samples under the PAB treatment had lower concentration of cells and chl a for most of the time, however, at the end of the experiment they had similar values as the PA and P treatments. During the experiment carried out with T. fluviatilis (Fig. 3c and d) a rather similar growth was determined in all treatments during the first 2 days; afterwards, PN and PAN treatments had the highest chl a concentration (Fig. 3d) although the concentration of cells was the same in the three radiation treatments (Fig. 3c). Regardless of the nutrient treatment, P. micans samples (Fig. 3e and f) had a 1–2 days lag phase; afterwards the concentration of cells and chl a increased in all samples, with cultures exposed to the PN treatment having the highest values. Finally, in the experiment carried out with H. triquetra (Fig. 3g and f) the lag phase lasted 3 days and, afterwards, PN samples displayed the highest growth. Growth rates (l) for these cultures, based on chl a measurements are
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Fig. 3. Growth of the four phytoplankton species expressed as cell concentration (cells ml1) (a, c, e and g) and chl a concentration (lg chl a L1) (b, d, f and h): (a and b) Chaetoceros gracilis, (c and d) Thalassiosira fluviatilis, (e and f) Prorocentrum micans and (g and h) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
presented in Table 1. The lowest l were attained in the PAB treatment (non-enriched) with values of 0.52, 0.41, 0.44 and 0.49 day1 in C. gracilis, T. fluviatilis, P. micans
and H. triquetra, respectively. On the other hand, the highest l values were determined in enriched cultures receiving only PAR – 0.85; 1.06, 0.97 and 0.69 day1 in C. gracilis, T. fluviatilis, P. micans and H. triquetra, respectively. Nevertheless, enriched cultures had significantly higher l (P < 0.05) than non-enriched cultures only in the PAB treatments. When considering the same nutrient condition, the PAB treatments had significantly lower l (P < 0.05) than the P treatments in the four phytoplankton species. Similar conclusions on growth rates as those from chl a measurements were obtained from cell concentration data (data not shown). Daily variations of photochemical quantum yield (Y) for the four species studied are shown in Fig. 4. The general pattern for all species was of a significant decrease of Y at or close to local noon in all treatments, with a significant recovery during the afternoon and night. However, the extent of the Y decrease as well as that of recovery was different among species. In C. gracilis (Fig. 4a) Y decreased significantly throughout the experiment, from an initial value of 0.7 to 0.1 in non-enriched cultures and to 0.4–0.5 in enriched cultures at the end of the experimental period. During the first day, samples exposed to UVR had significantly lower quantum yield than those exposed only to PAR, however, during the night of the second day and at the end of the experiment there were no significant differences in Y among the radiation treatments in the nonenriched cultures, while the Y in the enriched samples in the PN had significantly higher values than in the PABN and PAN treatments. Similarly, Y values of T. fluviatilis (Fig. 4b) at the end of the experiment decreased significantly from the initial value of 0.6 to 0.3–0.5 in enriched samples in the PABN and PAN/PN treatments, respectively, and to 0.1 in all radiation treatments in non-enriched cultures. In the experiment carried out with P. micans (Fig. 4c) there were also differences in Y, with enriched cultures having significantly higher values than non-enriched cultures. Moreover, samples exposed in the PABN had significantly lower Y values (i.e., 0.3) than in the PAN/PN treatments (i.e., 0.5–0.6) at the end of the experiment. In addition, the noon time Y value that was 0 during the first 3 days, increased to >0.1 as the experiment progressed. Finally, in the experiment carried out with H. triquetra
Table 1 Growth rates (l day1, calculated from chl a measurements) during experiments carried out with Chaetoceros gracilis, Thalassiosira fluviatilis, Prorocentrum micans and Heterocapsa triquetra exposed to different nutrient/radiation treatments Treatment/culture
Chaetoceros gracilis
Thalassiosira fluviatilis
Prorocentrum micans
Heterocapsa triquetra
PAB PA P PABN PAN PN P (l = lN)
0.52 (0.01) 0.64 (0.02) 0.89 (0.01) 0.62 (0.01)* 0.59 (0.01) 0.85 (0.01) <0.05
0.41 (0.02) 1.02 (0.03) 1.05 (0.03) 0.68 (0.02)* 1.08 (0.01) 1.06 (0.01) <0.05
0.44 (0.03) 0.77 (0.01) 0.75 (0.01) 0.50 (0.02)* 0.74 (0.03) 0.97 (0.01)* <0.05
0.49 (0.02) 0.61 (0.03) 0.63 (0.01) 0.61 (0.03)* 0.65 (0.02) 0.69 (0.01)* <0.05
Radiation treatments are denoted by PAB (PAR + UV-A + UV-B), PA (PAR + UV-A) and P (PAR only); N denotes cultures that received additional nutrients. The asterisks indicate significant differences between nutrient treatments (i.e., comparing the same radiation treatment).
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Fig. 4. Daily variations of photochemical quantum yield (Y) during the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
(Fig. 4d) there was a significant inhibition at the end of the day that was also evident during early morning during the first days. However, the Y value increased with time and at the end of the experiment enriched samples in the PAN and PN and non-enriched samples in the P treatments had significant higher Y value (>0.5) than the rest of the samples; this value was even higher than that measured at the beginning of the experiment (i.e., 0.4). The decrease in quantum yield at local noon (calculated from the Y values of Fig. 4) throughout the duration of the experiments is presented in Fig. 5. C. gracilis was the only species that had significant lower decrease of Y in the samples in the P treatment as compared those that received additionally UVR, regardless of the nutrient treatment, for the first 3 days of experimentation (Fig. 5a). However, at the end of the experimentation P samples were those with higher decrease of Y (i.e., ca. 100%). In T. fluviatilis
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Fig. 5. Decrease in photochemical quantum yield (%, relative to t0) at local noon throughout the duration of the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
(Fig. 5b), the decrease in quantum yield diminished with time and no significant differences were observed among nutrient/radiation treatments during the first 3 days of experimentation. After that, and towards the end of the experiment, samples with nutrient addition had significant lower decrease of Y than those that did not receive nutrients. In P. micans (Fig. 5c) the decrease of Y diminished continuously throughout the experiment while in H. triquetra (Fig. 5d) it was rather constant during the first 4 days and afterwards decreased. We further studied the potential mechanisms by which organisms could adapt to the conditions imposed in the experiments (i.e., through the presence of UV-absorbing compounds). The initial spectral absorption characteristics (O.D. mg chl a1) of the four species used in our experiments are presented in Fig. 6. All species had characteristic
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Fig. 6. Initial spectral absorption characteristics (O.D. mg chl a1) of Chaetoceros gracilis (triangles); Thalassiosira fluviatilis (circles); Prorocentrum micans (squares) and Heterocapsa triquetra (diamonds).
peaks of chl a at 440 and at 665 nm. Carotenoids (kmax = 470 nm) were present in T. fluviatilis, P. micans and H. triquetra, but they were barely noticeable in C. gracilis. UV-absorbing compounds (maximum absorption in the range of 310–360 nm) were especially abundant in P. micans, but they were present in very low concentrations (or virtually absent) in the other three species tested. The temporal variability in concentration of these UVabsorbing compounds during the experiments is shown in Fig. 7. In general, there was an increasing trend of concentration with time in all species tested; however, the extent of this increase was variable among species and according to the treatment imposed on the samples. In C. gracilis (Fig. 7a) this trend was especially evident during the first 4 days of experimentation and afterwards, its concentration decreased. The highest amounts of UV-absorbing compounds were determined in enriched cultures, whereas P and PA samples had a similar concentration to that at the beginning of the experiment. In T. fluviatilis (Fig. 7b) samples in the PABN and PAN and PAB treatments increased significantly the concentration of UV-absorbing compounds throughout the experiment. However and regardless of the nutrient treatment, samples exposed only to visible radiation had approximately the same concentration throughout the experiment. In P. micans (Fig. 7c) the increase of UV-absorbing compounds was evident only in samples exposed to full solar radiation, especially in enriched samples; in the other treatments, their concentration remained similar (and relatively low) throughout the experiment. A similar behaviour was observed in H. triquetra (Fig. 7d) with PAB samples increasing significantly the concentration of UV-absorbing compounds towards the end of the experiment, especially in enriched samples. The relationship between the decrease in quantum yield (from Fig. 5) and the concentration of UV-absorbing compounds (from Fig. 7) is presented in Fig. 8 (only significant relationships (P < 0.05) having R2 between 0.6
Fig. 7. Daily variations in concentration of UV-absorbing compounds (estimated by the peak height at 334 nm) during the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
and 0.94 are plotted). When comparing the four species, it was observed that samples in different treatments displayed significant different relationships. In C. gracilis (Fig. 8a) only samples in the PABN treatment had a significant negative relationship between the decrease in quantum yield and the increased concentration of UVabsorbing compounds. In T. fluviatilis (Fig. 8b) this relationship was observed in the PABN treatment, and in both nutrient treatments receiving UV-A and PAR (i.e., PA and PAN). Finally, in P. micans (Fig. 8c) only those treatments receiving full solar radiation (i.e., PAB and PABN) had a negative relationship between the decrease in quantum yield and the increased concentration of UV-absorbing compounds whereas in H. triquetra (Fig. 8d) this relationship was observed in the PA, PAN, P and PN treatments.
M.A. Marcoval et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 78–87
Fig. 8. Decrease in photochemical quantum yield (%) at local noon as a function of concentration of the UV-absorbing compounds throughout the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. The full lines indicate the best fit and the dotted lines are the confidence limits.
4. Discussion Many studies devoted to assessing the impact of solar UVR upon natural phytoplankton populations have highlighted the variability in responses mainly because they are species-specific, with some species being relatively tolerant and others sensitive [1]. However, in combination with other stress factors, species can display different responses than when considering solely UVR, because their interaction can be either antagonistic or synergic. In this study we compared the responses to both, solar radiation and nutrient addition in four phytoplankton species which are commonly found in marine environments. Our results can be summarized as follows: regardless of the nutrient condition, all species displayed some
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degree of stress when exposed to solar radiation, as seen in both growth (Fig. 3) and the photochemical quantum yield (Figs. 4 and 5). However, some evidence of acclimation was observed towards the end of the experimental period, especially in the dinoflagellates. Another feature of our data is the variability in responses among the species studied even though radiation (Figs. 1 and 2) and nutrient conditions were rather similar in the four experiments, clearly emphasizing the fact that responses are species-specific. In principle, one might think that the decrease in both growth and photochemical quantum yield (Figs. 3–5) as observed in our experiments mainly occurs because of exposure to high solar radiation levels (i.e., high doses, Fig. 1) and irradiances (Fig. 2) at which the organisms where exposed. These high radiation levels are characteristic of the Patagonia area [24] and they are due to a combination of high heliophany and long day-light period [25] during summer. In fact, these radiation levels during summer are comparable to those received in tropical areas during mid-spring/mid-autumn [26,27]) (however, the ratio UV-B to PAR is higher in tropical areas than in our study site). The reduction of both growth and photochemical quantum yield as effectively occurs in our experiments (Figs. 3–5) can be partially related to these high radiation levels; moreover, this seems to be a common feature of phytoplankton organisms, as seen in studies carried out with natural populations as well as with monospecific cultures [1]. Still, the observed responses in our experiments represent the net result between damage/inhibition and repair/ acclimation mechanisms. Nutrient addition in our experiments gave hints on their key role at the physiological level by allowing species to better cope with exposure to solar radiation. This was clearly seen by comparing growth and the photochemical quantum yield in enriched and non-enriched conditions; higher values were observed in samples that received supplemental nutrients (Figs. 3 and 4), particularly in the PAB treatments where enriched cultures had significantly higher growth rates (P < 0.05), than non-enriched cultures (Table 1). Moreover, the importance of nutrient addition in favoring species to acclimate to solar radiation was evident in dinoflagellates that had a lower decrease in quantum yield at the end of the experiment than at the beginning (Fig 5). Thus our data support the fact that the supplement of nutrients are indeed associated with the synthesis of UV-absorbing compounds, which are thought to be one of the main mechanisms to cope with solar UVR [7]. As compared to other taxa (i.e., chlorophytes or cyanobacteria) both diatoms and dinoflagellates are considered relatively resistant to UVR [28]. Nevertheless, their apparent resistance is thought to be caused by different mechanisms: In diatoms, their silicon skeleton seems to play a role in absorbing UVR and thus conferring some protection to the cell [29]; in dinoflagellates, the synthesis of UV-absorbing compounds (mainly MAAs) seems to be
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the most important mechanism for UV protection. However, the degree and role of one or another protection mechanism may vary between taxa and/or species. For example, working with Antarctic diatoms Helbling et al. [8] found differences between centric and pennates, with the former being more resistant than the latter; this difference was thought to be due to the synthesis of MAAs in the centric diatoms but absent in pennates. In dinoflagellates, a variety of responses have been observed [30–32] and nutrient deficiency has been associated with an increase in UV sensitivity of species as compared to those grown in nonlimited conditions [3]. Our data presented here show that UV-absorbing compounds levels are much lower in diatoms than in dinoflagellates. Nevertheless, diatoms significantly increased the concentration of UV-absorbing compounds towards the end of the experiments, with samples that received additional nutrient having a higher concentration of them than non-enriched samples (Fig. 7). Nutrient addition has been previously shown to enhance the synthesis of UV-absorbing compounds [13,29]. However, in our experiments, as well as in those previous studies, the initial nutrient concentration was not limited in the samples, and moreover, in those treatments that did not received additional nutrients both, cell and chl a concentration increased throughout the experiments (Fig. 3). It is plausible, however, that cells give priority for the use of nutrients to other metabolic processes rather than to the synthesis of UV-absorbing compounds. A hint of this could be inferred from the relatively higher growth rates in the diatoms (especially T. fluviatilis) as compared to dinoflagellates (Table 1). Nevertheless, our data clearly show that the decrease in quantum yield was less (i.e., better performance) with increasing concentration of UV-absorbing compounds (Fig. 8) although this was variable among species and radiation/ nutrient treatments. We cannot rule out the contribution of other ‘‘protecting’’ mechanisms such as an active xanthophyll cycle or self-shading as the cultures grew. In fact, the xanthophyll cycle have been shown to be an effective protective mechanism in the diatom Thalassiossira weisflogii [33]. In that study, the concentration of MAAs increased after 16–22 days of exposing cells to UV-B, and it was thought to occur after the cells recovered from the initial UVR stress involving the xanthophyll cycle. In our experiments, we do not have information on xanthophyll pigments, but UVabsorbing compounds increased, at least in some treatments, from the beginning of the experiments. It is possible that longer exposure periods might render higher concentrations of UV-absorbing compounds and hence cells are more effectively protected. However, in the case of the diatoms, growth curves (Fig. 3) had a plateau after the exponential growth. At this point (i.e., end of the exponential growth) self-shading might have a role in protecting the cells as was seen in studies carried out with the cyanobacterium Arthorspira platensis [34]. Nevertheless, our data on photochemical quantum yield do not show a recovery
during this period, thus suggesting that if this mechanism is active its role is rather insignificant. Overall, our data suggest that under the experimental conditions imposed on the samples, the photochemical quantum yield and growth of diatoms are more affected by solar radiation than in dinoflagellates are. Thus, under conditions of nutrient enrichment, as may occur by river input or re-suspension by mixing, dinoflagellates might outcompete diatoms by synthesizing important amounts of UV-absorbing compounds and hence have a higher fitness under UVR stress. 5. Abbreviations MAAs PAR UV-B UV-A UVR Y
mycosporine like amino acids photosynthetically active radiation ultraviolet B ultraviolet A ultraviolet radiation photochemical quantum yield
Acknowledgments This work was supported by the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica – ANPCyT (PICT 2003-13388 and PICT 2005-32034), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas – CONICET (PIP 2004-5157), PNUD-GEF (B-C-39) and Fundacio´n Playa Unio´n. We thank D.P. Ha¨der who kindly provided some cultures used in the experiments; J.I. Albarracı´n helped with culture maintenance. We also thank the comments and suggestions of two anonymous reviewers that helped to improve this manuscript. This is in partial fulfillment of the Ph.D. thesis of MAM. This is contribution number 109 of the Estacio´n de Fotobiologı´a Playa Unio´n. References [1] V.E. Villafan˜e, K. Sundba¨ck, F.L. Figueroa, E.W. Helbling, in: E.W. Helbling, H.E. Zagarese (Eds.), UV effects in aquatic organisms and ecosystems, Royal Society of Chemistry, 2003, pp. 357–397. [2] A.G.J. Buma, P. Boelen, W.H. Jeffrey, in: E.W. Helbling, H.E. Zagarese (Eds.), UV Effects In Aquatic Organisms And Ecosystems, The Royal Society of Chemistry, Cambridge, 2003, pp. 291–327. [3] E. Litchman, P.J. Neale, A.T. Banaszak, Increased sensitivity to ultraviolet radiation in nitrogen-limited dinoflagellates: photoprotection and repair, Limnol. Oceanogr. 47 (2002) 86–94. [4] K. Shelly, S. Roberts, P. Heraud, J. Beardall, Interactions between UV-B exposure and phosphorus nutrition. I. Effects on growth, phosphate uptake, and chlorophyll fluorescence, J. Phycol. 41 (2005) 1204–1211. [5] P.C. Frost, M.A. Xenopoulos, Ambient solar ultraviolet radiation and its effects on phosphorous flux into boreal lake phytoplankton communities, Can. J. Fish. Aquat. Sci. 59 (2002) 1090–1095. [6] L. Aubriot, D. Conde, S. Bonilla, R. Sommaruga, Phosphate uptake behavior of natural phytoplankton during exposure to solar ultraviolet radiation in a shallow coastal lagoon, Mar. Biol. 144 (2004) 623– 631.
M.A. Marcoval et al. / Journal of Photochemistry and Photobiology B: Biology 89 (2007) 78–87 [7] A.T. Banaszak, in: E.W. Helbling, H.E. Zagarese (Eds.), UV Effects In Aquatic Organisms And Ecosystems, The Royal Society of Chemistry, Cambridge, 2003, pp. 329–356. [8] E.W. Helbling, B.E. Chalker, W.C. Dunlap, O. Holm-Hansen, V.E. Villafan˜e, Photoacclimation of antarctic marine diatoms to solar ultraviolet radiation, J. Exp. Mar. Biol. Ecol. 204 (1996) 85–101. [9] P.J. Neale, A.T. Banaszak, C.R. Jarriel, Ultraviolet sunscreens in Gymnodinium sanguineum (Dinophyceae); mycosporine-like amino acids protect against inhibition of photosynthesis, J. Phycol. 34 (1998) 928–938. [10] F. Garcia-Pichel, A model for internal self-shading in planktonic organisms and its implications for the usefulness of ultraviolet sunscreens, Limnol. Oceanogr. 39 (1994) 1704–1717. [11] W.M. Bandaranayake, Mycosporines: are they nature’s screens? Nat. Prod. Rep. (1998) 159–172. [12] W.C. Dunlap, J.M. Shick, Ultraviolet radiation-absorbing mycosporine-like amino acids in coral reef organisms: a biochemical and environmental perspective, J. Phycol. 34 (1998) 418–430. [13] N. Korbee Peinado, R.T. Abdala Dı´az, F.L. Figueroa, E.W. Helbling, Ammonium and UV radiation stimulate the accumulation of mycosporine like amino acids in Porphyra columbina (Rhodophyta) from Patagonia, Argentina, J. Phycol. 40 (2004) 248–259. [14] P. Huovinen, J. Matos, I. Sousa-Pinto, F.L. Figueroa, The role of ammonium in photoprotection against high irradiance in the red alga Grateloupia lanceola, Aquat. Bot. 84 (2006) 308–316. [15] R.R.L. Guillard, J.H. Ryther, Studies of marine planktonic diatoms. I. Cyclotella nana Husted, and Detonula confervacea (Cleve) Gran, Can. J. Microbiol. 8 (1962) 229–239. [16] F.L. Figueroa, S. Salles, J. Aguilera, C. Jime´nez, J. Mercado, B. Vin˜egla, A. Flores-Moya, M. Altamirano, Effects of solar radiation on photoinhibition and pigmentation in the red alga Porphyra leucosticta, Mar. Ecol. Prog. Ser. 151 (1997) 81–90. [17] O. Holm-Hansen, B. Riemann, Chlorophyll a determination: improvements in methodology, Oikos 30 (1978) 438–447. [18] O. Holm-Hansen, C.J. Lorenzen, R.W. Holmes, J.D.H. Strickland, Fluorometric determination of chlorophyll, J. Cons. Int. Explor. Mer. 30 (1) (1965) 3–15. [19] W.C. Dunlap, G.A. Rae, E.W. Helbling, V.E. Villafan˜e, O. HolmHansen, Ultraviolet-absorbing compounds in natural assemblages of Antarctic phytoplankton, Antarct. J. US 30 (1995) 323–326. [20] B. Tartarotti, R. Sommaruga, The effect of different methanol concentrations and temperatures on the extraction of mycosporinelike amino acids (MAAs) in algae and zooplankton, Arch. Hydrobiol. 154 (2002) 691–703. [21] B.E. Genty, J.M. Briantais, N.R. Baker, Relative quantum efficiencies of the two photosystems of leaves in photorespiratory and nonphotorespiratory conditions, Plant Physiol. Biochem. 28 (1989) 1–10.
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[22] E. Weis, A. Berry, Quantum efficiency of photosystem II in relation to the energy dependent quenching of chlorophyll fluorescence, Biochim. Biophys. Acta 894 (1987) 198–208. [23] J.H. Zar, Biostatistical Analysis, Prentice Hall, Englewood Cliffs, NJ, 1984. [24] E.W. Helbling, E.S. Barbieri, M.A. Marcoval, R.J. Gonc¸alves, V.E. Villafan˜e, Impact of solar ultraviolet radiation on marine phytoplankton of Patagonia, Argentina, Photochem. Photobiol. 81 (2005) 807–818. [25] V.L. Orce, E.W. Helbling, Latitudinal UVR-PAR measurements in Argentina: extent of the ‘‘ozone hole’’, Global Planet Change 15 (1997) 113–121. [26] E.W. Helbling, K. Gao, H. Ai, Z. Ma, V.E. Villafan˜e, Differential responses of Nostoc sphaeroides and Arthrospira platensis to solar ultraviolet radiation exposure, J. Appl. Phycol. 18 (2006) 57–66. [27] V.E. Villafan˜e, K. Gao, P. Li, G. Li, E.W. Helbling, Vertical mixing within the epilimnion modulates UVR-induced photoinhibition in tropical freshwater phytoplankton from southern China, Freshwater Biol. 52 (2007) 1260–1270. [28] S. Demers, S. Roy, R. Gagnon, C. Vignault, Rapid light-induced changes in cell fluorescence and in xanthophyll-cycle pigments of Alexandrium excavatum (Dinophyceae) and Thalassiosira pseudonana (Bacillariophyceae): a photo-protection mechanism, Mar. Ecol. Prog. Ser. 76 (1991) 185–193. [29] W.H. van de Poll, M.A. van Leeuwe, J. Roggeveld, A.G.J. Buma, Nutrient limitation and high irradiance acclimation reduce PAR and UV-induced viability loss in the Antarctic diatom Chaetoceros brevis (Bacillariophyceae), J. Phycol. 41 (2005) 840–850. [30] J.I. Carreto, M.O. Carignan, G. Daleo, S.G. De Marco, Occurence of mycosporine-like aminoacids in the red-tide dinoflagellate Alexandrium excavatum: UV-photoprotective compounds? J. Plankton. Res. 12 (5) (1990) 909–921. [31] M. Klisch, D.P. Ha¨der, Mycosporine-like amino acids in the marine dinoflagellate Gyrodinium dorsum: induction by ultraviolet irradiation, Photochem. Photobiol. 55 (2002) 178–182. [32] H. Taira, S. Aoki, B. Yamanoha, S. Taguchi, Daily variation in cellular content of UV-absorbing compounds mycosporine-like amino acids in the marine dinoflagellate Scrippsiella sweeneyae, J. Photochem. Photobiol. B Biol. 75 (2004) 145–155. [33] L. Zudaire, S. Roy, Photoprotection and long-term acclimation to UV radiation in the marine diatom Thalassiosira weissflogii, J. Photochem. Photobiol. B Biol. 62 (2001) 26–34. [34] H. Wu, K. Gao, V.E. Villafan˜e, T. Watanabe, E.W. Helbling, Effects of solar UV radiation and photosynthesis of the filamentous cyanobacterium, Arthrospira platensis, Appl. Environ. Microbiol. 71 (2005) 5004–5013.
Journal of Photochemistry and Photobiology B: Biology 89 (2007) 78–87 www.elsevier.com/locate/jphotobiol
Interactive effects of ultraviolet radiation and nutrient addition on growth and photosynthesis performance of four species of marine phytoplankton M. Alejandra Marcoval
a,b,c
, Virginia E. Villafan˜e
a,b
, E. Walter Helbling
a,b,*
a
c
Estacio´n de Fotobiologı´a Playa Unio´n, Casilla de Correos No. 15, 9103 Rawson, Chubut, Argentina b Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina Universidad Nacional de Mar del Plata, Estacio´n Costera J. J. Na´gera, Ruta 11 Km 24, 7600 Chapadmalal, Buenos Aires, Argentina Received 6 August 2007; received in revised form 13 September 2007; accepted 13 September 2007 Available online 19 September 2007
Abstract Experiments (6–8 days) were carried out during the austral summer of 2005 in Chubut, Argentina (43° S, 65° W) to determine the interactive effects of solar UVR (280–400 nm) and nutrient addition on growth and chlorophyll fluorescence of four species of marine phytoplankton – the diatoms Thalassiosira fluviatilis Hustedt and Chaetoceros gracilis Schu¨tt, and the dinoflagellates Heterocapsa triquetra (Ehrenberg) Stein and Prorocentrum micans (Ehrenberg). Samples were incubated under three radiation treatments (two sets of each radiation treatment): (a) samples exposed to full solar radiation (PAR + UVR, PAB treatment, 280–700 nm); (b) samples exposed to PAR and UV-A (PA treatment, 320–700 nm) and (c) samples exposed only to PAR (P treatment, 400–700 nm). At the beginning of the experiments, nutrients (i.e., NaPO4H2 and NaNO3) were added to one set of samples from each radiation treatment (‘‘N’’ cultures) whereas in the other, the nutrients concentration was that of the culture medium. At all times, the lowest growth rates (l) were determined in the PAB treatments, where enriched cultures had significantly higher l (P < 0.05) than non-enriched cultures. Daily cycles of photochemical quantum yield (Y) displayed a pattern of relatively high values early in the morning with a sharp decrease at noon; recovery was observed late in the afternoon. In general, higher Y values were determined in enriched cultures than in non-enriched cultures. As the experiments progressed, acclimation (estimated as the difference between Y at noon and that at time zero) was observed in all species although in variable degree. All species displayed some degree of UVR-induced decrease in the photochemical quantum yield, although it was variable among treatments and species. However, this effect decreased with time, and this pattern was more evident in the dinoflagellates, as the concentration of UV-absorbing compounds increased. Thus, under conditions of nutrient enrichment as may occur by river input or by re-suspension by mixing, dinoflagellates outcompete with diatoms because they may have a higher fitness under UVR stress. Ó 2007 Elsevier B.V. All rights reserved. Keywords: Chaetoceros gracilis; Heterocapsa triquetra; Nutrients; Photosynthesis; Prorocentrum micans; Phytoplankton; Thalassiosira fluviatilis; Ultraviolet radiation; UV-absorbing compounds
1. Introduction Solar ultraviolet radiation (UVR, 280–400 nm) is a natural stress factor that has the potential to affect negatively * Corresponding author. Address: Estacio´n de Fotobiologı´a Playa Unio´n, Casilla de Correos No. 15, 9103 Rawson, Chubut, Argentina. Tel.: +54 2965 498019; fax: +54 2965 496269. E-mail address: [email protected] (E.W. Helbling).
1011-1344/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2007.09.004
phytoplankton organisms by reducing both growth and photosynthetic rates [1] and by damaging the DNA molecule [2]. Studies have shown that in combination with other abiotic factors (e.g., nutrient status/availability, temperature, mixing) the responses of phytoplankton to UVR can be potentiated. Particularly, when considering the interactive effects of UVR and nutrient status, a study [3] demonstrated the increased sensitivity to UVR when dinoflagellates grew in nutrient-limited conditions. Shelly
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et al. [4] showed for a chlorophyte species that the decline in growth rates and fluorescence parameters (i.e., effective quantum yield) after phosphorus starvation was significantly faster in the presence of UVR. Frost and Xenopoulos [5] working in boreal lakes of Canada, also showed that phytoplankton growth was co-regulated by phosphorus limitation and UVR suppression, with highest growth rates found in high-phosphorous, low-UVR treatments. Also, Aubriot et al. [6] showed that the phosphorous nutritional status modulated the sensitivity to UVR of natural assemblages from a southern Atlantic Ocean coastal lagoon. Even the negative effects caused by UVR, exposure of phytoplankton to short wavelengths of the solar spectrum has been frequently associated with the synthesis/accumulation of UV-absorbing compounds such as mycosporine like amino acids – MAAs [7]. The presence of these nitrogenous compounds (with absorption maxima between 310 and 360 nm) represents an acclimation mechanism against UVR stress, as seen in studies evaluating the photosynthetic performance of diatoms [8] and dinoflagellates [9]. These compounds are commonly found in relatively large species of dinoflagellates and diatoms [7]; on the other hand, for small cells the useful concentration would be too high and osmotically disadvantageous [10]. It is known that the synthesis and posterior accumulation of MAAs depends not only on the radiation climate (i.e., quality and quantity) but also on other environmental variables, such as salinity, temperature, or nutrient availability [11,12]. For example, studies carried out by Korbee Peinado et al. [13] revealed that not only the quality and quantity of the energy received induced the MAAs synthesis in the rhodophyte Porphyra columbina, but also high concentration of ammonium significantly increased the content of MAAs. However, for Grateloupia lanceola it was found [14] that although the addition of ammonium benefited the recovery of photosynthetic activity, the content of MAAs did not follow the same pattern hence, it could not be concluded that this was the cause of enhanced recovery. In any case, it is clear that the overall impact of UVR upon phytoplankton – expressed as the balance between the net (negative) effect and the acclimation capacity (e.g., through the presence of potentially protective compounds) – depends not only on solar radiation exposure but also on its interaction with other environmental variables. The aim of our study is to evaluate the interactive effects of solar UVR and nutrient addition on growth and photochemical performance of four species of marine phytoplankton – the diatoms Thalassiosira fluviatilis Hustedt and Chaetoceros gracilis Schu¨tt, and the dinoflagellates Heterocapsa triquetra (Ehrenberg) Stein and Prorocentrum micans (Ehrenberg). We further explored the ability of these organisms to potentially acclimate to the radiation conditions imposed throughout the experiment by the synthesis of UV-absorbing compounds. The species chosen are of ecological relevance as they are
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either an important component of marine phytoplankton communities and/or they are potentially toxic in coastal and oceanic waters. 2. Materials and methods 2.1. Organisms and culture conditions The phytoplankton species used in this study were the diatoms T. fluviatilis Hustedt (mean cell size 10 lm) and C. gracilis Schu¨tt (mean cell size 5 lm) and the dinoflagellates H. triquetra (Ehrenberg) Stein (mean cell size 20 lm) and P. micans (Ehrenberg) (mean cell size 50 lm) which were obtained from the Algae Culture Collection at Estacio´n de Fotobiologı´a Playa Unio´n (EFPU). C. gracilis, T. fluviatilis and P. micans were originally isolated from the Argentinean Sea whereas H. triquetra was obtained from the North Sea. Cells were kept in seawater-enriched medium f/2 [15] in an illuminated chamber (Sanyo model ML 350) at 20° C using a 12L:12D photoperiod with PAR (Photosynthetically Active Radiation, 400–700 nm) irradiance of 70 W m2 until experimentation (see below). The experiments were carried out at EFPU (43° S, 65° W), Chubut, Argentina, during the austral summer of 2005 during the periods: February 2–8, February 11–16, February 28– March 7 and March 14–20 for experiments performed with C. gracilis, T. fluviatilis, P. micans and H. triquetra, respectively. 2.2. Experimentation/sampling protocol For each experiment (i.e., with each species) aliquots of the monospecific culture were put in twelve 4-l UVtransparent containers (Plexiglas UVT, GS 2458, Ro¨hm and Haas, Darmstadt, Germany). Six of these containers received supplemental nutrients (i.e., 0.6 and 0.7 mM of NaPO4H2 and NaNO3, respectively) – (‘‘N’’) cultures, whereas in the other six the nutrient concentration was that of the original culture medium. Three radiation treatments were imposed on the samples, with two containers from each nutrient treatment exposed either to: (1) full solar radiation (UVR, 280–400 nm, and PAR, 400–700 nm) – uncovered containers – PAB treatment; (2) UV-A (320–400 nm) and PAR – containers covered with UV cut-off filter foil (Montagefolie, No. 10155099, Folex) (50% transmission at 320 nm) – PA treatment; and (3) only PAR – containers covered with Ultraphan film (UV Opak, Digefra) (50% transmission at 395 nm) – P treatment (the spectra of these materials are published in Figueroa et al. [16]). The containers were placed in a bath with running water as temperature control (17– 20 °C) and exposed to natural radiation for 6–8 days. At the beginning of each experiment and then, on a daily basis, samples were taken to determine chlorophyll a (chl a) concentration, absorption spectral characteristics and cell concentration (see below). Additionally, samples were collected three times during the day (i.e., 8 am, 1 pm
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and 5 pm) to determine fluorescence parameters (see below). 2.3. Analyses and measurements The analyses and measurements were conducted as described below: 2.3.1. Chlorophyll a (chl a) Chl a concentration was measured by filtering 50–100 ml of sample onto a Whatman GF/F filter (25 mm) and extracting the photosynthetic pigments in absolute methanol [17]. The fluorescence of the methanolic extract was measured using a Turner Designs fluorometer (model TD700) before and after acidification; chl a concentration was calculated from these readings [18] (it should be noted that the fluorometer is routinely calibrated against spectrophotometric readings). 2.3.2. UV-absorbing compounds UV-absorbing compounds were determined by filtering 50–100 ml of sample onto a Whatman GF/F filter (25 mm) and extracting these compounds in absolute methanol overnight. The estimation of concentration of UVabsorbing compounds was done by peak analysis of the scans (250–750 nm) using a Hewlett Packard spectrophotometer (model HP 8453E). The peak height at 334 nm was considered as an estimate of UV-absorbing compounds concentration [19]. We are aware that other studies (e.g., [20]) established the use of 20% methanol as the best extraction solvent for these compounds. Nevertheless, and since we were limited by the volume of samples, we considered that this procedure was appropriate for the purposes and aim of our investigation, even it might slightly underestimate the amount of UV-absorbing compounds. Once scanned, the same sample was used to determine the chl a concentration fluorometrically.
2.3.5. Radiation measurements and other atmospheric parameters Incident solar radiation was continuously measured using a broad band ELDONET radiometer (Real Time Computers Inc., Germany) that measures UV-B (280– 315 nm), UV-A (315–400 nm) and PAR (400–700 nm) and stores the data with a frequency of one reading per minute. In addition, continuous monitoring of other atmospheric parameters (e.g., temperature, humidity and wind speed and direction) was carried out using a meteorological station (Oregon Scientific model WMR-918). 2.3.6. Statistics/treatment of data The duration of each experiment (i.e., 6–8 days) together with the rapid changes in radiation levels that normally occur in the study area did not allow for repetition of experiments under similar radiation conditions. However, and within each experiment, treatments were done in duplicate, and duplicate samples were collected for each analysis/measurement. In the case of photochemical yield measurements, however, samples were measured 10 times with the PAM fluorometer. The data are reported as the mean and half range between duplicates. The non-parametric Kruskal Wallis test [23] was used to assess for significant differences between the samples exposed to different treatments using a 95% confidence limit. The decrease of the photochemical quantum yield at each wavelength interval (i.e., photochemical quantum yield in the PAB and PA treatments relative to that in the P control) over the incubation period was calculated as Decrease by UV-B ¼ ½ðY P Y PAB Þ ðY P Y PA Þ=ðY P Þ 100 Decrease by UV-A ¼ ðY P Y PA Þ=ðY P Þ 100 where YP, YPA, and YPAB are the photochemical quantum yield in the P, PA, and PAB treatments, respectively. 3. Results
2.3.3. Cell counts Aliquots of cell cultures (10 ml) were fixed with buffered formalin (final concentration in the sample = 0.4% of formaldehyde) and counted using an inverted microscope (Leica model DM IL) with a 1-ml Sedwick–Rafter chamber. 2.3.4. Photochemical quantum yield A portable pulse amplitude modulated fluorometer (Water-ED PAM, Walz, Germany) was used to determine in vivo chlorophyll fluorescence of the photosystem II. The photochemical effective quantum yield (Y) was calculated using the equations of Genty et al. [21] and Weis and Berry [22] as Y ¼ ðF 0m F t Þ=F 0m ¼ F 0v =F 0m where F 0m is the maximal fluorescence induced by a saturating light pulse (ca. 5300 lmol photons m2 s1 in 0.8 s), and Ft the current steady state fluorescence induced by weak actinic light in light-adapted cells.
Total ozone column concentrations and daily doses of solar radiation during the study period – February 2– March 20, 2005 (Julian days 33–79) are shown in Fig. 1. Ozone column concentration over Playa Unio´n (data obtained from http://toms.gsfc.nasa.gov) was variable but relatively high throughout the study period, with maximum and minimum ozone concentrations of 298 and 241 Dobson Units (D.U.) on Julian days 79 and 57, respectively (Fig. 1a). Daily doses of PAR, UV-A and UV-B (Fig. 1b–d) were variable due to differences in cloud cover but nevertheless, there was a clear decreasing trend towards the end of the study period. UV-B daily doses varied between 11 and 42 kJ m2 (Fig. 1b) whereas UV-A between 400 and 1500 kJ m2 (Fig. 1c). PAR followed the same trend as UVR wavebands, with daily doses ranging between 2100 and 10,500 kJ m2 (Fig. 1d). The ratio of UV-B to PAR during the study period ranged between 0.0033 and 0.0051, on Julian days 68 and 50, respectively.
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Fig. 1. (a) Total ozone column concentration (in Dobson Units, D.U.) during the study period (Julian days 33–79) and (b–d) Daily doses of solar radiation (in kJ m2) (Julian days 33–79). (b) UV-B, 280–315 nm; (c) UVA, 315–400 nm; (d) PAR, 400–700 nm. The experiments with Chaetoceros gracilis, Thalassiosira fluviatilis, Prorocentrum micans and Heterocapsa triquetra were carried out from February 2 to February 8, 2005, February 11 to February 16, 2005, February 28 to March 6, 2005 and March 14 to March 20 for, respectively.
The daily variations of PAR irradiance during the four experiments are presented in Fig. 2. Maximum PAR irradiance levels were higher (350 W m2) during experiments carried out with C. gracilis (Fig. 2a) and T. fluviatilis (Fig. 2b) than during those carried out with dinoflagellates (300 W m2) (Fig. 2c and d). In general, PAR irradiance conditions (and also that of UV-A and UV-B, data not shown) were rather similar within each experiment, except for the last 2 days of the C. gracilis experiment (Fig. 2a) where cloud cover resulted in ca. 50% reduction in daily doses. Growth (expressed as cells and chl a concentration) of C. gracilis, T. fluviatilis, P. micans and H. triquetra exposed to different radiation/nutrient treatments is presented in Fig. 3. The most evident feature was the lack of a lag phase in the diatoms (Fig. 3a–d), with the exception of C. gracilis samples exposed to full radiation. In the other treatments, however, the concentration of C. gracilis cells and chl a
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Fig. 2. Daily variations of PAR irradiance (in W m2) during the experiments carried out with (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra.
increased as soon as the experiment started (Fig. 3a and b). In samples under the P and PA treatments, the cultures that received additional nutrients had higher cells and chl a concentrations than those that did not receive them. Regardless of the nutrient treatment, the samples under the PAB treatment had lower concentration of cells and chl a for most of the time, however, at the end of the experiment they had similar values as the PA and P treatments. During the experiment carried out with T. fluviatilis (Fig. 3c and d) a rather similar growth was determined in all treatments during the first 2 days; afterwards, PN and PAN treatments had the highest chl a concentration (Fig. 3d) although the concentration of cells was the same in the three radiation treatments (Fig. 3c). Regardless of the nutrient treatment, P. micans samples (Fig. 3e and f) had a 1–2 days lag phase; afterwards the concentration of cells and chl a increased in all samples, with cultures exposed to the PN treatment having the highest values. Finally, in the experiment carried out with H. triquetra (Fig. 3g and f) the lag phase lasted 3 days and, afterwards, PN samples displayed the highest growth. Growth rates (l) for these cultures, based on chl a measurements are
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Fig. 3. Growth of the four phytoplankton species expressed as cell concentration (cells ml1) (a, c, e and g) and chl a concentration (lg chl a L1) (b, d, f and h): (a and b) Chaetoceros gracilis, (c and d) Thalassiosira fluviatilis, (e and f) Prorocentrum micans and (g and h) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
presented in Table 1. The lowest l were attained in the PAB treatment (non-enriched) with values of 0.52, 0.41, 0.44 and 0.49 day1 in C. gracilis, T. fluviatilis, P. micans
and H. triquetra, respectively. On the other hand, the highest l values were determined in enriched cultures receiving only PAR – 0.85; 1.06, 0.97 and 0.69 day1 in C. gracilis, T. fluviatilis, P. micans and H. triquetra, respectively. Nevertheless, enriched cultures had significantly higher l (P < 0.05) than non-enriched cultures only in the PAB treatments. When considering the same nutrient condition, the PAB treatments had significantly lower l (P < 0.05) than the P treatments in the four phytoplankton species. Similar conclusions on growth rates as those from chl a measurements were obtained from cell concentration data (data not shown). Daily variations of photochemical quantum yield (Y) for the four species studied are shown in Fig. 4. The general pattern for all species was of a significant decrease of Y at or close to local noon in all treatments, with a significant recovery during the afternoon and night. However, the extent of the Y decrease as well as that of recovery was different among species. In C. gracilis (Fig. 4a) Y decreased significantly throughout the experiment, from an initial value of 0.7 to 0.1 in non-enriched cultures and to 0.4–0.5 in enriched cultures at the end of the experimental period. During the first day, samples exposed to UVR had significantly lower quantum yield than those exposed only to PAR, however, during the night of the second day and at the end of the experiment there were no significant differences in Y among the radiation treatments in the nonenriched cultures, while the Y in the enriched samples in the PN had significantly higher values than in the PABN and PAN treatments. Similarly, Y values of T. fluviatilis (Fig. 4b) at the end of the experiment decreased significantly from the initial value of 0.6 to 0.3–0.5 in enriched samples in the PABN and PAN/PN treatments, respectively, and to 0.1 in all radiation treatments in non-enriched cultures. In the experiment carried out with P. micans (Fig. 4c) there were also differences in Y, with enriched cultures having significantly higher values than non-enriched cultures. Moreover, samples exposed in the PABN had significantly lower Y values (i.e., 0.3) than in the PAN/PN treatments (i.e., 0.5–0.6) at the end of the experiment. In addition, the noon time Y value that was 0 during the first 3 days, increased to >0.1 as the experiment progressed. Finally, in the experiment carried out with H. triquetra
Table 1 Growth rates (l day1, calculated from chl a measurements) during experiments carried out with Chaetoceros gracilis, Thalassiosira fluviatilis, Prorocentrum micans and Heterocapsa triquetra exposed to different nutrient/radiation treatments Treatment/culture
Chaetoceros gracilis
Thalassiosira fluviatilis
Prorocentrum micans
Heterocapsa triquetra
PAB PA P PABN PAN PN P (l = lN)
0.52 (0.01) 0.64 (0.02) 0.89 (0.01) 0.62 (0.01)* 0.59 (0.01) 0.85 (0.01) <0.05
0.41 (0.02) 1.02 (0.03) 1.05 (0.03) 0.68 (0.02)* 1.08 (0.01) 1.06 (0.01) <0.05
0.44 (0.03) 0.77 (0.01) 0.75 (0.01) 0.50 (0.02)* 0.74 (0.03) 0.97 (0.01)* <0.05
0.49 (0.02) 0.61 (0.03) 0.63 (0.01) 0.61 (0.03)* 0.65 (0.02) 0.69 (0.01)* <0.05
Radiation treatments are denoted by PAB (PAR + UV-A + UV-B), PA (PAR + UV-A) and P (PAR only); N denotes cultures that received additional nutrients. The asterisks indicate significant differences between nutrient treatments (i.e., comparing the same radiation treatment).
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Fig. 4. Daily variations of photochemical quantum yield (Y) during the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
(Fig. 4d) there was a significant inhibition at the end of the day that was also evident during early morning during the first days. However, the Y value increased with time and at the end of the experiment enriched samples in the PAN and PN and non-enriched samples in the P treatments had significant higher Y value (>0.5) than the rest of the samples; this value was even higher than that measured at the beginning of the experiment (i.e., 0.4). The decrease in quantum yield at local noon (calculated from the Y values of Fig. 4) throughout the duration of the experiments is presented in Fig. 5. C. gracilis was the only species that had significant lower decrease of Y in the samples in the P treatment as compared those that received additionally UVR, regardless of the nutrient treatment, for the first 3 days of experimentation (Fig. 5a). However, at the end of the experimentation P samples were those with higher decrease of Y (i.e., ca. 100%). In T. fluviatilis
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Fig. 5. Decrease in photochemical quantum yield (%, relative to t0) at local noon throughout the duration of the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
(Fig. 5b), the decrease in quantum yield diminished with time and no significant differences were observed among nutrient/radiation treatments during the first 3 days of experimentation. After that, and towards the end of the experiment, samples with nutrient addition had significant lower decrease of Y than those that did not receive nutrients. In P. micans (Fig. 5c) the decrease of Y diminished continuously throughout the experiment while in H. triquetra (Fig. 5d) it was rather constant during the first 4 days and afterwards decreased. We further studied the potential mechanisms by which organisms could adapt to the conditions imposed in the experiments (i.e., through the presence of UV-absorbing compounds). The initial spectral absorption characteristics (O.D. mg chl a1) of the four species used in our experiments are presented in Fig. 6. All species had characteristic
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Fig. 6. Initial spectral absorption characteristics (O.D. mg chl a1) of Chaetoceros gracilis (triangles); Thalassiosira fluviatilis (circles); Prorocentrum micans (squares) and Heterocapsa triquetra (diamonds).
peaks of chl a at 440 and at 665 nm. Carotenoids (kmax = 470 nm) were present in T. fluviatilis, P. micans and H. triquetra, but they were barely noticeable in C. gracilis. UV-absorbing compounds (maximum absorption in the range of 310–360 nm) were especially abundant in P. micans, but they were present in very low concentrations (or virtually absent) in the other three species tested. The temporal variability in concentration of these UVabsorbing compounds during the experiments is shown in Fig. 7. In general, there was an increasing trend of concentration with time in all species tested; however, the extent of this increase was variable among species and according to the treatment imposed on the samples. In C. gracilis (Fig. 7a) this trend was especially evident during the first 4 days of experimentation and afterwards, its concentration decreased. The highest amounts of UV-absorbing compounds were determined in enriched cultures, whereas P and PA samples had a similar concentration to that at the beginning of the experiment. In T. fluviatilis (Fig. 7b) samples in the PABN and PAN and PAB treatments increased significantly the concentration of UV-absorbing compounds throughout the experiment. However and regardless of the nutrient treatment, samples exposed only to visible radiation had approximately the same concentration throughout the experiment. In P. micans (Fig. 7c) the increase of UV-absorbing compounds was evident only in samples exposed to full solar radiation, especially in enriched samples; in the other treatments, their concentration remained similar (and relatively low) throughout the experiment. A similar behaviour was observed in H. triquetra (Fig. 7d) with PAB samples increasing significantly the concentration of UV-absorbing compounds towards the end of the experiment, especially in enriched samples. The relationship between the decrease in quantum yield (from Fig. 5) and the concentration of UV-absorbing compounds (from Fig. 7) is presented in Fig. 8 (only significant relationships (P < 0.05) having R2 between 0.6
Fig. 7. Daily variations in concentration of UV-absorbing compounds (estimated by the peak height at 334 nm) during the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. Each data point is the mean value and the vertical lines indicate the half range around the mean.
and 0.94 are plotted). When comparing the four species, it was observed that samples in different treatments displayed significant different relationships. In C. gracilis (Fig. 8a) only samples in the PABN treatment had a significant negative relationship between the decrease in quantum yield and the increased concentration of UVabsorbing compounds. In T. fluviatilis (Fig. 8b) this relationship was observed in the PABN treatment, and in both nutrient treatments receiving UV-A and PAR (i.e., PA and PAN). Finally, in P. micans (Fig. 8c) only those treatments receiving full solar radiation (i.e., PAB and PABN) had a negative relationship between the decrease in quantum yield and the increased concentration of UV-absorbing compounds whereas in H. triquetra (Fig. 8d) this relationship was observed in the PA, PAN, P and PN treatments.
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Fig. 8. Decrease in photochemical quantum yield (%) at local noon as a function of concentration of the UV-absorbing compounds throughout the experiments carried out with: (a) Chaetoceros gracilis; (b) Thalassiosira fluviatilis, (c) Prorocentrum micans and (d) Heterocapsa triquetra. The symbols indicate the different nutrient/radiation treatments imposed on the samples: diamonds: samples exposed to full radiation (PAR + UVR); circles: samples exposed to PAR + UV-A; squares: samples exposed to PAR. Open symbols indicate samples without addition of nutrients whereas black symbols indicate samples that received additional nutrients. The full lines indicate the best fit and the dotted lines are the confidence limits.
4. Discussion Many studies devoted to assessing the impact of solar UVR upon natural phytoplankton populations have highlighted the variability in responses mainly because they are species-specific, with some species being relatively tolerant and others sensitive [1]. However, in combination with other stress factors, species can display different responses than when considering solely UVR, because their interaction can be either antagonistic or synergic. In this study we compared the responses to both, solar radiation and nutrient addition in four phytoplankton species which are commonly found in marine environments. Our results can be summarized as follows: regardless of the nutrient condition, all species displayed some
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degree of stress when exposed to solar radiation, as seen in both growth (Fig. 3) and the photochemical quantum yield (Figs. 4 and 5). However, some evidence of acclimation was observed towards the end of the experimental period, especially in the dinoflagellates. Another feature of our data is the variability in responses among the species studied even though radiation (Figs. 1 and 2) and nutrient conditions were rather similar in the four experiments, clearly emphasizing the fact that responses are species-specific. In principle, one might think that the decrease in both growth and photochemical quantum yield (Figs. 3–5) as observed in our experiments mainly occurs because of exposure to high solar radiation levels (i.e., high doses, Fig. 1) and irradiances (Fig. 2) at which the organisms where exposed. These high radiation levels are characteristic of the Patagonia area [24] and they are due to a combination of high heliophany and long day-light period [25] during summer. In fact, these radiation levels during summer are comparable to those received in tropical areas during mid-spring/mid-autumn [26,27]) (however, the ratio UV-B to PAR is higher in tropical areas than in our study site). The reduction of both growth and photochemical quantum yield as effectively occurs in our experiments (Figs. 3–5) can be partially related to these high radiation levels; moreover, this seems to be a common feature of phytoplankton organisms, as seen in studies carried out with natural populations as well as with monospecific cultures [1]. Still, the observed responses in our experiments represent the net result between damage/inhibition and repair/ acclimation mechanisms. Nutrient addition in our experiments gave hints on their key role at the physiological level by allowing species to better cope with exposure to solar radiation. This was clearly seen by comparing growth and the photochemical quantum yield in enriched and non-enriched conditions; higher values were observed in samples that received supplemental nutrients (Figs. 3 and 4), particularly in the PAB treatments where enriched cultures had significantly higher growth rates (P < 0.05), than non-enriched cultures (Table 1). Moreover, the importance of nutrient addition in favoring species to acclimate to solar radiation was evident in dinoflagellates that had a lower decrease in quantum yield at the end of the experiment than at the beginning (Fig 5). Thus our data support the fact that the supplement of nutrients are indeed associated with the synthesis of UV-absorbing compounds, which are thought to be one of the main mechanisms to cope with solar UVR [7]. As compared to other taxa (i.e., chlorophytes or cyanobacteria) both diatoms and dinoflagellates are considered relatively resistant to UVR [28]. Nevertheless, their apparent resistance is thought to be caused by different mechanisms: In diatoms, their silicon skeleton seems to play a role in absorbing UVR and thus conferring some protection to the cell [29]; in dinoflagellates, the synthesis of UV-absorbing compounds (mainly MAAs) seems to be
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the most important mechanism for UV protection. However, the degree and role of one or another protection mechanism may vary between taxa and/or species. For example, working with Antarctic diatoms Helbling et al. [8] found differences between centric and pennates, with the former being more resistant than the latter; this difference was thought to be due to the synthesis of MAAs in the centric diatoms but absent in pennates. In dinoflagellates, a variety of responses have been observed [30–32] and nutrient deficiency has been associated with an increase in UV sensitivity of species as compared to those grown in nonlimited conditions [3]. Our data presented here show that UV-absorbing compounds levels are much lower in diatoms than in dinoflagellates. Nevertheless, diatoms significantly increased the concentration of UV-absorbing compounds towards the end of the experiments, with samples that received additional nutrient having a higher concentration of them than non-enriched samples (Fig. 7). Nutrient addition has been previously shown to enhance the synthesis of UV-absorbing compounds [13,29]. However, in our experiments, as well as in those previous studies, the initial nutrient concentration was not limited in the samples, and moreover, in those treatments that did not received additional nutrients both, cell and chl a concentration increased throughout the experiments (Fig. 3). It is plausible, however, that cells give priority for the use of nutrients to other metabolic processes rather than to the synthesis of UV-absorbing compounds. A hint of this could be inferred from the relatively higher growth rates in the diatoms (especially T. fluviatilis) as compared to dinoflagellates (Table 1). Nevertheless, our data clearly show that the decrease in quantum yield was less (i.e., better performance) with increasing concentration of UV-absorbing compounds (Fig. 8) although this was variable among species and radiation/ nutrient treatments. We cannot rule out the contribution of other ‘‘protecting’’ mechanisms such as an active xanthophyll cycle or self-shading as the cultures grew. In fact, the xanthophyll cycle have been shown to be an effective protective mechanism in the diatom Thalassiossira weisflogii [33]. In that study, the concentration of MAAs increased after 16–22 days of exposing cells to UV-B, and it was thought to occur after the cells recovered from the initial UVR stress involving the xanthophyll cycle. In our experiments, we do not have information on xanthophyll pigments, but UVabsorbing compounds increased, at least in some treatments, from the beginning of the experiments. It is possible that longer exposure periods might render higher concentrations of UV-absorbing compounds and hence cells are more effectively protected. However, in the case of the diatoms, growth curves (Fig. 3) had a plateau after the exponential growth. At this point (i.e., end of the exponential growth) self-shading might have a role in protecting the cells as was seen in studies carried out with the cyanobacterium Arthorspira platensis [34]. Nevertheless, our data on photochemical quantum yield do not show a recovery
during this period, thus suggesting that if this mechanism is active its role is rather insignificant. Overall, our data suggest that under the experimental conditions imposed on the samples, the photochemical quantum yield and growth of diatoms are more affected by solar radiation than in dinoflagellates are. Thus, under conditions of nutrient enrichment, as may occur by river input or re-suspension by mixing, dinoflagellates might outcompete diatoms by synthesizing important amounts of UV-absorbing compounds and hence have a higher fitness under UVR stress. 5. Abbreviations MAAs PAR UV-B UV-A UVR Y
mycosporine like amino acids photosynthetically active radiation ultraviolet B ultraviolet A ultraviolet radiation photochemical quantum yield
Acknowledgments This work was supported by the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica – ANPCyT (PICT 2003-13388 and PICT 2005-32034), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas – CONICET (PIP 2004-5157), PNUD-GEF (B-C-39) and Fundacio´n Playa Unio´n. We thank D.P. Ha¨der who kindly provided some cultures used in the experiments; J.I. Albarracı´n helped with culture maintenance. We also thank the comments and suggestions of two anonymous reviewers that helped to improve this manuscript. This is in partial fulfillment of the Ph.D. thesis of MAM. This is contribution number 109 of the Estacio´n de Fotobiologı´a Playa Unio´n. References [1] V.E. Villafan˜e, K. Sundba¨ck, F.L. Figueroa, E.W. Helbling, in: E.W. Helbling, H.E. Zagarese (Eds.), UV effects in aquatic organisms and ecosystems, Royal Society of Chemistry, 2003, pp. 357–397. [2] A.G.J. Buma, P. Boelen, W.H. Jeffrey, in: E.W. Helbling, H.E. Zagarese (Eds.), UV Effects In Aquatic Organisms And Ecosystems, The Royal Society of Chemistry, Cambridge, 2003, pp. 291–327. [3] E. Litchman, P.J. Neale, A.T. Banaszak, Increased sensitivity to ultraviolet radiation in nitrogen-limited dinoflagellates: photoprotection and repair, Limnol. Oceanogr. 47 (2002) 86–94. [4] K. Shelly, S. Roberts, P. Heraud, J. Beardall, Interactions between UV-B exposure and phosphorus nutrition. I. Effects on growth, phosphate uptake, and chlorophyll fluorescence, J. Phycol. 41 (2005) 1204–1211. [5] P.C. Frost, M.A. Xenopoulos, Ambient solar ultraviolet radiation and its effects on phosphorous flux into boreal lake phytoplankton communities, Can. J. Fish. Aquat. Sci. 59 (2002) 1090–1095. [6] L. Aubriot, D. Conde, S. Bonilla, R. Sommaruga, Phosphate uptake behavior of natural phytoplankton during exposure to solar ultraviolet radiation in a shallow coastal lagoon, Mar. Biol. 144 (2004) 623– 631.
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