UV radiation and its effects on P-uptake in arctic diatoms

Journal of Experimental Marine Biology and Ecology 411 (2012) 45–51

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UV radiation and its effects on P-uptake in arctic diatoms Dag O. Hessen a,⁎, Helene Frigstad b, c, Per J. Færøvig a, Marcin W. Wojewodzic a, Eva Leu d a

University of Oslo, Dept. Biology, P.O. Box 1066 Blindern, 0316 Oslo, Norway Geophysical Institute, University of Bergen, Allégaten 70, 5007 Bergen, Norway c Bjerknes Centre for Climate Research, University of Bergen, Allégaten 55, 5007 Bergen, Norway d Norwegian Polar Institute, N-9296 Tromsø, Norway b

a r t i c l e

i n f o

Article history: Received 7 July 2011 Received in revised form 25 October 2011 Accepted 28 October 2011 Available online 25 November 2011 Keywords: Arctic diatoms Nutrient uptake Solar radiation Stoichiometry

a b s t r a c t Due to severe alterations of the physical conditions in Arctic ice-covered ecosystems (decrease of sea ice, thinning of the ozone layer), the underwater light climate is changing both with respect to its intensity and spectral composition. It is commonly observed that phytoplankton respond differently to photosynthetically active radiation (PAR, 400-700 nm) and ultraviolet radiation (UVR, 280–400 nm) in terms of their elemental stoichiometry, where high levels of PAR tend to increase the ratios of carbon to phosphorus (C:P), while UVR has the opposite effect. Since this has importance not only for elemental cycling and Psequestration, but also for the algal food quality for grazers, it is of considerable interest to reveal the role of different spectral regimes of elemental uptake and stoichiometry. There are ambiguous evidence as to whether UVR stimulates or reduces the uptake of inorganic P, and to test this for three common, arctic marine diatoms; Porosira glacialis, Thalassiosira sp. Synedropsis hyperborean, we performed 33P-uptake assays with exposure to either PAR alone or PAR + UVR. Neither of the species showed strong P-uptake responses to UVR exposure, yet the two former species had a negative trend, while Synedropsis, showed a slight increase in its P-uptake under moderate UVR exposure. The latter species also had a remarkably fast P-uptake kinetics. These mixed results indicate a species-specific complex role of P in the algal response to UVR induced light stress, where not only species affinities but also ambient conditions may yield different outcomes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The cycling of key elements like carbon (C), nitrogen (N) and phosphorus (P) in marine systems depends to a large extent on productivity and fate of marine autotrophs. Light is the key factor regulating the absolute and relative uptake of these elements in autotrophs, both with respect to its intensity or quantity, but also in terms of its spectral properties. Recent studies have demonstrated an inverse effect of ultraviolet radiation (UVR, 280–400 nm) and photosynthetically active radiation (PAR, 400–700 nm) with regard to elemental ratios, notably C:P, in phytoplankton (Hessen et al., 2008). Under high PAR intensities but low levels of inorganic phosphorus (P), a disproportionate fixation of C relative to P has been demonstrated for freshwater phytoplankton, yielding an increased cellular C:P-ratio (Hessen et al., 2002; Sterner et al., 1997; Urabe and Sterner, 1996; Urabe et al., 2002). A skewed uptake of C relative to P or N commonly causes an accumulation of C rich storage compounds (Berman-Frank and Dubinsky, 1999), which may have consequences for autotroph C-sequestration as well as the transport of biomass and energy through the food web. (Sterner and Hessen, 1994; Sterner and Elser, 2002). UVR and P combined may also affect algal fatty

⁎ Corresponding author. Tel.: + 47 22854553. E-mail address: [email protected] (D.O. Hessen). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.10.028

acid composition, especially the highly unsaturated fatty acids which are crucial for zooplankton nutrition (cf. Leu et al., 2006a,b; VillarArgaiz et al., 2009). In contrast, UVR seems to promote reduced C:P-ratios both in freshwater and marine phytoplankton (Hessen et al., 2008; Leu et al., 2006a, 2007), yet the mechanisms behind this are not fully understood. Whereas the capacity of UVR to reduce photosynthetic activity and thus C-uptake is clearly documented (Neale et al., 2003), its role in nutrient uptake and cellular stoichiometry is less well understood. Phytoplankton cells exposed to UVR undergo a series of physiological changes that influence cell volume and intracellular morphology, as well as biochemical pathways and products, eventually affecting nutrient uptake as well. Reduced uptake of inorganic N under UVstress has been reported by some authors (Braune and Döhler, 1994; Döhler and Biermann, 1987), while other studies found rather weak or even no effects of UV on C:N ratios in phytoplankton (Fauchot et al., 2000; Mousseau et al., 2000). Wängberg et al. (1998) reported decreased photosynthesis but increased P-uptake in marine phytoplankton communities under UV exposure. Recent field studies have also demonstrated decreased C:P-ratios in phytoplankton and epilithic communities in lakes under UV-stress (Tank et al., 2003; Watkins et al., 2001; Xenopoulos et al., 2002), although in situ experiments suggest that this effect is weak under low ambient P concentrations (Frost and Xenopoulos, 2002).

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D.O. Hessen et al. / Journal of Experimental Marine Biology and Ecology 411 (2012) 45–51

The response to UVR in general differs between taxa and species, and typically small species of diatoms are more vulnerable than large species (e.g. Karentz et al. 1991). Also their nutritional status and not the least the intensity or dose-rate of UVR may affect the net uptake of nutrients. Hessen et al. (1995) observed increased uptake of 33P in flagellates under low to moderate UV exposure, and accredited this to increased demands for P for nucleotide or membrane repair. At very high doses of UV, the uptake of P was impaired, however, in line with the findings of Aubriot et al. (2004). These observations suggest that not only the photon flux per se, but also the PAR:UV ratio may play a crucial role for productivity and biomass of autotrophs, and also govern their nutritional quality in terms of elemental composition. This in turn will affect nutrient cycling and trophic transfer efficiency to higher trophic levels. To the extent that UVR affect P-uptake and growth rate, it should also be assumed to affect RNA and RNA:protein ratio (cf. Sterner and Elser, 2002), and hence we included an assays on RNA:protein dynamics related to light exposure. Both with regard to the physiological response in the phytoplankton cells, and due to its relevance for the uptake and cycling of C and P in the sea and nutrient competition between autotrophs and prokaryotic heterotrophs, it is of considerable interest to reveal if the reported decrease in C:P under UVR exposure relates primarily to reduced C-fixation, or whether the exposed cells also have an enhanced uptake of P. This is accentuated by the continued ice loss in the Arctic as well as ozone anomalies and minima (Manney et al., 2011). To test for a potential stimulating effect of UVR on P-uptake in high latitude diatoms, we performed radioactive 33P-tracer uptake experiments with and without UVR for three common diatom species isolated from the high Arctic. 2. Materials and methods The unialgal cultures used in this experiment were isolated from waters around the Svalbard archipelago in 2007. Two of the three species are pelagic, centric diatoms that are usually occurring in chain-forming colonies: Porosira glacialis, and Thalassiosira sp., where the latter has a rather small cell size for diatoms (~10 μm). The third species, Nitzschia frigida: is a pennate diatom, most often occurring as an epiphyte on the common sea ice algae species. All cultures were kept at 3 °C in f/10 media (a fivefold dilution of f/2 medium, Guillard, 1975). In the treatment with surplus phosphorus (marked + P), 1 mg L − 1 NaH2PO4 according to the f/10 concentrations was added, while the medium for the algae grown under P limitation (marked −P) contained only background concentrations of P found in filtered seawater that was used for media preparation. All other nutrients were added as in f/10. A light:dark cycle of 16:8 h was applied, with 55 μmol m − 2 s − 1 of PAR. The experimental protocol followed that of Leu et al. (2007), and details of experimental setup can be found here. In brief, the experiments were conducted in a thermostatically controlled room at 3− 4 °C, using 500 mL quartz bottles (i.e. UV-transparent) covered with different cutoff foils (see below). Photosynthetically active radiation (PAR) was provided by one daylight fluorescent tube (OSRAM Lumilux de luxe 36W/950 daylight) under a 16 h light:8 h dark cycle, and measured with a 4 π sensor (QSL-100, Biospherical Instruments, Inc.). UVR was provided by two Q-Panel UVA-340 fluorescent tubes (Q-Panel Lab Products, Cleveland, USA), and measured with a IL 1400A radiometer (International Lights, Inc., Newburyport, MA, USA) equipped with a SPS 300 sensor (UVB) and a SUL 033 sensor for UVA. Since the SUL 033 sensor has no sharp cutoff at 320 nm, we used Mylar foil for measuring the exact UVA intensities. The whole setup was coated with aluminum foil, in order to provide a light field as homogenous as possible. PAR exposure lasted daily from 5:00 a.m. to 9 p.m. In the middle of this daylight period, cultures were exposed to UVR for 8 h from 9 a.m.

to 5 p.m. Two different irradiation treatments were applied in three replicates each: (1) no UVR (referred to as PAR), shielded from UVA and UVB radiation by an Ultraphan 400 foil (Digefra, Munich, Germany) (2) PAR + UVR (referred to as UV), covered with cellulose acetate (Tamboer & Co Chemie B.V., Heemstede, Netherlands) to correct for the ca. 10% absorption of Ultraphan 400 in the PAR spectrum. The absorption spectra for these foils can be found in Leu et al. 2007. In the UV experiments, we applied UVR intensities near 50% of maximum surface UVR-levels at a latitude of 80° N, the latitude from which the algae was isolated, at noon in mid-summer (referred to as low light), and an elevated level twice of this (i.e. corresponding to peak surface values), denoted high light (cf. Leu et al., 2006b). Measurements of applied UVA and UVB intensities were carried out with a IL 1400A radiometer (International Lights, Inc., Newburyport, MA, USA), PAR-levels ranged between 56 and 64 μmol m− 2 s− 1. For UVA the average measured intensity was 11.3 W m − 2, UVB was 1.0 W m − 2. The high UV treatments had close to 200 μmol m − 2 s − 1 PAR, and the UV-was increased correspondingly, so that the ratio between PAR, UVA, and UVB was roughly: 2:1:0.1 under both radiation regimes. The difference in radiation intensity was obtained by changing distance to the lamps. To ensure equal exposure for all treatments, the flasks were exchanged randomly between the 18 positions marked on the shelf every day. Since the initial counts showed no signs of enhanced P-uptake under UVR exposure in neither Porosira, nor Thalassiosira grown under surplus P conditions, we did not run experiments with elevated levels of P for these species (i.e. only the −P culture was used). In Synedropsis, we found signs of an elevated P-uptake under UVR under both conditions (P replete and P depleted). Hence, we also performed a test with elevated levels of P to see if this effect was either weakened or stimulated by increased access to P. Due to the extremely fast kinetics for P-uptake in this species, we run an additional experiment with higher time resolution to study the initial response in more detail. The cultures were diluted with the respective medium (+P or −P) prior to the start of the experiment to the final concentration. The algal concentration at the start of the experiment was in the range of 1–2 mg C L − 1 or 30–40 μg chlorophyll a L − 1. For labeling of the cultures we added 10 μL of carrier-free 33P ortophosphate (Amersham BF1003) yielding initial specific activity of 10 μCi l− 1 in the algal cultures, representing tracer amounts relative to 31P in the media even at ambient concentrations of P (−P), 33P would represent an insignificant change in total P. Quenching was low and counting efficiency exceeded 90% for all cultures, meaning that the reported counts per minute (CPM) mL− 1 should be equivalent to uptake of 33P. The radiotracer was added one hour after UV-lamps were turned on. Generally 15 mL from each flask was filtered on 25 mm GF/F filters and washed after 1 h, 6 h and 21 h, yet some experiments have additional samples (see Table 1 for an overview). After filtration each GF/F filter was placed in a 20 mL scintillation vial, and 1 mL of filtrate was added into another 20 mL scintillation vial. 20 mL liquid scintillation cocktail (Ultima Gold: High Flash-point, Universal LSCcocktail, Perkin Elmer cat. No: 6013329) was added to both vials, and the activity was counted on a Packard TriCarb liquid scintillation counter. Due to the very fast uptake kinetics of Synedropsis, we performed a second set of experiments with higher time resolution for this species, with sampling after 10, 30, 50, 70 and 100 min. Since a major part of P in unicellular organisms are allocated to ribosomes which are the site of protein synthesis, short-term UVR-effects on P-uptake or growth rates would presumably be reflected in changed levels of RNA. We thus run assays on two of the species, Porosira and Synedropsis, to check for changes in RNA quantities during the course of the experiment under PAR and PAR +UV exposure. Ten milliliter from each triplicated treatment was collected at the beginning, after 6 and 21 h of the experiment. Samples were centrifuged at 5000 g for

D.O. Hessen et al. / Journal of Experimental Marine Biology and Ecology 411 (2012) 45–51 Table 1 Overview of treatments applied and sampling intervals (in hours) for each species. Note that − P actually represents background P. −P

+P

Light

3. Results The tested species revealed somewhat mixed responses, but gave in general little support to the hypothesis of enhanced uptake of P under UVR exposure (cf. Table 2 for statistics). For Porosira low UVR yielded no difference between the PAR and UVR treatments, while there was a decrease in P-uptake (RM-ANOVA, light: p= 0.007) under the high UVR treatment (Fig. 1). This species also showed a significant light× time interaction (RM-ANOVA, light ×time: pb0.001), where the difference in P-uptake between the PAR and UVR exposure increased over time. A similar response was found for Thalassiosira, where there were no effects at low UVR, while a reduced uptake (RM-ANOVA, light: p =0.028) was observed under enhanced UVR (Fig. 2). The activity in the filtrate

0.678 0.007*

Thalassiosira sp. PAR vs. UVR PAR vs. high UVR

1.1 11.2

0.349 0.028*

3.0 0.4 1.0

0.154 0.543 0.371

8.7 170.7

0.041* 0.000*

S. hyperborea− P PAR vs. UVR PAR vs. high UVR PAR vs. high UVR short-term S .hyperborea + P PAR vs. UVR PAR vs. high UVR

PAR > HUV

996.8 6614.5

0.000* 0.000*

0.1 0.9437 31.1 b0.001*

PAR > HUV

2492.7 126.6

0.000* 0.000*

1.6 3.6

0.256 0.075

6.3 9.0 429.4

0.022* 0.008* 0.000*

2.1 11.3 4.7

0.178 0.004* 0.009*

22.3 782.8

0.000* 0.000*

6.1 0.024* 79.2 b0.001*

PAR > UV PAR > HUV

P

mirrored the uptake in the particulate fraction in all experiments (data not shown). The 33P-uptake kinetics was rather slow for both species, and saturated uptake of P clearly exceeded 20 h for both species at this low temperature. For Synedropsis, however, we recorded an increased uptake of P under UVR (Fig. 3), with the strongest effect at low UVR, however due to the variability between the replicates this difference was not significant. This species had a remarkably fast P-uptake kinetics, hence we performed a second set of experiments with higher time resolution for the high UVR-treatment at −P (Fig. 4) and also a second essay with higher concentrations of P (denoted +P), shown in Fig. 5. These experiments revealed that the half-saturation constant for P-uptake in this species was less than 1 h, and thus strikingly faster compared to the other two diatoms that also are assumed to be “cold-adapted” species. The high time-resolution experiment (performed at − P) showed an increased uptake of P in the high UVR treatment at the end of the experiment, however this difference was not significant. However there was a significant interaction effect

20000

PAR UVR High UVR

15000

5 min, supernatants were gently aspirated and remained samples were stored at −80 °C until analysis. RNA was extracted and quantified similarly to the RiboGreen method (Gorokhova and Kyle, 2002) and the obtained values were normalized to the protein content measured in the corresponding extracts. Briefly, RNA from the sample was extracted by 2 min sonication in an ice-cold-maintained cuphorn (Brandson 101147048) with 60 μL of 1% (v/w) N-lauroysarcosine (Sigma, L-5125). Immediately 300 μL of ice-cold TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH = 7.5) was added to the sample. Homogenates were dispensed in duplicates (75 μL) into RNAse free wells (655076, Greiner Bio-One, USA) where one of the subsample was exposed to half-an-hour RNAse digestion (0.1 μg, A7973, Promega). After this step 75 μL of 100× diluted RiboGreen dye (R-11490, Molecular Probe, USA) was dispensed to all wells, mixed for following 5 min and the fluorescence signal was measured at 535 nm after previous excitation at 480 nm (BioTek FL x 800, BioTek, USA). Difference in a fluorescence signal between undigested and digested with RNAse samples was related to RNA content in the assayed sample. To translate fluorescence values to RNA content (μg mL− 1) and to control for a proper digestion of RNA during the quantification, commercially available RNA standards were included (R11490, Molecular Probes). Bicinchoninic acid method was used to measure proteins from all extracts (Smith et al. 1985) using BCA kit (Termo Scientific Pierce, 23227) and following the manufacture protocol. To test the hypothesis that UVR increase the cellular uptake of P, we conducted one-way repeated measures analysis of variance (RM-ANOVA) with time as within-factor (three levels, except for Synedropsis in the short-term experiment (5 levels) and PAR vs. High UVR at + P (four levels)) and light (two levels: PAR and UVR) as between-factor for each species and light intensity. For all three species the RM-ANOVAs were performed on the PAR vs. UVR and PAR vs. high UVR at low P concentration. In addition for Synedropsis these same treatment combinations were tested for the + P cultures. To test if UVR affected the RNA:protein ratio, a RM-ANOVA was also performed on the PAR vs UVR (between-factor) and time (within-factor_ 3 levels) for Porosira and Synedropsis grown at -P. Prior to statistical analyses all data were tested for homogeneity of variances using the robust Brown–Forsythe Levene-type test, based on the absolute deviations from the median. The confidence level of each test was set to 95%. All analyses were performed with the statistical software R (R Development Core Team, 2011) on untransformed data.

0.2 26.4

F

10000

0.5, 2, 20 1, 6, 21

P. glacialis PAR vs. UVR PAR vs. high UVR

P

5000

0.5, 1, 6, 21

Light x Time

F

0

1, 6, 21

250000

1, 6, 21 (Short-term: 10, 30, 50, 70, 100 min) 1, 6, 21 1, 6, 21

P

200000

1, 6, 21

Time

F

150000

Par vs. high UVR

Uptake

PAR vs UVR

100000

Par vs. high UVR

50000

PAR vs UVR

0

Porosira Thalassiosira

Table 2 Results of one-way RM-ANOVA on UVR response of Porosira glacialis, Thalassiosira sp. and Synedropsis hyperborea. Significant effects denoted by asterisk.

33P

Synedropsis

47

0.5

2.5

20

Time (Hours)

1

6

21

Time (Hours)

Fig. 1. Uptake of 33P (counts pr min) in P. glacialis over the course of the experiment for PAR vs. UVR and PAR vs high UVR. Bars and vertical lines represents mean ± SD (n = 3).

D.O. Hessen et al. / Journal of Experimental Marine Biology and Ecology 411 (2012) 45–51

20000

35000

20000

48

30000

PAR UVR High UVR

15000

Uptake 33P

1

6

21

1

Time (Hours)

6

21

Time (Hours) 0

0

0

5000

5000

5000

10000

10000

20000 15000

Uptake

25000

15000

High UVR

10000

33P

PAR

Fig. 2. Uptake of 33P (counts pr min) in Thalassiosira sp. over the course of the experiment for PAR vs. UVR and PAR vs high UVR. Bars and vertical lines represents mean± SD (n= 3).

10

30

50

70

100

Time (Minutes)

apparently slightly higher concentrations of RNA after 6 h of UVRexposure in Porosira, the differences were insignificant between light regime (RM-ANOVA, light: p = 0.557) and only slightly significant for time. (p= 0.036). For Synedropsis the responses were similar for the PAR and UVR (RM-ANOVA, light: p = 0.969), with a significant drop is the RNA:protein ratio after 6 hour period of light exposure (RM-

3000

2500

21

2000

Uptake

1000

33P

1000 500

1

6

21

Time (Hours)

Fig. 3. Uptake of 33P (counts pr min) in S. hyperborea at −P over the course of the experiment for PAR vs. UVR and PAR vs high UVR. Bars and vertical lines represents mean±SD (n=3).

0

0

0 6

Time (Hours)

1500

2000

25000 20000 15000 10000 5000

5000 0 1

4000

3000

PAR UVR High UVR

PAR UVR High UVR

30000

25000 20000

Uptake

15000 10000

33P

Fig. 4. High time-resolution uptake of 33P (counts pr min) in S. hyperborea at low P over the course of the experiment for PAR vs high UVR. Bars and vertical lines represents mean ± SD (n = 3).

35000

between light and time (RM-ANOVA, light × treatment: p = 0.009). The +P experiment showed lower P-uptake both for the UVR and high UVR exposure (RM-ANOVA, light: p = 0.041 for PAR vs. UVR and p b0.001 for PAR vs. high UVR) relative to the PAR alone in S. hyperborea. The difference in P-uptake also increased over time for both the UVR and high UVR exposure, as shown by significant light × time interactions (RM-ANOVA, light × treatment: p = 0.024 for PAR vs UVR and p b0.001 for PAR vs high UVR, cf. Table 2). The lack of any strong response in 33P-uptake under UVR was confirmed by the RNA-assays (Table 3 and Fig. 6). Although there were

1

6

21

Time (Hours)

0.5

1

6

21

Time (Hours)

Fig. 5. Uptake of 33P (counts pr min) in S. hyperborea at +P over the course of the experiment for PAR vs. UVR and PAR vs high UVR. Bars and vertical lines represent mean±SD (n=3).

D.O. Hessen et al. / Journal of Experimental Marine Biology and Ecology 411 (2012) 45–51 Table 3 Results of one-way RM-ANOVA on UVR response of Porosita glacialis and Synedropsis hyperborean on RNA:protein ratio. Light

Time

Light x Time

F

P

F

P

F

P

P.glacialis PAR vs. UVR

0.41

0.557

5.17

0.036 *

2.24

0.169

S.hyperborea PAR vs. UVR

0.0017

0.969

8.62

0.010 *

0.068

0.935

ANOVA, time: p = 0.010) for both PAR and UVR exposure, probably reflecting an active protein synthesis under light for this fast-growing species. 4. Discussion Light levels and spectral properties both regulate the uptake of inorganic C and nutrients in autotrophs, and thus determine to a large extent the cellular stoichiometry (in this context the C:N:P-ratio). The algal response to UVR includes typically reduced photosynthetic activity and thus reduced C-fixation (cf. Neale et al., 2003), while there are ambiguous reports on the effect of UVR on nutrient uptake (Hessen et al., 2008). The reduced C:P-ratio that frequently have been observed under UVR-stress both in freshwater (Leu et al. 2006a; Tank et al., 2003; Watkins et al., 2001, Xenopoulos et al., 2002) and marine algae (Hessen et al. 2008; Leu et al., 2007) could be caused by reduced C-uptake, increased P-uptake or both. Previous experiments (Leu et al., 2007) with other marine diatoms (Thalassiosira antarctica, Chaetoceros socialis and Bacterosira bathuomphala) yielded reduced C:P-ratios under UVR (while no effect on C:N), and the authors suggested that this response could reflect an enhanced P-uptake. The three arctic diatoms tested in the current study did not support this hypothesis however, neither did the RNA-assays for Porosira and Synedropsis.

A

B

49

Reduced uptake of inorganic N under UV-stress has been reported by some authors (Braune and Döhler, 1994; Döhler and Biermann, 1987), while other studies found rather weak or even no effects of UV on C:N ratios in phytoplankton (Fauchot et al., 2000; Mousseau et al., 2000). Similarly, while several studies have demonstrated a decrease in cellular C:P-content under UVR-stress, there are mixed evidence for the role on UVR on P-uptake, especially for natural systems (Sereda et al., 2011). There are different uptake mechanisms and enzymes involved for N and P in autotrophs, and these may respond differently to UVR. There is clearly also a reciprocal effect of ambient concentrations of P and UVR (Medina-Sánchez et al., 2006, Villar-Argaiz et al., 2009, Xenopoulos et al., 2002), but enhanced levels of P may both mask or enforce the negative effects of UVR (Carrillo et al., 2008). Typically, experiments are run in media with nutrient concentrations that are much higher than ambient conditions, and typically C:P-ratios, also in response of UVR, decrease with decreased ambient levels of P (Frost and Xenopoulos, 2002, Xenopoulos and Frost, 2003). Likewise, uptake of P has been found to depend on levels of UVR (Aubriot et al., 2004; Hessen et al., 1995). The interpretation of this would be that low to moderate doses of UVR could stimulate an increased uptake of P for photorepair, or could be used to compensate for a decreased rate of protein synthesis under UVR-stress by allocating more P into ribosomes. Under higher levels of UVR exposure, however, the uptake of nutrients could be impaired by membrane damage or by disruption of alkaline phosphatase activity (cf. Sereda et al., 2011). The reason for these and previous inconsistencies with regard to nutrient uptake responses may partly be attributed to strong species-specific differences in UVR response (Delgado-Molina et al., 2009; Karentz et al., 1991), as well as the fact that the responses are quite subtle in that they depend on a suite of experimental or ambient conditions. E.g. the time course and light source as well as light quality and intensity, time for photorepair, algal cell size, physiological status, growth phase, ambient nutrient conditions and temperature. Finally, there is always a risk that culturing conditions in the absence of UVR could yield different responses compared to UVRadapted cultures, especially if UVR-protection is a costly and thus inducible trait. A full factorial test on all parameters that potentially might influence the cellular uptake of P and C:P-ratios is unachievable, and certainly beyond the scope of this study. Nevertheless, our experiments suggest that UVR does not trigger an enhanced P-uptake, in support of the findings of Carrillo et al. 2008. Thus the frequently observed up-regulation of cellular C:P under UVR-stress seem most likely to be due to impaired fixation of CO2, or enhanced cellular leakage of organic C. Since P is a scarce element being crucial for nucleic acids and certain membrane lipids, it may very well be that most species have evolved mechanisms to protect and regulate P-uptake under UVR-stress. Given the importance of nutrient uptake and elemental ratios in the elemental flux and the marine food web, the variability in underwater spectral properties both due to natural variability and expected periods of ozone-minima and likely extended loss of ice-cover, there is a definite need for testing a wider range of species and a wider range of spectral properties and UV doses to reveal the nature of carbon and nutrient uptake in autotrophs. We also need to use different approaches that will allow us to understand better the exact physiological role of P in the algal response to light stress and UVR exposure.

Acknowledgements

Fig. 6. RNA:protein ratio (μg μg− 1) in (A) P. glacialis and (B) S. hyperborea at high P during the course of the experiment for PAR vs. UV. Bars and vertical lines represents mean ± SD (n = 3).

This study was financed by grants from the Norklima Program under The Norwegian Council of Sciences and Letters to projects MERCLIM and CATCHMENT. We are most thankful to two anonymous reviewers for their most helpful and constructive comments. [SS]

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Appendix A

Table A1 Uptake of

33

P (in counts per minute) of Porosira glacialis, Thalassiosira sp and Synedropsis hyperborea for all treatment combinations. Means ± SD.

P. glacialis PAR vs UVR PAR UVR PAR vs high UVR PAR High UVR Thalassiosira sp. PAR vs UVR PAR UVR PAR vs high UVR PAR High UVR

t=1

t=2

t=3

13,164 ± 3706 17,432 ± 1498

58,724 ± 13,489 64,382 ± 4662

213,221 ± 24,861 218,904 ± 9593

3277 ± 130 3081 ± 57

9678 ± 425 8516 ± 166

18,815 ± 631 16,615 ± 213

9097 ± 355 9285 ± 363

25,742 ± 328 24,734 ± 650

32,225 ± 737 31,877 ± 1099

6341 ± 269 5704 ± 55

14,456 ± 769 13,753 ± 1503

18,477 ± 1435 14,578 ± 1791

18,351 ± 880 20,824 ± 2710

15,766 ± 6221 22,030 ± 1499

29,377 ± 3081 27,927 ± 1814

24,005 ± 3731 26,804 ± 1764

9205 ± 360 8947 ± 152

11,632 ± 273 11,585 ± 133

1255 ± 21 1019 ± 138

2253 ± 279 1509 ± 153

1468 ± 38 1507 ± 41

2417 ± 140 2230 ± 49

S. hyperborea PAR vs UVR at − P PAR 14,381 ± 2710 UVR 13,333 ± 357 PAR vs high UVR at − P PAR 28,752 ± 1076 High UVR 24,158 ± 520 PAR vs high UVR at − P short-term PAR 5411 ± 103 High UVR 5343 ± 165 PAR vs UVR at + P PAR 712 ± 78 UVR 1052 ± 456 PAR vs high UVR at + P PAR 1320 ± 49 High UVR 1413 ± 53

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t=4

t=5

14,597 ± 803 14,106 ± 816

14,126 ± 630 15,729 ± 266

3819 ± 102 2709 ± 12

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