Ultraviolet radiation negatively affects growth and food quality of the pelagic diatom Skeletonema costatum

Journal of Experimental Marine Biology and Ecology 383 (2010) 164–170

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Ultraviolet radiation negatively affects growth and food quality of the pelagic diatom Skeletonema costatum Sarah Nahon a,⁎, François Charles b, François Lantoine a, Gilles Vétion b, Karine Escoubeyrou b, Martin Desmalades b, Audrey M. Pruski a a b

UPMC Univ Paris 06, UMR 7621, LOBB, Observatoire Océanologique, F-66651, Banyuls/mer, France CNRS, UMR 7621, LOBB, Observatoire Océanologique, F-66651 Banyuls/mer, France

a r t i c l e

i n f o

Article history: Received 1 July 2009 Received in revised form 30 November 2009 Accepted 5 December 2009 Keywords: Amino acids Fatty acids Mediterranean Sea Nutritional quality Phytoplankton UV radiation

a b s t r a c t There are now compelling observations of the ecological impacts of global change on marine ecosystems. A predicted 10% increase in UVB doses at the Earth's surface has been shown to impair indirectly the reproductive output of marine copepods. Wild females of Calanus helgolandicus fed with UVB-irradiated diatoms produced fewer eggs and unhealthy offspring exhibiting a large proportion of high lethal naupliar deformities. By reproducing the same irradiative stress on the pelagic diatom Skeletonema costatum, we looked for potential modifications of the algal properties (cell size, biovolume, chlorophyll content) and biochemical characteristics (fatty acid and amino acid contents). Our results confirmed that the metabolism of S. costatum is adversely affected by enhanced UVB exposure: cell growth was reduced and the biochemical characteristics, and thus the algal nutritional quality, were significantly altered. UVB irradiation reduced cell division, leading to cell elongation and thus increasing the biovolume. Meanwhile, the amino acid and fatty acid contents did not increase concomitantly to the cell enlargement and were thus diluted in the cell. Because of this dilution, the irradiated S. costatum represents a poorer diet for its potential consumers. Moreover, UVB dramatically affects the relative contribution of certain essential fatty acids such as eicosapentanoic acid (EPA), which are essential for the development of marine invertebrates. © 2010 Elsevier B.V. All rights reserved.

1. Introduction There is now growing evidence that ocean biota has undergone significant changes in recent years. Increased water temperature, enhanced ultraviolet-B (UVB) radiation, upper-ocean acidification, and other pervasive human disturbances may affect all levels of ecological hierarchies and a broad array of marine ecosystems (Walther et al., 2002). Shifts in phenology, distribution, abundance and structure of marine communities and their cycles are already observable (Häder et al., 1998; Beaugrand et al., 2002; Hays et al., 2005; Molis and Wahl, 2009). In the north-western Mediterranean Sea, such ecological changes have recently been suggested for the phytoplankton community structure; Skeletonema costatum, formerly described as the dominant species of the local diatom blooms (Jacques, 1969), is now superseded by other diatoms species Chaetoceros spp. and Pseudo-nitzschia calliantha (Charles et al., 2005). Diatom blooms are at the base of a short and efficient food chain to fish stocks via zooplankton (Cushing, 1989). Consequently, any modification in the diversity of algal diets may lead, through

⁎ Corresponding author. Tel.: +33 4 68 88 73 94; fax: +33 4 68 88 73 95. E-mail address: [email protected] (S. Nahon). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2009.12.006

bottom–up processes, to major alterations in the energy flow to higher trophic levels (Müller-Navarra et al., 2000). In zooplankton, reproductive success is affected by the quantity and nutritional quality of maternal diets (Kleppel et al., 1998b). The nutritional deficiency hypothesis (Pond et al., 1996; Jonasdottir et al., 2002) states that lack of essential compounds in marine copepod diets induces a decrease of egg production, hatching success, and larval survival. Even if the role of food constituents in decoupling egg production, egg hatching and larval survival has still not been clearly demonstrated (Pohnert et al., 2002; Jonasdottir et al., 2005) and remains controversial (Dutz et al., 2008), food components such as highly unsaturated fatty acids seem to have a common importance for egg production and growth (Sargent and Whittle, 1981; Brett and Müller-Navarra, 1997). Change in phytoplankton nutritional quality may be linked not only to diversity but also to the life history of the algal cells (Thompson et al., 1990). For instance, UVB radiation is known to affect the cellular mechanisms involved in fatty acids production (Döhler and Biermann, 1994; Goes et al., 1994; Wang and Chai, 1994; Skerratt et al., 1998). However, whereas direct detrimental effects of UV have been widely reported for marine phytoplankton and zooplankton (Nahon et al., 2008; Nahon et al., 2009; Pruski et al., 2009), the potential effects of UVB radiation on trophic interactions have barely been investigated. Recently, Kouwenberg and Lantoine

S. Nahon et al. / Journal of Experimental Marine Biology and Ecology 383 (2010) 164–170

(2007) have evidenced the indirect effects of near future increase UVB radiation on the reproductive output of a common copepod, Calanus helgolandicus. These authors suggested that reduced egg production and high frequency naupliar abnormalities could be explained by a deficiency in essential components of the UVB-stressed maternal diet. The present study was designed to examine this hypothesis. The effect of UVB radiation on S. costatum was evaluated for predicted future UVB conditions in the surface layer of the NW Mediterranean Sea (Kouwenberg and Lantoine, 2007). By reproducing the same irradiative stress, we report changes in cell growth, cell structure, amino acid and fatty acid composition of S. costatum after exposure to UVB radiation. 2. Materials and methods 2.1. Experimental design The incubator designed by Kouwenberg and Lantoine (2007) was used for the growth and exposure experiments. The incubator was separated into two identical boxes using a wooden partition, the whole covered with a coarse open grill at 0.4 m distance from the light sources below. Lighting conditions were identical to those used by Kouwenberg and Lantoine (2007) and consisted of 14 Philips ‘TL'D 18 W/965 Natural Daylight fluorescent tubes. The spectral irradiance of the light sources from both boxes was measured with the MACAM spectroradiometer (Fig. 1). Box 1 was fitted with two additional Philips ‘TL’ 20 W/12RS ultraviolet-B fluorescent tubes. The grill was covered with Schott WG 305 cut-off filters to stop UVC and the shorter UVB wavelengths that do not reach the Earth's surface. The irradiance at wavelengths above 320 nm reaching the cultures was kept identical with the aid of three layers of cellulose acetate film (0.13 mm thick) stretched on the grill of box 2. The strain of S. costatum used by Kouwenberg and Lantoine (2007) was grown in box 2 in a Pyrex® Erlenmeyer containing 1.5 L of f/2 medium (Guillard, 1975) under a 14/10 h light/dark cycle at 18 °C. Initially, the stock culture was kept in exponential growth phase by daily dilutions. After 8 days of exponential growth, the stock culture was diluted and then 300 mL of this stock culture was added to each of ten 500 mL Erlenmeyer flasks for the experiments. Five of the flasks used were Pyrex® Erlenmeyers and five were made of quartz glass (100% UVB transmittance). Control cultures were grown without UVB in Pyrex® Erlenmeyers, and the “UV-irradiated” cultures were placed

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in quartz Erlenmeyers and received additional UVB irradiation, 4 h per day in the middle of the light cycle. The daily UVB dose was 17.3 kJ m− 2 which is between the 4 h dose for the measured noon underwater UVB irradiance at 1 m depth (15.3 kJ m− 2) and the predicted 4 h noon underwater dose at 1 m depth for 10% ozone depletion (19.2 kJ m− 2). Both the measured and modeled doses were based on mean values for mid-day light regimes in June (Kouwenberg and Lantoine, 2007). 2.2. Estimation of cell growth, biovolume and chlorophyll concentration Cultures were grown during 4 days and harvested the next morning for measuring cell volume, chlorophyll concentration and the biochemical analyses. Every day, cells from each culture were counted under a microscope and photographed using a computer-linked video camera. Images were processed and analysed with the software Visilog 6.0 (Noesis, France). Algal concentration was determined using a Malassez counting slide on 4 replicates every morning to allow for the processes of DNA dark repair and photorepair after the UVB exposure. Assuming an elliptical cylindrical shape, cell volume was estimated from cell length and width measured on 100 live cells. Chlorophyll concentration was determined spectrofluorimetrically. Two milliliters from each culture were filtered on a Whatman GF/F filter (47 mm diameter). Photosynthetic pigments were extracted overnight in acetone 90%, at 5 °C in total darkness. The fluorescence of the supernatant was measured on an LS 55 spectrofluorimeter (Perkin Elmer Inc., USA) according to the method developed by Neveux and Lantoine (1993) to accurately determine 6 pigments: chlorophylls a, b, and c, and the three associated phaeopigments. 2.3. Assessment of the nutritional value of S. costatum For total hydrolysable amino acid (THAA) composition, 50 mL of each culture were filtered onto pre-combusted (450 °C) Whatman GF/F glass fiber filters (47 mm diameter). The samples were then lyophilized and stored at −20 °C until analysis. THAA extraction and analysis has been detailed in Pusceddu et al. (2005). Briefly, filters were submitted to a strong acid hydrolysis in vacuum sealed ampoules (2 mL of 6N HCl, 100 °C, 24 h). Homoserine (100 µL, 0.5 mM) was added prior to hydrolysis as an internal standard. An aliquot of the hydrolysates (200 µL) was neutralised with NaOH (6 N, 200 µL), buffered with H3BO3 (0.4 M, 400 µL, pH 8), centrifuged (681g, 2 min), filtered through a 0.2 µm-syringe filter (Uptidisc RC 13 mm, Interchrom) into a HPLC

Fig. 1. Spectral irradiance for Skeletonema costatum growth cultures (Kouwenberg and Lantoine, 2007); (a) fluorescent spectral irradiance for the S. costatum experimental cultures, additionally exposed to UVB during 4 h day− 1 (Philips ‘TL'D 18 W/965 + Philips ‘TL’ 20 W/12RS); (b) fluorescent spectral irradiance for the S. costatum control cultures in the laboratory, grown without UVB (Philips ‘TL'D 18 W/965).

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autosampler vial. Primary amino acids were derivatized with orthophtaldialdehyde and β-mercaptoethanol (Mopper and Lindroth, 1982) in 0.4 M borate buffer (pH 8), S-methyl cysteine being added to the reaction mixture as an external standard. Compound separation was performed by reverse phase high performance liquid chromatography on a Gynkotek-Dionex system equipped with a microsorb reverse phase C18 column (250× 4.6 mm, Varian). Non-linear binary gradients of methanol-acetate buffer were used with a constant flow rate of 1 mL min− 1 (total run time was 60 min). The isoindole derivatives were detected by fluorescence at 450 nm using an excitation wavelength of 335 nm. Amino acid identification and quantification were achieved by comparing retention times and peak areas of a standard amino acids solution to unknown samples. The amino acid standard solution was prepared from the SIGMA AAS18 mixture and 8 individual amino acids. The concentrations of 14 amino acids were calculated and expressed as weight per biovolume of algae (fg µm− 3) to facilitate comparison of chemical composition among cells of different sizes (Jonasdottir, 1994). Note that tryptophane and cysteine are not quantified by this method; the former is destroyed during the hydrolysis step and the latter produces derivatives with minimal fluorescence. Aspartic acid and glutamic acid peaks include the contribution of the corresponding deaminated amino acids, glutamine and asparagine. For fatty acids composition, 150 mL of each culture were filtered and treated as described above. The samples were then lyophilized and stored at −20 °C until analysis. Fatty acids were extracted and methylated using a direct transesterification procedure with minor modifications of the protocols described by Lewis et al. (2000) and Indarti et al. (2005). Briefly, filters were placed in Teflon-lined screw cap vials with 8 mL of a cold transesterification solution [methanol: hydrochloric acid:chloroform (1.7:0.3:2, v/v/v)]. Chloroform was supplemented with an antioxidant (50 mg mL− 1 of buthylhydroxytoluene, Christie, 2003) and the extractions vials were spiked with 10 µL of nonadecanoid acid (1 mg mL− 1) as an internal standard. The samples were vortexed and placed in a preheated oven at 90 °C for 90 min with occasional shaking by hand (3 to 4 times). The extraction vials were allowed to cool on ice before addition of 2 mL cold UV-oxidised reagentgrade (Milli-Q) water. The extracts were centrifuged (1328g, 5 min) and the lower organic phase containing the fatty acid methyl esters (FAMEs) was transferred in another tube. The upper aqueous phase was rinsed with 2 mL of hexane-chloroform (4:1) and centrifuged (1328g, 5 min). This procedure was repeated twice and the pooled organic phases were rinsed with a 2% solution of potassium carbonate (4 mL). Following centrifugation (1328g, 5 min), an aliquot of the organic phase (9.5 mL) was evaporated to dryness in a rotary evaporator (Savant Speed Vac system) at room temperature and FAMEs were redissolved in 100 µL of hexane prior to analysis. Fatty acids as methyl esters were analysed using a Varian 3900 gas chromatograph (GC) coupled to a Saturn 2100T ion-trap mass spectrometer (MS). One microliter of sample was injected in a split/ splitless injector maintained at 260 °C with a split ratio of 100:1. The carrier gas was helium (constant column flow 1 mL min− 1). FAME separation was performed on a fuse silica capillary Factor Four VF-23ms column (30 m × 0.25 mm ID, 0.25 µm thickness) from Varian using the following temperature program: 165 °C (45 min isothermal), 200 °C (rate: 2 °C min− 1, 12.5 min isothermal). Total run time was 75 min. The manifold, transfer line and the ion trap were set at 50 °C, 230 °C and 230 °C, respectively. Mass spectra (m/z 50 to m/z 450) were recorded at a rate of 0.9 scans per second with ionization energy of 70 eV electron impact. FAMEs in samples were identified by comparison of retention times and mass spectra with authentic commercial standards: Qualmix Fish Synthetic (Ladoran Fine Chemicals, INTERCHIM, France), Supelco 37, PUFA n°1 and n°3 (SUPELCO, France). Qualitatively, the contribution of each individual fatty acid was determined on the basis of the reconstructed integrated chromatogram and expressed as the percentage of the specific compound area to the total peak area (% of total fatty acids). Using a standard containing known amounts of FAMEs (Supelco

37), calibration curves were generated for 37 individual FAMEs by plotting the surface area of the quantifying ion as a function of FAME concentration. Fatty acid concentrations in the cultures were calculated using these calibration curves, corrected with the internal standard and expressed as weight per biovolume of algae (fg µm− 3). 3. Results 3.1. Effect of UV radiation on cell growth, biovolume and chlorophyll concentration As shown in Kouwenberg and Lantoine (2007), UVB exposure reduced algal growth. During the 4 days of growth, cell concentration in the controls (no UVB) increased steadily from 3.8 × 10 5 to 14.9× 105 cells mL− 1, while in cultures exposed to UVB the mean cell number reached only 7.2× 105 cells mL− 1. The average growth rate significantly decreased with values of 0.34 ± 0.03 and 0.18 ± 0.01 day− 1 in the control and UVB-irradiated cultures, respectively (p b 0.01, Mann– Whitney test). Moreover, under UVB exposure, cell volume increased (Fig. 2). At the end of the experiment, the average biovolume of UVirradiated cells was 404 ± 173 µm3 compared to a significantly lower cell volume of 181 ± 88 µm3 for diatoms from the control cultures (p b 0.01, Mann–Whitney test). The increase in biovolume was mainly due to the elongation of the UV-irradiated diatoms. Finally, UVB exposure reduced the chlorophyll concentration. After 4 days of growth, chlorophyll a concentration in controls was slightly higher than in UVirradiated cultures (Table 1, p b 0.01, Mann–Whitney test). When expressed as cell content, irradiated diatoms contained greater amounts of chlorophylls a and c (chla and chlc) than non-irradiated cells, but this trend was reversed when chlorophyll content was expressed per unit of cell volume. Spectrofluorometric analysis of the acetone extracts indicated that the UV radiation regime did not alter the chla:chlc ratio or induce the degradation of chlorophyll pigments. 3.2. Assessment of the nutritional value of S. costatum The concentrations of the fourteen total hydrolysable amino acids identified in S. costatum are given in Fig. 3. The THAA composition of S. costatum was dominated by glutamate, aspartate and leucine, and each accounted for more than 10% of the THAA pool. The essential amino acids (arginine, histidine, isoleucine, leucine, lysine, threonine, phenyalanine and valine) represented nearly one half of the THAA pool (~47.5%). Among these essential amino acids, leucine was the

Fig. 2. Cell volume for Skeletonema costatum under control and UVB treatment on the fourth day. Data are represented by box and whiskers plots as following: the top and bottom of the box mark the 25th and 75th percentiles, the whiskers show the extreme values, and closed circles correspond to median values (n = 100 cells). Significant differences between control and treatment are marked as follow: ⁎⁎ p b 0.01, Mann– Whitney test.

S. Nahon et al. / Journal of Experimental Marine Biology and Ecology 383 (2010) 164–170 Table 1 Effects of UV stress on pigment composition in Skeletonema costatum (mean ± SD, n = 5). Significant differences between treatment and control are marked as follow: ⁎⁎ p b 0.01 (Mann–Whitney test), nd: not detectable. Control ng cell Chla Chlc Chla/Chlc Phaeopigments

−1

1.30 ± 0.09⁎⁎ 0.18 ± 0.01⁎⁎ 7.22 ± 0.12 nd

UVB ng cell− 1 1.92 ± 0.08⁎⁎ 0.27 ± 0.01⁎⁎ 7.13 ± 0.04 nd

most concentrated and histidine the least abundant. No typical bacterial amino acids (β-alanine, α-amino butyric acid, β-glutamic acid, γ-butyric acid) were detected in the samples. UVB exposure affected neither the relative contribution of each amino acid to the THAA pool nor the proportion of essential amino acids. Conversely, an overall reduction by one-quarter (on average) was observed for all the amino acids in the irradiated culture. THAA concentration significantly decreased with values of 70.3 ± 3.9 and 53.1 ± 2.7 fg µm− 3 in the control and UV-irradiated cultures, respectively (p b 0.05, Mann– Whitney test). Twelve fatty acids were identified in S. costatum; their respective contributions to the fatty acid pool are given as percent area for both treatments in Fig. 3. Fatty acid profiles were dominated by two saturated fatty acids (SFAs) C14:0 and C16:0, one monounsaturated fatty acid (MUFA) C16:1ω7, and two polyunsaturated fatty acids (PUFAs) C16:3ω4 and C20:5ω3. These fatty acids accounted for about 80% of the fatty acids in both the control and UV-irradiated cultures. UVB exposure affects fatty acid content. An apparent fatty acid accumulation was observed in the irradiated cells with a 1.5-fold increase of the cellular concentration (data not presented). However, fatty acid content per unit of biovolume decreased from 6.01 to 4.00 fg µm− 3 (p b 0.05, Mann–Whitney test) as a result of the concomitant elongation of the irradiated diatoms. The more pronounced effect was found for PUFA content, which decreased significantly from 35.1% to 28.0% of the fatty acids (p b 0.01, Mann–Whitney test; Fig. 4). This was primarily due to EPA (C20:5ω3), whose strong depletion in concentration accounted for 22.1% of the total decrease in fatty acids per unit of biovolume. A substantial decrease of two other PUFAs, namely C18:4ω3 and C16:3ω4, was also observed. The proportions of MUFAs and to a lesser extent the SFAs, increased in the irradiated diatoms conversely to the decrease of the PUFA content (p b 0.05, Mann–Whitney test). Still, individual fatty acid concentrations were lower in the irradiated culture (Table 2), at the exception of C15:0 and C18:0, which were present in trace amounts and did not vary between treatments. The most prominent decreases were

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found for even numbered short-chain saturated fatty acids (C14:0 and C16:0), one MUFA (C16:1ω7) and one PUFA (C20:5ω3), which respectively explained 43.6, 6.7, 26.0 and 22.0% of the global decrease in fatty acids. The ω3:ω6 ratio also significantly decreased with values of 11.7 and 7.8 for the control and UV-irradiated cultures, respectively (p b 0.01, Mann–Whitney test). Branched fatty acid isomers in C15 and C17, which are considered of bacterial origin, were not found in the cultures. 4. Discussion In marine invertebrates, the influence of maternal diet on fertility and hatching is now well established (Harrison, 1990). For instance, egg production and viability are both related to the presence of certain essential components in the food of female copepods (Pond et al., 1996; Broglio et al., 2003; Arendt et al., 2005). Many studies have stressed that solar UVB radiation may impair the nutritional value of phytoplankton and therefore affects higher trophic levels in the food-web (Hessen et al., 1997; Van Donk et al., 1997). However, experimental evidence for such a “bottom–up” effect mainly concerns freshwater ecosystems (see citations above). Recently, Kouwenberg and Lantoine (2007) have experimentally shown that UV radiation indirectly affects the success of reproduction in the marine pelagic copepod C. helgolandicus possibly via the modification of the biochemical composition of the diatom S. costatum used as the food source in their experiment. We therefore reproduced the first part of their study, which consisted in irradiating a culture of S. costatum daily with UVB. The nature and magnitude of S. costatum's response to UVB were comparable to the effects reported by Kouwenberg and Lantoine (2007): a marked decline in growth rate due to the inhibition of cell division and the increase of cell volume that was mainly explained by the elongation of the cells. We can thus reasonably assume that the biochemical changes will be similar for both studies. We also noted that phaeopigments remained low confirming the expectation that the culture was not senescent after four consecutive days of UV-treatment. In addition, amino acids and branched fatty acids specific to bacteria were not detected in either culture, indicating that bacterial contamination and proliferation were limited. These results are additional evidence that C. helgolandicus was fed on healthy cultures. Since the C. helgolandicus were allowed an unlimited food supply, the copepods most certainly have ingested diatoms until satiety (Kouwenberg and Lantoine, 2007). Hence, the cell number holds less significance for the copepod metabolism than the ingested biovolume and thus the nutriment intake. Our results suggest that the irradiated diatoms represented a “poorer” diet than the control culture. The total amino acid content, which roughly corresponds to the protein concentration, and the total fatty acid content were significantly

Fig. 3. Effects of UV on amino acid concentration in Skeletonema costatum. Amino acids are expressed in fg µm− 3 (mean ± SD, n = 5). ASP: aspartate; GLU: glutamate; SER: serine; HIS: histidine; GLY: glycine; THR: threonine; ARG: arginine; ALA: alanine; TYR: tyrosine; VAL: valine; PHE: phenyalanine; ILE: isoleucine; LEU: leucine; LYS: lysine and ●: essential amino acids. Significant differences between control and treatment are marked as follow: ⁎ p b 0.05, Mann–Whitney test.

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Fig. 4. Effects of UV on fatty acid composition in Skeletonema costatum. Fatty acids are expressed as percentage of total fatty acids (mean ± SD, n = 5). Significant differences between control and treatment are marked as follow: ⁎ p b 0.05 and ⁎⁎ p b 0.01, Mann–Whitney test.

lower in the irradiated diatoms. Maternal investment in egg production is related to nutriments and energy availability during oogenesis. The reduced fecundity of the females fed on irradiated diatoms (Kouwenberg and Lantoine, 2007) may be attributed, at least partly, to the lower concentration in amino acids and fatty acids, two crucial elements for vitellogenesis and egg maturation (Harrison, 1990; Von Elert and Stampfl, 2000; Lacoste et al., 2001). This is in agreement with field and laboratory results suggesting that for copepods, food quantity affects egg production, while food quality mainly impairs hatching success (Pond et al., 1996; Jonasdottir et al., 1995). Since hatching success remained high for the 7 first days of the experiments, the observed higher mortality of females fed on irradiated diatoms (Kouwenberg and Lantoine, 2007) may be explained by the draining of their internal lipid reserve during vitellogenesis (Hirche and Kattner, 1993). Nutritional adequacy of copepod maternal food has been shown to be as important for successful reproduction as food quantity (Guisande et al., 1999; Von Elert and Stampfl, 2000). In particular, their diet has to meet a high requirement in essential nutriments necessary for complete ontogenesis. For C. helgolandicus, experimental evidence has confirmed that the biochemical composition of the eggs, notably the amino and fatty acid pools, is influenced by the maternal diet (Jonasdottir, 1994; Koski et al., 1998; Guisande et al., 2000; Lacoste et al., 2001). Empirical studies have shown that egg production is correlated to dietary levels in several amino acids (glutamate, and three essential amino acids methionine, arginine and histine, Kleppel et al., 1998a) and suggested

Table 2 Effects of UV stress on fatty acid concentrations in Skeletonema costatum. Fatty acids are expressed in fg µm− 3 (mean ± SD, n = 5). Significant differences between control and treatment are marked as follow: ⁎ p b 0.05, ns: non significant, Mann–Whitney test. Fatty acid

C14:0 C15:0 C16:0 C18:0 Σ SFA C16:1ω7 C18:1ω9 cis Σ MUFA C18:2ω6 cis C20:5ω3 ω3/ω6 ratio Σ PUFA Total

Control

UVB

fg µm− 3

fg µm− 3

2.40 ± 0.30 0.05 ± 0.01 0.91 ± 0.07 0.02 ± 0.00 3.39 ± 0.35 1.89 ± 0.21 0.11 ± 0.01 2.01 ± 0.20 0.11 ± 0.01 0.85 ± 0.12 7.51 ± 0.88 0.97 ± 0.13 6.37 ± 0.67

1.37 ± 0.14 0.05 ± 0.00 0.73 ± 0.04 0.02 ± 0.00 2.17 ± 0.18 1.28 ± 0.20 0.09 ± 0.01 1.39 ± 0.20 0.09 ± 0.01 0.32 ± 0.05 3.56 ± 0.56 0.42 ± 0.05 3.97 ± 0.36

Statistic

⁎ ns ⁎ ns ⁎ ⁎ ns ⁎ ⁎ ⁎ ⁎ ⁎ ⁎

that an amino acid limitation could affect copepod reproduction in the field (Guisande et al., 2000). Based on the reported composition of C. helgolandicus eggs (Guisande et al., 2000), S. costatum would represent a well-balanced amino acid source for the copepods, providing high amounts of essential amino acids that may be stored in the eggs and used during the development of the nauplii. Since UVB irradiation did not affect the amino acid composition of the diatom, we can rule out the possibility that the impairment of embryonic development was due to an amino acid deficiency in the irradiated diet. On the other hand, our results underlined significant modifications in the fatty acid profile after UVB exposure. Even if similar alterations in phytoplankton have been reported (Wang and Chai, 1994; Skerratt et al., 1998), the debate about UV radiation effects on nutritional quality remains open (Leu et al., 2006). Several parameters such as species-specific differences in UV sensitivity, conditions of growth, irradiation mode and UV doses may explain some of these discrepancies. Fatty acids are usually given in percent area for practical reasons (i.e., commercial quantitative standards of certain fatty acids are not available), however percent area cannot account for weight percentages because fatty acids do not respond uniformly (Dodds et al., 2005). Moreover, since percentages take into account all the compounds, each individual compound is affected by the variation of the other compounds. Our results (Table 2 and Fig. 4) clearly illustrate that the choice of the unit may lead to different interpretations. Dietary deficiencies are not simply explained by fatty acid imbalance in the food (e.g. ω3/ω6 ratio), but also limitations arising when the supply of one or several compounds is below the animal's requirements (Jonasdottir, 1994). Quantitative and qualitative data on fatty acid composition are therefore both crucial to assess nutritional value and potential deficiencies (Brett and MüllerNavarra, 1997). UV radiation may affect the nutritional quality of phytoplanktonic algae by the disruption of fatty acid synthesis and/or the oxidation of the double bonds in PUFAs (Döhler and Biermann, 1994; Goes et al., 1994; Wang and Chai, 1994; Skerratt et al., 1998). In the present study, the effects of UVB were not restricted to the PUFAs; concentrations of other major compounds including myristic, palmitic and palmitoleic acids also decreased. Although the nutritional requirements for copepod reproduction are not precisely defined, a general need for highly unsaturated fatty acids of the ω3 series, in particular EPA and DHA, has been reported for marine invertebrates (Harrison, 1990). Egg production is for instance, positively related with dietary ΣPUFA:ΣFA and ω3:ω6 in copepods (Jonasdottir, 1994). Accordingly, both ratios were significantly lower in the irradiated diatoms than in the controls and female copepods that received the irradiated food produced fewer eggs (Kouwenberg and Lantoine, 2007). Hatching success was also impaired in Kouwenberg and Lantoine's feeding experiment. This was not unexpected in view of the general

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pattern of PUFA dependence for normal embryogenesis in crustaceans (Jonasdottir and Kiorboe, 1996). In the calanoid copepod Arcatia tonsa, Broglio et al. (2003) reported experimental results suggesting that a minimal amount of certain PUFAs (C18:3ω3, C20:5ω3 and C22:6ω3) is needed to obtain over 60% hatching success. There were only two ω3 PUFAs in our culture of S. costatum: EPA, a major membrane constituent in copepods (Albers et al., 1996), and stearidonic acid (C18:4ω3), which may serve as a precursor for the 22 moiety when DHA is absent in the diet (Guschina and Harwood, 2006). The abnormal development of the nauplii obtained from the female copepods fed on irradiated diatoms (Kouwenberg and Lantoine, 2007) may be related to ω3 dietary deficiencies since both EPA and a potential precursor for DHA were depleted. Apart from amino and fatty acids, other biochemical compounds such as vitamins (Guerin et al., 2001), carotenoïds (Lotocka et al., 2004) and sterols (Ederington et al., 1995) may be essential for copepods reproduction. These were not considered in the present study. The same is true for the negative effect of polyunsaturated aldehydes (Pohnert, 2005). Polyunsaturated aldehydes are derived from the oxidative transformation of PUFAs (Pohnert, 2000), these secondary metabolites cause hatching impairment (D'Ippolito et al., 2002) and larval abnormality in copepods (Ianora et al., 2004). Given that UV radiation may lead to photooxidative stress, it would be of interest to determine whether UVB stress contributes to the production of polyunsaturated aldehydes in irradiated algae. 5. Conclusion The present study was designed to explain Kouwenberg and Lantoine's finding that reproduction of the pelagic copepod C. helgolandicus was impaired when the copepods were fed on diatoms irradiated with UVB (2007). Our results support the hypothesis, that UVB radiation has indirectly affected the reproductive success of the copepods through the modification of the food nutritional quality. An overall dilution of all components (fatty acids and amino acids) was observed concomitantly to the increase of the diatom size and certain essential components for oogenesis and embryogenesis were strongly depleted under UVB. Furthermore, the results underline that in order to fully assess the impact of UV radiation on the fluxes of matter and energy in marine food webs, the detrimental effects of UVB on the quality of the primary resources should not be the only parameter to be considered. For example, it is acknowledged that some of the material grazed by copepods may be lost as dissolved organic carbon (DOC) by the mechanism of “sloppy feeding” which is expected to depend on the relative size of the prey (Moller, 2005). It would be interesting to quantify the loss of DOC, and in particular essential nutriments, associated to the increase in cell size under UVB radiation. Acknowledgements This work is a contribution to the UV joint action and has been supported by the Laboratoire d'Océanographie Biologique de Banyuls (LOBB/UMR-CNRS 7621) and the Université Pierre et Marie Curie. S.N. was supported by a MENRT grant from the French Ministry of Education and Research. We acknowledge J. Guarini for her revision of the manuscript language. [ST] References Albers, C.S., Kattner, G., Hagen, W., 1996. The compositions of wax esters, triacylglycerols and phospholipids in Arctic and Antarctic copepods: evidence of energetic adaptations. Marine Chemistry 55, 347–358. Arendt, K.E., Jonasdottir, S.H., Hansen, P.J., Gärtner, S., 2005. Effects of dietary fatty acids on the reproductive success of the calanoid copepod Temora longicornis. Marine Biology 146, 513–530. Beaugrand, G., Reid, P.C., Ibanez, F., Lindley, J.A., Edwards, M., 2002. Reorganization of North Atlantic marine copepod biodiversity and climate. Science 296 (5573), 1692–1694.

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