Dual control of the levels of photoprotective compounds by ultraviolet radiation and temperature in the freshwater copepod Boeckella antiqua

Journal of Plankton Research, Jul 2008

Photoprotective compounds (PPCs), such as carotenoids and mycosporine-like amino acids (MAAs), confer photoprotection to aquatic organisms against harmful ultraviolet-B (UVB) radiation. The natural variability of these compounds in zooplankton has been related to temperature, radiation and diet, but the ultimate mechanisms regulating the observed patterns in the field are still unclear. In this study, we analysed the variability of carotenoids and MAAs in a population of the calanoid copepod Boeckella antiqua in a shallow pond located in Northern Patagonia (Argentina). During our field survey, carotenoids and MAAs in B. antiqua varied without a clear seasonal pattern. Nevertheless, both groups of PPCs reached their maxima during spring and minima during summer. Inverse relationships were found between carotenoid concentrations versus temperature and irradiance. For MAAs, the same relationships were not significant. Tolerance experiments showed that mortality of B. antiqua was significantly influenced both by temperature and UVB dose, being more vulnerable at high temperature. We further investigated the effect of radiation regime and temperature on the bioaccumulation of PPCs in controlled laboratory experiments. We found that the concentrations of PPCs could be experimentally modified by manipulating radiation exposure and temperature. In addition, by breaking down the bioaccumulation processes into uptake and elimination, we were able to show that (i) the uptake rate was stimulated by photosynthetically active radiation (PAR)+UVA exposure and (ii) both uptake and elimination rates increased with temperature. Thus, the net accumulation (i.e. the balance between uptake and elimination), which ultimately dictates the concentration observed in an animal, could be either positive or negative depending on the specific combination of radiation exposure and temperature. The dual regulation of PPCs by radiation exposure and temperature should be considered in future efforts to reconstruct or predict the photoprotective responses of aquatic organisms to the past or future climate scenarios.

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Dual control of the levels of photoprotective compounds by ultraviolet radiation and temperature in the freshwater copepod Boeckella antiqua

Photoprotective compounds (PPCs), such as carotenoids and mycosporine-like amino acids (MAAs), confer photoprotection to aquatic organisms against harmful ultraviolet-B (UVB) radiation. The natural variability of these compounds in zooplankton has been related to temperature, radiation and diet, but the ultimate mechanisms regulating the observed patterns in the field are still unclear. In this study, we analysed the variability of carotenoids and MAAs in a population of the calanoid copepod Boeckella antiqua in a shallow pond located in Northern Patagonia (Argentina). During our field survey, carotenoids and MAAs in B. antiqua varied without a clear seasonal pattern. Nevertheless, both groups of PPCs reached their maxima during spring and minima during summer. Inverse relationships were found between carotenoid concentrations versus temperature and irradiance. For MAAs, the same relationships were not significant. Tolerance experiments showed that mortality of B. antiqua was significantly influenced both by temperature and UVB dose, being more vulnerable at high temperature. We further investigated the effect of radiation regime and temperature on the bioaccumulation of PPCs in controlled laboratory experiments. We found that the concentrations of PPCs could be experimentally modified by manipulating radiation exposure and temperature. In addition, by breaking down the bioaccumulation processes into uptake and elimination, we were able to show that (i) the uptake rate was stimulated by photosynthetically active radiation (PAR)+UVA exposure and (ii) both uptake and elimination rates increased with temperature. Thus, the net accumulation (i.e. the balance between uptake and elimination), which ultimately dictates the concentration observed in an animal, could be either positive or negative depending on the specific combination of radiation exposure and temperature. The dual regulation of PPCs by radiation exposure and temperature should be considered in future efforts to reconstruct or predict the photoprotective responses of aquatic organisms to the past or future climate scenarios. - PATRICIA ELIZABETH GARCIA1, ALEJANDRA PATRICIA PE REZ1, MARIA DEL CARMEN DIE GUEZ1*, MARCELA ANDREA FERRARO2 AND HORACIO ERNESTO ZAGARESE2 1LABORATORIO DE FOTOBIOLOGIA, CENTRO REGIONAL UNIVERSITARIO BARILOCHE, UNIVERSIDAD NACIONAL DEL COMAHUE, QUINTRAL 1250, (8400) SAN CARLOS DE BARILOCHE, RIO NEGRO, ARGENTINA AND 2LABORATORIO DE ECOLOGIA Y FOTOBIOLOGIA ACUA TICA, INSTITUTO DE INVESTIGACIONES BIOTECNOLO GICAS, INSTITUTO TECNOLO GICO DE CHASCOM US (IIB-INTECH), CC 164 (B7130IWA) CHASCOM US, BUENOS AIRES, ARGENTINA I N T R O D U C T I O N Photoprotective compounds (PPCs) are ubiquitous chemicals in aquatic organisms. On an evolutionary time scale, the acquisition of PPCs is regarded as a precondition for the colonization of ultraviolet (UV) exposed habitats (Cockell and Blaustein, 2001). On the basis of the premise that PPCs are produced in response to UV radiation, several authors have attempted These two groups of experiments were repeated with minor modifications in different years (2004 and 2006). In the 2004 experiments, we tested the tolerance to UVB and the bioaccumulation of PPCs at two different temperatures (108C and 188C). In the 2006 experiments, we extended the temperature range and added intermediate temperatures. In these experiments, both the tolerance to UVB and bioaccumulation of PPCs were assessed at 58C, 88C, 128C, 168C and 208C. Collectively, the whole series of experiments virtually covered all the ecophysiological temperature range of the species. UVB tolerance experiments a previous study showed that B. antiqua from Lake Los Juncos had relatively high MAAs levels (Perez et al., 2006) when compared with other Boeckella species (Tartarotti et al., 2004). The lake was visited on 24 occasions, from June 2002 to October 2003. On each sampling date, pH, conductivity, dissolved oxygen and temperature were measured, and water samples were collected using a van Dorn bottle for chlorophyll a, total solids (TS), and absorbance of dissolved fraction. Zooplanktons were collected by horizontal tows with a 45 mm mesh net. The dominant calanoid in Lake Los Juncos is B. antiqua. This copepod was selected as model organism for our study because it was found to be the only copepod species present throughout the year, and it can be maintained for several months in laboratory cultures for experimentation (Zagarese et al., 1997; Perez et al., 2006). In addition, this copepod has moderate to high levels of carotenoids and MAAs and the accumulation of both compounds can be experimentally induced upon exposure with UVA (Perez et al., 2006). Collections of B. antiqua for the analysis of PPCs were made on 18 occasions during the study period. In the laboratory, 150 adult copepods were sorted in three Eppendorf vials and kept at 2208C. Each batch was subsequently analysed for MAAs, carotenoids and dry weight. Diffuse attenuation coefficients (kd) were calculated using the optical density at 320 and 440 nm of filtered (GF/F, Whatmanw) water samples using the equations of Morris et al. (Morris et al., 1995). The incident solar radiation was measured continuously using a GUV-511 radiometer (Biospherical Instruments, Inc.) located at a nearby site. The measurements were integrated over time for UV and PAR wavelengths and averaged to obtain mean daily fluences. Mean downwelling fluences (Hl) for the water column were calculated following Ferrero et al. (Ferrero et al., 2006) as follows: Fig. 1. Radiation conditions in the UVB tolerance and bioaccumulation experiments performed in 2004 and 2006 with B. antiqua. (Philips), in order to provide favorable conditions for photoreactivation (Fig. 1). In all cases, mortality proportions were adjusted by the Abbott formula (Newman, 1995) to account for the differences in control mortality between temperatures. Bioaccumulation experiments The bioaccumulation of MAAs and carotenoids in B. antiqua was studied in longer-term incubations. In the 2004 experiments, batch cultures of 150 B. antiqua individuals were incubated for 10 days at two temperatures (108C and 188C), and either exposed to PAR+UVA or maintained in the dark. Each of these four treatments (two temperatures 2 radiation conditions) was run on six replicates. In the 2006 experiments, batches of 200 B. antiqua individuals were incubated for 10 days at five temperatures (58C, 88C, 128C, 168C and 208C) and either exposed to PAR+UVA or maintained in the dark. Each of these 10 treatments (five temperatures two radiation conditions) was run on three replicates. The B. antiqua individuals used in the experiments were collected from Laguna Los Juncos on the same day of the experiments. In both years, the dark treatments were wrapped with aluminum foil, whereas the exposed treatments were irradiated with 1.75 W m22 of UVA plus 110 mmol quanta m22 s21 of PAR, under a photoperiod of 14 h light: 10 h dark (Fig. 1). The animals were maintained in 2 L UV transparent acrylic containers (Plasmatic, Spain) filled with filtered (GF/F) water containing about 1 104 cells mL21 of C. reinhardii. The culture medium was replaced by half every 2 days. At the end of the experiments, all individuals were counted, sorted into separate Eppendorf vials and stored for 1 day at 2208C until PPCs extraction. The experiments were run in a test chamber Sanyo MLR5. The assessment of dry weight and initial MAAs and carotenoid concentrations in B. antiqua was carried out on triplicate samples preserved at 2208C. Each initial sample consisted of 150 (2004 experiments) or 200 individuals (2006 experiments). Analytical procedures Copepod dry weights were measured on samples dried for 2 days at 708C and weighed on an analytical balance (OHAUS AP 310). MAAs and carotenoids were sequentially extracted from copepod samples. Extraction of MAAs was performed in 3 mL of 20% aqueous methanol according to Sommaruga and Garca Pichel (Sommaruga and Garca Pichel, 1999) and Laurion et al. (Laurion et al., 2000). After centrifugation and filtration (Whatmann GF/F) the extracts were scanned in a UV visible spectrophotometer (Hewlett Packard P 8453-E, 1 cm path-length quartz cuvette). MAAs concentration was normalized and expressed as optical density per unit of copepod mass (i.e. OD mg DW21 of B. antiqua). The pellets obtained after centrifugation were re-suspended using 3 mL of 96% ethanol to extract carotenoids, following Byron (Byron, 1982) and Hessen (Hessen, 1993). When necessary, the pellet was re-extracted in fresh solvent. The loss of Boeckellas carotenoids by applying the sequential extraction technique was estimated by comparing the carotenoid concentration of Boeckella obtained by direct ethanol 96% extraction and by sequential extraction using 20% methanol followed by 96% ethanol. The mean loss of carotenoids in the sequential extractions was estimated as 20% (+10%) assuming 100% as the carotenoid concentration obtained by direct extraction in separate samples. Initial and final batch samples of Boeckella from the different treatments of the bioaccumulation experiments were treated similarly. Finally, the extracts obtained were scanned in a UV visible spectrophotometer (1 cm path-length quartz cuvette). Carotenoid concentrations were calculated as in Hessen (Hessen, 1993) and expressed as mg mg DW21: where D is the absorbance at 474 nm (corrected by chlorophyll a), V the extract volume, E the extinction coefficient (2500) and W the sample dry weight. Carotenoid concentrations in seston samples were determined after Wetzel and Likens (Wetzel and Likens, 2000), whereas MAAs concentrations were determined from methanolic extracts as explained above. The uptake and elimination rates for carotenoids and MAAs were estimated from the results of the bioaccumulation experiments performed in 2004 and 2006. As a first approximation, we assumed (i) that both processes (i.e. uptake and elimination) obey first-order kinetics and (ii) that uptake is induced only when the copepods are exposed to visible and/or UV radiation. The second assumption (i.e. null uptake in the dark) was adopted for simplicity, but it could be easily relaxed by incorporating a baseline uptake level in the dark. More specifically, for each assayed temperature, we derived the elimination constants using data from the dark treatments, and the net-gain constants from the radiation-exposed treatments. Finally, the uptake constants were calculated as the sum of net-gain plus elimination constants, as follows: Elimination rateq Net-gain ratek q LnPPC i Ln PPC dark;f Dt LnPPC i Uptake ratek Net-gain rate Elimination rate k where [PPC]I is the initial concentration of PPC (either carotenoids or MAAs) at the beginning of the experiment, [PPC]dark, f the final PPC concentration in copepods kept in dark, [PPC]exposed, f the final PPC concentration in copepods exposed to PAR+UVA and Dt the duration of bioaccumulation trials, i.e. 10 days. The relationships between fluences, temperature, sestonic carotenoids and MAAs in B. antiqua in seasonal samples were analysed using simple regression analyses. Two-way analysis of variance was applied to analyse single and combined effects of temperature and radiation for both UVB tolerance and bioaccumulation of PPCs. The relationships between bioaccumulation rates (for both MAAs and carotenoids) and temperature from the 2006 experiments were fitted to simple linear models using regression analysis (Zar, 1999). R E S U LT S Seasonal variation of environmental conditions and patterns of PPCs in B. antiqua During the study period, the depth at the centre of Laguna Los Juncos averaged (+1 SD) 0.8 + 0.3 m. Mean temperature (+1 SD) was 10.3 + 5.48C, and the ice-cover period lasted about 20 days from late June to early July. The temperature pattern recorded during the study is shown in Fig. 2a. Mean pH and conductivity were 9.8 + 0.66 and 462 + 109 mS cm21, respectively. Dissolved oxygen concentration was always high averaging 10.5 + 1.84 mg L21. Total phosphorus (P) and total nitrogen (N) concentrations measured on selected sampling dates were high: 900 and 3000 mg L21, respectively. Mean chlorophyll a concentration was 4.97 + 4.34 mg L21. Water transparency was low, due to high mean levels of TS (4.2 + 1.6 mg L21) and high DOC concentrations (30.29 + 18.07 mg L21). Kd320 averaged 66.21 + 35 m21, whereas that at 440 nm kd440 was 7.63+5 m21. The seasonal pattern of the mean daily fluence at 320 nm in the water column is shown in Fig. 2a. Higher fluence levels were recorded during spring and summer (i.e. October February). The fluence at 320 nm varied between 14 and 180 J m22 and that of PAR between 2 and 13 mol photons m22. The seasonal patterns of carotenoids and MAAs in B. antiqua of Laguna Los Juncos are shown in Fig. 2b. The concentrations of both types of compounds varied greatly between sampling dates, even within the same season. The MAAs present in B. antiqua have been identified by HPLC, using appropriate standards as mycosporine-glycine, porphyra-334, shinorine and Fig. 2. Seasonal patterns of variation in: (a) temperature and mean daily fluence (H320) in the water column of Laguna Los Juncos and (b) concentrations of PPCs in B. antiqua from Laguna Los Juncos. MAA-332 (Perez et al., 2006). Carotenoids in B. antiqua ranged between 1.4 mg mg DW21 (October) and 3.5 mg mg DW21 (August) and the mean concentration of these compounds was 2.52 + 0.6 mg mg DW21 (mean + 1 SD) (Fig. 2b). During the study period, MAAs concentration in B. antiqua varied between 0.01 OD mg DW21 in January and 0.05 OD mg DW21 in September, whereas the mean content of these compounds was 0.03 + 0.009 OD mg DW21 (Fig. 2b). The concentration of carotenoids in B. antiqua was inversely related to water temperature (r2 = 0.42, F = 10.98, p = 0.005), as well as to the mean daily fluence at 320 nm (r2 = 0.44, F = 11.12, p = 0.005). Conversely, MAAs concentration did not show any obvious relationship with fluence or temperature (P . 0.05). The carotenoid content in B. antiqua was unrelated to the concentration of carotenoids in its food (i.e. bioseston) (P = 0.32). On the other hand, the concentrations of MAAs in seston samples were always below the detection limit of the spectrophotometric method and therefore did not relate to MAAs content in the copepod. Assessment of B. antiqua tolerance to UVB at different temperatures In the first tolerance experiments (2004) the mortality of B. antiqua was significantly influenced by both UVB dose and temperature. In this case, mortality was higher at 188C than at 108C (F = 22.4, P , 0.001, n = 48) at both levels of UVB radiation. Mortality increased at higher UVB doses (F = 7.59, P , 0.01, n = 48), but the interaction between dose and temperature was not statistically significant (F = 1.4, P = 0.24, n = 48) (Fig. 3a). In the second tolerance experiments (2006), the copepod mortality was influenced significantly by UVB exposure (F = 24.2, P , 0.001, n = 120), but not by temperature (F = 0.50, P = 0.73, n = 120). The mortality of B. antiqua increased with UVB dosage (F = 7, p , 0.0001) and was negligible at the lowest assayed doses (t = 0.95, P = 0.35, n = 80; Fig. 3b). Bioaccumulation of PPCs as affected by radiation exposure and temperature The results from the first bioaccumulation experiment (2004) showed that both radiation and temperature affected the concentrations of PPCs in B. antiqua. MAAs concentration was significantly influenced by both the radiation and temperature treatment (F = 117.68, P = 0.001 and F = 5.75, P = 0.027, n = 24, respectively). Overall, the concentration of MAAs was higher in PAR+UVA than in the dark treatment (t = 10.85, Fig. 3. Proportions of dead individuals of B. antiqua exposed experimentally to different levels of UVB radiation: (a) Experiment 2004 at 108C and 188C and (b) Experiment 2006 at 58C, 88C, 128C, 168C and 208C (mean + 1 SD). In all cases, mortality proportions were adjusted by the Abbott formula (see text) to account for the differences in control mortality between temperatures. P , 0.0001, n = 24) and at 108C versus at 188C (t = 2.4, P = 0.027) (Fig. 4a). Within the same radiation treatment, MAAs concentrations were similar at 108C and 188C (P . 0.105). Within each temperature treatment, the concentration of MAAs was higher in PAR+UVA than in the dark (t = 7.9, P , 0.001, n = 12 at 108C and t = 7.5, P , 0.001, n = 12 at 188C). The concentration of carotenoids in B. antiqua was significantly affected by the radiation regime (F = 47.24, P , 0.001, n = 24), whereas the effect of temperature was not significant (F = 0.38, P = 0.55, n = 24). However, the interaction of main factors resulted significant, suggesting that the effect of radiation depends on the temperature at which the copepods were maintained. In this case, post hoc contrasts showed that the concentration of carotenoids was significantly higher at 188C than at 108C within the PAR+UVA treatment (t = 3.85, P = 0.001, n = 12), whereas the pattern was inverse in the dark treatment (t = 2.98, P = 0.007, n = 12). At 108C carotenoid concentrations were similar in PAR+UVA and dark Fig. 4. Variation in PPCs concentration in B. antiqua in the bioaccumulation experiments at different temperatures and radiation regimes (PAR+UVA and dark). (a) Final MAAs and (b) carotenoid concentrations resulting from the 2004 experimental set-up and (c) final MAAs and (d) final carotenoid concentrations resulting from the 2006 experimental set-up. treatments (t = 1.44, P = 0.16, n = 12), whereas at 188C the concentration of carotenoids was significantly higher in the PAR+UVA treatment (t = 8.27, P , 0.001, n = 12) (Fig. 4b). In the second series of experiments (2006), the accumulation of PPCs in B. antiqua were both influenced by temperature and radiation (Fig. 4c and d). In the case of the MAAs, the concentration was affected significantly by temperature (F = 8.9, P , 0.001, n = 30) and radiation (F = 539.3, P , 0.001, n = 30). The interaction between main factors was also significant, revealing that the effects of radiation were influenced by the temperature at which the copepods were maintained (F = 23.1, P , 0.001, n = 30). Within each temperature level, the concentration of MAAs was significantly higher in PAR+UVA compared to the dark treatment (P , 0.001). Within the PAR+UVA treatment significant differences were found between most temperatures levels (0.005 , P , 0.02) excluding the pairs 88C and 128C and 168C and 208C. In the dark treatment, however, MAAs concentrations were significantly different at 5 and 208C (t = 3.4, P = 0.003; Fig. 4c). The concentration of carotenoids in B. antiqua was related to temperature (F = 33.1, P , 0.001, n = 30) and radiation (F = 200.9, P , 0.001, n = 30). The interaction between temperature and radiation was also significant (F = 31.3, P , 0.001, n = 30), once again suggesting that the effect of radiation under the influence of temperature (Fig. 4d). The concentration of carotenoids was significantly higher in the PAR+UVA compared with the dark treatment at four out of the five temperature levels tested (P , 0.0001), i.e. except at 58C (P = 0.29). Within the PAR+UVA treatment, the comparisons between carotenoid concentration of B. antiqua at 8 208C, 5 128C and 128 208C were not significant (P . 0.05). Within the dark treatment, the carotenoid levels were similar at most temperatures tested, although a significant difference in the amounts of pigments in B. antiqua was found between incubations at 88C and 208C. The uptake (k), elimination rate (q) and net gain of MAAs and carotenoids calculated from the results of the previous experiments are shown in Table I. The uptake rates of PPCs calculated for both series of experiments were positive (except for one case in which it was near zero) and were usually higher than elimination rates. In the 2004 experiments, however, the elimination rates of carotenoids exceeded the uptake rate. Elimination rates were negative at lower temperatures and positive at the mid and higher temperatures of the range (Table I). Negative values of the elimination rates in complete darkness indicate that there was a basal uptake of MAAs and carotenoids, even in the absence of induction by radiation exposure. The uptake, elimination and net gain rates of MAAs increased linearly with temperature (Fig. 5a). On the other hand, the uptake and elimination rates of carotenoids also increased with temperature; however, the net gain rate was not significantly related to this variable (Fig. 5b). D I S C U S S I O N A N D C O N C L U S I O N S Laguna Los Juncos is a small, shallow lake located in the cold temperate region of Northern Patagonia. The Bioaccumulation rates MAAs (day21) Carotenoids (day21) k (uptake), q (elimination) and k2q (net gain). Fig. 5. Relationship between B. antiquas PPCs bioaccumulation rates (uptake, elimination and net gain) and temperature estimated for the 2006 bioaccumulation experiment: (a) MAAs rates versus temperature and (b) carotenoids rates versus temperature. lake is subject to large seasonal variations in temperature and fluences (Fig. 2a). In addition, it is affected by the hydrological cycle resulting in appreciable fluctuations of water depth. During our field survey, the concentrations of MAAs in B. antiqua did not show a distinct seasonal pattern (Fig. 2a) and no significant relationship could be established between the levels of these compounds, water temperature and mean fluence in the water column. Also, the MAAs content of B. antiqua could not be related to the biosestonic MAAs, the potential source in Laguna Los Juncos, since they were undetectable by means of the spectrophotometric method applied. On the other hand, carotenoid concentrations tended to be higher in spring and lower by late summer, but this trend displayed a great deal of variability within short periods of time (Fig. 2b). Nevertheless, the concentration of carotenoids did, however, show significant negative relationships with temperature and mean fluence. Similar inverse relationships between carotenoid concentration and temperature have been reported in earlier studies. Based on such evidence, Ringelberg (Ringelberg, 1980) and Byron (Byron, 1982) raised doubts about the actual significance of carotenoids in photoprotection. However, several experimental studies have demonstrated that higher carotenoid concentrations alleviate the potential damage induced by exposure to solar or artificial radiation (Hairston, 1976; Hansson, 2000). On the other hand, although the natural patterns of MAAs concentration with temperature or fluence have been less studied, there is a broader consensus within the scientific community regarding their photoprotective function (Shick and Dunlap, 2002; Sommaruga and Augustin, 2006; Tartarotti and Sommaruga, 2006). Tartarotti and Sommaruga (Tartarotti and Sommaruga, 2006) reported higher levels of MAAs in phytoplankton and also in the copepod Cyclops abyssorum in summer compared to the ice-cover period in an Alpine Lake. These authors found that phytoplanktonic MAAs correlated significantly with the incident solar radiation, the water transparency to UV and also with water temperature. Furthermore, they observed that MAAs content of C. abyssorum was correlated with phytoplanktonic MAAs. From a photoprotection perspective, one may expect that the levels of PPCs would track the variations in radiation exposure experienced by the zooplankton (Moeller et al., 2005; Sommaruga and Augustin, 2006; Tartarotti and Sommaruga, 2006). As mentioned above, this is not always the case. Particularly, in our study, we found no support for a direct relationship between PPCs concentration and mean water column radiation. It has been argued that the efficiency of PPCs may decrease with decreasing temperature or that the efficiency of other mechanisms that alleviate the effects of radiation, such as enzymatic repair, may be lower at lower temperatures. Either alone or in combination, the two previous phenomena could account for the lack of a direct relationship between PPCs concentration and the radiation exposure conditions. We have already pointed out that a logical extension of these arguments must be that the organisms should be less tolerant to UVB radiation at low than at high temperature. Previous studies suggest that this is not always the case (Hairston, 1979b) and in fact, our tolerance experiments demonstrated that the vulnerability of B. antiqua to UVB exposure either increased (2004) with or was unaffected (2006) by temperature. It must be emphasized that the temperature range used in our experiments (58C or 208C) encompasses virtually the whole ecophysiological range of the species (Fig. 2a). In the bioaccumulation experiments, the concentration of carotenoids either decreased (2004) or increased (2006) over time. This may be related to differences in the initial levels of these compounds at the start of the experiments, as demonstrated for yeasts (Libkind et al., 2004), but this possibility is one that remains to be explored in future work with B. antiqua. On the other hand, the concentration of MAAs increased, particularly in the treatment exposed to PAR+UVA, confirming the findings reported by Perez et al. (Perez et al., 2006). These results were unexpected, as the levels of MAAs in the alga C. reinhardii used as food are nil, or at least below HPLC detection limits (Perez et al., 2006). Besides, animals are thought to be unable to de novo synthesize MAAs because they supposedly lack the shikimate pathway. The latter statement, however, should be taken with caution. As Shick and Dunlap (Shick and Dunlap, 2002) has put it the oft-repeated dogma that animals lack this pathway apparently stems from the inability of vertebrates (variously given as animals, vertebrates, fish, mammals and humans in literature accounts) to synthesize essential aromatic amino acids, which they must obtain from their diets [. . .], and is not based on empirical evidence such as failures to detect activities of enzymes of the shikimate pathway or DNA sequences encoding these enzymes. Alternatively, the source of MAAs in B. antiqua may be the various endosymbionts and parasites that often infest free-living copepods (Harris, 1993; Carman and Dobbs, 1997). The latter explanation appears more likely. For example, Yakovleva and Baird (Yakovleva and Baird, 2005) found that accumulation of MAAs in the coral Goniastrea retiformis occurs in the absence of zooxanthellae and other dietary input, but is inhibited when the larvae were treated with the antibiotic rifampicin, indicating a possible contribution of prokaryotes associated with the animal tissue. In fact, our recent experiments (unpublished data) suggest that accumulation of MAAs in B. antiqua is inhibited when the copepods are treated with antibiotics. A similar pattern of uncoupling between diet and MAAs content has been reported in the coral Stylophora pistillata in which the levels of these compounds were related to the symbiotic zooxanthellae Symbiodinium while dietary MAAs were of little quantitative importance (Shick et al., 2005). More recently, Starcevic et al (Starcevic et al., 2008) found molecular evidence establishing horizontal transfer of ancestral genes of the shikimic acid pathway into the sea anemone, Nematostella vectensis, genome from both bacterial and eukaryotic (dinoflagellate) donors. Nevertheless, regardless of the actual origin of the MAAs present in B. antiqua, it seems clear that the irradiance and thermal conditions induced their net accumulation in our experiments, although the effects were less evident in the field survey. In designing the bioaccumulation experiments, we departed from the traditional concept according to which the concentrations of PPCs must be fine-tuned to the recent history of environmental conditions experienced by the organisms. This concept places the accent on PPCs concentrations and implicitly assumes that they are (almost) instantly adjusted to the prevailing environmental conditions. In contrast, we focused on what factors affected the uptake and elimination rates of PPCs. We found that, for both carotenoids and MAAs, the uptake and elimination rates increased with temperature. These findings are in agreement with predictions based on metabolic kinetics (i.e. that metabolic rates increase with temperature), but are neutral as regards to the adaptive significance of PPCs. According to our view, the concentration of PPCs reflects the balance between uptake and elimination over a certain period of time. Both process rates are influenced by environmental conditions, but the actual concentrations do not necessarily correspond with the present requirements of the organisms. However, as a deficiency in PPCs may be disadvantageous to organisms that may be suddenly exposed to harmful levels of solar radiation, we suggest that certain threshold concentration is probably maintained. In other words, we believe that, in the absence of inhibitory factors (i.e. visual hunters), PPCs may be accumulated in excess [i.e. luxury consumption (Sterner and Elser, 2002)] at places or periods in which net accumulation is favored. The negative values of the elimination rates at the lower end of the temperature range assayed (Table I) falsify our initial assumption that uptake is induced only when the copepods are exposed to visible and/or UV radiation. Our results actually show that a certain amount of PPCs uptake can be achieved in the absence of any source of radiation. Moreover, this basal dark-uptake appears to increase as the temperature decreases. This is a very interesting observation that deserves to be explored in more detail. Here, we only want to point out that this observation reconciles a number of seemingly paradoxical observations reporting that copepods display their strongest red coloration right before ice melt in spring or early summer, i.e. after several months of living under conditions of low temperature and dim light (Hansson, 2004; Tartarotti and Sommaruga, 2006). The demonstration of a dual regulation of PPCs by radiation exposure and temperature has significant implications for reconstructing (Leavitt et al., 2003) or predicting the photoprotective responses of aquatic organisms to the past or future climate scenarios. The general trend showing that PPCs concentrations are expected to decrease with temperature tacitly implies that global warming may jeopardize the effectiveness of PPCs to counteract the damaging effects of increased UV exposure due to stratospheric ozone depletion. Moreover, an increase in temperature may, by itself, generate photo-toxicity stress in cold adapted populations, as has been suggested for tropical corals (Lesser and Farrell, 2004; Ferrier-Page`s et al., 2007). AC K N O W L E D G E M E N T S We thank the staff of Estancia San Ramo n for allowing us to work in Laguna Los Juncos. P. Fasoli, A. Busto, G. Perotti, D. Milano, D. An o n Suarez and J. Lancelotti are specially acknowledged for their cooperation in the logistics and field assistance. Funding was provided by ANPCyT (PICT CONICET (PIP 6451) and UNC (B001).


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Patricia Elizabeth García, Alejandra Patricia Pérez, María del Carmen Diéguez, Marcela Andrea Ferraro, Horacio Ernesto Zagarese. Dual control of the levels of photoprotective compounds by ultraviolet radiation and temperature in the freshwater copepod Boeckella antiqua, Journal of Plankton Research, 2008, 817-827, DOI: 10.1093/plankt/fbn041