Phenotypic and molecular responses of copepods to UV radiation stress in a clear versus a glacially turbid lake.

Zooplankton are exposed to multiple environmental stressors in alpine lakes. However, phenotypic and molecular responses of copepods to different environmental conditions, including ultraviolet radiation (UVR), are still not fully understood. Here, we tested whether gene expression patterns vary within the same species, Cyclops abyssorum tatricus, but in populations from different environments (a clear vs. a glacially turbid lake) when exposed to UVR. Moreover, we wanted to examine potential seasonal variation (summer vs. autumn) in copepod gene expression.We measured photoprotective compounds (mycosporine-like amino acids and carotenoids) and antioxidant capacities in two copepod populations and studied gene expression of heat shock proteins (hsps) as indicator of stress after UVR exposure in the laboratory.Compared with the copepod population from the clear lake, the population from the turbid lake showed lower mycosporine-like amino acid, but higher carotenoid concentrations that decreased over the season. Antioxidant capacities (both lipophilic and hydrophilic) were higher in autumn than in summer. The hsp60 and hsp90 genes were constitutively expressed, regardless of habitat origin and season, while hsp70 was upregulated after exposure to UVR (up to 2.8-fold change). We observed stronger upregulation of hsp70 gene expression in autumn for the turbid and summer for the clear lake, with highest gene expression 24 hr post-UVR exposure (up to 10.2-fold change in the turbid and 3.9-fold in the clear lake).We show how variation in phenotypic traits modulates hsp gene expression patterns, specifically hsp70 gene expression. Rapidly induced defences against cellular stress may improve survival in harsh environments such as alpine lakes, especially since these sensitive ecosystems may experience further changes in the future.

The influence of predator community composition on photoprotective traits of copepods.

Trait expression of natural populations often jointly depends on prevailing abiotic environmental conditions and predation risk. Copepods, for example, can vary their expression of compounds that confer protection against ultraviolet radiation (UVR), such as astaxanthin and mycosporine-like amino acids (MAAs), in relation to predation risk. Despite ample evidence that copepods accumulate less astaxanthin in the presence of predators, little is known about how the community composition of planktivorous fish can affect the overall expression of photoprotective compounds. Here, we investigate how the (co-)occurrence of Arctic charr (Salvelinus alpinus) and threespine stickleback (Gasterosteus aculeatus) affects the photoprotective phenotype of the copepod Leptodiaptomus minutus in lake ecosystems in southern Greenland. We found that average astaxanthin and MAA contents were lowest in lakes with stickleback, but we found no evidence that these photoprotective compounds were affected by the presence of charr. Furthermore, variance in astaxanthin among individual copepods was greatest in the presence of stickleback and the astaxanthin content of copepods was negatively correlated with increasing stickleback density. Overall, we show that the presence and density of stickleback jointly affect the content of photoprotective compounds by copepods, illustrating how the community composition of predators in an ecosystem can determine the expression of prey traits that are also influenced by abiotic stressors.

Spatiotemporal dynamics of different CO2 fixation strategies used by prokaryotes in a dimictic lake.

The Calvin-Benson-Bassham (CBB) cycle and the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle are two inorganic carbon assimilation pathways widely used by prokaryotic autotrophs in lakes. We investigated the effect of mixing periods and stable water stratification patterns on the trajectories of both CO2 fixation strategies in a dimictic lake (Piburger See), because information on the spatiotemporal dynamics of prokaryotes using these pathways in freshwater ecosystems is far from complete. Based on a quantitative approach (droplet digital PCR) of genes coding for key enzymes in different CO2 assimilation pathways, nine depths covering the entire water column were investigated on a monthly basis for one year. Our data show that the abundance of photoautotrophs and obligate chemolithoautotrophs preferentially using form IA RubisCO was determined by seasonal variations. Highest numbers were observed in summer, while a strong decline of prokrayotes using RubisCO form IA was measured between December and May, the period where the lake was mostly covered by ice. The spatiotemporal distribution patterns of genes coding for RubisCO form IC genes, an enzyme usually used by facultative autotrophs for CO2 assimilation, were less pronounced. Bacteria harboring RubisCO form II were dominating the oxygen limited hypolimnion, while nitrifying Thaumarchaeota using the HP/HB cycle were of minor importance in the lake. Our data reveal that the seasonal heterogeneity, which is determined by the dimictic thermal regime of the lake, results in pronounced spatiotemporal changes of different CO2 assimilation pathways with depth-dependent environmental parameters as key factors for their distribution.

Contrasting diurnal patterns in antioxidant capacities, but not in expression of stress protein genes among copepod populations from clear versus glacially fed alpine and subalpine lakes.

Short-term changes in environmental conditions largely influence planktonic organisms, but their responses will depend on the habitat characteristics. Here we studied diurnal patterns in antioxidative metabolites (lipophilic and hydrophilic antioxidant capacities) and in the expression of stress protein genes (heat shock proteins, hsp) of copepods to identify short-term stress responses in clear and turbid alpine lakes, as well as in less transparent subalpine ones. Cyclops abyssorum tatricus showed diurnal variation in antioxidant capacities with maxima around noon in clear, but not in glacially fed, turbid lakes. Low fluctuations of these metabolites were also observed in another copepod, Acanthodiaptomus denticornis. Although levels of hsp genes differed between populations living in clear or glacially fed lakes, there was no diurnal rhythmicity in gene expression. Our data show that when planktonic organisms may be at greatest risk of oxidative damage, such as during the daytime in high UV radiation environments, they activate antioxidant responses. Conversely, in less transparent lakes, the physiological response seems to be unnecessary. The difference in gene expression levels suggests an ecological, albeit not acute, role of these genes in copepods experiencing daily environmental fluctuations.

The Impact of UV Radiation on Paramecium Populations from Alpine Lakes.

Paramecium populations from a clear and a glacier-fed turbid alpine lake were exposed to solar simulated ultraviolet (UVR) and photosynthetically active radiation (PAR) at 8 and 15 °C. The ciliates were tested for DNA damage (comet assay), behavioral changes, and mortality after UVR + PAR exposure. High DNA damage levels (~58% tail DNA) and abnormal swimming behavior were observed, although no significant changes in cell numbers were found irrespective of the lake origin (clear, turbid), and temperatures. We conclude that environmental stressors such as UVR and their effects may influence the adaptation of ciliates living in alpine lakes.

Distribution and UV protection strategies of zooplankton in clear and glacier-fed alpine lakes.

Zooplankton, a group of aquatic animals important as trophic link in the food web, are exposed to high levels of UV radiation (UVR) in clear alpine lakes, while in turbid glacier-fed lakes they are more protected. To study the interplay between behavioral and physiological protection responses in zooplankton from those lakes, we sampled six lakes of different UVR transparency and glacial turbidity. Copepods were absent in the upper water layers of the clearest lake, while in glacier-fed lakes they were more evenly distributed in the water column. Across all lakes, the weighted copepod mean depth was strongly related to food resources (chlorophyll a and rotifers), whereas in the fishless lakes, glacial turbidity largely explained the vertical daytime distribution of these organisms. Up to ~11-times (mean 3.5) higher concentrations of photo-protective compounds (mycosporine-like amino acids, MAAs) were found in the copepods from the clear than from the glacier-fed lakes. In contrast to carotenoid concentrations and antioxidant capacities, MAA levels were strongly related to the lake transparency. Copepods from alpine lakes rely on a combination of behavioral and physiological strategies adapted to the change in environmental conditions taking place when lakes shift from glacially turbid to clear conditions, as glacier retreat proceeds.

Ciliate community structure and interactions within the planktonic food web in two alpine lakes of contrasting transparency.

Climate warming is accelerating the retreat of glaciers and recently, many 'new' glacial turbid lakes have been created. In the course of time, the loss of the hydrological connectivity to a glacier causes, however, changes in their water turbidity and turns these ecosystems into clear ones.To understand potential differences in the food-web structure between glacier-fed turbid and clear alpine lakes, we sampled ciliates, phyto-, bacterio- and zooplankton in one clear and one glacial turbid alpine lake, and measured key physicochemical parameters. In particular, we focused on the ciliate community and the potential drivers for their abundance distribution.In both lakes, the zooplankton community was similar and dominated by the copepod Cyclops abyssorum tatricus and rotifers including Polyarthra dolichoptera, Keratella hiemalis, Keratella cochlearis and Notholca squamula. The phytoplankton community structure differed and it was dominated by the planktonic diatom Fragilaria tenera and the cryptophyte alga Plagioselmis nannoplanctica in the glacial turbid lake, while chrysophytes and dinoflagellates were predominant in the clear one.Ciliate abundance and richness were higher in the glacial turbid lake (∼4000-27 800 Ind L-1, up to 29 species) than in the clear lake (∼570-7150 Ind L-1, up to eight species). The dominant species were Balanion planctonicum, Askenasia cf. chlorelligera, Urotricha cf. furcata and Mesodinium cf. acarus. The same species dominated in both lakes, except for Mesodinium cf. acarus and some particle-associated ciliates, which occurred exclusively in the glacial turbid lake. The relative underwater solar irradiance (i.e. percentage of PAR and UVR at depth) significantly explained their abundance distribution pattern, especially in the clear water lake. In the glacial turbid lake, the abundance of the dominating ciliate taxa was mainly explained by the presence of predatory zooplankton.Our results revealed an unexpected high abundance and richness of protists (algae, ciliates) in the glacial turbid lake. This type of lake likely offers more suitable environmental conditions and resource niches for protists than the clear and highly UV transparent lake.

UV-induced DNA damage in Cyclops abyssorum tatricus populations from clear and turbid alpine lakes.

Zooplankton from clear alpine lakes thrive under high levels of solar UV radiation (UVR), but in glacially turbid ones they are more protected from this damaging radiation. Here, we present results from experiments done with Cyclops abyssorum tatricus to assess UV-induced DNA damage and repair processes using the comet assay. Copepods were collected from three alpine lakes of differing UV transparency ranging from clear to glacially turbid, and exposed to artificial UVR. In addition, photoprotection levels [mycosporine-like amino acids (MAAs) and lipophilic antioxidant capacity] were estimated in the test populations. Similar UV-induced DNA damage levels were observed among the copepods from all lakes, but background DNA damage (time zero and dark controls) was lowest in the copepods from the glacially turbid lake, resulting in a higher relative DNA damage accumulation. Most DNA strand breaks were repaired after recovery in the dark. Low MAA concentrations were found in the copepods from the glacially turbid lake, while the highest levels were observed in the population from the most UV transparent lake. However, the highest lipophilic antioxidant capacities were measured in the copepods from the lake with intermediate UV transparency. Photoprotection and the ability to repair DNA damage, and consequently reducing UV-induced damage, are part of the response mechanisms in zooplankton to changes in water transparency caused by glacier retreat.

UV radiation and freshwater zooplankton: damage, protection and recovery.

While many laboratory and field studies show that zooplankton are negatively affected when exposed to high intensities of ultraviolet radiation (UVR), most studies also indicate that zooplankton are well adapted to cope with large variations in their UVR exposure in the pelagic zone of lakes. The response mechanisms of zooplankton are diverse and efficient and may explain the success and richness of freshwater zooplankton in optically variable waters. While no single behavioural or physiological protection mechanism seems to be superior, and while several unexplained and contradictory patterns exist in zooplankton UVR ecology, recent increases in our understanding are consistent with UVR playing an important role for zooplankton. This review examines the variability in freshwater zooplankton responses to UVR, with a focus on crustacean zooplankton (Cladocera and Copepoda). We present an overview of UVR-induced damages, and the protection and recovery mechanisms freshwater zooplankton use when exposed to UVR. We review the current knowledge of UVR impact on freshwater zooplankton at species and community levels, and discuss briefly how global change over the last three decades has influenced the UVR milieu in lakes.

Sublethal stress: Impact of solar UV radiation on protein synthesis in the copepod Acartia tonsa.

Aquatic organisms respond to environmental challenges such as thermal stress with the rapid induction of highly conserved polypeptides known as stress proteins or heat shock proteins (Hsps). Solar ultraviolet radiation (UVR, 280-400 nm) is an important environmental stressor in marine ecosystems. Here, we present results of experiments conducted with the marine copepod Acartia tonsa to follow the de novo protein synthesis and measure the level of constitutive and inducible isoforms of the Hsp70 gene family of stress proteins after UV exposure. Animals were collected from Tampa Bay, Florida (USA), and exposed to solar radiation (full spectrum), UV-A (320-400 nm) and PAR (400-700 nm), or PAR only, for periods of 0.5-4 h. Controls were kept in the dark. Protein synthesis was robust under all treatments when the copepods were exposed to low solar radiation intensities. Conversely, high solar radiation intensities (both UV-B and UV-A) caused an overall suppression in the protein synthesis of the copepods with no detectable induction of stress-inducible isoforms of Hsps. Immunochemical assays (western blotting) showed that UVR increased levels (3.5-4-fold increase compared to the dark control) of the constitutively expressed 70 kDa heat-shock (Hsc70) protein in A. tonsa, without indication of inducible isoform upregulation.

Seasonal and ontogenetic changes of mycosporine-like amino acids in planktonic organisms from an alpine lake.

We studied the quantitative and qualitative mycosporine-like amino acid (MAA) composition in phytoplankton and the copepod Cyclops abyssorum tatricus from an alpine lake over a 15-month period. Up to eight MAAs were identified in the samples, with shinorine being predominant. The MAAs occurred year round and showed a strong seasonal pattern. Compared with ice-cover periods, concentrations during the summer were on average 3.6 and 3.0 times higher in phytoplankton and C. abyssorum tatricus, respectively. During the summer, the contents of MAAs in phytoplankton decreased with depth, suggesting their photoprotective role. Chlorophyll a-specific concentrations of MAAs in phytoplankton correlated significantly with the incident solar radiation and ultraviolet (UV) water transparency (r(2) ≤ 0.36), however, the strongest relationship was found with water temperature (r(2) = 0.67). In zooplankton, highest contents of MAAs were found in eggs, nauplii, and young copepodids, presumably providing a high level of photoprotection for progeny. Proportions of the dominant MAAs in the copepod showed seasonal and ontogenetic variations, which were consistent with relative changes in the predominant MAA, but not other abundant MAAs, in phytoplankton. Considering a time lag of approximately 1 month between the synthesis and subsequent accumulation of these compounds, MAA concentrations in late copepodid to adult life stages were significantly correlated to those in phytoplankton. Annual patterns in MAAs with high concentrations during periods of elevated environmental stress are consistent with the idea that these compounds play an important role in protecting aquatic organisms against UV damage.

Mycosporine-like amino acids in planktonic organisms living under different UV exposure conditions in Patagonian lakes.

Mycosporine-like amino acids (MAAs) were studied in zooplankton from 13 Argentinian lakes covering a broad range in altitude, maximum depth and physico-chemical properties of the water. Four to nine different MAAs (predominantly porphyra-334 and shinorine) were found in the copepods Boeckella gibbosa, B. gracilipes, B. meteoris and Parabroteas sarsi, and in the ciliate Stentor amethystinus, while MAAs were undetectable in the cladoceran Daphnia middendorffiana. Among the different copepods, maximum MAA concentrations accounted for 0.25-1.31% of the dry weight, and contents were generally about three to seven times (up to 43 times) higher in the animals living in the clearest lakes compared to those occurring in low-UV systems. This variability in the content of MAAs was related to the lake altitude (r(2) = 0.71), and the fraction of the water column to which 1% of the surface UV radiation at 320 nm penetrated (r(2) = 0.57). Our data therefore underscore the role of MAAs as sunscreens to decrease the potential negative effects of solar radiation, but they also indicate that other environmental factors besides UV transparency play a role in determining MAA concentrations. One lake was selected to obtain additional information on the qualitative composition of MAAs in seston of <100 µm between two sampling sites and over a 2 month study period (austral summer). Six different MAAs were detected in the samples, with porphyra-334 and palythine being predominant. In the copepods collected simultaneously, there was low variation in MAA concentrations between the two sites and over time. Thus, our results suggest that under similar UV exposure conditions MAA contents of planktonic organisms show low temporal variation.