Sensitivity and acclimation to UV radiation of zoospores from five species of Laminariales from the Arctic
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- Wiencke, C., Clayton, M.N. & Schoenwaelder, M. Marine Biology (2004) 145: 31. doi:10.1007/s00227-004-1307-9
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Spores of five Laminariales from Arctic Spitsbergen were exposed in the laboratory to photosynthetically active radiation (PAR; 400–700 nm), PAR+UVA radiation (UVAR; 320–400 nm) and PAR+UVAR+UVB radiation (UVBR; 280–320 nm). Subsequently, germination was monitored over periods of 3, 6 and 9 days. The investigated species were the upper sublittoral Saccorhiza dermatodea, the upper to mid-sublittoral Alaria esculenta and Laminaria digitata, the mid-sublittoral L. saccharina and the lower sublittoral L. solidungula. The germination capacity decreased sharply after 16 h exposure to PAR+UVAR+UVBR in all species. However, S. dermatodea was able to recover from the damaging effects of UVBR. There was also a small increase in percentage germination of A. esculenta 6–9 days after the treatment. No recovery was evident in the other species. After 8 h exposure to PAR+UVA+UVB, L. digitata recovered completely, and L. saccharina and L. solidungula, partially. The only species susceptible to PAR+UVAR was L. solidungula. One prominent cytological feature of UVR-exposed spores was the enlargement of phenolic vesicles (physodes) (particularly seen in S. dermatodea and A. esculenta), which may have a protective function against UVR. Pilot experiments under natural irradiance conditions indicate that the PAR component of solar radiation exerts an additional stress. Overall the data show that zoospores of the species from the upper sublittoral are less sensitive to UVR or have the capacity to recover from UV stress in contrast to species from deeper waters, probably due to their UV protective and repair capabilities.
Stratospheric ozone depletion is evident not only over Antarctica (Solomon 1999; Staehelin et al. 2001), there is also a great potential for ozone losses over the Arctic (Gathen et al. 1995; Rex et al. 2002). According to the latest predictions (Knudsen et al., unpublished data), ozone losses over the Arctic are likely to increase until 2010 to 2020 and decrease only slightly by 2030. Reductions in the thickness of the ozone column lead to enhanced levels of UVB radiation (UVBR) at the earth’s surface, and shifts the solar spectrum to shorter wavelengths (Roy et al. 1990). UVBR also penetrates into the water. For example, on the Arctic island of Spitsbergen biologically significant UVBR levels are recorded down to 8 m water depth (Hanelt et al. 2001; van de Poll et al. 2002d).
In macroalgae, UVBR causes molecular damage to nucleic acids and proteins and inhibits important metabolic processes (Franklin and Forster 1997). UV exposure can show photoinhibition or even photodamage to photosynthesis (Dring et al. 1996a; Hanelt et al. 1997; Bischof et al. 1998a, 2000). Cyclobuthane-pyrimidine dimers are formed in the DNA, thus blocking DNA and RNA polymerases and consequently inhibiting genome replication and expression (van de Poll et al. 2002a, 2002b). In addition, UVBR exerts indirect effects mediated by oxidative stress (Foyer et al. 1994; Aguilera et al. 2002). On the other hand, there is considerable potential for acclimation to UVBR in the macrothalli of seaweeds. Saccorhiza dermatodea, Alaria esculenta and Laminaria saccharina collected over a depth gradient on Spitsbergen exhibited a different UV sensitivity in relation to photosynthesis, with the most tolerant individuals growing in shallow waters (Bischof et al. 1998b). Maximum quantum yield of photosynthesis of A. esculenta acclimates to enhanced levels of UV radiation (UVR) within a few days (Bischof et al. 1999).
The acclimation potential is based on various repair and protective mechanisms. The D1 protein of photosystem II undergoes a permanent turn-over cycle after radiative damage (Campbell et al. 1998; Máté et al. 1998). DNA damage can be repaired by light-dependent photolyases and light-independent nucleotide excision repair (Pakker et al. 2000a, 2000b; Poll et al. 2002b). Oxidative stress is counteracted by enzymatic defense systems and scavenging by antioxidants (Collen and Pedersen 1996; Aguilera et al. 2002; Dummermuth et al. 2003). UVR-absorbing substances may prevent damage. In red algae, mycosporine-like amino acids were invoked to protect cellular molecules, structures and processes against the damaging effects of UVR (Karsten et al. 1998, 1999; Cockell and Knowland 1999). In brown algae phlorotannin (polyphenolic)-containing vesicles, the so-called physodes, may play a role in chemical UV defense. Phlorotannins strongly absorb in the UVB (and UVC) region of the spectrum and their formation is inducible by UVBR (Pavia et al. 1997; Pavia and Brock 2000; Schoenwaelder 2002a).
The balance between the various negative effects of UVBR and the repair and protective mechanisms is indicated by the integrative parameters growth and reproduction. Growth in species of the eulittoral/upper sublittoral is initially inhibited by UVBR, but can acclimate to a considerable extent (Altamirano et al. 2000; Han et al. 2003). Similarly, growth of Fucus distichus from Spitsbergen is not significantly reduced by UVR, whereas growth of sporophytes of S. dermatodea, L. digitata and L. saccharina shows considerable inhibition after exposure to the full solar spectrum (Aguilera et al. 1999). Also, growth rates of young and mature sporophytes of the upper sublittoral L. digitata from Helgoland are less sensitive to UVR compared to mid-sublittoral L. saccharina and L. hyperborea (Dring et al. 1996b).
The reproductive cells and the early developmental stages of many organisms are more susceptible to UVR when compared to the adult stages (summarized by Coelho et al. 2000). Motility of spores from L. saccharina (Makarov and Voskoboinikov 2001) and phototaxis of swarmers of Scytosiphon lomentaria and Petalonia fascia (Flores-Moya et al. 2002) is reduced by UVBR. Huovinen et al. (2000) pointed out that microtubules might be affected by UVBR as nuclear division and translocation of the nucleus in zoospores of Macrocystis pyrifera are inhibited. In L. digitata from Spitsbergen the loss of zoospore viability is positively correlated to DNA damage and photodamage of the photosynthetic apparatus (Wiencke et al. 2000). Moreover, zoospores of species from the upper shore were generally more susceptible to UVR than species occurring at greater depths (Wiencke et al. 2000). The published data on UV sensitivity of spores of Laminariales from Spitsbergen are so far limited to A. esculenta, L. digitata and L. saccharina.
The present study was conducted to describe the UVR susceptibility of zoospores of five species of Laminariales occurring in the Kongsfjord on the west coast of Spitsbergen, a glacial fjord in the Arctic, in relation to their depth distribution. Saccorhiza dermatodea grows in the uppermost sublittoral, followed by A. esculenta, L. digitata and L. saccharina. The endemic Arctic L. solidungula grows almost exclusively in the lower sublittoral (Hop et al. 2002; Vögele et al., unpublished data). Species from this region might be particularly affected due to the ozone losses over the Arctic and the related increase in UVBR levels (Groß et al. 2001; Dahlback 2002). Moreover, we studied for the first time the ability of these small developmental stages to repair UV-induced damage and also to examine the protective potential of phlorotannin containing physodes formed during and after exposure to artificial UVR in the laboratory. Additionally, we conducted for the first time a pilot study on the performance of spores under natural irradiance conditions.
Materials and methods
Fertile specimens of Saccorhiza dermatodea, Alaria esculenta, Laminaria digitata, L. saccharina and L. solidungula were collected in August 2002 by SCUBA diving in Kongsfjorden close to Ny Ålesund (78°55′N, 11°56′E) on the west coast of Spitsbergen. An overview on the physical environment and the ecosystem of the Kongsfjord was given by Svendsen et al. (2002) and Hop et al. (2002). Thallus parts with sori were blotted with tissue paper and kept overnight or for a few days in a wet chamber in dim light at a temperature of 5–7°C. Spores were released by flooding the tissue with filtered seawater in a Petri-dish. The initial spore concentration was determined by use of a Thoma chamber (Brand, Germany) and was usually in the range between 30,000 and 80,000 spores ml−1. For the experiments two to four drops from these suspensions were put into Petri-dishes 5 cm in diameter containing 11 ml of Provasoli-enriched seawater (Starr and Zeikus 1987). In this way the density of spores per unit area was similar in all experiments.
Spores obtained from five individual sporophytes of A. esculenta, L. digitata, and L. saccharina were separately exposed for 8 or 16 h to artificial UVR. In S. dermatodea spores came from two individual sporophytes and were mixed before UVR exposure; in L. solidungula only one fertile sporophyte was available. UVR was generated by UVA-340 fluorescent tubes (Q-Panel, Cleveland, Ohio, USA), emitting a radiation similar to the solar spectrum in the wavelength range below 340 nm. Photosynthetically active radiation (PAR) was additionally provided by daylight fluorescent tubes (Osram Lumilux Deluxe, L36 W/12–950). UV measurements were performed using a Solar Light PMA 2100 broadband radiometer equipped with the UVA sensor PMA 2110 and the UVB sensor PMA 2106 (Solar Light, Philadelphia, Pa., USA). As the spectral range of the UVA sensor extends into the UVB region of the spectrum and vice versa, UVA measurements were taken below a Schott WG 320 filter (Schott, Mainz, Germany). UVBR was determined after subtraction of measured irradiance under the filter from the irradiance without the filter. PAR was measured with a Li-Cor LI 1000 data logger (Li-Cor, Lincoln, Neb., USA) equipped with a LI-190 SA cosine corrected flat-head sensor. The irradiances applied were: 28.8±5.05 µmol photons m−2 s−1 PAR, 8.22±0.64 W m−2 UVAR and 1.27±0.12 W m−2 UVBR. To study the effect of different wavelength ranges, the Petri dishes were covered with three different cut-off filters: (1) Ultraphan URT 300 foil (Digefra, Munich, Germany) for exposure to the full spectrum, (2) Folex PR Montage Folie (Dr. Schleussner, Dreieich, Germany) for exposure to PAR and UVAR and (3) Ultraphan URUV farblos (Digefra) for exposure to PAR. The spectra under these radiation regimes correspond to those used by Wiencke et al. (2000). After exposure the spore suspensions were put under dim white daylight at a temperature of 10°C for 3, 6 and 9 days to monitor germination rates. Germination was determined microscopically by use of an Axioplan microscope (Zeiss, Göttingen, Germany) equipped with a 25×seawater immersion objective. A spore was classified as germinated if at least a germ-tube was formed. We did not differentiate between dead and living, but germinated and not germinated spores. In each sample about 300 spores were counted and the percentage of germinated and non-germinated spores determined. Dead spores and their remains were clearly visible within the time periods under study.
For photography, cover slips were placed in extra Petri dishes and spore suspensions were added. These dishes were then treated in the same way as the others. At various times cover slips with spores/germlings were taken out of the dish and used for preparation of microscopic slides. Microscopy of the various developmental stages was performed using oil immersion objectives mounted on the Axioplan microscope. Micrographs were taken digitally using a Nikon Coolpix camera.
For the outdoor experiments, spore suspensions of A. esculenta, L. saccharina, L. digitata and L. solidungula were exposed in Petri dishes to surface solar radiation outside the laboratory. Temperatures varied in these experiments between about 5 and 15°C. To estimate the effect of UVR, some of the Petri dishes with spores of A. esculenta were covered with a Schott GG 400 filter (Schott, Mainz Germany). All other dishes were covered with Schott WG 280 filters to stop possible evaporation and salinity changes in the medium. UVBR was measured in the field by use of a spectroradiometer with a 32-channel photon counting detector equipped with a 2 π diffusor (Hanken and Tüg 2002). UVAR and PAR were measured using a spectroradiometer with a 256-channel photodiode array detector and a cosine diffusor (ISITEC Bremerhaven, Germany).
Germination rates were similar or only slightly lower after 8- and 16-h exposures to UVAR in addition to PAR and 3 days post-culture in S. dermatodea, A. esculenta L. digitata and L. saccharina (Figs. 1, 2). In contrast, in L. solidungula, germination rates were strongly reduced in most cases after the UVAR+PAR exposure. Even after 6 days of post-culture germination rates of only 20–30% were recorded in the UVAR+PAR experiment compared to 55% and 70% in the PAR condition (Fig. 1, 2).
Additional exposure to UVBR for 8 h resulted in a similar pattern in L. solidungula for the two post-culture times tested. However, after the 16-h exposure to the full spectrum almost no germination was found after 3 and also after 6 days of post-culture (Fig. 2). Exposure to the full spectrum had a similar effect in L. saccharina and L. digitata. Only an extremely low germination rate was recorded after the 16-h exposure to the full spectrum under all three post-culture times (Fig. 2). In the 8-h experiment, however, there was only a small difference in germination rates under PAR+UVAR+UVBR compared with PAR+UVAR and a post-culture time of 3 days (Fig. 1). After 6 days of post-culture the germination rate increased, indicating a considerable recovery in the spores, which did not germinate within the first 3 days of post-culture (Fig. 1).
Alaria esculenta and S. dermatodea were the species most tolerant to UVBR. After a 16-h exposure to the full spectrum and 3 days of post-culture a germination rate of 21% and 40% was recorded, respectively (Fig. 2). Additionally there was a great potential for recovery, as germination rates increased slightly to 38% after 9 days of post-culture in A. esculenta. In S. dermatodea values measured after 6 days of post-culture were similar to those obtained after exposure to PAR and PAR+UVAR (Fig. 2). In the 8-h experiment there were no differences in germination rates between the three conditions in either species (Fig. 1).
Germination of spores (means±SD; n=5) of Laminariales 6–7 days after exposure to solar radiation for the time periods given. Photosynthetically active radiation (PAR) and UVR doses taken from Table 2. In Alaria, germination was additionally recorded after UV exclusion
GG400 (solar radiation, UV-depleted)
WG 280 (full solar radiation)
WG 280 (full solar radiation)
WG 280 (full solar radiation)
WG 280 (full solar radiation)
Comparison between PAR and UVR doses for the dates of the outdoor experiments in Table 1 and those from the laboratory experiments. The field PAR doses were calculated by an extrapolation of the UVA region using a standard atmospheric model. Values for the experiment with L. solidungula on 13 August 2002 were divided by 2 as we used shade-cloth excluding 50% of the incident radiation in this particular experiment
Dates and/or times
Field experiments (2002)
The most important results of this study are, firstly, that spores of the species growing in shallow water, especially Saccorhiza dermatodea, are much more tolerant to UVR than species from the mid-to lower sublittoral, such as Laminaria solidungula. Secondly, there is a considerable recovery from UVR-induced damage during post-culture without UVR. Thirdly, zoospores contain several large physodes after UV exposure that probably contribute to protecting the spores against UVR-induced damage due to their content of UV-absorbing phlorotannins.
The differential sensitivity to UVAR and UVBR of brown algal zoospores from species inhabiting different water depths has been demonstrated for L. saccharina, L. digitata, Alaria esculenta and for Chordaria flagelliformis from Spitsbergen and for various Laminariales from southern Spain (Wiencke et al. 2000). Similar data were also obtained by Lüning (1980) on the upper sublittoral L. digitata, L. saccharina and the mid-sublittoral L. hyperborea from Helgoland, results not seen by Dring et al. (1996b) on the same species. The data presented here clearly demonstrate this relationship between UV sensitivity and depth distribution. S. dermatodea occurs in the upper sublittoral of the Kongsfjord, and is common at depths between 1.5 and 5.5 m. It is the most tolerant species against UVR. A. esculenta and L. digitata are common at depths between 1.5 and 12.5 m (Vögele et al., unpublished data; Hop et al. 2002). These species and L. saccharina exhibit a strongly reduced UVR tolerance compared to S. dermatodea. L. saccharina grows predominantly between 1.5 and 15.5 m. The upper distribution limit of S. dermatodea, A. esculenta and L. digitata is 0.5 m, that of L. saccharina 1.5 m. The species most susceptible to UVR is the endemic Arctic deep water alga L. solidungula. This species occurs at depths between 10.5 and 15.5 m in the middle zone of the Kongsfjord. These differences in the sensitivity of brown algal spores against UVR are comparable to the differential UVR tolerance of photosynthesis in macrothalli of species growing at different water depths as shown, for example, by Dring et al. (1996a), Hanelt et al. (1997), Bischof et al. (1998a) and Karsten et al. (2001). In all these studies, photosynthesis in species from shallow waters was much more UVR tolerant than species from deeper waters.
The reasons for the UVR-induced spore mortality are, among others, damage to the photosynthetic apparatus, to the DNA and, possibly, to the microtubules. The latter possibility was invoked by Huovinen et al. (2000) for UVR-exposed zoospores of Macrocystis pyrifera. Damage of the photosynthetic apparatus was demonstrated in zoospores of L. digitata from Spitsbergen (Wiencke et al. 2000). Interspecific differences in DNA damage were found after exposure to the full spectrum in L. digitata, L. saccharina and A. esculenta from Spitsbergen (Wiencke et al. 2000). Whereas cyclobuthane-pyrimidine dimer formation as an indicator of DNA damage was greatly enhanced after exposure to the full spectrum in the first two species, in A. esculenta from shallower waters it was negligible and comparable to the controls only exposed to PAR. In a detailed experiment with L. digitata spores the formation of cyclobuthane-pyrimidine dimers was positively correlated with the UVB dose (Wiencke et al. 2000).
The recovery from UVR-induced damage has been shown here for the first time for spores during post-culture without UVR by the increase of germination rates after 6 and 9 days in S. dermatodea and A. esculenta. Undoubtedly, this recovery must be the result of repair processes. Repair of the D1 protein in the reaction center of photosystem II and UVBR-induced differential transcription of psbA genes encoding the D1 protein has been demonstrated by Campbell et al. (1998) and Máté et al. (1998). Repair of UVR-induced damage of the DNA has been shown recently for macrothalli of a number of tropical, temperate and Arctic marine macroalgae (Pakker et al 2000b; van de Poll et al. 2002a, 2002b, 2002c, 2002d). Although no data are available on DNA repair processes in the unicellular propagules of marine algae, it is reasonable to assume that the repair mechanisms would be operating in these developmental stages.
The increase in the number and size of the physodes in the UVR-exposed zoospores, especially in S. dermatodea, A. esculenta, but also in L. digitata and L. saccharina, is described here for the first time and makes quantitative studies pressing. It is regarded as a protective reaction against UVR. Physodes contain UVR-absorbing phlorotannins (Ragan and Glombitza 1986; Schoenwaelder 2002a) and represent, therefore, an effective protective mechanism. In support of this explanation, Peckol et al. (1996) found higher contents of phlorotannins in Fucus vesiculosus thalli collected in the high intertidal compared to a population from the lower intertidal. Highest concentrations occurred in early summer and were lowest in winter. In Homosira banksii, phlorotannins are released from physodes in the sun-exposed cortical cells and form a protective layer for the underlying tissue (Schoenwaelder 2002b). The most striking support for this hypothesis is the induction of phlorotannin formation after UVR exposure in Ascophyllum nodosum (Pavia et al. 1997). Overall, it is reasonable to claim that physodes in the zoospores of the Laminariales studied here absorb UVR and protect the protoplasm of the three species occurring in shallow water (S. dermatodea, Alaria esculenta, L. digitata) against the damaging effects of UVR, a hypothesis for which we will provide additional evidence in an accompanying paper (Clayton et al. 2004). The observed change in color of the physodes in S. dermatodea spores after 3 days is presumably due to changes in the phlorotannins, the significance of which is not clear.
One major limitation of laboratory experiments is the generally low level of PAR compared to the high light conditions in the field. This is also the case in our laboratory experiments. For example, spores of A. esculenta exposed to solar radiation depleted of UVR showed a germination rate of only 42% (Table 1) compared to 96% (Fig. 1) in the PAR treatment in the laboratory (8 h exposure, 6 days post culture ) with about 10 times lower PAR doses (Table 2). However, additional UVR lowered the germination rate in the outdoor experiment to only 3% (Table 1). In L. saccharina, there is no difference at all between laboratory and outdoor experiments: Under similar UVAR conditions and the dissimilar PAR+UVBR conditions outdoors and in the laboratory (16 h exposure, 6 days post culture), there was no germination at all (Tables 1, 2, Fig. 2). The germination rate of L. digitata under natural irradiance conditions seems to be considerably depressed to only 5% (Table 1) compared to a germination rate of 92% under the 16 times lower PAR dose and a 5 times higher UVBR dose in the laboratory (8 h exposure, 6 days post culture; Fig. 1, Table 2). In L. solidungula similar germination rates were obtained in the 8-h exposure and 6-days post-culture laboratory experiment and outdoors under about 5 times higher PAR doses and about 3 times lower UVR doses during the outdoor experiment (Tables 1, 2; Fig. 1). Beside the differences in PAR between field and laboratory, another limitation of laboratory experiments are the usually much higher UVBR doses, which in our experiments were about 10 times higher than in the field (Table 2). Clearly, more data are necessary to characterize better the performance of algae in the field and experiments at different water depths with close monitoring of the radiation conditions are indispensable. According to the presently available data biologically effective UVBR goes down to about 8 m in the Kongsfjord and exhibits a considerable seasonal variation due to strong changes in water transparency (Hanelt et al. 2001; van de Poll et al. 2002d). To estimate the ecological effect of enhanced UVBR the seasonal changes of UVR stress have to be put in the context of the timing of spore release in the various species and the patterns of spore dispersal.
The detection of recovery from UVR damage and the unexpected acclimation potential of the zoospores from shallow water species especially in S. dermatodea and A. esculenta diversifies our scenario about the ecological implications of the UVR susceptibility of spores. Certainly, spores are the life-history stages most sensitive to UVR (Dring et al. 1996b; Wiencke et al. 2000). But according to our results it will be necessary to test the acclimation potential to UVR, which includes a detailed study of the UVR-protective and repair mechanisms.
This work was performed at the Ny Ålesund International Research and Monitoring Facility on Spitsbergen (Svalbard). The authors are grateful to M. Schwanitz, C. Daniel, M. Assmann, H. Wessels and H. Schmidt for providing samples by SCUBA diving, as well as to the staff at Koldewey Station, especially H. Pötschick. The experiments comply with the current laws of Germany and Norway.