Vertical Migration and Motility Responses in Three Marine Phytoplankton Species Exposed to Solar Rad

Aug 11, 04:00 AM

Current Headlines: By Richter, Peter R Hader, Donat-P; Goncalves, Rodrigo J; Marcoval, M Alejandra; Et al

ABSTRACT Diurnal vertical migration in the water column and the impact of solar radiation on motility were investigated in three marine phytoplankton species: Tetraselmis suecica, Dunaliella salina and Gymnodinium chlorophorum. Cells were exposed to solar radiation either in ultraviolet radiation (UVR, 280-400 nm) transparent Plexiglas tubes (45 cm length, 10 cm diameter) or in quartz tubes under three radiation treatments: PAB (280700 nm), PA (320-700 nm) and P (400-700 nm). The three species displayed different behavior after exposure to solar radiation. Tetraselmis suecica was insensitive to UVR and under high solar radiation levels, cells accumulated preferentially near the surface. Exposure experiments did not indicate any significant changes in swimming speed nor in the percentage of motile cells after 5 h of exposure. On the other hand, D. salina was sensitive to UV-B displaying a significant decrease in swimming speed and percentage of motile cells after 2-3 h of exposure. Moreover, D. salina cells migrated deep in the water column when irradiance was high. The response of G. chlorophorum was in between that of the other two species tested, with a slight (but significant) decrease in swimming speed and percentage of motile cells in all radiation treatments after 5 h of exposure. While G. chlorophorum cells were more or less homogenously distributed in the water column, a slight (but significant) avoidance response to high radiation was observed at local noon, with cells migrating deep in the water column. Our data clearly indicate that these sub-lethal effects of solar radiation are species-specific and they might have important implications for the aquatic ecosystem.

INTRODUCTION

Solar radiation is the energy source for photosynthesis that drives almost all life forms on Earth. However, it can produce deleterious effects on organisms, especially the short wavelengths of the solar spectrum (i.e. ultraviolet radiation [UVR], 280-400 nm). In fact, the effects of solar UVR on cells, organisms and ecosystems have been the focus of many investigations (1-3), especially since the discovery of the Antarctic ozone "hole" (4-6) that kindled the concern about increased levels of UV-B (280-315 nm) reaching the Earth's surface. Studies have shown that UVR can severely harm aquatic organisms by reducing both photosynthetic and growth rates (7) and damaging the DNA molecule through the formation of cyclobutane pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts (8,9). Other UVR-induced effects such as the formation of reactive oxygen species (10) and damage to proteins (11) have been frequently cited in the literature.

Solar UVR is also known to produce sublethal effects on motile unicellular organisms by causing the loss or reduction of movement (12-17). On the other hand, it triggers avoidance mechanisms that allow organisms to escape towards shaded and thus protected environments. Indeed, avoidance mechanisms seem to be a common strategy against exposure to high radiation levels, as seen in the benthic diatom Gyrosigma balticum which displayed a downward movement (18). In phytoplankton organisms, avoidance can also be achieved by means of circadian rhythms that cause the cells to swim down at noon to depths where radiation intensities are low, as occur in some dinoflagellates (19).

In this study, we investigated the effects of solar radiation on vertical migration and motility of three phytoplankton species - the dinoflagellate Gymnodinium chlorophorum Elbrachter et Schnepf and the chlorophytes Dunaliella salina (Dunal) Teodoresco and Tetraselmis suecica (Kylin) Butcher. The three species have a wide distribution in the World's oceans. While D. salina is found in varied ecosystems (brackish and marine), G. chlorophorum forms dense blooms in coastal areas; T. suecica is a common food source used for aquaculture purposes. In particular, we assessed how solar radiation conditions affected the vertical distribution of cells in the water column, and which portion of the solar spectra was most effective in causing sub-lethal effects (i.e. changes in swimming speed and number of motile cells).

MATERIALS AND METHODS

Organisms and culture conditions. The phytoplankton species used in the experiments were the dinoflagellate G. chlorophorum Elbrachter et Schnepf (mean cell size 5 [mu]m) and the chlorophytes D. salina (Dunal) Teodoresco (mean cell size 11 [mu]m) and T. suecica (Kylin) Butcher (mean cell size 25 [mu]m). Cultures of these species were obtained from the phytoplankton collection at the Estacion de Fotobiologia Playa Union (EFPU), where they have been maintained since 1998 in a growth chamber. Cells were grown in seawater-enriched medium f/2 (20) in an illuminated chamber (Sanyo model ML 350) at 20[degrees]C under a 12 L:12 D photoperiod with an irradiance (photosynthetically active radiation [PAR] 400-700 nm) of 200 W m^sup -2^. When the organisms reached the exponential growth phase they were used for the experimentation as described below. The experiments were carried out at the EFPU (43[degrees]S. 65[degrees]W), Chubut, Argentina, during September and October 2004.

Experimentation. Two types of experiments were conducted to assess the impact of solar radiation on vertical migration and motility in the three phytoplankton species as described below:

1. Vertical migration experiments: Samples were dispensed into duplicate UV-transparent Plexiglas tubes (45 cm length, 10 cm diameter) that were closed at the bottom. The tubes containing the samples were placed inside a tank (3 m diameter, 1.2 m height) filled with water to stabilize the temperature (20 +- 2[degrees]C) and exposed to solar radiation from the surface down to 40 cm depth (height of the water column inside the tubes). The tubes were labeled at 5-cm intervals to enable sampling at defined depths. Each experiment lasted one light-day. and every hour, from early morning (0600-0800 h) to late afternoon (1500-1900 h). aliquots of 2 mL of samples were taken from each depth (i.e. from 0 to 40 cm depth for every 5 cm) with a syringe attached to a long silicone tube. Fresh medium was added very gently to the column (to avoid mixing) to replace the removed liquid. Sampling was always done at the same position in the column to avoid possible effects of lateral cell movement. Cell concentration at each depth was obtained using an image analysis system (see below) immediately after sampling ( < 5 min). In G. chlorophorum experiments, and due to the relatively low number of cells per field, samples were fixed with formaline (final concentration 0.4% formaldehyde) and counted in an inverted microscope using a Sedgwick Rafter chamber.

2. Motility experiments: Cell suspensions were dispensed into 50- mL quartz tubes with tight lids and exposed in duplicates to solar radiation during 5 h (centered on local noon), inside a water bath (just beneath the surface) for temperature control (20 +- 2[degrees]C). The quartz tubes were wrapped with different cut-off filter films to obtain three radiation treatments: (1) P treatment. PAR (400-700-nm)-tubes covered with Ultraphan UV Opak. Difrega. Munich. Germany; (2) PA treatment, PAR + UV-A-(320-700-nm)-tubes covered with Montagefolie, Nr. 10155099, Folex, Dreieich, Germany; and (3) PAB treatment-uncovered tubes, samples exposed to full spectra of solar radiation. The transmission spectra of the filter films are published elsewhere (21). Every 30 min and throughout the incubation period, aliquots of 2 mL of samples were drawn after gentle mixing and immediately measured by image analysis (see below).

Analyses and measurements. The analytical procedure for each determination/measurement was as follows:

1. Absorption characteristics: Before and after the 5-h irradiation time a variable volume of cell suspension (28-50 mL) was collected by centrifugation (15 min. 1750 g) and the pellet was extracted with 7 mL of absolute MeOH (60 min at 45[degrees]C). After extraction, the samples were centrifuged ( 1 5 min, 1750 g) and the supernatant measured with a spectrophotometer (Hewlett Packard model HP-8453E) doing a scan from 250 to 750 nm. Chl a concentration was calculated from the optical density (OD) using the equation of Porra (22).

2. Radiation measurements: Solar radiation was constantly monitored during the whole experimental period using a broadband filter radiometer European Light Dosimeter Network (ELDONET) (Real Time Computers Inc., Mohrendorf, Germany) (23) that is permanently installed on the roof of the EFPU (24). The radiometer has channels for PAR, UV-A (315-400 nm) and UV-B and sensors for surface temperature (25). A submersible ELDONET radiometer (with similar characteristics as to that previously described) was used to obtain information of the underwater radiation field in the incubation tanks/columns. The mean irradiances at the sampling depths were calculated using the incident solar radiation data and the attenuation coefficient.

3. Image analysis: Cell counts, as well as movement analysis were performed using the cell tracking system WinTrack 2000 (Real Time Computers Inc., Mohrendorf, Germany) (26). The system uses a video A/ D flash converter (Meteor, Matrox, Canada) in a PCI slot of an IBM- compatible computer which digitizes the analog video images from a CCD camera mounted on an inverted microscope (Leica DM IL). The digitized images are transferred to the computer memory where objects are detected by brightness differences between cells and background. With the "count module" of the program it is possible to count all objects in the field of vision, which fulfill user defined criteria (e.g. size and velocity). Before measurements the field of view is calibrated with an object micrometer so that the software can directly calculate physical values (cell size, cells per volume) from the obtained data. For determination of the cell density a cuvette with defined depth (200 [mu]m) was used. At least 10 fields of view were counted for each sample. Movement analysis was performed with the "track module" of the software. After calibration of the field of view, the movement vectors of all motile cells on screen were determined by subsequent analysis of five consecutive video frames (movement vectors of the objects from frame I1 to frame 5). The individual cell tracks were pooled together to calculate a variety of parameters, of which in this study only motility and velocity of the cells were taken into account. To exclude the evaluation of nonmotile cells, which were in the size range of the observed cells, the software was set to measure movement only on cells with a speed > 10 [mu]m s^sup -1^. In all experiments, the movement of cells was visually monitored to avoid any mistakes of data acquisition of the obtained cell tracks by the software. Tetraselmis suecica and D. salina were monitored with a 10 x objective, while for the smaller G. chlorophorum a 20 x objective was used.

4. Statistics: All experiments were done twice, with duplicate samples in each treatment. Results from all experimentation days were pooled and data are shown together (with no date distinction) as mean and standard deviation. An ANOVA test was used to determine significant differences between each radiation treatment with a confidence level of 95%. Samples were counted a variable number of times/fields depending on the measurement done. When best fitted curves are presented (i.e. proportion of cells as a function of irradiance, Fig. 3) the probability, R^sup 2^ and P-values are given in the text.

RESULTS

Vertical migration experiments

The density of cells (as percentage of the total cells in each column) of the three cultures used in our experiments as a function of time and depths are shown in Fig. 1 . The mean cell density of G. chlorophorum in the columns was 7 x 10^sup 7^ cells L^sup -1^, whereas the concentration for D. salina and T. suecica were 3.8 x 10^sup 8^ and 2.5 x 10^sup 8^ cells L^sup -1^, respectively. The mean daily doses during the experiments were, 5.9 MJ m^sup -2^ (SD = 1.8); 895 kJ m^sup -2^ (SD = 262); and 21.4 kJ m^sup -2^ (SD = 6.1) for PAR, UV-A and UV-B, respectively. The mean attenuation coefficients in the water column were 1.9, 3.4 and 7.2 m^sup -1^ for PAR. UV-A and UV-B, respectively. In G. chlorophorum experiments (Fig. 1A) cells were more or less homogenously distributed in the 40 cm water column, although there were some variations throughout the day, with cells migrating to the deeper part of the column at or close to noontime. Dunaliella salina (Fig. 1B) also migrated downward throughout the day and a relatively few cells were observed in the upper 20-25 cm of the water column. A complete opposite behavior was observed in T. suecica (Fig. 1C) that moved upwards in the water column so that most of the cells were found at the surface.

We used two extreme exposure irradiances (i.e. at the surface and at depth [40 cm]) to study in detail the variations of cell distribution as a function of time during the daily cycle (Fig. 2). A relatively high proportion of G. chlorophorum cells were found at the surface (Fig. 2A) during the morning (13%) and afternoon (12%) but the concentration decreased significantly (P < 0.05) towards noon, to reach 5% of the total cell in the column. Cell concentration at 40 cm was significantly higher than that at the surface from 1100 to 1300 h, reaching maximum values of 17%. The proportion of D. salina cells at the surface (Fig. 2B) was similar throughout the day, varying between 5% to 10% of the total cell numbers, with a slight but significant (P < 0.05) decrease around local noon when compared with early morning or late afternoon. There was a clear pattern of cells migrating downwards from 0800 h, reaching maximum values (> 40% in the deepest layer) at noon-early afternoon, and starting to migrate upwards again after 1600 h, as seen by the decrease in the percentage of cells at 40 cm. In T. suecica experiments (Fig. 2C) there were no significant differences in the concentration of cells at the surface and at 40 cm during early morning (up to 0900 h) and in the early evening (1800h). However, there were significant differences (P < 0.05) throughout the rest of the day with cells migrating to the surface layer reaching a maximum concentration of 35% of the total cells at 1400 h.

Figure 1. Surface plots of the percentage of cells (relative to the total) as a function of the time of the day and depth in the water column. (A) Gymnodinium chlorophorum, (B) Dunaliella salina and (C) Tetraselmis suecica.

Figure 2. Mean percentage of cells (relative to the total) at the surface and at the bottom of the Plexiglas columns as a function of the time of the day. (A) Gymnodinium chlorophorum, (B) Dunaliella salina and (C) Tetraselmis suecica. Open symbols indicate cells at the surface while solid symbols indicate cells at the bottom. Error bars show the standard deviation.

The relative cell concentration (as percentage of the total cells in the column) determined at all depths and time, was plotted against the mean PAR irradiance received in situ (calculated from the incident radiation and the attenuation coefficient) during the hour preceding sampling (Fig. 3). As expected, there were significant differences among species at different irradiances (P < 0.05). G. chlorophorum had a rather similar distribution at all irradiances (Fig. 3A) although a slight (R^sup 2^ = 0.17) but significant decrease in cell concentration (P < 0.05) was determined towards high irradiances. On the other hand, D. salina had high concentrations (~45%) at low irradiances (<25 W m^sup -2^) but decreased to < 15% at irradiances > 50 W m^sup -2^ (Fig. 3B). Finally, the proportion of T. suecica cells was relatively low, 5- 15% at irradiances < 120 W m^sup -2^, but afterwards it increased significantly (R^sup 2^ = 0.49, P < 0.01).

Figure 3. Mean percentage of cells (relative to the total) at different depths in the water column as a function of the PAR irradiance (in W m^sup -2^). (A) Gymnodinium chlorophorum, (B) Dunaliella salina and (C) Tetraselmis suecica. The solid lines indicate the best fit while the broken lines indicate the 95% confidence limits.

Motility experiments

In the experimental set up used for this investigation (see Materials and Methods section), with the tubes containing the samples at the water surface, cells were unable to migrate vertically and thus they were exposed to maximum radiation levels. Mean irradiance values during motility experiments were 275 (SD = 39), 41.1 (SD = 6.5) and 1.1 (SD = 0.26) W m^sup -2^, for PAR, UV-A and UV-B, respectively. It was seen that swimming speed was differentially affected in the three species tested (Fig. 4). Gymnodinium chlorophorum had a general decrease in swimming speed in all radiation treatments (Fig. 4A), from its initial value of 18 [mu]m s^sup -1^ to significantly (P < 0.05) lower values of ~13 [mu]m s^sup -1^ (PAB treatment) or 15 [mu]m s^sup -1^ (P treatment); samples under the PA treatment had intermediate values. In D. salina (Fig. 4B) no significant changes were determined for the PA and P treatments when compared with the initial value of 42 [mu]m s^sup - 1^; however, cells in the PAB treatment were significantly inhibited (P < 0.05) after 3 h of exposure and reduced their swimming speed to 26 [mu]m s^sup -1^ at the end of the experiment. Finally, swimming speed of T. suecica (Fig. 4C) was not significantly affected at all in any of the radiation treatments and remained close to the initial value of 55 [mu]m s^sup -1^.

Figure 4. Mean swimming speed (in [mu]m s^sup -1^) of the three species tested at maximum irradiance (i.e. at the surface) as a function of exposure time. (A) Gymnodinium chlorophorum, (B) Dunaliella salina and (C) Tetraselmis suecica. Error bars show the standard deviation. Note the different scales in each panel.

Similar to the results obtained for swimming speed, the percentage of motile cells varied throughout the duration of the experiment (Fig. 5). In G. chlorophorum (Fig. 5A) the maximum percentage of motile cells was 57% at the start of the experiments but this value decreased significantly (P < 0.05) after 5 h of exposure, with the lowest values determined in the PAB (42%) and the highest in the P treatment (49%). Samples under the PA treatment had intermediate values. In D. salina (Fig. 5B) the percentage of motile cells was very high, close to 100%, but it decreased significantly in the PAB treatment (P < 0.05) after 3.5 h of exposure, reaching a value as low as 61% at the end of the experiment. On the other hand, the percentage of motile cells under the P and PA treatments remained similar to those determined at the beginning of the experiment. Finally, no significant changes were observed in T. suecica exposed to different radiation treatments and the mean percentage of motile cells varied between 84% and 94% (Fig. 5C).

Figure 5. Mean percentage of motile cells (relative to the total) as a function of exposure time. (A) Gymnodinium chlorophorum, (B) Dunaliella salina and (C) Tetraselmis suecica. Error bars show the standard deviation.

The absorption spectra of the methanol extract of the cultures at the beginning and after 5 h of exposure to solar radiation are shown in Fig. 6. There were differences in the OD among species as well as within radiation treatments. In G. chlorophorum, the lowest absorption at all wavelengths was determined in the PAB treatment whereas the highest was in the P treatment; OD under the PA treatment presented intermediate values (Fig. 6A). In D. salina (Fig. 6B), a significant reduction in the absorption was determined in samples under the PAB treatment, when compared with those under PA and P that had similar values. On the contrary, samples of T. suecica (Fig. 6C) under the PAB treatment had significantly higher absorption than those under P, whereas cells under the PA treatment had intermediate values. DISCUSSION

Phytoplankton are important primary producers that contribute a large share of carbon uptake on Earth (27,28) and constitute the most important basis of the food webs in aquatic ecosystems. Due to the depletion of the ozone layer caused by anthropogenic chlorofluorocarbons (CFCs) (29) the ambient level of UV-B reaching the Earth's surface has been increasing in recent years, predominantly in the polar regions (30,31) and this has motivated an increasing number of investigations devoted to evaluate the impact of UVR on living organisms. Nowadays, extensive literature reports many UVR-induced effects on phytoplankton organisms (3) but relatively few studies have been devoted to evaluate the effects of solar radiation on diverse motility parameters of these autotrophs (32,33). Here, we specifically determined how solar radiation conditions affected the vertical distribution of cells in the water column, and which portion of the solar spectra was most effective in causing changes in swimming speed and number of motile cells. Our study was conducted during spring, time of maximum ozone depletion over Patagonia (24). Maximum solar irradiances, however, are attained later during the summer period. In our study, we found variable responses depending on the species tested; for vertical distribution. no pronounced response was observed in the small- sized G. chlorophorum whereas a "negative" response to solar radiation was found in D. salina. On the other hand, a "positive" response to solar radiation was determined in T. suecica.

Figure 6. Absorption spectra of methanol extracts of the three species tested at the start (broken line) and after 5 h of exposure to solar radiation under PAB (squares), PA (circles) and P (triangles) treatments. (A) Gymnodinium chlorophorum. (B) Dunaliella salina and (C) Tetraselmis suecica. Error bars show the standard deviation drawn at 20 nm intervals.

Ultraviolet radiation can have a great impact on organisms exposed to solar radiation (34,35); however, they have evolved different ways to counteract the damage produced. In order to avoid or minimize the deleterious radiation it first has to be detected by the exposed organism. Many algal photoreceptors described so far are not sensitive in the UVR range (36) and thus can not be used to assess a response to enhanced UVR. However, studies conducted with Euglena gracilis (37,38) found a receptor with absorption in the blue and UVR region (with a strong peak at 280 nm) due to compounds such as flavins and pterins. Chlamydomonas and other green algae use the rhodopsin-type chlamyopsins for light perception in the blue- green region (39). These algae would not detect an increase of UV-B that might not he accompanied by a PAR increment (e.g. one effect of the ozone-depleted atmosphere). In any case, once radiation has been detected, vertical migration is one of the mechanisms which enable cells to escape by moving to deeper and more-protected positions in the water column. For example, in the natural environment, migrations of several meters have been found in phytoplankton during the course of a day (40). In this study, and due to the limited length of our columns (i.e. 40 cm), cells were prevented from migrating deeper to escape the still relatively high irradiances (at least for PAR and UV-A) that were 47%, 25% and 5.6% of the surface irradiance for PAR, UV-A and UV-B, respectively. However, the water column height was enough to detect clear patterns for each species.

Under natural conditions D. salina thrives in habitats exposed to high solar radiation, probably due to the fact that the species is capable of synthesizing high amounts of carotenoids to protect themselves from high solar radiation (41-43). In our experiments, however, carotenoids in the cells exposed to PAB and PA did not differ from their initial concentration (Fig. 6B) while the overall spectra decreased in cells exposed to PAB. It should be noted, however, that in our experiments the cells were already preacclimated to relatively high irradiances (200 W m^sup -2^) of PAR (but not UVR). Therefore, part of the observed response might be due to the preacclimated condition of the cells. Carotenoids might have provided only partial protection that was not enough to cope with the effects of solar radiation on speed (Fig. 4B), motility (Fig. 5B) and downward migration of cells (Figs. 1B and 2B). Nevertheless, the decrease in swimming speed and motile cells occurred after 3 h of exposure to full solar radiation, so this might have little impact on the swimming behavior observed in this species throughout the day. While D. salina is only UV-B tolerant when adapted to high light conditions (44), it can be shown from our results that T. suecica might have pronounced scavenging and protection mechanisms even when cultured under moderate radiation conditions. A hint to this could be inferred from the facts that this species shows no changes in swimming speed (Fig. 4C) after exposure, and that cells were distributed in surface waters even during maximal irradiance (i.e. at noon. Figs 1C and 2C). However, we can not rule out that no UV sensing mechanisms were operative. Nevertheless, exposure to solar UVR did not affect motility (Fig. 5C) nor pigment concentration (i.e. no photobleaching was detected. Fig. 6C). It is very likely that the cells were attracted by light and not by other factors such as CO2 because in darkness (early in the morning or late afternoon) they were equally distributed in the water column (Fig. 1C). The mechanisms by which T. suecica cells protect themselves from high irradiances were not investigated here, but probably high levels of antioxidants such as tocopherol might have played an important role. Other studies (43) carried out with this species indicated an increased production of tocopherol at high irradiances. Overall, the resistance of T. suecica as found in this study is in agreement with other investigations that showed that this species is indeed extremely tolerant to UV-B stress (44).

In contrast to the other species mentioned above, solar radiation seems to have relatively little impact on the variables measured in G. chlorophorum, as cells were homogenously distributed in the water column (Figs. 1A and 2A), as well as under different irradiances (Fig. 3A). Nevertheless, a small, but significant impact was observed with cells migrating away from the surface at noon (Fig. 2A). or showing a decrease in swimming speed after 5 h of maximum exposure to solar radiation (Figs. 4A and 5A). It should be noted that in other Gymnodinium species (G. sanguineum) (45) significant amounts of mycosporine-like amino acids (MAA) have been found, which probably served as sunscreens or antioxidants (46-48) and hence provided protection for the cells. The spectra of cells used in this study did not indicate any considerable concentrations of MAA (Fig. 6A). The small cell size of G. chlorophorum (i.e. when compared with that of the chlorophytes investigated here) determines that this species might be more sensitive to UVR (e.g. DNA damage). In fact, it has been demonstrated that in small species the synthesis and accumulation of these UV-absorbing compounds would not be useful for the cells (49). Indeed, in G. chlorophorum, damage to the motility apparatus and/or photoreceptors of the cells might have impaired the ability of the cells to avoid deleterious levels of UV-B by vertical migration, as seen in studies carried out with related species (50).

Overall, our study emphasizes that responses to solar radiation are highly species-dependent. UVR-tolerant species, such as T. suecica might tend to dominate or replace more sensitive organisms in habitats when the increased UVR is the main factor controlling ecological succession. Such changes in the species composition, in turn, might also affect higher trophic levels in the food web (34) leading to a potential change in the dynamics of the ecosystem.

Acknowledgements-We thank the comments and suggestions of two anonymous reviewers and from the Associate Editor of Photochemistry and Photobiology (Dr. P. Neale) that helped to improve our manuscript. We also thank M. Klisch Tor critical discussion and valuable suggestions. The study was partially supported by the DLR (ARG 04/ZO2 to DPH). Secyt-BMBF (AL/PA04-BI/029 to EWH). CONICET (PIP 2005/06-5157 to EWH and VEV) and Fundacion Playa Union (Argentina). This is Contribution N[degrees] 91 of Estacion de Fotobiologia Playa Union.

[dagger] This invited paper is part of the Symposium-in-Print: UV Effects in Aquatic and Terrestrial Environments.

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Peter R. Richter1, Donat-P. Hader1, Rodrigo J. Goncalves2,3, M. Alejandra Marcoval2,3,

Virginia E. Villafane2,3 and E. Walter Helbling*2,3

1 Institut fur Biologie, Friedrich-Alexander-Universitat, Erlangen, Germany

2 Estacion de Fotobiologia Playa Union, Rawson, Chubut, Argentina

3 Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Argentina

Received 28 August 2006; accepted 18 January 2007; DOI: 10.1111/ j.1 751 -1097.2007.00076.x

* Corresponding author email: whelbling@efpu.org.ar (E. Walter Helbling)

(c) 2007 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/07

Copyright American Society for Photobiology Jul/Aug 2007

(c) 2007 Photochemistry and Photobiology. Provided by ProQuest Information and Learning. All rights Reserved.