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Chlorophyll fluorescence imaging analysis of the responses of Antarctic bottom-ice algae to light and salinity during melting
Journal of Experimental Marine Biology and Ecology 399 (2011) 156–161
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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
Chlorophyll fluorescence imaging analysis of the responses of Antarctic bottom-ice algae to light and salinity during melting K.G. Ryan a,⁎, M.L. Tay a, A. Martin a,b, A. McMinn b, S.K. Davy a a b
School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand Institute of Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania, Australia
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Article history: Received 7 July 2010 Received in revised form 5 January 2011 Accepted 11 January 2011 Keywords: Algae Antarctic Chlorophyll fluorescence Imaging PAM Salinity Sea ice
a b s t r a c t Bottom-ice algae within Antarctic sea ice were examined using chlorophyll fluorescence imaging. The detailed structure of the bottom-ice algal community growing in the platelet and congelation layers of solid pieces of sea ice was evident for the first time in chlorophyll imaging mode. Strands of fluorescence representing algal cells were clearly visible growing upward into brine channels in a fine network. Images of effective quantum yield (ФPSII) revealed that the ФPSII of algae embedded in the sea ice was approximately 0.5. Furthermore, ФPSII decreased slightly with distance from the ice–water interface. The response of Antarctic sea ice algae to changes in irradiance and salinity, and the effects of slowly warming and melting the ice block sample were examined using this system. The ФPSII of bottom-ice algae decreased as irradiance increased and salinities decreased. Bottom-ice algae appear to be most vulnerable to changes in their environment during the melting process of the ice, and this suggests that algae from this region of the ice may not be able to cope with the stress of melting during summer. Chlorophyll fluorescence imaging provides unprecedented imagery of chlorophyll distribution in sea ice and allows measurement of the responses of sea ice algae to environmental stresses with minimal disruption to their physical habitat. The results obtained with this method are comparable to those obtained with algae that have been melted into liquid culture and this indicates that previous melting protocols reveal meaningful data. In this chlorophyll imaging study, rapid light curves did not saturate and this may prevent further use of this configuration. © 2011 Elsevier B.V. All rights reserved.
1. Introduction At its greatest extent, Antarctic sea-ice covers 19 million km2 (Arrigo and Thomas, 2004). The phytoplankton, protists and bacteria growing within sea-ice exert a strong influence in the Antarctic marine environment, and the bottom or interstitial, communities can reach biomass levels of over 300 mg Chlorophyll-α m−2 in the austral summer (Palmisano and Sullivan, 1983; Kirst and Wiencke, 1995). Their contribution to the primary production of the area is substantial (McMinn et al., 2000) and the overall contribution of ice algae to total primary production in ice-covered regions of the Southern Ocean is estimated at ~25% (Arrigo et al., 1997). The physicochemical conditions for the sea ice microbial community (SIMCO) are highly variable. There are strong gradients of light, temperature, salinity, and nutrient concentration within the ice column (Arrigo and Sullivan, 1992) and while microbes are found throughout the ice profile, bottom-ice communities are dominant in fast ice around most of Antarctica (Palmisano and Sullivan, 1983;
⁎ Corresponding author. Tel.: + 64 4 463 6083; fax: + 64 4 463 5331. E-mail address: [email protected] (K.G. Ryan). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.01.006
McMinn and Ashworth, 1998; Trenerry et al., 2002; Fiala et al., 2006; Ryan et al., 2006). This is not the case for some 15–30% of the pack ice around Antarctica where surface communities can dominate (Arrigo and Thomas, 2004). The SIMCO must endure increased salinities during the freezing process where salinities can exceed 100‰ (Vargo et al., 1985; Gleitz and Thomas, 1992; Ralph et al., 2007), and temperatures may decrease well below the freezing point of sea water (Thomas et al., 2008). Annual fast ice often reaches over 2 m thickness at the end of winter and the light levels for the bottom-ice algal community, even at midday rarely reach N15 μmol photons m−2 s−1 (Ryan and Beaglehole, 1994). The bottom-ice algal community may be amongst the most shade adapted photosynthetic organisms on earth (Thomas and Dieckmann, 2002). Photosynthesis has been recorded in these organisms at irradiances of less than 1 μmol photons m−2 s−1 (Palmisano and Sullivan, 1983; McMinn et al., 2003). Their Ek (onset of light saturation) is often b15 μmol photons m−2 s−1, and they generally become photoinhibited at b20 μmol photons m−2 s−1 (Palmisano et al., 1985; Kirst and Wiencke, 1995; McMinn et al., 2003) although this light level is rarely achieved under annual fast ice. They are also able to rapidly acclimate to diurnal changes in irradiance (McMinn et al., 2003). These data suggest that the bottom-ice algae in the Ross Sea region may never
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be exposed to saturating irradiances, and are therefore continually lightlimited whilst retained within the ice. The bottom-ice algal community nevertheless, grows well in this low light environment, where they are acclimated to normal salinity sea water with a regular supply of nutrients from the surrounding sea water (Garrison, 1991). During melting of the ice in summer, salinities surrounding bottom-ice algae decrease as the ice melts (Meguro et al., 1967; Gleitz and Thomas, 1992). In addition, algal cells may be exposed to high irradiances during melting near the water surface. Bottom-ice algae appear to be particularly vulnerable to low salinity stress, although they are less vulnerable to higher salinities (Ralph et al., 2007). Furthermore, different ice algal species respond in different ways with some species being quite halotolerant while other species may be more sensitive to reduced salinities (Ryan et al., 2004). These observations indicate that some species of bottom-ice algae may undergo considerable photosynthetic stress during the process of melting into the hyposaline melt water at the ice edge during summer. Previous studies have examined the responses of sea ice algae to a range of experimental stimuli including light (Palmisano et al., 1985), ultraviolet-B radiation (Ryan, 1992; Ryan and Beaglehole, 1994; McMinn et al., 1999, 2003), temperature (Ralph et al., 2005), and salinity (Vargo et al., 1985; Arrigo and Sullivan, 1992; Ralph et al., 2007) focusing on bottom-ice algae. Most studies on the stress responses of sea ice algae have involved releasing cells from the ice matrix by melting, and exposing them to artificial culture techniques. It is possible that this practice may modify or damage cellular physiology and ideally, algae should be studied in situ. Some in situ studies have been conducted using oxygen electrodes (McMinn et al., 2000; Trenerry et al., 2002; McMinn et al., 2010) and chlorophyll fluorescence techniques (e.g. Pulse Amplitude Modulation, PAM) (Kühl et al., 2001; McMinn et al., 2003) to estimate primary productivity. The latter technique offers considerable possibilities for the study of sea ice physiology as they provide a rapid non-destructive analysis of photosynthetic activity (McMinn et al., 2003). The first in situ measurements of sea ice algal photosynthesis were made using a PAM fluorometer in Greenland fast ice by Kühl et al. (2001). Diurnal changes in photosynthetic activity were observed for the first time by McMinn et al. (2003), who demonstrated that bottomice algae respond rapidly to changes in ambient irradiances. However, the possibilities for physiological experimentation are limited with in situ measurements, and of course there is the constant risk of losing expensive equipment while deployed under the ice. The recent development of chlorophyll imaging fluorometers offers new possibilities for the physiological study of ice algae, and verification of the results of prior studies using isolated cultures. The Imaging-PAM (I-PAM, Walz, Germany) system permits the observation and study of solid samples of sea ice in controlled environments. Variations in temperature, salinity, light, UVB radiation, and nutrients are now all possible using this system. In this study we demonstrate for the first time detailed images of the distribution of chlorophyll-α in bottom-ice samples and document in situ changes in the photosynthetic activity of the algae as they progress through melting under different light and salinity levels. These studies ultimately will help us understand some of the physical stresses imposed on sea ice algae during melting at the ice edge.
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of cells several centimeters from the bottom surface and above the platelet layer. 2.2. Sample collection Samples were collected during the austral summers of 2006 and 2007 from sea ice near Gondwana Station in Terra Nova Bay, Antarctica (74° 38.67′S, 164° 12.47′ E). Holes 20 cm in diameter were drilled into the ice using a powered ice auger to within approximately 30 cm from the bottom surface. The last section was removed using an ice coring drill (Kovaks, USA). The ice core was shrouded with a black blanket during extraction from the sea ice to avoid light shock to the bottom-ice algae. The ice samples were then immediately placed in individual black polythene bags and then inside a cool box, and transported 1 km back to the field laboratory. During this transfer no brine drained from the core samples. 2.3. Experimental setup Each core sample was cut into two pieces, 7 cm × 6 cm × 1.5 cm thick, to include the bottom-ice algae. The longest dimension of the block corresponded to the vertical axis of the ice. The blocks were then placed into individual dark plastic containers with clear plastic covers, and covered with 0.22 μm filtered sea water, collected from beneath the sea ice at the site where the sea ice was obtained. For each experiment, the samples were placed in a temperature controlled water bath at −3 ± 0.2 °C, with an artificial light source comprising a 4 × 4 array of 50 W (Philips) halogen lights 80 cm above the water bath. This lamp array generated some heat and this was dispersed using 2 small fans. A white Perspex sheet approximately 2 mm thick was mounted immediately below the lamps as a light diffuser and as an additional heat absorber. Periodically, the sample box was taken from the water bath and examined using an Imaging PAM (I-PAM, Walz, GmbH, Effeltrich, Germany). The I-PAM instrument was maintained in a cold box at approximately 0 °C. 2.4. Bottom-ice algal irradiance experiment using Imaging PAM (I-PAM) The two mirror blocks of ice, obtained from the one core, were placed in the water bath that was pre-cooled to −3 °C and were exposed to intensities of either 40 μmol photons m−2 s−1 or 270 μmol photons m−2 s−1. These light conditions were obtained using neutral density filters that were attached directly to the clear plastic cover of each box and were maintained at these irradiances for the duration of the experiment. Three additional replicate blocks of sea ice were obtained from a further three ice cores. During the course of the experiment, the ice cores were allowed to melt by slowly raising the temperature of the water bath (final recorded temperature after eight hours was 5 °C). At two hour intervals, Rapid Light Curves (RLCs) were recorded using the I-PAM. However the curves obtained did not saturate and this data is therefore not included. The blocks were not dark adapted. The experiment was then repeated with new ice cores obtained from the same site and using irradiances of 1 μmol photons m−2 s−1 and 100 μmol photons m−2 s−1, which were obtained by adjusting the neutral density filters.
2. Materials and methods 2.5. Bottom-ice algal salinity experiments using I-PAM 2.1. Study site The sea ice around the study sites at Gondwana Station in Terra Nova Bay, Antarctica, was single year ice, approximately 2.1 to 2.6 m thick. The bottom surface of sea ice at Terra Nova Bay was covered with a 1–2 cm layer of platelet ice that eventually consolidated to become an integral part of the sea ice structure. Most of the chlorophyll in the bottom of the core sample was concentrated in a relatively narrow band
Samples were prepared as described above and placed in their boxes in the water bath at temperature of −1.5 °C under a light intensity of 1 μmol photons m−2 s−1. Each ice block was floated in water of different salinities: 0 ppt (i.e. distilled freshwater), 12 ppt, 24 ppt, and 36 ppt (i.e. full strength sea water). The ice blocks were then allowed to melt over the course of the experiment, with a temperature of 2.5 °C at the end of the experiments and all blocks were completely melted. Water
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temperatures and I-PAM RLCs were taken at 30 min intervals up to the end of the experiment (four hours). The experiment was then repeated two more times using different ice cores, resulting in three replicates for each salinity treatment for this one irradiance level. The complete set of experiments was then repeated with light intensities of 20 μmol photons m−2 s−1 and 90 μmol photons m−2 s−1, so that there were three replicates for each salinity treatment at each light level. 2.6. Under ice light measurements A LiCor (Nebraska, USA) Li-192 2π underwater broad band quantum radiometer was deployed through a 20 cm hole through the sea ice. The radiometer was attached to the end of a 2.5 m long hinged arm and measured the radiation passing through the sea ice immediately under the ice. 2.7. Statistical tests Normality of data was assessed using the Anderson–Darling test. Mean values were compared using an analysis of variance (ANOVA, test statistic F) in the form of a generalised linear model (Quinn and Keough, 2002) with subsequent Tukey's pairwise comparisons. 3. Results The bottom-ice algal community at Terra Nova Bay during these experiments was dominated by diatoms, particularly Nitzschia stellata, Entomoneis kjellmannii, Berkelya adeliensis, Navicula sp, and Fragillariopsis curta. The under-ice irradiance of 1 μmol photons m−2 s−1 was measured using a 2π quantum sensor at solar noon on a clear sunny day. Additional lower quality sensors were embedded in and under the ice for the full duration of the field season (data not shown). These instruments showed that this irradiance under the ice was reached at mid day each day. The weather during the field season was predominantly clear and fine. Sea ice cores were removed from the sea ice and transferred to the field laboratory in the dark in a cold insulated box. During this transfer no brine drained from the cores. During the subsequent cutting of the cores into the blocks for incubation, a minimal level of brine drained from the cores at the cut edges. 3.1. Imaging PAM observations of solid sea ice sections The sample blocks were floated in filtered sea water at −1.5 °C in small opaque boxes with lids of different light transmission qualities.
At intervals, the lid was removed and the whole box was placed in an I-PAM chlorophyll fluorometer and images of the sea ice algae within the ice were recorded under various modes. Fig. 1a shows a typical ice sample from the bottom of the core imaged using natural fluorescence, F0, (F mode), which illustrates clearly the distribution of chlorophyll-α within the ice. A region 1–2 cm thick from the ice– water interface represents the platelet layer, which is a relatively loosely aggregated layer of small platelets of new ice formed on the very bottom of the core. The chlorophyll-α is most concentrated in a 2 cm thick band inside the platelet layer. Further within the ice, strands of chlorophyll-α can be seen growing up into brine channels within the ice. The resolution of this image is remarkable, as such detail is not visible to the naked eye. Immediately following a saturating flash of actinic light, a second image of this ice sample was recorded illustrating ФPSII (Fig. 1b). Here it can be see that ФPSII values of 0.5 exist over large regions of the core, and that higher levels of ФPSII do not necessarily correspond to regions of higher or lower chlorophyll-α concentration. The highest levels of ФPSII appear to be in the algae growing within the platelet layer and that cell photosynthetic health decreases with distance from the ice– water interface. The fate of each block of ice can be followed over time by placing the box containing the ice back into the water bath under known conditions and repeatedly measuring ФPSII and other parameters. At each measurement we attempted to record RLCs for each sample, however, we were unable to make the RLCs saturate, and therefore we could not derive the RLC parameters (data not shown).
3.2. Bottom-ice algal irradiance High light intensity had a negative effect on the photosynthesis of bottom-ice algae (Fig. 2), with ФPSII decreasing significantly with increasing light intensity (F60,3 = 15.64, p b 0.001). The final ФPSII of samples cultured at 1 μmol photons m−2 s−1 (~ 0.4) was approximately four times higher than those of algae exposed to 270 μmol photons m−2 s−1, (0.1) (p b 0.0001) with ФPSII of samples exposed to 40 μmol photons m−2 s−1 and 100 μmol photons m−2 s−1 intermediate between these. There was a sudden drop in ФPSII when the algae were first exposed to 40, 100, and 270 μmol photons m−2 s−1 light intensities. There was a gradual decline in ФPSII of samples for all light intensities during melting of the ice core, which was induced about two hours after the algae samples were first exposed to the different light treatments. The ice begins to melt about three to four hours after the start of the experiment and after this at intermediate irradiances, the algae begin to acclimate.
Fig. 1. I-PAM images of bottom-ice algae growing in sea ice. The bottom of each image is the ice water interface, where there is a 1–2 cm thick layer of platelet ice. a. Image of initial (F′) fluorescence showing the distribution of chlorophyll-α. The highest density of chlorophyll-α is concentrated in a dense layer of algal biomass immediately above the platelet layer. Strands of algae can be seen growing vertically up into the ice in brine channels. b. ФPSII image of the same section of sea ice. Note the relatively even distribution of fluorescence. The colour bar at the bottom is an arbitrary scale of intensity from 0 to 1.0.
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Fig. 2. ФPSII of bottom-ice algae and the temperature of the melting ice cores over time. The units, μE refer to μmol photons m−2 s−1. Data shows means and standard error of the mean; n = 3.
3.3. Bottom-ice algal salinity Samples melted into the highest salinity sea water (36 and 24‰) were barely affected by the melting process at low light levels (Fig. 3a). There was a highly significant effect on the ФPSII of the algae as salinity decreased (F252,3 = 43.04, p b 0.001). There was also a highly significant effect on the ФPSII of the algae as irradiance increased (F252,3 = 25.68, p b 0.001) (Fig. 3a–c). Thus, the effect of salinity on the photosynthesis of the algae became more pronounced as light levels increased. At the lowest salinity treatment of 0‰, there was no ФPSII from the algal samples after three hours at low light levels (1 and 20 μmol photons m−2 s−1), and after only two and a half hours at the high light level (90 μmol photons m−2 s−1). The ФPSII of ice samples melting into the highest salinity sea water (36 ppt and 24 ppt) at highest irradiances dropped to less than half their original values during the melting process. Intermediate salinities and moderate irradiances had intermediate effects. 4. Discussion To our knowledge, this is the first observation of bottom sea ice algae using an Imaging PAM. The chlorophyll image (Fig. 1a) shows the distribution of sea ice algae with unprecedented clarity. The algal biomass in this ice is concentrated in a narrow band 2 cm thick above the 1–2 cm thick platelet layer on the very bottom of the ice and it is possible to see algal growing up into brine channels in the ice. All of the algae in this section of ice core exhibited a relative high ФPSII of about 0.5 (Fig. 1b), and this verifies that the extraction and cutting process has not damaged the cells. This image thus illustrates the photosynthetic health of the algae while still embedded in the sea ice, at close to in situ conditions. 4.1. Irradiance The light level recorded under the ice at midday at this site was approximately 1 μmol photons m−2 s−1, and is similar to others recorded in fast ice in the Ross Sea region (McMinn et al., 1999; Ralph et al., 2005). In some instances, midday under-ice irradiances of up to 55 μmol photons m−2 s−1 have been recorded in fast ice (McMinn et al., 2000) but this is unusually high. The bottom-ice algae in this study were therefore highly adapted to low light. In Fig. 2a, the initial ФPSII value for all samples was approximately 0.5. This value is considerably lower than that normally found in healthy plant leaves (Schreiber et al., 1994) but is similar to other in situ measurements of sea ice algae (Kühl et al., 2001; McMinn et al., 2003). Comparable values have also been recorded in cultured bottom-ice algae, (Ryan
Fig. 3. ФPSII of bottom-ice algae over time at different salinities and different light conditions. a: low irradiance (1 μmol photons m−2 s−1); b: medium irradiance (20 μmol photons m−2 s−1); and c: high irradiance (90 μmol photons m−2 s−1). Temperatures are not included because the profile varied slightly for each replicate. Melting occurred in these samples at approximately 2 h. Data shows means and standard error of the mean; n = 3.
et al., 2004; Ralph et al., 2007; McMinn et al., 2007), and all three methods of PAM analysis (in situ, water PAM and I-PAM) appear to provide relevant measures of photosynthetic activity. When ice samples were incubated under an irradiance of 1 μmol photons m−2 s−1, ФPSII dropped slightly to 0.4 during the melting process but this was not statistically significant. This result is similar to that obtained from samples of pack ice melting in the dark by Ryan et al. (2004) and indicates that there is little stress due to melting to the photosynthetic apparatus under low light conditions. These
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comparable results in both protocols give confidence that real values are being recorded. When the algae were exposed to higher irradiances during the melting process, the ФPSII was decreased by a factor of 4. Irradiances of 100 and 270 μmol photons m−2 s−1 are equivalent to ambient conditions recorded ~ 0.6 m and 0.3 m from the ice surface at midday, according to in situ measurements in adjacent sections of ice (Ryan et al., 2006). At these irradiances, ФPSII dropped significantly to less that 0.1 (Fig. 2, p b 0.0001). Once the ice was melted, there was a gradual increase in ФPSII, but only at intermediate irradiances, which may indicate that the algae were most vulnerable to physical stresses during the melting process and recovered once they were in a fully liquid medium. The melting process involves an increase in temperature and it is possible that photosynthetic parameters may have been influenced by temperature as well as light (Arrigo and Thomas, 2004; Ryan et al., 2004). However, because all samples were melted with the same temperature regime, we conclude that temperature change was not the major influence on the differences observed here. 4.2. Salinity The bottom-ice algae in this study were acclimated to sea water salinities of approximately 36‰. The ФPSII of bottom-ice algal samples melted into normal or near normal salinity water (36 and 24‰) under 1 μmol photons m−2 s−1 irradiance conditions, did not change during the melting process (p N 0.05) (Fig. 3a). This plot is similar to the low light plot of Fig. 2 and serves as an independent verification of that result. We conclude from these data that bottom-ice algae remain photosynthetically active during the melting process, as long as they are melted into near normal salinities under low light conditions. However, photosynthetic health declined during melting as the salinity of the bathing solution decreased (Fig. 3a), and when the samples were melted out into fresh water, ФPSII rapidly decreased to zero. All lower salinity solutions were diluted from sea water and therefore have reduced added nutrients and this may have influenced survival. However the responses are so rapid that it is unlikely for a lack of nutrients in the water to have influenced survival and the response is more likely to be due to the change in osmolarity. These results confirm and extend those of Ryan et al. (2004) where pack ice algae exposed to low salinity (10‰) in the dark had low quantum yields and did not recover in the subsequent 5 days of culture. At the lowest irradiance in the current study (1 μmol photons m−2 s−1), the algae were able to withstand salinities as low as 12‰, however, higher irradiances induced greater salinity stress. Thus, when ice samples were melted while exposed to higher irradiances, algae could not tolerate salinities lower than 24 ppt. This result confirms the observations of Ralph et al. (2007) who also showed a combined effect of light intensity and saline concentrations. As described by Arrigo and Sullivan (1992), temperature, salinity and light are physically coupled in sea ice. They found that light intensity and salinity act independently in their effects on the growth of sea ice algae, and our observations are consistent with this. Particularly in the moderate irradiance experiment (Fig. 3b), there was a trend upward once the sample had melted (see also Ryan et al., 2004). We suggest that the process of melting is the most stressful for these algae and once melted they may be able to acclimate to their new environment. These observations have important implications for culture work and physiological studies on these organisms. Sea ice algae should be melted in low light into normal strength sea water to maintain physiological activity. The results of this study have implications for the fate of sea ice algae during melting of ice in summer, where they may be exposed to salinities as low as one third normal strength sea water during ice melt (Weeks and Ackley, 1982). In addition, once algae have been released into the water at the ice edge, they may be exposed to considerably higher irradiances. Together the combined effects of
higher light and lower salinity may mean that bottom-ice algae may die at the ice edge during or after melting. Their contribution to bloom events may be only as a secondary food source for krill and other grazers rather than contributing directly to primary productivity. IPAM fluorometry provides unprecedented imagery of chlorophyll distribution in sea ice and allows measurement of the responses of sea ice algae to environmental stresses with minimal disruption to their physical habitat. The results obtained with this method are similar to those obtained with algae that have been melted out of the ice into liquid culture. It is unfortunate that RLCs did not saturate and this may prevent further use of this configuration. Acknowledgements We are grateful to the Latitudinal Gradient Project and Antarctica NZ for logistic support especially Brian Staite, Rob Teesdale and Shulamit Gordon. For assistance at Gondwana Station we thank Daniel McNaughtan and Libby Liggins. 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Chlorophyll fluorescence imaging analysis of the responses of Antarctic bottom-ice algae to light and salinity during melting K.G. Ryan a,⁎, M.L. Tay a, A. Martin a,b, A. McMinn b, S.K. Davy a a b
School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand Institute of Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania, Australia
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Article history: Received 7 July 2010 Received in revised form 5 January 2011 Accepted 11 January 2011 Keywords: Algae Antarctic Chlorophyll fluorescence Imaging PAM Salinity Sea ice
a b s t r a c t Bottom-ice algae within Antarctic sea ice were examined using chlorophyll fluorescence imaging. The detailed structure of the bottom-ice algal community growing in the platelet and congelation layers of solid pieces of sea ice was evident for the first time in chlorophyll imaging mode. Strands of fluorescence representing algal cells were clearly visible growing upward into brine channels in a fine network. Images of effective quantum yield (ФPSII) revealed that the ФPSII of algae embedded in the sea ice was approximately 0.5. Furthermore, ФPSII decreased slightly with distance from the ice–water interface. The response of Antarctic sea ice algae to changes in irradiance and salinity, and the effects of slowly warming and melting the ice block sample were examined using this system. The ФPSII of bottom-ice algae decreased as irradiance increased and salinities decreased. Bottom-ice algae appear to be most vulnerable to changes in their environment during the melting process of the ice, and this suggests that algae from this region of the ice may not be able to cope with the stress of melting during summer. Chlorophyll fluorescence imaging provides unprecedented imagery of chlorophyll distribution in sea ice and allows measurement of the responses of sea ice algae to environmental stresses with minimal disruption to their physical habitat. The results obtained with this method are comparable to those obtained with algae that have been melted into liquid culture and this indicates that previous melting protocols reveal meaningful data. In this chlorophyll imaging study, rapid light curves did not saturate and this may prevent further use of this configuration. © 2011 Elsevier B.V. All rights reserved.
1. Introduction At its greatest extent, Antarctic sea-ice covers 19 million km2 (Arrigo and Thomas, 2004). The phytoplankton, protists and bacteria growing within sea-ice exert a strong influence in the Antarctic marine environment, and the bottom or interstitial, communities can reach biomass levels of over 300 mg Chlorophyll-α m−2 in the austral summer (Palmisano and Sullivan, 1983; Kirst and Wiencke, 1995). Their contribution to the primary production of the area is substantial (McMinn et al., 2000) and the overall contribution of ice algae to total primary production in ice-covered regions of the Southern Ocean is estimated at ~25% (Arrigo et al., 1997). The physicochemical conditions for the sea ice microbial community (SIMCO) are highly variable. There are strong gradients of light, temperature, salinity, and nutrient concentration within the ice column (Arrigo and Sullivan, 1992) and while microbes are found throughout the ice profile, bottom-ice communities are dominant in fast ice around most of Antarctica (Palmisano and Sullivan, 1983;
⁎ Corresponding author. Tel.: + 64 4 463 6083; fax: + 64 4 463 5331. E-mail address: [email protected] (K.G. Ryan). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.01.006
McMinn and Ashworth, 1998; Trenerry et al., 2002; Fiala et al., 2006; Ryan et al., 2006). This is not the case for some 15–30% of the pack ice around Antarctica where surface communities can dominate (Arrigo and Thomas, 2004). The SIMCO must endure increased salinities during the freezing process where salinities can exceed 100‰ (Vargo et al., 1985; Gleitz and Thomas, 1992; Ralph et al., 2007), and temperatures may decrease well below the freezing point of sea water (Thomas et al., 2008). Annual fast ice often reaches over 2 m thickness at the end of winter and the light levels for the bottom-ice algal community, even at midday rarely reach N15 μmol photons m−2 s−1 (Ryan and Beaglehole, 1994). The bottom-ice algal community may be amongst the most shade adapted photosynthetic organisms on earth (Thomas and Dieckmann, 2002). Photosynthesis has been recorded in these organisms at irradiances of less than 1 μmol photons m−2 s−1 (Palmisano and Sullivan, 1983; McMinn et al., 2003). Their Ek (onset of light saturation) is often b15 μmol photons m−2 s−1, and they generally become photoinhibited at b20 μmol photons m−2 s−1 (Palmisano et al., 1985; Kirst and Wiencke, 1995; McMinn et al., 2003) although this light level is rarely achieved under annual fast ice. They are also able to rapidly acclimate to diurnal changes in irradiance (McMinn et al., 2003). These data suggest that the bottom-ice algae in the Ross Sea region may never
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be exposed to saturating irradiances, and are therefore continually lightlimited whilst retained within the ice. The bottom-ice algal community nevertheless, grows well in this low light environment, where they are acclimated to normal salinity sea water with a regular supply of nutrients from the surrounding sea water (Garrison, 1991). During melting of the ice in summer, salinities surrounding bottom-ice algae decrease as the ice melts (Meguro et al., 1967; Gleitz and Thomas, 1992). In addition, algal cells may be exposed to high irradiances during melting near the water surface. Bottom-ice algae appear to be particularly vulnerable to low salinity stress, although they are less vulnerable to higher salinities (Ralph et al., 2007). Furthermore, different ice algal species respond in different ways with some species being quite halotolerant while other species may be more sensitive to reduced salinities (Ryan et al., 2004). These observations indicate that some species of bottom-ice algae may undergo considerable photosynthetic stress during the process of melting into the hyposaline melt water at the ice edge during summer. Previous studies have examined the responses of sea ice algae to a range of experimental stimuli including light (Palmisano et al., 1985), ultraviolet-B radiation (Ryan, 1992; Ryan and Beaglehole, 1994; McMinn et al., 1999, 2003), temperature (Ralph et al., 2005), and salinity (Vargo et al., 1985; Arrigo and Sullivan, 1992; Ralph et al., 2007) focusing on bottom-ice algae. Most studies on the stress responses of sea ice algae have involved releasing cells from the ice matrix by melting, and exposing them to artificial culture techniques. It is possible that this practice may modify or damage cellular physiology and ideally, algae should be studied in situ. Some in situ studies have been conducted using oxygen electrodes (McMinn et al., 2000; Trenerry et al., 2002; McMinn et al., 2010) and chlorophyll fluorescence techniques (e.g. Pulse Amplitude Modulation, PAM) (Kühl et al., 2001; McMinn et al., 2003) to estimate primary productivity. The latter technique offers considerable possibilities for the study of sea ice physiology as they provide a rapid non-destructive analysis of photosynthetic activity (McMinn et al., 2003). The first in situ measurements of sea ice algal photosynthesis were made using a PAM fluorometer in Greenland fast ice by Kühl et al. (2001). Diurnal changes in photosynthetic activity were observed for the first time by McMinn et al. (2003), who demonstrated that bottomice algae respond rapidly to changes in ambient irradiances. However, the possibilities for physiological experimentation are limited with in situ measurements, and of course there is the constant risk of losing expensive equipment while deployed under the ice. The recent development of chlorophyll imaging fluorometers offers new possibilities for the physiological study of ice algae, and verification of the results of prior studies using isolated cultures. The Imaging-PAM (I-PAM, Walz, Germany) system permits the observation and study of solid samples of sea ice in controlled environments. Variations in temperature, salinity, light, UVB radiation, and nutrients are now all possible using this system. In this study we demonstrate for the first time detailed images of the distribution of chlorophyll-α in bottom-ice samples and document in situ changes in the photosynthetic activity of the algae as they progress through melting under different light and salinity levels. These studies ultimately will help us understand some of the physical stresses imposed on sea ice algae during melting at the ice edge.
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of cells several centimeters from the bottom surface and above the platelet layer. 2.2. Sample collection Samples were collected during the austral summers of 2006 and 2007 from sea ice near Gondwana Station in Terra Nova Bay, Antarctica (74° 38.67′S, 164° 12.47′ E). Holes 20 cm in diameter were drilled into the ice using a powered ice auger to within approximately 30 cm from the bottom surface. The last section was removed using an ice coring drill (Kovaks, USA). The ice core was shrouded with a black blanket during extraction from the sea ice to avoid light shock to the bottom-ice algae. The ice samples were then immediately placed in individual black polythene bags and then inside a cool box, and transported 1 km back to the field laboratory. During this transfer no brine drained from the core samples. 2.3. Experimental setup Each core sample was cut into two pieces, 7 cm × 6 cm × 1.5 cm thick, to include the bottom-ice algae. The longest dimension of the block corresponded to the vertical axis of the ice. The blocks were then placed into individual dark plastic containers with clear plastic covers, and covered with 0.22 μm filtered sea water, collected from beneath the sea ice at the site where the sea ice was obtained. For each experiment, the samples were placed in a temperature controlled water bath at −3 ± 0.2 °C, with an artificial light source comprising a 4 × 4 array of 50 W (Philips) halogen lights 80 cm above the water bath. This lamp array generated some heat and this was dispersed using 2 small fans. A white Perspex sheet approximately 2 mm thick was mounted immediately below the lamps as a light diffuser and as an additional heat absorber. Periodically, the sample box was taken from the water bath and examined using an Imaging PAM (I-PAM, Walz, GmbH, Effeltrich, Germany). The I-PAM instrument was maintained in a cold box at approximately 0 °C. 2.4. Bottom-ice algal irradiance experiment using Imaging PAM (I-PAM) The two mirror blocks of ice, obtained from the one core, were placed in the water bath that was pre-cooled to −3 °C and were exposed to intensities of either 40 μmol photons m−2 s−1 or 270 μmol photons m−2 s−1. These light conditions were obtained using neutral density filters that were attached directly to the clear plastic cover of each box and were maintained at these irradiances for the duration of the experiment. Three additional replicate blocks of sea ice were obtained from a further three ice cores. During the course of the experiment, the ice cores were allowed to melt by slowly raising the temperature of the water bath (final recorded temperature after eight hours was 5 °C). At two hour intervals, Rapid Light Curves (RLCs) were recorded using the I-PAM. However the curves obtained did not saturate and this data is therefore not included. The blocks were not dark adapted. The experiment was then repeated with new ice cores obtained from the same site and using irradiances of 1 μmol photons m−2 s−1 and 100 μmol photons m−2 s−1, which were obtained by adjusting the neutral density filters.
2. Materials and methods 2.5. Bottom-ice algal salinity experiments using I-PAM 2.1. Study site The sea ice around the study sites at Gondwana Station in Terra Nova Bay, Antarctica, was single year ice, approximately 2.1 to 2.6 m thick. The bottom surface of sea ice at Terra Nova Bay was covered with a 1–2 cm layer of platelet ice that eventually consolidated to become an integral part of the sea ice structure. Most of the chlorophyll in the bottom of the core sample was concentrated in a relatively narrow band
Samples were prepared as described above and placed in their boxes in the water bath at temperature of −1.5 °C under a light intensity of 1 μmol photons m−2 s−1. Each ice block was floated in water of different salinities: 0 ppt (i.e. distilled freshwater), 12 ppt, 24 ppt, and 36 ppt (i.e. full strength sea water). The ice blocks were then allowed to melt over the course of the experiment, with a temperature of 2.5 °C at the end of the experiments and all blocks were completely melted. Water
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temperatures and I-PAM RLCs were taken at 30 min intervals up to the end of the experiment (four hours). The experiment was then repeated two more times using different ice cores, resulting in three replicates for each salinity treatment for this one irradiance level. The complete set of experiments was then repeated with light intensities of 20 μmol photons m−2 s−1 and 90 μmol photons m−2 s−1, so that there were three replicates for each salinity treatment at each light level. 2.6. Under ice light measurements A LiCor (Nebraska, USA) Li-192 2π underwater broad band quantum radiometer was deployed through a 20 cm hole through the sea ice. The radiometer was attached to the end of a 2.5 m long hinged arm and measured the radiation passing through the sea ice immediately under the ice. 2.7. Statistical tests Normality of data was assessed using the Anderson–Darling test. Mean values were compared using an analysis of variance (ANOVA, test statistic F) in the form of a generalised linear model (Quinn and Keough, 2002) with subsequent Tukey's pairwise comparisons. 3. Results The bottom-ice algal community at Terra Nova Bay during these experiments was dominated by diatoms, particularly Nitzschia stellata, Entomoneis kjellmannii, Berkelya adeliensis, Navicula sp, and Fragillariopsis curta. The under-ice irradiance of 1 μmol photons m−2 s−1 was measured using a 2π quantum sensor at solar noon on a clear sunny day. Additional lower quality sensors were embedded in and under the ice for the full duration of the field season (data not shown). These instruments showed that this irradiance under the ice was reached at mid day each day. The weather during the field season was predominantly clear and fine. Sea ice cores were removed from the sea ice and transferred to the field laboratory in the dark in a cold insulated box. During this transfer no brine drained from the cores. During the subsequent cutting of the cores into the blocks for incubation, a minimal level of brine drained from the cores at the cut edges. 3.1. Imaging PAM observations of solid sea ice sections The sample blocks were floated in filtered sea water at −1.5 °C in small opaque boxes with lids of different light transmission qualities.
At intervals, the lid was removed and the whole box was placed in an I-PAM chlorophyll fluorometer and images of the sea ice algae within the ice were recorded under various modes. Fig. 1a shows a typical ice sample from the bottom of the core imaged using natural fluorescence, F0, (F mode), which illustrates clearly the distribution of chlorophyll-α within the ice. A region 1–2 cm thick from the ice– water interface represents the platelet layer, which is a relatively loosely aggregated layer of small platelets of new ice formed on the very bottom of the core. The chlorophyll-α is most concentrated in a 2 cm thick band inside the platelet layer. Further within the ice, strands of chlorophyll-α can be seen growing up into brine channels within the ice. The resolution of this image is remarkable, as such detail is not visible to the naked eye. Immediately following a saturating flash of actinic light, a second image of this ice sample was recorded illustrating ФPSII (Fig. 1b). Here it can be see that ФPSII values of 0.5 exist over large regions of the core, and that higher levels of ФPSII do not necessarily correspond to regions of higher or lower chlorophyll-α concentration. The highest levels of ФPSII appear to be in the algae growing within the platelet layer and that cell photosynthetic health decreases with distance from the ice– water interface. The fate of each block of ice can be followed over time by placing the box containing the ice back into the water bath under known conditions and repeatedly measuring ФPSII and other parameters. At each measurement we attempted to record RLCs for each sample, however, we were unable to make the RLCs saturate, and therefore we could not derive the RLC parameters (data not shown).
3.2. Bottom-ice algal irradiance High light intensity had a negative effect on the photosynthesis of bottom-ice algae (Fig. 2), with ФPSII decreasing significantly with increasing light intensity (F60,3 = 15.64, p b 0.001). The final ФPSII of samples cultured at 1 μmol photons m−2 s−1 (~ 0.4) was approximately four times higher than those of algae exposed to 270 μmol photons m−2 s−1, (0.1) (p b 0.0001) with ФPSII of samples exposed to 40 μmol photons m−2 s−1 and 100 μmol photons m−2 s−1 intermediate between these. There was a sudden drop in ФPSII when the algae were first exposed to 40, 100, and 270 μmol photons m−2 s−1 light intensities. There was a gradual decline in ФPSII of samples for all light intensities during melting of the ice core, which was induced about two hours after the algae samples were first exposed to the different light treatments. The ice begins to melt about three to four hours after the start of the experiment and after this at intermediate irradiances, the algae begin to acclimate.
Fig. 1. I-PAM images of bottom-ice algae growing in sea ice. The bottom of each image is the ice water interface, where there is a 1–2 cm thick layer of platelet ice. a. Image of initial (F′) fluorescence showing the distribution of chlorophyll-α. The highest density of chlorophyll-α is concentrated in a dense layer of algal biomass immediately above the platelet layer. Strands of algae can be seen growing vertically up into the ice in brine channels. b. ФPSII image of the same section of sea ice. Note the relatively even distribution of fluorescence. The colour bar at the bottom is an arbitrary scale of intensity from 0 to 1.0.
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Fig. 2. ФPSII of bottom-ice algae and the temperature of the melting ice cores over time. The units, μE refer to μmol photons m−2 s−1. Data shows means and standard error of the mean; n = 3.
3.3. Bottom-ice algal salinity Samples melted into the highest salinity sea water (36 and 24‰) were barely affected by the melting process at low light levels (Fig. 3a). There was a highly significant effect on the ФPSII of the algae as salinity decreased (F252,3 = 43.04, p b 0.001). There was also a highly significant effect on the ФPSII of the algae as irradiance increased (F252,3 = 25.68, p b 0.001) (Fig. 3a–c). Thus, the effect of salinity on the photosynthesis of the algae became more pronounced as light levels increased. At the lowest salinity treatment of 0‰, there was no ФPSII from the algal samples after three hours at low light levels (1 and 20 μmol photons m−2 s−1), and after only two and a half hours at the high light level (90 μmol photons m−2 s−1). The ФPSII of ice samples melting into the highest salinity sea water (36 ppt and 24 ppt) at highest irradiances dropped to less than half their original values during the melting process. Intermediate salinities and moderate irradiances had intermediate effects. 4. Discussion To our knowledge, this is the first observation of bottom sea ice algae using an Imaging PAM. The chlorophyll image (Fig. 1a) shows the distribution of sea ice algae with unprecedented clarity. The algal biomass in this ice is concentrated in a narrow band 2 cm thick above the 1–2 cm thick platelet layer on the very bottom of the ice and it is possible to see algal growing up into brine channels in the ice. All of the algae in this section of ice core exhibited a relative high ФPSII of about 0.5 (Fig. 1b), and this verifies that the extraction and cutting process has not damaged the cells. This image thus illustrates the photosynthetic health of the algae while still embedded in the sea ice, at close to in situ conditions. 4.1. Irradiance The light level recorded under the ice at midday at this site was approximately 1 μmol photons m−2 s−1, and is similar to others recorded in fast ice in the Ross Sea region (McMinn et al., 1999; Ralph et al., 2005). In some instances, midday under-ice irradiances of up to 55 μmol photons m−2 s−1 have been recorded in fast ice (McMinn et al., 2000) but this is unusually high. The bottom-ice algae in this study were therefore highly adapted to low light. In Fig. 2a, the initial ФPSII value for all samples was approximately 0.5. This value is considerably lower than that normally found in healthy plant leaves (Schreiber et al., 1994) but is similar to other in situ measurements of sea ice algae (Kühl et al., 2001; McMinn et al., 2003). Comparable values have also been recorded in cultured bottom-ice algae, (Ryan
Fig. 3. ФPSII of bottom-ice algae over time at different salinities and different light conditions. a: low irradiance (1 μmol photons m−2 s−1); b: medium irradiance (20 μmol photons m−2 s−1); and c: high irradiance (90 μmol photons m−2 s−1). Temperatures are not included because the profile varied slightly for each replicate. Melting occurred in these samples at approximately 2 h. Data shows means and standard error of the mean; n = 3.
et al., 2004; Ralph et al., 2007; McMinn et al., 2007), and all three methods of PAM analysis (in situ, water PAM and I-PAM) appear to provide relevant measures of photosynthetic activity. When ice samples were incubated under an irradiance of 1 μmol photons m−2 s−1, ФPSII dropped slightly to 0.4 during the melting process but this was not statistically significant. This result is similar to that obtained from samples of pack ice melting in the dark by Ryan et al. (2004) and indicates that there is little stress due to melting to the photosynthetic apparatus under low light conditions. These
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comparable results in both protocols give confidence that real values are being recorded. When the algae were exposed to higher irradiances during the melting process, the ФPSII was decreased by a factor of 4. Irradiances of 100 and 270 μmol photons m−2 s−1 are equivalent to ambient conditions recorded ~ 0.6 m and 0.3 m from the ice surface at midday, according to in situ measurements in adjacent sections of ice (Ryan et al., 2006). At these irradiances, ФPSII dropped significantly to less that 0.1 (Fig. 2, p b 0.0001). Once the ice was melted, there was a gradual increase in ФPSII, but only at intermediate irradiances, which may indicate that the algae were most vulnerable to physical stresses during the melting process and recovered once they were in a fully liquid medium. The melting process involves an increase in temperature and it is possible that photosynthetic parameters may have been influenced by temperature as well as light (Arrigo and Thomas, 2004; Ryan et al., 2004). However, because all samples were melted with the same temperature regime, we conclude that temperature change was not the major influence on the differences observed here. 4.2. Salinity The bottom-ice algae in this study were acclimated to sea water salinities of approximately 36‰. The ФPSII of bottom-ice algal samples melted into normal or near normal salinity water (36 and 24‰) under 1 μmol photons m−2 s−1 irradiance conditions, did not change during the melting process (p N 0.05) (Fig. 3a). This plot is similar to the low light plot of Fig. 2 and serves as an independent verification of that result. We conclude from these data that bottom-ice algae remain photosynthetically active during the melting process, as long as they are melted into near normal salinities under low light conditions. However, photosynthetic health declined during melting as the salinity of the bathing solution decreased (Fig. 3a), and when the samples were melted out into fresh water, ФPSII rapidly decreased to zero. All lower salinity solutions were diluted from sea water and therefore have reduced added nutrients and this may have influenced survival. However the responses are so rapid that it is unlikely for a lack of nutrients in the water to have influenced survival and the response is more likely to be due to the change in osmolarity. These results confirm and extend those of Ryan et al. (2004) where pack ice algae exposed to low salinity (10‰) in the dark had low quantum yields and did not recover in the subsequent 5 days of culture. At the lowest irradiance in the current study (1 μmol photons m−2 s−1), the algae were able to withstand salinities as low as 12‰, however, higher irradiances induced greater salinity stress. Thus, when ice samples were melted while exposed to higher irradiances, algae could not tolerate salinities lower than 24 ppt. This result confirms the observations of Ralph et al. (2007) who also showed a combined effect of light intensity and saline concentrations. As described by Arrigo and Sullivan (1992), temperature, salinity and light are physically coupled in sea ice. They found that light intensity and salinity act independently in their effects on the growth of sea ice algae, and our observations are consistent with this. Particularly in the moderate irradiance experiment (Fig. 3b), there was a trend upward once the sample had melted (see also Ryan et al., 2004). We suggest that the process of melting is the most stressful for these algae and once melted they may be able to acclimate to their new environment. These observations have important implications for culture work and physiological studies on these organisms. Sea ice algae should be melted in low light into normal strength sea water to maintain physiological activity. The results of this study have implications for the fate of sea ice algae during melting of ice in summer, where they may be exposed to salinities as low as one third normal strength sea water during ice melt (Weeks and Ackley, 1982). In addition, once algae have been released into the water at the ice edge, they may be exposed to considerably higher irradiances. Together the combined effects of
higher light and lower salinity may mean that bottom-ice algae may die at the ice edge during or after melting. Their contribution to bloom events may be only as a secondary food source for krill and other grazers rather than contributing directly to primary productivity. IPAM fluorometry provides unprecedented imagery of chlorophyll distribution in sea ice and allows measurement of the responses of sea ice algae to environmental stresses with minimal disruption to their physical habitat. The results obtained with this method are similar to those obtained with algae that have been melted out of the ice into liquid culture. It is unfortunate that RLCs did not saturate and this may prevent further use of this configuration. Acknowledgements We are grateful to the Latitudinal Gradient Project and Antarctica NZ for logistic support especially Brian Staite, Rob Teesdale and Shulamit Gordon. For assistance at Gondwana Station we thank Daniel McNaughtan and Libby Liggins. 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