The coralline alga, Corallina officinalis, is a widely distributed intertidal species in temperate coastal regions. It is usually exposed to high fluctuations of light intensity, light quality, temperature, and desiccation, all of which affect the temporal and spatial distribution as well as the morphology and the metabolism of this alga. In laboratory experiments we examined the effects of different light intensities (50, 100, and 200 μmol photons m-2 s-1) on photosynthesis, calcification, photosynthetic pigment contents (chlorophyll a and carotenoids), and growth rate of C. officinalis to clarify its photoacclimation strategies. Net photosynthesis, calcification and dissolution rates based on weight were not sensitive to irradiance. Although, photosynthesis and calcification did not clearly respond to light intensity, photosynthetic pigment contents were significantly lower at higher light intensities. In addition, higher irradiances induced significant enhancement of gross photosynthesis based on chlorophyll a. As a result, the specific growth rate was significantly stimulated by high light intensity. Our results suggest that photoacclimation of C. officinalis to different light conditions may be regulated to optimize growth.
Coralline algae (Corallinaceae, Rhodophyta) are the important calcifying organisms in the marine environment and contribute to the formation of calcareous structures on the ocean floor (Adey and Macintyre 1973, Steneck 1986). Many experiments have been conducted on the responses of coralline algae to future ocean conditions (e.g., Noisette et al. 2013
Light is one of the most important factors that affect distribution, morphology and physiological activities of corallines. Coralline algae can survive with limited light, e.g., a rhodophycean coralline found at 286 m off the Bahamas in about 0.008 μmol photons m-2 s-1-1 (Littler et al. 1985). Other species of Corallinales dominate the tropical shores where full sunlight can reach 2,300 μmol photons m-2 s-1 (Lawson 1966). Saturating irradiance for photosyn-thesis of the tropical coralline alga,
In temperate regions, many coralline algae are adapted to low light intensity. These include the non-articulated species,
In addition to photosynthesis, calcification is another important physiological process that coralline algae require for growth. Calcification is a light-stimulated metabolic process for most coralline algae (see Borowitzka 1977, El Haikali et al. 2004). This is because active photosynthetic CO2 and HCO3- uptake increases pH and CO32- ions as well the calcite and aragonite saturation state of seawater on the diffusive boundary layer of the thallus surface (Larkum et al. 2003). Generally, active photosynthetic activity increases pH, and the relative fraction of carbon species changes (i.e., increase of CO32- and decrease of CO2 and HCO3-). Dark respiration also causes the reverse carbon chemical changes; thus calcification is reduced. Most calcifying organisms are very sensitive to seawater pH, because they produce some type of organic cover layer to separate their skeleton from ambient seawater. For example, coralline algae build up the skeletons with aragonite or calcite crystals within the organic cell wall material, which are more easily affected by pH changes (Borowitzka 1981, Morse et al. 2007). Several previous studies showed enhanced calcification when coralline algae are exposed to high light conditions. The free-living coralline alga,
Previous studies of photoacclimation kinetics of coralline algae were conducted using fluorescence measurements to consider light stress, but changes in calcification and photosynthesis were not documented (e.g., Wilson et al. 2004). Furthermore, photo-physiological adaptation strategies of many coralline algal species were not clearly identified in the previous studies. Hence, in this study, we evaluated photosynthesis and calcification in the articulated coralline alga,
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Sample collection and pre-incubation
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Light acclimation experiment
Samples were cultured at three irradiances: low (50 μmol photons m-2 s-1), medium (100 μmol photons m-2 s-1), and high (200 μmol photons m-2 s-1) at 18℃ for 10 days. These irradiances were used because in initial samples saturation occurred at approximately 200 μmol photons m-2 s-1 (unpublished data). The three irradiances were established by varying the distance between sample positions and the light source (four 36-W daylight fluorescent lamp with reflector, Dulux L 36W/865; Osram, Munich, Germany). At each light level, three 500-mL beakers were used as three replicates to cultivate three specimens (approximately 0.5 g each) per beaker as sub-samples. All of the beakers were put in the same water bath (30 L water volume) to maintain a stable temperature among different light levels. Turnover time of seawater in each beaker was less than 3 min, and a full exchange of seawater was performed every other day to prevent depletion of nutrients and calcium ions. After 10 days, the experimental endpoints were determined for each specimen.
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Photosynthesis and calcification measurement
One random specimen in each beaker was chosen for both light and dark incubation to measure photosynthesis and calcification. The light incubation took place in a 70-mL cell culture flask (Corning, Corning, NY, USA) over a 2 h time period. The sample was then removed from the bottle and acclimated in a water bath in the dark to avoid abrupt light changes. Dark incubation started after the acclimation period, and lasted for 2 h in another cell culture flask with the sample from the light incubation. Dissolved oxygen concentrations in the bottles were measured to estimate photosynthesis (oxygen production) before and after the incubation period, and were measured with a Clark-type oxygen microelectrode (OX25; Unisense, Aarhus, Denmark) connected to a picoammeter (PA2000; Unisense). The oxygen microelectrode had an outside tip diameter of 30 μm, a stirring sensitivity of 5% and a 90% response time less than 0.5 s. The oxygen microelectrode was two point calibrated using air-saturated and pure nitrogen-saturated seawater at the experimental temperature and salinity conditions. Net photosynthesis and respiration were determined as changes in oxygen concentration after 2 h incubations under the three lights levels and dark conditions, respectively, and these results were normalized to wet weight of coralline samples. Gross photosynthesis was calculated as the gross production of oxygen after incubation normalized to chlorophyll
The seawater was fixed with saturated HgCl2 to measure seawater carbon chemistry after incubation, and kept in the dark at 4℃ until analyzed for total alkalinity (
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Photosynthetic pigment contents
After the 10 day acclimation to different light conditions all coralline samples were frozen and stored at -18℃ before photosynthetic pigment analysis. About 0.1 g of specimen was ground and placed in absolute methanol at 4℃ in the dark 24 h for total extraction of photosynthetic pigments. The supernatant was then carefully pipetted to glass cuvettes. Chlorophyll
Specific growth rate (μ) was estimated by measuring changes in the wet weights of coralline sample after 4 and 10 days of cultivation in the different light conditions. Wet weights were measured after gently blotting the sample with paper tissue. Specific growth rate (μ) was calculated as:
SGR (μ) = ln (NT - N0)/(DT - D0)
, where
Separate one-way ANOVAs were applied to examine responses in photosynthetic rate, calcification and growth rate of
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Photosynthesis and respiration
In general, oxygen production and consumption were slightly higher than carbon assimilation and release. The photosynthetic quotient (PQ), which is an overall ratio
of oxygen production to carbon assimilation from wet weight (ww) and chlorophyll
and carbon assimilation rates between light treatments based on weight calculations (p > 0.05), and the values for net photosynthesis ranged from 17.76 to 21.18 μmol O2 g-1 ww h-1 and 15.38 to 21.35 μmol C g-1 ww h-1 (Fig. 1A & C). However, the oxygen consumption rate (respiration) in the low light condition was significantly lower than at the medium and high light conditions (F = 11.677, p < 0.05), but carbon release was not significantly changed by irradiance (p > 0.05). The O2 consumption rate under low light was 9 and 13% lower than at medium and high light, respectively. Respiration comprised approximately 37.9% of the net O2 production rate, and 37.6% of the net carbon assimilation rate regardless of light intensity. Gross oxygen production based on chlorophyll
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Calcification and dissolution
Rates of calcification (under light conditions) and dissolution (under dark condition) of
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Photosynthetic pigment contents and growth rate
The differences in chlorophyll
Specific growth rate was significantly affected by light intensity at the different times (F = 27.735, p < 0.001) (Fig. 4A). After 4 days exposure to different light levels, specific growth rates were 16.7 and 30.0% increased at medium and high light compared to low light. However, growth rate was significantly different only between low and high light (p < 0.05). Also,
after a 10 day acclimation (p < 0.05), and this was counter to the trend with photosynthetic pigments. The specific growth rates at medium and high light were 26.2 and 40.8% higher than that at low light after 10 days acclimation, respectively. Also, relative weight was 3.6% d-1 at low light compared to day one, and was 31.8 and 50.65% higher at the medium and high light conditions (Fig. 4B)
In this study, photosynthesis of the coralline alga
In contrast, respiration (oxygen consumption rate) was significantly lower at the low light intensity compared to the medium and high light intensities (Fig. 1A). Martin et al. (2006) also examined dark respiration in
Calcification was enhanced with increasing light but saturates at some point (Chisholm 2000, Marubini et al. 2001, Martin et al. 2006). In this study, high light intensity had no effect on the calcification of
Until now, it has been impossible to explain whether dissolution serves a biological function. The seeming paradox is that coralline algae are simply unable to maintain conditions that favor the precipitation of CaCO3 in the dark. The production of spore chambers and the sloughing of outer epithelial cells (see Garbary et al. 2013 for review) may contribute to the dissolution of CaCO3 (Chisholm 2000). In this study there was no obvious spore production during the experiment (personal observation). Therefore, decalcification may be a result of other physiological processes of coralline algae. It is unknown whether decalcification continuously occurred under both light and dark conditions or only in the dark condition. Regardless, dark calcification was so low that the dissolution rate exceeded the carbonate fixation rate. The dissolution of
The growth rate of
In short,