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Shannon Point Marine Center and Department of Biology, Western Washington University, Bellingham, Washington 98225-9160
* To whom correspondence should be addressed. E-mail: Gisele.Muller-Parker{at}wwu.edu
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(13C) and 5 (15N) enriched and zoochlorellate anemone tissue was 6 (13C) and 8 (15N) enriched over their respective symbionts, suggesting that zoochlorellate anemones receive less nutrition from their symbionts than do zooxanthellate individuals. The disparity between predicted contributions from the algal carbon budgets and the stable isotopic composition suggests that short-term measures of algal contributions may not reflect actual nutritional inputs to the host. Isotopic data support the hypothesis of substantial reliance on external food sources. This additional nutrition may allow both algae to persist in this temperate intertidal anemone in spite of differences in seasonal photosynthetic carbon contributions. |
Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDISCUSSIONLiterature Cited |
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Given these environmental extremes in irradiance and temperature, it is of interest to know how these two algal symbionts differ in their productivity and contributions to the host anemone during the summer and winter seasons. Studies have shown that zoochlorellae are maintained at higher densities and have higher maximum photosynthetic rates under conditions of low light and low temperature (Saunders and Muller-Parker, 1997; Engebretson and Muller-Parker, 1999), and that zooxanthellate individuals of A. elegantissima sustain higher photosynthetic rates than zoochlorellate individuals under summertime conditions of high light and temperature (Verde and McCloskey, 2001, 2002, 2007). Zooxanthellae and zoochlorellae may respond differently to seasonal fluctuations in environmental parameters because they differ in their physiological tolerances to temperature and irradiance (O'Brien, 1980; Saunders and Muller-Parker, 1997; Engebretson and Muller-Parker, 1999). Verde and McCloskey (2007) recently determined that in the spring and summer the net productivity of zooxanthellate A. elegantissima was greater than that of zoochlorellate A. elegantissima, and they calculated a higher potential contribution of photosynthetic carbon from zooxanthellae than from zoochlorellae in all seasons. It is also possible that during low light conditions in winter, the nutritional relationship of both algae with their host may shift from mutualistic to parasitic with respect to carbon.
The purpose of this study was to examine how symbiont populations and the productivity of zooxanthellae and zoochlorellae isolated from A. elegantissima vary with seasonal changes in environmental parameters, and to compare the nutritional contributions of both algae by estimating the amount of photosynthetic carbon available for translocation to the host and by using stable isotopes to examine the relative importance of allocthonous versus translocated carbon to the anemone diet. Since the delta13C and delta15N signatures of an organism are related to those of its diet, the relative contribution of photosynthetic carbon and heterotrophically derived food to the diet of A. elegantissima may be deduced by comparing the isotopic signatures of symbiotic anemones with those of nonsymbiotic counterparts, because the latter are exclusive heterotrophs.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDISCUSSIONLiterature Cited |
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0.2 g wet weight) were collected. At Shannon Point Marine Center, anemones were separated by color, cleaned of rock and shell debris, and placed in a flow-through natural seawater table until analysis. The conditions in the seawater table approximated those of the submerged habitat. The seawater temperature was the same (supplied from the same body of water), and anemones were exposed to diffuse natural light supplied by large windows next to the seawater table. Light or olive-colored anemones (with low or mixed algal symbiont populations) were removed, and the remaining green and brown anemones were selected haphazardly for analysis. For each collection, anemones were processed in six sets of four individuals at a time (two zooxanthellate and two zoochlorellate anemones). Three sets of anemones were homogenized on the first and on the second day after collection, for a total of 24 anemones. All anemones were processed within 48 h of collection.
To isolate the algae from animal tissue, anemones were homogenized in 5-µm filtered seawater, using a 60-ml glass tissue homogenizer and a motorized Teflon pestle. Subsamples were frozen for later determination of algal density and mitotic index (MI; the number of cells with complete cleavage furrows), and for protein analysis. The remaining homogenate was centrifuged in a tabletop swinging bucket centrifuge at about 1600 x g; the animal supernatant was discarded and the algal pellet resuspended in filtered seawater by vortexing. This process of centrifugation and resuspension was repeated three times. The final algal suspension was filtered through a 25-µm Nitex screen to remove residual animal tissue. Of the final algal suspension, 5 to 10 ml was filtered onto a 25-mm GF/C filter under gentle vacuum pressure (10 mm Hg) for later analysis of chlorophyll pigments, and 3 ml was used for immediate measurements of algal 14C-fixation rates. A sample was also taken and frozen for later cell counts.
Photosynthesis-Irradiance (P-I) measurements
The protocol of Bachman and Muller-Parker (2007) was followed, with slight modifications. 14C bicarbonate (2 µCi) was added to freshly isolated algae suspended in 3 ml of filtered seawater. The algal-14C mixture was allocated as 0.2-ml subsamples into 12 glass mini-scintillation vials (7-ml volume) and placed in a temperature-controlled photosynthetron (CHPT Industries). The temperature of the photosynthetron was set to the 2004–2005 average temperature for the month of collection (see Fig. 1). Vials were exposed to a gradient of irradiances ranging from 0 to about 1200 µmol photons · m–2 · s –1, as measured by a Biospherical Instruments QSL-101 quantum scalar sensor immersed in water in a glass vial placed into each position of the photosynthetron. After 0.5 h, incubations were terminated by acidification of each sample with 1 mol l–1 HCl in a fume hood overnight, followed by neutralization with 1 mol l–1 NaOH. Disintegrations per minute (DPM) of each sample was counted in a Packard 1900 TR scintillation counter, and the photosynthetic rate of the algae at each irradiance was calculated from the DPM data and the total inorganic carbon content of the seawater. The total inorganic carbon content of seawater at each incubation temperature was determined from a linear curve relating total inorganic carbon content with seawater temperature. This curve was derived from pH and alkalinity values of a series of seawater samples (practical salinity = 30) ranging in temperature from 5 to 30 °C, using the methods and tables in Parsons et al. (1984). The maximum photosynthetic rate (Pmax cell), and photosynthetic efficiency (
) were derived from the P-I data of algae from each anemone by using a hyperbolic tangent function in Sigma Plot 9.0.
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To gauge algal division rates, MI was tallied per subsample of 1000 cells and reported as percent cells dividing. For the biomass and carbon content of the algae, the diameter of 100–150 cells (about 10 per anemone sampled) was measured with a calibrated ocular micrometer at 400x magnification. Zooxanthellae and zoochlorellae from summer (July 2004) and winter (December 2004 and February 2005) were measured, and cell volumes calculated assuming spherical algae. The equations of Muscatine et al. (1983) and Menden-Deuer and Lessard (2000; equation for protist plankton excluding diatoms) were used, respectively, to derive animal and algal biomass ratios and the carbon content of zooxanthellae and zoochlorellae.
For chlorophyll analysis, filters containing zooxanthellae or zoochlorellae were ground to a pulp in a 10-ml glass tissue homogenizer with a motorized Teflon pestle, using the appropriate solvent (100% HPLC-grade acetone and 100% HPLC-grade methanol for zooxanthellae and zoochlorellae, respectively). Samples were analyzed as described in Seavy and Muller-Parker (2002), using the equations of Jeffrey and Humphrey (1975) for zooxanthellae and Holden (1976) for zoochlorellae.
Carbon translocation and photosynthetic carbon budgets
The percentage of photosynthetically fixed 14C transferred from algae to anemone host during the winter months was determined from carbon translocation experiments; the incubation protocols of Engebretson and Muller-Parker (1999) were used to allow direct comparison of our winter values with their summer values. Zooxanthellate and zoochlorellate anemones (0.13 + 0.02 g; n = 8) were collected from Shannon Point Beach on 26 December 2004, and the experiment was conducted the next day. Anemones (n = 3 of each type) were incubated with 14C bicarbonate (2–6 µCi) for 1.5 h under light-saturating irradiance (400 µmol photons · m–2 · s–1) at 9 °C. A fourth anemone of each type served as a dark control. After incubation, anemones were rinsed thoroughly and resubmerged in 5 ml of filtered seawater for a 1.75-h period of dark phase before the algal and animal fractions were homogenized and isolated, as described above. Three 0.5-ml subsamples were taken from the homogenate (total symbiosis), combined supernatant (animal fraction), and resuspended pellet (algal fraction) from each anemone and pipetted into 7-ml plastic scintillation vials. Immediately after collection, samples were acidified with 0.3 ml of 6 mol l–1 HCl and placed on a shaker table overnight to remove un-incorporated 14C. Samples were then neutralized with the addition of 0.3 ml of 6 mol l–1 NaOH; 4 ml of EcoScint scintillation fluid was added; and the DPM of the homogenate, animal, and algal fractions was counted by the liquid scintillation counter.
Percent translocation of 14C from algae to anemone host was calculated by dividing the DPM of the animal fraction by the total homogenate DPM. Heterotrophic 14C fixation was accounted for by subtracting the calculated DPM of the dark control anemone samples from the corresponding anemone samples incubated in the light.
To calculate carbon budgets, summer and winter daily productivities of zooxanthellae and zoochlorellae were derived from the equation P = Pmax tanh ( · I · Pmax–1) using data from the average P-I curves of isolated algae for each season (summer: July 2004; winter: Dec. 2004 and Feb. 2005) and data from the average hourly irradiance levels (I) experienced during an average summer or winter day for 2004–2005 in Anacortes, Washington. Local values of sea-surface irradiance were obtained from data collected continuously by Shannon Point Marine Center using a LiCor 1400 datalogger and LI-190SA quantum sensor. Daily productivities, expressed on the basis of anemone protein biomass, were calculated for the average hourly sea-surface irradiance (100% of available light) and for 50%, 25%, 15%, 10%, 5%, 3%, and 1% of the average hourly sea-surface irradiance. This range of light levels was selected to encompass the possible ranges experienced by the algae in hospite because of light attenuation by seawater and anemone tissues, and also to account for possible differences in photosynthesis of symbionts in hospite and freshly isolated.
Stable isotopic signatures of C and N
Zooxanthellate, zoochlorellate, and nonsymbiotic anemones were collected from Shannon Point Beach on the collection dates listed previously, and also on 17 August 2004 and 19 July 2005. The day following collection, six zooxanthellate, six zoochlorellate, and five nonsymbiotic A. elegantissima were selected haphazardly for analysis. These anemones averaged 0.15 ± 0.03 g wet weight, with an oral disk diameter of 8.20 ± 0.70 mm (n = 100 ± SE). Each anemone was prodded to contract, forcing expulsion of undigested food particles, and was frozen at –70 °C until processing.
Frozen anemones were cut in half from oral to aboral end; one half serving as a sample of anemone tissue (nonsymbiotic anemones) or anemone tissue plus algal symbionts (zooxanthellate and zoochlorellate anemones). To obtain algae, the other half of each symbiotic anemone was ground in a 10-ml glass tissue homogenizer with a motorized Teflon pestle, and the algae were isolated using repeated centrifugation as described above. The final algal pellets were resuspended in less than 1 ml of deionized water in microfuge tubes and frozen at –70 °C before lyophilization. Lyophilized samples were ground to a fine powder and sent to the Washington State University stable isotope laboratory where they were analyzed for 13C and 15N isotope signatures by flow-through mass spectrometry. Values are reported in parts per thousand () relative to the Peedee belemnite (PDB) limestone and nitrogen in the atmosphere.
Statistical analyses
A two-way analysis of variance (ANOVA) was used to examine algal density, MI, and productivity. The factors examined were month of year (5 levels: July, Oct., Dec., Feb., Apr.) and algae (2 levels: zooxanthellae and zoochlorellae). One-way multivariate analysis of variance (MANOVA) was used to analyze changes in chlorophylls a and b for zoochlorellae and chlorophylls a and c for zooxanthellae over time. A one-way ANOVA was used to determine whether percent carbon translocation differed for zooxanthellate and zoochlorellate anemones.
All data were checked for the assumptions of equal variance and a normal distribution. Data that failed to meet the assumption of equal variance were natural-log or square-root transformed. When transformed data still failed to meet the assumption of equal variance or equality of covariance matrices, the severity of the violation was determined (Hartley's Fmax) and alpha adjusted to 0.01 (Underwood, 1981). Otherwise all data were analyzed at the significance level of 5%.
The statistical software package SPSS was used for all analyses. When a significant effect was found for month, a priori contrasts were used to determine if summer (July) values were significantly different from winter (Dec. and Feb.) values. If a significant month x alga interaction was found, simple main effect contrasts were used to determine the reason for the significant interaction. All other contrasts were done using Scheffé's S Test. The critical difference, 
(S), that contrast means must exceed to be declared significant was calculated manually.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDISCUSSIONLiterature Cited |
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Algal biomass parameters, pigments, and photosynthetic parameters
Overall, anemones collected for the productivity and algal biomass measurements averaged 0.17 ± 0.02 g wet weight, with an oral disk diameter of 7.9 ± 0.49 mm (n = 120, ± SE). There was no significant difference in the protein biomass of zooxanthellate and zoochlorellate anemones, which averaged 9.56 ± 0.95 mg protein and 8.72 ± 0.81 mg protein, respectively (n = 60, ± SE).
Zoochlorellate anemones had significantly higher algal densities than zooxanthellate individuals (Fig. 2; P < 0.001; sq. rt. transformed; Fmax= 11.06; adj. 0.01). Although month of collection had a significant effect on algal density (P < 0.001), a contrast comparing the pooled densities of zooxanthellae and zoochlorellae in the summer (July) and the winter (Dec. and Feb.) did not yield a significant difference. The significant effect of month was probably the result of the elevated density of zoochlorellae in December (Fig. 2); however, due to low power, an a posteriori contrast did not find this density significantly different from that of zoochlorellae in anemones collected in all other months ((S) = 27.57). Winter densities of zooxanthellae (Dec. and Feb.) also were not significantly different from April densities ((S) = 16.06).
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adj. 0.01). While chlorophyll c exhibited smaller seasonal changes than chlorophyll a (Fig. 3a), neither pigment showed a significant difference between summer and winter concentrations (chl a, P = 0.087; chl c, P = 0.511). Month of year also had a significant effect on chlorophyll a and b concentrations in zoochlorellae (P < 0.001, Pillai's trace; ln transformed; adj. 0.01). Both pigments followed the same seasonal trend, with highest concentrations of chlorophyll a and b during fall through winter (Oct., Dec., and Feb.; Fig. 3b). The concentrations of chlorophyll a and b were significantly higher in winter (Dec. and Feb.) than during July (P < 0.001; P = 0.002, respectively).
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), revealed that zooxanthellae and zoochlorellae exhibit similar photosynthetic efficiencies (P = 0.226; two-way ANOVA) with month of year having a significant effect (P < 0.001). The photosynthetic efficiency of both algae declined gradually from July 2004 to April 2005 and was lowest during April (Fig. 5a). The photosynthetic efficiency of both algae was about 2-fold higher in July than during December and February (P = 0.020).
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(S) = 1.00). Figure 6 compares the daily sea-surface irradiance levels (and 75%, 50%, 25%, 15%, 10%, and 5% of the average daily distributions of sea-surface irradiance) at the collection site during July 2004 and winter (Dec. 2004, Feb. 2005) with the Ik for zooxanthellae and zoochlorellae obtained from the P-I curves for these months. In the summer, light-saturated photosynthetic rates (at irradiance levels > Ik) may occur for more than 6 h each day at irradiance levels as low as 10% of ambient sea-surface levels (Fig. 6a). During winter, the comparison shows that Pmax for both algae may occur at irradiances 15% of ambient sea-surface for 4 h.
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A comparison of the potential benefits of hosting photosynthetic zooxanthellae and zoochlorellae must account for the carbon required for algal growth and respiration, with any extra fixed carbon potentially available to the anemone or to the alga for storage. Since zooxanthellae are larger and therefore contain 3 (summer) and 2 (winter) times the carbon content of zoochlorellae (Table 1), a dividing zooxanthella requires more carbon for growth. The amount of carbon (C) utilized for growth (Cµ) depends on algal MI and the duration of cytokinesis (td). While MI is measured easily and is 6 times higher for zoochlorellae (Table 1), td in hospite has not been measured directly. Using algal density and expulsion rates of A. elegantissima collected from the neighboring San Juan Islands in Washington State, McCloskey et al. (1996) estimated the td of zooxanthellae and zoochlorellae to be 28 and 69 h, respectively. Use of these estimates, with a much longer td (69 h) for zoochlorellae, results in similar C-specific growth (Cµ) for both symbionts during winter and summer (Table 1). When the Cµ is normalized for the respective density of zooxanthellae and zoochlorellae within the host anemone, the carbon required for algal growth in a zoochlorellate anemone is 4 times greater than that required in a zooxanthellate anemone (Table 1).
Although the daily productivity (P; pg C fixed · alga–1 · d–1) of a zooxanthella at 50% of the average hourly irradiance is over twice that of a zoochlorella during summer, the two algae exhibit comparable daily productivities during winter (Table 1). When daily productivity is expressed on the basis of the density of the alga in the anemone, the high biomass of zoochlorellae means that the productivity of zoochlorellate anemones is about 2 and 4 times that of zooxanthellate anemones during summer and winter, respectively (Table 1; Fig. 7).
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3% of sea-surface levels during summer, and for daily photon flux densities 15% of sea-surface levels during winter (Fig. 7a, b). When Cµ is subtracted from the daily productivity calculated for 50% of the average hourly sea-surface irradiance (P - Cµ), 7.2 (summer) and 0.9 (winter) micrograms of the daily C fixed per milligram protein of zooxanthellate anemone and 10.2 (summer) and 1.8 (winter) micrograms of the daily C fixed per milligram protein of zoochlorellate anemone is provided in excess of algal growth requirements (Table 1). Some of this carbon is used for algal respiration, and the rest is potentially available for translocation to the anemone. These calculations, based on short-term photosynthetic rates of freshly isolated symbionts, show that zoochlorellate anemones may be supplied with 30%–50% more photosynthetic carbon than zooxanthellate anemones at 50% sea-surface irradiance levels.
Symbiont contribution to anemone diet estimated by stable isotopes, delta13C and delta15N
Figure 8 shows the annual distributions of delta13C and delta15N values of nonsymbiotic anemone tissue, zooxanthellate and zoochlorellate anemone tissue, and isolated algal symbionts. Zooxanthellae and zoochlorellae show distinctly different seasonal patterns in their delta13C and delta15N values (Fig. 8a). This difference between symbionts is reflected in the delta values of symbiotic anemones, which differ from nonsymbiotic individuals. The delta13C and delta15N values of zooxanthellate and zoochlorellate anemones are intermediate between those of their respective symbionts and those of nonsymbiotic anemones.
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in nonsymbiotic anemones and 0.95 in symbiotic anemones. Nonsymbiotic anemone tissue had the highest delta13C values, followed by zooxanthellate anemone tissue, and then zoochlorellate anemone tissue (–16.27, –17.41, and –18.77, respectively; Table 2). Compared to the anemones, the zooxanthellae and zoochlorellae exhibited low delta13C and delta15N values (Fig. 8a; Table 2). The delta13C of zoochlorellae (–24.93) was substantially lower than that of zooxanthellae (–19.67) throughout the year (Table 2). Zooxanthellae did not exhibit a strong seasonal signal, whereas the delta13C values of zoochlorellae were more depleted during the winter and spring (Fig. 8a).
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, 11.34, and 10.30, respectively; Fig. 8; Table 2). The delta15N values of the isolated algae are much lower than values obtained from the symbiotic anemones (Fig. 8a). Although the mean delta15N of zooxanthellae (5.15) is greater than that of zoochlorellae (3.35) (Table 2), during most of the year the two algae had similar values (Fig. 8a). |
DISCUSSION |
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TOPAbstractIntroductionMaterials and MethodsResultsDISCUSSIONLiterature Cited |
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The density of zoochlorellae in the anemone host was almost 4 times that of zooxanthellae. Although slightly greater, this difference in symbiont densities is consistent with that observed in previous studies with anemones collected from the same region, with densities of zoochlorellae from 1.3, 3, and 2 times higher than densities of zooxanthellae (studies by, respectively, McCloskey et al., 1996; Engebretson and Muller-Parker, 1999; Verde and McCloskey, 2002). However, because zoochlorellae are smaller, the proportional biomass of symbionts in a host anemone may be similar (Verde and McCloskey, 2002) or lower than that of zooxanthellae (Table 1). Although some seasonal fluctuation in algal density was observed, population densities of both algal symbionts in A. elegantissima were similar between summer and winter, in spite of large differences in temperature and irradiance. Although Verde and McCloskey (2007) found a significant seasonal change in the densities of zooxanthellae and zoochlorellae in A. elegantissima, two other field studies (Dingman, 1998; Farrant et al., 1987) found that densities of zooxanthellae in A. elegantissima and Capnella gaboensis, a temperate soft coral, are constant and do not differ significantly between winter and summer. The demonstration of similar symbiont populations in summer and winter for zoochlorellae in addition to zooxanthellae supports the hypothesis of Muller-Parker and Davy (2001) that temperate algal symbioses exhibit less fluctuation in algal densities than tropical ones.
Consistent population densities of symbionts may result from balanced growth of the algae and of the host during summer and winter, or may result from seasonal adjustments in algal expulsion rates, since A. elegantissima controls symbiont density primarily by expelling excess algae (McCloskey et al., 1996). Algal expulsion is related to the percentage of algae dividing (MI). Although the MI of zooxanthellae and zoochlorellae show similar seasonal patterns, only zooxanthellae exhibited a significant seasonal difference, with a higher percentage of cells dividing in July than in December and February. Verde and McCloskey (2007) obtained similar results. The elevated MI of zooxanthellae during the summer months may result from a greater supply of photosynthetic carbon available for algal growth during this season, as well as from the high thermal and light tolerances of zooxanthellae (Saunders and Muller-Parker, 1997; Engebretson and Muller-Parker, 1999).
During all months, the percentage of zoochlorellae dividing was about 6 times greater than that of zooxanthellae (20% and 3%). The higher MI for zoochlorellae has been reported previously, but the values of MI for zooxanthellae and zoochlorellae differ among studies. McCloskey et al. (1996) report MI values of 5.64% and 1.68%, and Verde and McCloskey (1996) report values of 7.34% and 0.34%, for zoochlorellae and zooxanthellae respectively. Values comparable to those observed in this study were obtained for zooxanthellae isolated from A. elegantissima in California (4.69%) and Washington (2.88%; Wilkerson et al., 1983), and for zoochlorellae isolated from A. xanthogrammica in British Columbia, Canada (20%; O'Brien and Wyttenbach, 1980). The difference between the MI of algae in the anemones collected from the same area by Verde and McCloskey and this study may be related to the size and developmental stage of the host. Smith (1986) found that the photosynthetic capacity and MI of zooxanthellae in the tropical juvenile anemone Aulactinia stelloides were double those of zooxanthellae in adult anemones, and that algal densities were higher in juveniles than in adults. Anemones in the study by McCloskey et al. (1996) were 1.5 to 1.0 g wet weight compared to about 0.2 g wet weight in this study. Since younger and smaller invertebrates have higher nutrient excretion rates (Smith, 1986), the elevated MI and population densities observed in this study might be due to the greater availability of inorganic nutrients for the symbionts in the small anemones.
The increase in chlorophyll a and b concentrations of zoochlorellae during winter is consistent with photoacclimatization to the seasonal reduction in irradiance and with the results obtained by Verde and McCloskey (2007). Lack of seasonal variation in the chlorophyll content of zooxanthellae is supported by observations of constant chlorophyll a concentrations in zooxanthellae isolated from the temperate soft coral Capnella gaboensis (Farrant et al., 1987), and by two laboratory studies with A. elegantissima that found zooxanthellae maintained constant chlorophyll content whereas zoochlorellae increased with decreasing irradiance (Verde and McCloskey, 2001, 2002). These findings are in sharp contrast with those for tropical associations, in which anemones and corals exhibit distinct seasonal fluctuations in the density, photosynthetic capacity, and pigment concentrations of zooxanthellae (Muller-Parker, 1987; Fagoonee et al., 1999; Fitt et al., 2000; Warner et al., 2002).
The light-saturated productivity (Pmax cell) and photosynthetic efficiency () of both zooxanthellae and zoochlorellae are significantly higher in July than during December and February. Verde and McCloskey (2007) observed similar trends, with zooxanthellate and zoochlorellate anemones exhibiting significantly higher productivity during the spring and summer months. The smaller seasonal decline in the Pmax of zoochlorellae from summer to winter might result from depressed photosynthesis in the summer that is alleviated by a return to cool, low light conditions favoring photosynthesis by this alga (Verde and McCloskey, 2001, 2002). Although the trend of decreasing photosynthetic efficiency with seasonal reduction in light appears counterintuitive, the actual amount of light received by the symbionts is unknown and may be affected by anemone behaviors that may change seasonally, including orientation and phototactic behavior (Pearse, 1974), attachment of gravel/shell debris, and expansion/retraction of tentacles (Shick and Dykens, 1984), as well as differences in the distribution of symbionts within different body regions (Dingman, 1998). Additionally, a lack of seasonal adjustment in photosynthetic efficiency might indicate that the symbionts utilize host carbon to satisfy their metabolic requirements during the winter.
During the high-light and high-temperature conditions of July, the productivity of zooxanthellae was considerably elevated over that of zoochlorellae, with Pmax values of 2 and 0.8 pg C · cell–1 · h–1, respectively. This trend of elevated productivity of isolated zooxanthellae is consistent with that observed in other summer studies (2 and 3 pg C · zooxanthella–1 · h–1 and 0.5 and 1.25 pg C fixed · zoochlorella–1 · h–1; McFarland and Muller-Parker, 1993; Augustine and Muller-Parker, 1998, respectively). Similarly, the productivity of zooxanthellate A. elegantissima during the summer is much higher than that of zoochlorellate individuals, especially at irradiances exceeding 100 µmol photons · m–2 · s–1 (Verde and McCloskey, 2002; Secord and Muller-Parker, 2005). While high light-saturated productivity during the summer implies that a greater amount of photosynthate may be available for zooxanthellate anemones during this season, the Pmax (cell) of zooxanthellae and zoochlorellae was greatly reduced and similar during the rest of the year (fall, winter, spring), suggesting that any advantage is limited to summer conditions.
Algal contribution to host anemone diet
Carbon translocation rates, carbon budgets, and seasonal fluctuations in C and N stable isotopes were examined and compared to determine the possible nutritional benefits to the anemone of hosting one symbiont over the other. The short-term measures based on the photosynthetic contributions of the two algae do not correlate with the stable isotopic composition of the host. Results of the 14C method indicate that during the winter the percentage of carbon translocated from symbiont to host tissues was similar for zooxanthellate and zoochlorellate anemones. The average value of 48% translocated C is consistent with that obtained in other 14C studies of carbon translocation in A. elegantissima in California (Trench, 1971a). Although the percentage of carbon translocated during winter is higher than that observed during summer (30%; Engebretson and Muller-Parker, 1999), the actual amount of carbon translocated during the summer will be much greater since both algae are more productive during the summer.
Davy et al. (1996) modeled carbon budgets for temperate subtidal symbiotic anemones during summer and concluded that most temperate host anemones and zoanthids need to feed heterotrophically to obtain their carbon requirements at other times of the year. In this study we compared the seasonal productivity of isolated algal symbionts and constructed carbon budgets for them on an anemone biomass basis for winter and summer light conditions and for a series of light-reduction scenarios in each season because the actual light levels received by the algae in the host are unknown. We used 50% light reduction as the basis for comparing carbon budgets of zooxanthellae and zoochlorellae, since productivity was measured using isolated algae and Davies (1991) showed that daylight variations (e.g., 50% light reduction) have a significant effect on the energy budgets of shallow-water corals. Comparing the daily productivity and C-specific growth, C budgets show that the pools of C available for translocation are slightly larger for zoochlorellate anemones than for zooxanthellate anemones during both summer and winter, and that fixed C exceeds Cµ for most light levels (Fig. 7).
The C budgets suggest a low potential for the algae to be carbon parasites on their host, and the greater potential availability of fixed C for zoochlorellate anemones contradicts the overall conclusions from C budgets constructed for A. elegantissima by Verde and McCloskey (1996, 2001, 2002, 2007) that zooxanthellae provide a significantly greater amount of fixed C to the host. Although we used the same estimates of td as Verde and McCloskey, we determined the productivity of isolated zooxanthellae and zoochlorellae in the laboratory, constructing P-I curves for freshly isolated symbionts and using the P-I curves, daily irradiance levels, and algal density to derive productivity on an anemone basis. Whereas Verde and McCloskey measured the productivity of substantially larger (175 mg protein) whole anemones exposed to natural levels of sea-surface irradiance, our small anemones (10 mg protein), had a 4-fold higher density of zoochlorellae than of zooxanthellae. This large difference in algal density resulted in a much greater contribution of photosynthetic carbon to the host calculated for zoochlorellae than for zooxanthellae. Additionally, possible differences in the distribution of zooxanthellae and zoochlorellae in different body regions or changes in anemone posture affecting light levels received in hospite may also contribute to the differences between our results and those of Verde and McCloskey (2007).
Stable isotopic comparisons of delta13C and delta15N signatures of symbionts and hosts were used as a proxy to examine the potential carbon contributions of zooxanthellae and zoochlorellae to their hosts. In contrast with the carbon budgets derived from short-term measures of photosynthetic rates and estimated growth rates, stable isotopes serve as a long-term index of carbon assimilation (Muscatine et al., 1989) and allow comparisons with nonsymbiotic counterparts. Zooxanthellae and zoochlorellae exhibit distinctly different patterns in delta13C and delta15N values (Fig. 8a). This difference in the signatures of symbionts is repeated in the delta values of symbiotic anemones, which are different from those of nonsymbiotic individuals. The delta13C and delta15N values of zooxanthellate and zoochlorellate anemones are intermediate between those of nonsymbiotic anemones and their respective symbionts, indicating that both external food sources and translocated products contribute to the diet of the host anemone.
Nonsymbiotic individuals of A. elegantissima were most enriched in delta13C and delta15N. The delta13C values were similar to values obtained for Puget Sound estuarine and marine littoral epibenthic crustaceans (–16.2 ± 2.5; Simenstad and Wissmar, 1985), which are a likely food source (Sebens, 1981a). The carbon and nitrogen isotopic signatures of nonsymbiotic, zooxanthellate, and zoochlorellate anemones did not vary seasonally (Fig. 8). Compared to the anemones, both zooxanthellae and zoochlorellae exhibited low delta13C and delta15N values, which varied seasonally. The intermediate delta values of symbiotic anemones—between those of their respective symbionts and nonsymbiotic anemones—combined with a lack of a seasonal signal in anemone tissues, may indicate that feeding rates increase in proportion to possible increases in carbon translocation during the summer, so that the relative contribution of autotrophic inputs from the algae remains the same year-round.
Although zooxanthellae and zoochlorellae are both intracellular photosynthetic symbionts, the delta13C values of zooxanthellae are more similar to those of Puget Sound phytoplankton (–20.3 ± 1.4; Carpenter and Peterson, 1989) while the delta13C values of zoochlorellae are on average 5 lower. The large difference in delta13C values of zooxanthellae and zoochlorellae may result from differences in carbon fixation and photosynthetic pathways (Wong and Sackett, 1978). Zooxanthellae possess Form II Rubisco (Whitney et al., 1995); discrimination against 13CO2 relative to 12CO2 by Form II Rubisco is less than that observed in plants and green algae with Form I Rubisco. Alternatively, the two symbionts may primarily utilize different carbon sources. Due to the 4-fold higher density and higher growth rate of zoochlorellae in the host, this symbiont may be forced to rely to a greater extent on host-derived carbon sources (e.g., depleted CO2 from animal respiration or depleted host organic carbon via heterotrophic uptake), whereas zooxanthellae may utilize enriched CO2 diffusing into the animal host. In a survey of tropical corals sampled at various depths ranging from 1 m to 50 m in Jamaica and Eilat, Muscatine et al. (1989) obtained delta13C values for zooxanthellae ranging from –9.63 to –19.21. The zooxanthellae in A. elegantissima have delta13C values similar to the lowest values, obtained for the algal symbionts from large-polyp corals sampled from deep waters.
Zoochlorellae show a larger seasonal variation than zooxanthellae in delta13C, with the most depleted values occurring during spring and late winter. Similar seasonal fluctuations have been observed in maple leaves, marine eelgrass, macroalgae, and phytoplankton (Lowdon and Dyck, 1974; Stephenson et al., 1984; Simenstad and Wissmar, 1985; Goering et al., 1990). Possible causes of seasonal fluctuations in 13C include seasonal shifts in the isotopic composition of carbon sources, differential storage of isotopically different biochemical components (i.e., depleted lipids vs. enriched amino acids; Stephenson et al., 1984), and changes in isotopic fractionation due to temperature (Sackett et al., 1965). The observed seasonal trend in delta13C of zoochlorellae may be due to their greater C requirements for growth during winter, resulting in utilization of the internal CO2 pool faster than it can be replenished and thus lower fractionation (Swart et al., 2005).
The seasonal variation observed in the delta15N signatures of symbionts may be explained by a shift in nitrogen source or a change in the alga's nitrogen requirements. The very low delta values in zoochlorellae during February (similar to that of atmospheric nitrogen, 0.00) suggest that zoochlorellae utilize more of the isotopically light N waste metabolites (Adams and Sterner, 2000) excreted by A. elegantissima during the winter, while zooxanthellae may utilize an enriched external source such as nitrate.
The intermediate delta13C and delta15N values of zooxanthellate and zoochlorellate anemones indicate that symbiotic anemones receive nutrition from both external (heterotrophic) and internal (translocated algal products) sources; however, the delta values of the symbiotic anemones are more similar to those of the nonsymbiotic anemones than they are to those of their respective symbionts. A combination of the enriched state of zooxanthellate and zoochlorellate anemones and the lack of seasonal variation in these and nonsymbiotic anemones suggest that A. elegantissima relies primarily on external food sources, regardless of symbiotic condition.
Although A. elegantissima derives most of its nutrition from heterotrophic sources, stable isotope data suggest that in terms of carbon, zooxanthellae may be the more favorable symbionts for the anemone to host. Zooxanthellate anemone tissue was only 2 (13C) and 5 (15N) enriched over zooxanthellae, while zoochlorellate anemone tissue was 6 (13C) and 8 (15N) enriched over zoochlorellae. This greater disparity in the delta13C and delta15N values of zoochlorellate anemones and zoochlorellae, combined with the elevated delta15N signature of zoochlorellate anemones (1 higher than that of zooxanthellate anemones), suggests that more of the carbon fixed by zooxanthellae is translocated to the host in zooxanthellate anemones; zoochlorellate anemones receive less nutrition from their symbionts and rely to a greater extent on external food sources.
Although stable isotope data indicate that zooxanthellae may be "better" symbionts for nutritional purposes—supporting the results of Verde and McCloskey (1996, 2001, 2002, 2007) that zooxanthellate anemones receive more translocated carbon than zoochlorellate individuals—this conclusion contradicts that based on the C budgets, which showed that zoochlorellate anemones have slightly larger pools of C available for translocation during both summer and winter, and also contradicts the similar percent values for carbon translocation obtained for both zooxanthellate and zoochlorellate anemones (Engebretson and Muller-Parker, 1999). There are several possible reasons for this discrepancy. The productivity measurements were short-term, using isolated symbionts. Since zooxanthellae may undergo changes after removal from symbiosis (Trench, 1971b) and are also exposed directly to seawater, it is possible that the physiological performance of zooxanthellae and zoochlorellae measured in vitro does not reflect carbon fixation rates in hospite. Our aim was to directly compare the photosynthetic performance of these two algae on an individual cell basis, under the same conditions. Therefore, we used dilute (unshaded) suspensions of algae, comparing their photosynthetic rates under equivalent conditions of light and carbon dioxide supply to construct the P-I curves. Since the symbionts were isolated from the host using the same procedures, any effects on isolation from the host should apply to both zooxanthellae and zoochlorellae. Another possible reason for the discrepancy in carbon budgets constructed for intact symbioses and from photosynthetic measurements with isolated algae is that the distribution of the two symbionts in the animal host is not the same; zoochlorellae may be more abundant in the body column, and zooxanthellae more abundant in the tentacles (Dingman, 1998). Because these distributions may change seasonally and with anemone size, the photosynthetic contributions of the algae in vivo may differ accordingly. Since the duration of cytokinesis (td) has not been measured directly and has a substantial influence on the outcome of the carbon calculations, actual td measurements for zooxanthellae and zoochlorellae in A. elegantissima during summer and winter are also needed to resolve carbon budgets and any conclusions concerning the possible nutritional benefits of the two symbionts.
For tropical corals, stable isotopes are good predictors of the relative contributions of zooxanthellae to the host (Muscatine et al., 1989; Reynaud et al., 2002). For the temperate Anthopleura elegantissima, either the photosynthetic contributions are not retained by the anemones (lost as gametes, dissolved and particulate organic carbon), or translocated carbon composes a relatively small portion of the host anemone's diet, with the host deriving most of its nutrition via heterotrophy. Perhaps the abundant supply and utilization of external food sources allow the anemone host the flexibility of associating with both zoochlorellae and zooxanthellae, and enable the symbiosis to persist during unfavorable conditions when one or both algae may become carbon parasites. This flexibility would also allow the alga best able to grow under a particular set of temperature and light conditions to dominate in an anemone host. Alternatively, since anemones may receive the most translocated C during the summer months when planktonic food is also plentiful, the additional carbon could be allocated to asexual reproduction or gonad production and increased sexual output of symbiotic individuals (Muller-Parker and Davy, 2001). It is also possible that symbionts translocate some product of value to the host other than carbon. Although zooxanthellae translocate primarily glycerol, a carbon-rich compound (Trench, 1971a), zoochlorellae may provide the host anemone with N-rich amino acids (Minnick, 1984, cited in Verde and McCloskey, 2007).
The long-term advantages (fitness consequences) for the host of associating with different taxa of symbionts are unknown, and await studies that measure the relative contributions of each algal symbiont to the growth and reproductive condition of the host sea anemone. Field studies, such as those conducted by Sebens (1981a, b, 1982) for zooxanthellate A. elegantissima, are needed to compare the growth, survival, and sexual and asexual reproduction of natural populations of zoochlorellate, nonsymbiotic, and zooxanthellate A. elegantissima to determine whether there is an ecological advantage for these temperate anemones to be symbiotic—with zooxanthellae, with zoochlorellae, or with both.
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TOPAbstractIntroductionMaterials and MethodsResultsDISCUSSIONLiterature Cited |
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-proteobacteria. Proc. R. Soc. Lond. B. Biol. Sci. 259:271–275.[Medline]
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