Proceedings 9th International Coral Reef Symposium, Bali, Indonesia 23-27 October 2000, Vol. 1
Phosphorus uptake and allocation by Symbiodinium bermudense isolated from Aiptasia pallida R. Kelty1 and F. Lipschultz2 ABSTRACT The primary objective of this study was to compare the uptake, release , and allocation of dissolved inorganic phosphate in freshly isolated versus 24-hour isolated zooxanthellae. Symbiodinium bermudense isolated from Aiptasia pallida for 24 h incorporated 75 times more counts of 33P[orthophosphate] per cell than freshly isolated algae. 24-hour isolated cells excreted roughly twice as much labeled phosphorus as did freshly isolated algae. 33P entering the cells was incorporated into metabolic intermediates (ATP and oligonucleotides) and then redistributed into structural compounds. The distribution of new phosphate was similar for the 24-hour isolated and freshly isolated algae, but the 24-hour isolated algae transferred 33P from intermediate to structural pools much more quickly than freshly isolated algae. The ability of S. bermudense to take up 75 times more phosphate per cell after being isolated from the host for 24 hours indicates that nutrient history and uptake are tightly coupled; this would allow algal cells to rapidly increase uptake to take advantage of transient pulses of nutrients.
Keywords Zooxanthellae, Nutrient uptake, Symbiosis Introduction Phosphate uptake rates for cnidarians are usually calculated as the rate of depletion from the media (Pomeroy and Kuenzles 1969), Pomeroy et al. 1974, D’Elia 1977, Cates and McLaughlin 1979). However, because true uptake rates can be masked by simultaneous release, phosphate uptake is not accurately estimated by measuring its decrease in the water. More recent studies have quantified phosphate uptake as incorporation of radiolabeled-PO4-3 in corals and isolated zooxanthellae (D’Elia 1977, Muller-Parker et al. 1990, Sorokin 1989, Jackson and Yellowlees 1990). These studies have assessed the overall result of membrane transport of inorganic orthophosphate (Pi), but do not distinguish between assimilation into soluble phosphorus compounds and incorporation into macromolecules. No studies have specifically measured phosphorus allocation within the different metabolic compart-
ments. Studies on nutrient uptake and excretion by free-living and isolated zooxanthellae have contrasting results and have led the authors to draw different conclusions. Amphidinium carterae, Amphidinium klebsii, and Gymnodinium microadriaticum all followed Michaelis-Menten kinetics for phosphate uptake. However, Deane and O’Brien (1981) reported Michaelis constants (Km) ranging from 0.005 to 0.016 µM while Jackson and Yellowlees (1990) measured Km of 14 µM for Gymnodinium sp. Furthermore, Jackson and Yellowlees (1990) found that uptake was enhanced by light, whereas Deane and O’Brien (1981) observed greater uptake in the dark than the light. The contrasting results of these studies indicate that there is a need for more work in this area. A better understanding of the phosphorus uptake dynamics of isolated algae is needed to understand how the host and algal symbiont may relate with respect to this nutrient in the intact association. Methods Uptake of 33P[orthophosphate] by freshly isolated (< 1 h: FIZ) and recently isolated zooxanthellae (24 h: RIZ) and its
1 2
incorporation into a suite of metabolic intermediates was assessed in pulse experiments with zooxanthellae isolated from the tropical sea anemone Aiptasia pallida. Symbiotic A. pallida were collected from Walshingham Pond, Bermuda and maintained in a flow-through sea water system for four weeks. Isolation of Algae Anemones (4-8 mm basal diameter) were homogenized by sonication with a ‘Vibra Cell’ (Sonics and Materials) 2 mm microtip in chilled filtered (Whatman GF/E) surface Sargasso Seawater (FSSW) at 60% power until there were no tissue clumps. Examination of sonicated zooxanthellae under an epi-fluorescent microscope determined that this treatment did not disrupt algal cells. The homogenate was centrifuged at 3,200 rpm for four minutes; the animal supernatant was discarded and the algal pellet was resuspended in 15 ml FSSW. This procedure was repeated five times to produce a pellet of zooxanthellae with minimal amounts of animal tissue. Zooxanthellae density was determined by six haemocytometer counts and was 1.1 – 1.8 x 106 cells/ml. Half of the FIZ were immediately given a phosphorus spike and the remaining zooxanthellae were maintained in the incubator in FSSW for use after 24 h. Preparation of Freshly Isolated Zooxanthellae The suspension of FIZ was immediately transferred to an acid-washed glass flask and brought up to 25 ml with FSSW. The flask was maintained in an incubator at 25 °C with a 12:12 L:D photoperiod and irradiance levels of ~ 80 µmol m-2s-1. A stir bar provided continuous stirring. Preparation of Recently Isolated Zooxanthellae The algal suspension was filtered (20 µm), resuspended in 10 ml FSSW, and maintained in the incubator with continuous stirring provided by a stir bar. After 24 hours the algae were transferred to a glass flask and brought up to 25 ml with FSSW and exposed to the tracer.
National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Studies, 1305 East-West Highway, Silver Spring, MD 20910,
[email protected], fax – 301-713-4353, phone – 301-713-3020. Bermuda Biological Station for Research, St. George's GE01, Bermuda,
[email protected], fax – 441-297-8143, phone – 441-297-1880.
Biochemical fractionation One 1600 µl sample was removed for analysis at 10 time points up to 72 hours after 33P addition. Three 50µl aliquots of the algae were taken and quantified for 33P incorporation and total phosphorus in the cells. The remainder of each sample was then split into three replicates which were fractionated into metabolic intermediates (ATP and oligonucleotides), nucleic acids, lipids, and proteins following a protocol modified from Roberts et al. (1965) and Szmant-Froelich (1981). Cold 10% trichloroacetic acid (TCA) was added to each replicate in a 1:2 ratio, vortexed, and frozen overnight. These samples were then thawed and centrifuged at 4,000 rpm for five minutes; the cold-TCA soluble supernatant was taken as the metabolic intermediate fraction. The pellet was resuspended in 95% EtOH and heated to 45 °C for 45 minutes. The sample was centrifuged and the supernatant, which contained the lipid fraction, was removed and counted. Next, the pellet was resuspended in 5% TCA and heated to 100°C for 30 minutes. When centrifuged again, the resulting supernatant contained the nucleic acids and the pellet contained the protein. All samples were placed directly into 7 ml glass scintillation vials and counted in Packard Ultima Gold Ltd. cocktail with a Packard TriCarb scintillation counter. DPM were corrected for radioactive decay. Results
Both 24-hour isolated and freshly isolated zooxanthellae rapidly took up phosphate (Table 1). An average of 91% of the label in the total algal sample was recovered in the macromolecular fractions. 24-hour isolated zooxanthellae took up and retained more phosphate than FIZ. Within 30 minutes of the 33P spike, 24 -hour
Table 1. Distribution of total 33P in major constituents of Symbiodinium bermudense after 21 hours. 24-hour isolated zooxanthellae DPM % 568,184 1,419,416 320,252 23 236,734 17 809,790 57 19,002 1
medium total cell intermediates lipids nucleic acids proteins
Freshly isolated zooxanthellae DPM % 206,382 693,198 200,790 29 36,460 5 426,982 62 17,962 3
From the end of the pulse until the termination of the experiment 48 hours later, the number of counts in the cells remained relatively constant. FIZ maintained an average of 0.165 (+ 0.003 SE) DPM per zooxanthella. In contrast, after the initial drop between 2.5 and 3.5 hours, radioactivity in RIZ remained at about 0.700 (+ 0.004 SE) DPM per zooxanthella. Labeled phosphorus was released into the water by both 24-hour isolated and freshly isolated algae (Fig. 1). By the end of the experiment, the RIZ had released seven times more newly incorporated phosphate into the water, however, they also took up more phosphorus during the pulse. When release is expressed as a percentage of the total counts, RIZ algae excreted roughly twice as much labeled phosphorus as did freshly isolated algae. 3.0
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Uptake was determined by inoculating 10 ml of the algal suspensions with 40 µl of 1 mM K2PO4 and 150 µCi 33 P-orthophosphate (carrier free) for a final concentration of 4 µM K2PO4. The cells were maintained at 25°C at a light intensity of ~ 80 µmol m-2s-1and were stirred constantly. After three hours, the algae were washed and resuspended in FSSW. Unincorporated phosphate was removed by spinning down the algae (3,600 rpm for two minutes), pouring off the supernatant, and resuspending in non-enriched water. The algae were then rinsed five times in 5 ml FSSW (all spins for four minutes at 1,100 – 1,800 rpm). The final pellet was resuspended in 10 ml LNFSW, sonicated, returned to the rinsed flask, and brought back up to the original volume (minus the volume of samples) with FSSW. The concentration and isotopic content of Pi in the incubation water were measured immediately, three, and 72 hours after the initial spike. The radioactivity incorporated into the cells was determined by Cherenkov radiation measured on a Packard TriCarb scintillation counter in Packard Ultima Gold Ltd. cocktail. DPM were corrected for radioactive decay.
isolated algae had 75 times more counts per zooxanthella than FIZ (Fig. 1). Phosphate uptake by 24-hour isolated algae leveled off after less than one hour. Thereafter, no significant increase in uptake of radioactivity was observed and radioactivity declined slightly between 1.5 and 2.5 hours (Fig. 1). In contrast, 33P incorporation in FIZ increased throughout the pulse and had not attained apparent steady-state values after three hours. Furthermore, radioactivity in freshly isolated zooxanthellae continued to increase for 0.5 hours after the incubation water was replaced with FSSW (Fig. 1).
DPM per cell
Measurement of 33P[orthophosphate] uptake
time (hours)
Fig. 1. Counts per zooxanthella incorporated by 24-h isolated (solid squares) and freshly isolated algae (solid triangles) and counts per cell released into incubation water (hollow squares and triangles). Error bars show + one standard error.
Allocation Phosphate incorporation was biphasic. Upon entering the cell, 33P was first incorporated into metabolic intermediates, probably ATP and oligonucleotides, and then redistributed into structural compounds (lipids, nucleic acids, and proteins). Thirty minutes after receiving the phosphate pulse, the majority (54-65%) of the radioactivity was in metabolic intermediates. Subsequently however, radioactivity in the intermediate pool declined. This decline was matched by an increase in labeled nucleic acids and, to a lesser extent, lipids and proteins (Table 2). Approximately twenty-four hours into the incubation, about 60% of the newly incorporated phosphate was incorporated into nucleic acids. However the rates at which 24-hour isolated and freshly isolated algae incorporated new phosphate into nucleic acids differed. Incorporation into nucleic acids in the 24-hour isolated algae leveled off between four and 10 hours, then roughly doubled between 11 and 15 hours; the freshly isolated algae maintained a steady rate of incorporation for about 24 hours. Table 2. Distribution of counts (%) in macromolecular pools of freshly isolated and 24-hour Symbiodinium bermudense. N = 2 flasks for each sampling time.
Although the pathway of phosphate assimilation from intermediates to structural compounds was the same for RIZ and FIZ, allocation of 33P within the structural pools differed. RIZ moved new phosphate from intermediate to structural pools more quickly than freshly isolated algae (Fig. 2). Incorporation of new phosphate into structural compounds by freshly isolated algae lagged behind the availability of labeled intermediates (Fig. 2). Discussion The ability of S. bermudense to take up 75 times more phosphate per cell after being isolated from the host for 24 hours indicates that nutrient history and uptake are tightly coupled. Phosphate concentrations available to the zooxanthellae in situ are higher than phosphate concentrations in FSSW, and the cells respond rapidly to the change in nutrient availability. The ability to rapidly increase uptake capability would allow the algae to take advantage of transient pulses of nutrients. 100 90 80 70
24-hour Isolated Zooxanthellae time (hr) 0.5 1.5 2.5 3.5 7 11 15 21 27 48
intermediates 53.62 + 9.1 30.44 +7.6 33.85 +1.1 37.42 + 4.6 31.92 + 15.9 26.35 + 3.4 24.38 +1.3 22.66 + 5.4 20.35 + 0.4 32.39 + 2.1
lipids 12.10 + 5.0 14.74 + 0.9 15.65 + 6.2 13.97 + 3.9 12.60 + 0.3 12.58 + 1.1 19.59 + 6.7 16.75 + 1.9 15.06 + 3.0 10.81 + 1.4
60
nucleic acids 35.15 + 4.1 40.63 + 5.9 44.63 + 2.9 35.08 + 5.8 37.44 + 3.6 34.39 + 7.6 66.22 + 3.1 57.30 + 9.5 58.06 + 3.7 57.83 + 3.0
0.85 + 22.5 0.82 + 15.9 1.08 + 12.0 0.88 + 2.8 1.84 + 10.1 1.75 + 8.7 1.08 + 14.9 1.34 + 9.5 1.20 + 2.8 1.37 + 11.6
Freshly Isolated Zooxanthellae lipids
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proteins
time (hr) 0.5 1.5 2.5 3.5 7 11 15 21 27
intermediates
nucleic acids
proteins
48 72
39.61 + 4.9 5.41 + 4.1 58.37 + 2.05 2.14 + 2.6 40.99 + 5.9 11.65 + 8.6 51.04 + 4.0 2.34 + 5.8
63.83 + 17.1 6.02 + 3.6 7.72 + 9.4 2.95 + 13.9 48.32 + 18.0 10.40 + 25.3 9.59 + 9.0 3.99 + 10.2 54.12 + 9.3 10.50 + 22.8 14.66 + 7.6 29.15 + 5.9 52.88 + 15.7 10.20 + 7.2 17.68 + 5.8 18.77 + 11.2 41.55 + 17.1 9.10 + 2.9 40.12 + 9.8 10.98 + 17.3 38.25 + 2.2 4.76 + 1.4 50.69 + 11.2 5.90 + 1.0 28.80 + 3.2 5.23 + 6.6 61.25 + 6.2 2.58 + 6.5 27.26 + 7.6 6.12 + 7.9 63.81 + 8.4 2.69 + 8.2
40 30 20 10 0
10
20 time (hours)
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Fig. 2. Percent of counts incorporated into intermediates (open/dashed) and macromolecular compounds (solid) of freshly isolated (triangles) and 24-hour isolated zooxanthellae (squares). Error bars show + one standard error. These results are in agreement with those obtained in a study of phosphate uptake in Gymbiodinium microadriaticum isolated from the clam Tridacna maxima (Deane and O’Brien 1981). As in our study, isolated algae readily took up and had a high affinity for [32P]orthophosphate. 32P uptake by freshly isolated algae was not significantly different from uptake by zooxanthellae cultured in T. maxima haemolymph, which has phosphate concentrations of 0.05 µM. Algae in host haemolymph had a Km of 0.005 µM P and a Vmax of 0.009 pmol P/105 cells/10 minutes. Km and Vmax values for cultured algae were 0.007 µM P and 0.011 pmol P/105 cells/10 minutes respectively (Deane and O’Brien 1981). Because the phosphate concentrations in the media were the same, phosphate uptake characteristics did not change over time. Intracellular P pools: Movement from intermediates to macromolecular pools
24-hour isolated and freshly isolated algae differed in their assimilation of phosphate into metabolic intermediates and macromolecular compounds. In freshly isolated algae, incorporation of new phosphate into structural compounds lagged behind the availability of low molecular weight intermediates (LWM) (Fig. 2). Accumulation of ATP and metabolic intermediates during the first half hour, and again between four and seven hours, indicates that Pi was assimilated into LWM more quickly than it could go into macromolecules (Fig. 2). An important ecological implication of this is that Pi is taken up more rapidly than required for growth. After uptake meets total daily requirements, excess P can be stored (Fujita et al. 1988). Furthermore, the capability for rapid uptake would allow algae to take advantage of transient phosphorus pulses. Short term phosphate uptake in phosphorus-starved algae can be up to 100 times higher than the maximal growth rate (Harrison et al. 1989). Surge uptake also increases utilization of very low concentrations of nutrients (Parslow et al. 1985). RIZ immediately began incorporating new phosphate into structural compounds and did not experience a similar lag (Fig. 2). Assimilation of intermediates into macromolecules by RIZ increased between 0.5 and 1.5 hours then stabilized (Table 2). The decrease in metabolic intermediates between 0.5 and 1.5 hours indicates that Pi was incorporated into macromolecules more rapidly than it was transported into the cells. The observed decrease in uptake once the assimilatory process becomes active (after 1.5 hours) supports the notion of feedback inhibition (Fujita et al. 1988). The high level of newly incorporated phosphate released by the RIZ cells suggests that they had become N-limited; phosphate taken up could not be used for growth. Allocation within macromolecular pools The rapid assimilation into nucleic acids (Table 2) indicates that the algae grew in response to the phosphorus pulse. Rhee (1973) has shown that polyphosphates regulate the growth rate of free-living Scenedesmus sp. Increased phosphorus availability allowed in vitro zooxanthellae isolated from the clam Tridacna gigas to maintain a higher mean specific growth rate (Belda et al. 1993). 24-hour isolated zooxanthellae allocated more freshly incorporated phosphate to lipids (about 15%) than the FIZ (about 8%) (Table 2). Phospholipids are a primary form of phosphate storage (Cembella et al. 1984). The comparatively modest increase in the lipid pool of the freshly isolated algae may indicate that they are using the newly incorporated phosphate for growth or turnover rather than storage. This is supported by the high levels of radioactivity in the nucleic acid pool of freshly isolated algae (Table 2). Acknowledgements This work was partially funded by the American Museum of Natural History, PADI, and the Munson Foundation. It is contribution #1606 of the Bermuda Biological Station for Research.
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Proceedings 9th International Coral Reef Symposium Bali, Indonesia 23-27 Oktober 2000
