Cellular and ultrastructural changes in the endoderm of the temperate sea anemone Anemonia viridis as a result of increased temperature

Exposure of the temperate sea anemone Anemonia viridis Forskål to increased seawater temperature (from 16 to 26°C) reduced the lysosomal latency of coelenterate tissues. Lysosomes in the mesenterial filaments of anemones were destabilised by increased temperature, with greater destabilisation in heat-shocked symbiotic anemones than in heat-shocked aposymbiotic anemones in the early stages of the experiment. Lysosomal enzyme activity in zooxanthellae from heat-shocked symbiotic anemones was associated with the algal membranes and the cytoplasm of degenerate algal cells. While the relationship between host coelenterate and symbiotic alga may confer many benefits under normal conditions, comparison of the responses of symbiotic and aposymbiotic anemones to heat shock suggests that there may be disadvantages for symbiotic anemones under stress.     

Effect of feeding regime and irradiance on the photophysiology of the symbiotic sea anemone Aiptasia pulchella

To determine how the animal and algal components of the symbiotic sea anemone Aiptasia pulchella respond to changes in food availability and culture irradiance, sea anemones from a single clone were maintained at four irradiance levels (320, 185, 115, and 45 E m^-2 s^-1) and either starved or fed for 5 wk. Changes in protein biomass of sea anemones maintained under these conditions were not related to the productivity of zooxanthellae, since the protein biomass of fed A. pulchella decreased with increase in irradiance and there was no difference in protein biomass among starved sea anemones at the four irradiance levels. Except for the starved high-light sea anemones, the density of symbiotic zooxanthellae was independent of culture irradiance within both starved and fed. A. pulchella. Starved sea anemones contained over twice the density of zooxanthellae as fed sea anemones. Within both starved and fed individuals, chlorophyll per zooxanthella increased with decreasing culture irradiance while algal size remained constant (in fed sea anemones) at about 8.80 m diameter. Chlorophyll a: c ₂ ratios of zooxanthellae increased with decreasing culture irradiance in zooxanthellae from starved sea anemones but remained constant in zooxanthellae from fed sea anemones. As estimated from mitotic index data, the in situ growth rates of zooxanthellae averaged 0.007 d^-1 and did not vary with irradiance or feeding regime. Photosynthesis-irradiance (P-I) responses of fed A. pulchella indicated an increase in photosynthetic efficiency with decreasing culture irradiance. But there was no consistent pattern in photosynthetic capacity with culture irradiance. Respiration rates of fed sea anemones also did not vary in relation to culture irradiance. The parameter I _k , defined as the irradiance at which light-saturated rates of photosynthesis are first attained, was the only parameter from the P-I curves which increased linearly with increasing culture irradiance. The daily ratio of net photosynthesis to respiration for A. pulchella ranged from 1.6 to 2.8 for sea anemones maintained at the three higher irradiances, but was negative for those maintained at 45 E m^-2 s^-1. Since the final protein biomass was greatest for sea anemones maintained at the lowest irradiance, these results indicate that sea anemone growth cannot be directly related to productivity of zooxanthellae in this symbiotic association.     

Distribution of lipids between the zooxanthellae and animal compartment in the symbiotic sea anemoneAnemonia viridis: Wax esters,triglycerides and fatty acids

The temperate sea anemoneAnemonia viridis (Forskäl) contained about 11% lipid on a dry weight basis when maintained at light levels of about 10µE m^–2 s^–1 and a temperature of 10°C. Aposymbiotic forms of the anemone had similar lipid levels. These values are very low compared with tropical symbiotic Anthozoa in which lipid levels constitute up to 50% of dry weight. In symbioticA. viridis, <6% of total lipid consisted of the storage lipids, wax esters and triglycerides. Most of the triglyceride was stored in the animal tissues rather than the zooxanthellae. Zooxanthellae contained only small amounts of wax esters. An analysis was made of the wax ester, triglyceride and fatty acid composition of symbiotic anemones, isolated zooxanthellae and aposymbiotic anemones. Wax ester composition was similar in symbiotic and aposymbiotic forms. However, triglyceride composition differed. In particular trimyristin (C42) was found only within the symbiotic association. Fatty acids showed a high degree of unsaturation, and acids with both even and odd numbers of carbon atoms were found. The most abundant fatty acid was 16:0 in all samples, except for the total lipids from zooxanthellae in which the major fatty acid wastrans-18:1.     

Symbiont photosynthesis increases both respiration and photosynthesis in the symbiotic sea anemone Anemonia viridis

Dark respiration of the symbiotic sea anemone Anemonia viridis (Forskäl) was observed to increase by 34% when anemones were exposed to hyperoxic sea water (150% oxygen saturation) overnight, and by 39% after exposure to 6 h in the light at a saturating irradiance of 300 E m^-2 s^-1 at normoxia (100% oxygen saturation). No increase due to light stimulation was observed in aposymbiotic control anemones. In darkness, the oxygen concentration of the coelenteric fluid was hypoxic. However, within 10 min of anemones being illuminated, coelenteric fluid was hyperoxic, and it remained elevated throughout a 12 h light period. When measured over a 24 h period (12 h light: 12 h dark), the dark respiration rate increased gradually over the first 6 h of the light period until it was 35% above the dark night-time resting rate. It remained elevated throughout the remaining light period and for 2 h into the following dark period, after which it fell back to the resting rate. Gross photosynthesis (P ^gross) increased significantly when anemones were exposed to either hyperoxia (150% oxygen saturation) or 300 E m^-2 s^-1 at normoxia. This increase was not observed when symbiotic anemones were illuminated at a low-light intensity of 100 E m^-2 s^-1. The results of this study suggest that respiration in the dark is limited by oxygen diffusion and that normal respiration is restored in the daytime by utilisation of the oxygen released by photosynthesis. Furthermore, it appears that the increased respiration following exposure to high-light intensities provides a CO₂-rich intracellular environment which further enhances the photosynthetic rate of the zooxanthellae.     

Effects of feeding frequency and symbiosis with zooxanthellae on nitrogen metabolism and respiration of the coral Astrangia danae

Colonies of the temperate coral Astrangia danae occur naturally with and without zooxanthellae. Basal nitrogen excretion rates of nonsymbiotic colonies increased with increasing feeding frequency [average excretion rate was 635 ng-at N (mg-at tissue-N)^-1 h^-1]. Reduced excretion rates of symbiotic colonies were attributed to N uptake by the zooxanthellae. Nitrogen uptake rates of the zooxanthellae averaged 8 ng-at N (10⁶ cells)^-1 h^-1 in the dark and 21 ng-at N (10⁶ cells)^-1 h^-1 at 200 Ein m^-2 s^-1. At these rates the zooxanthellae could provide 54% of the daily basal N requirement of the coral if all of the recycled N was translocated. Basal respiration rates were 172 nmol O₂ cm^-2 h^-1 for starved colonies and 447 nmol O₂ cm^-2 h^-1 for colonies fed three times per week. There were no significant differences between respiration rates of symbiotic and nonsymbiotic colonies. N excretion and respiration rates of fed (symbiotic and nonsymbiotic) colonies increased greatly soon after feeding. N absorption efficiencies decreased with increasing feeding frequency. A N mass balance, constructed for hypothetical situations of nonsymbiotic and symbiotic (3×10⁶ zooxanthellae cm^-2) colonies, starved and fed 15 g-at N cm^-2wk^-1, showed that the presence of symbionts could double the N growth rate of feeding colonies, and reduce the turnover-time of starved ones, but could not provide all of the N requirements of starved colonies. Rates of secondary production, estimated from rates of photosynthesis and respiration were similar to those estimated for reef corals.     

The role of chemosensory behavior of Symbiodinium microadriaticum,intermediate hosts,and host behavior in the infection of coelenterates and molluscs with zooxanthellae

Symbiotic dinoflagellates, Symbiodinium microadriaticum (=zooxanthellae), may gain access to aposymbiotic hosts (i.e., those lacking zooxanthellae) by chemosensory attraction of the motile algae by the potential host or via an intermediate host. Laboratory experiments showed that motile zooxanthellae were attracted to intact aposymbiotic host animals, but not to starved symbiotic hosts. Fed symbiotic hosts and brine shrimp (Artemia sp.) nauplii also attracted motile zooxanthellae. The attraction of these zooxanthellae was directly correlated with nitrogen levels in the seawater surrounding the hosts; thus ammonia and possibly nitrate could be atractants. Brine shrimp nauplii, acting as intermediate hosts actively ingested both motile and non-motile zooxanthellae. the ingested zooxanthellae tended to remain morphologically unaltered during and after passage through the gut of the brine shrimp. Capture and ingestion of brine shrimp containing zooxanthellae by aposymbiotic scyphistomae of the jellyfish Cassiopeia xamachana led to infection of the scyphistomae with zooxanthellae. Zooxanthellae isolated from 17 different species of coelenterates and molluscs could be transferred via brine shrimp to the endodermal cells of the scyphistomae. However only 10 of these isolates persisted to establish a permanent association with C. xamachana. Scyphistomae in suspensions of motile zooxanthellae responded by a classical coelenterate feeding response, which may facilitate ingestion of the potential symbionts and establishment of a symbiosis.     

Oxygen as a limiting factor in phototaxis and in intraclonal spacing of the sea anemone Anthopleura elegantissima

Phototaxis in Anthopleura elegantissima, a sea anemone symbiotic with zooxanthellae, was investigated with special reference to oxygen as a possible controlling factor. Under high oxygen concentrations in seawater, movement towards light was not observed for symbiotic anamones as it was under normal oxygen concentrations. Both aposymbiotic and symbiotic anemones demonstrated movement towards high oxygen concentrations in seawater. Oxygen is, therefore, implicated as a controlling factor in phototaxis. Under laboratory conditions, increased intraclonal spacing occurred with low oxygen concentrations in seawater. In the field, individuals in symbiotic clones were spaced significantly closer than in aposymbiotic clones. Since intraclonal spacing is controlled by oxygen in the laboratory, spacing may also be affected in the field by oxygen; symbiotic clones may be spaced closer because they have better oxygen availability than do aposymbiotic clones.     

Photosynthesis-irradiance responses and photosynthetic periodicity in the sea anemone Aiptasia pulchella and its zooxanthellae

Sea anemones (Aiptasia pulchella) containing zooxanthellae (Symbiodinium microadriaticum) were maintained in a long-term laboratory culture on a 12 h light (100 E m^-2 s^-1):12 h dark cycle. Photosynthetic oxygen production was measured for the symbiotic association and for freshlyisolated zooxanthellae. Light utilization efficiencies () were similar for both sets of zooxanthellae, suggesting negligible shading of zooxanthellae by animal tissue in this association. Whereas freshly-isolated zooxanthellae were photoinhibited at high irradiances (800 to 1 800 E m^-2 s^-1), zooxanthellae in the host continued to function at photosynthetic capacity. Time of day may influence photosynthetic measurements in symbiotic organisms, as it was found that photosynthesis in A. pulchella followed a diel periodicity at both light-saturating (1 200 E m^-2 s^-1) and subsaturating (150 E m^-2 s^-1) irradiances. There was a peak period of photosynthesis between 12.00 and 14.00 hrs. Light stimulated dark respiration rates of A. pulchella. Dark respiration of sea anemones increased somewhat towards the end of the light cycle and was always greater after exposure to high irradiances.     

Copper uptake by the sea anemoneAnemonia viridis and the role of zooxanthellae in metal regulation

Anemonia viridis (Forskäl) were collected from south-west Scotland and south-west England in October 1988. When exposed to 0.05 and 0.2mg 1^–1 copper in sea water, anemones did not take up the metal in proportion to external concentrations. Results suggested thatA. viridis regulated copper by expelling symbiotic algae (or zooxanthellae) which were shown to accumulate copper. The use of aposymbiotic (non-zooxanthellate) anemones in similar metal-uptake experiments indicated that other mechanisms may also be involved in metal regulation. Mucus was produced byA. viridis when the anemone was exposed to copper, and it is proposed that mucus may be involved in the regulation process. The implication of this work on the use of coelenterates as biological indicators of environmental metal levels is discussed.     

Effect of light on the total lipid content and storage lipids of the symbiotic sea anemoneAnemonia viridis

In order to examine the effect of light level on the storage lipids of the symbiotic sea anemoneAnemonia virudis (Forskäl), anemones were exposed to three experimental light regimes of 10, 100 and 300 E m^-2s^-1. Anemones were fed once a week. After 30 d there were no significant differences in the total lipid levels between anemones at any of the light intensities. However, after 60 d lipids had increased in proportion to light level in both the animal-tissue and zooxanthellae compartments. The higher levels of total lipid were in part due to increases in storage lipid (wax esters and triglycerides). Wax ester levels increased in the animal tissues but remained constant in the zooxanthellae, whereas triglycerides increased in both compartments. In contrast to fed anemones, starved anemones which were maintained at 300 E m^-2s^-1 for 30 or 60 d did not show a statistically significant change in lipid levels at 60 d, although a slight increase in the lipid level was observed. However, there was a significant increase in the storage lipids, which suggested that the non-storage phospholipids and structural lipids had declined as a result of cellular catabolism. The composition of the wax esters and triglycerides of both fed and starved anemones was analysed and compositional changes were observed at higher light intensities.     

Effects of starvation,and light and dark on the energy metabolism of symbiotic and aposymbiotic sea anemones,Anthopleura elegantissima

Rates of oxygen and carbon-dioxide exhange were measured in symbiotic and aposymbiotic specimens of the sea anemone Anthopleura elegantissima while fed and starved under light or dark conditions. Respiratory quotients indicated that fed anemones switched from a carbohydrate to a fat catabolism when starved, with the exception that symbiotic individuals starved in the light showed a pronounced carbohydrate catabolism for over 1 month. The source of the carbohydrate was probably photosynthate translocated by the dinoflagellate Symbiodinium (=Gymnodinium) microadriaticum (Freudenthal) living in the anemones' tissues. The starved symbiotic anemones maintained in the light had lipid levels not significantly different from fed controls and 44 to 61% higher than starved aposymbiotic anemones after 1 month. Thus, the quality and quantity of the metabolic flux from the symbionts to the sea anemone were sufficient to conserve the host's lipid reserves.     

Evidence for facultative heterotrophy in cultured zooxanthellae

The locus of symbiotic dinoflagellates within host cells provides a habitat which could potentially be exploited by the alga through heterotrophic uptake of host-derived organic substrates. Using zooxanthellae (Symbiodinium sp.) isolated from the tropical sea anemone Aiptasia pulchella collected from Kaneohe Bay, Hawaii, the effect of various potential organic substrates on growth in vitro was assessed in Erdschreiber seawater medium (ES) supplemented with organic compounds. Zooxanthellae maintained at 5 to 7 E m^-2 s^-1 (below compensation irradiance) grew heterotrophically when supplied with 100 M glycerol, glycolate, acetate, malate, or propionate, and grew in darkness on 100 M propionate. Zooxanthellae exposed to irradiance below compensation were able to utilize carbon sources in the unsupplemented ES medium for slow growth, but generally the growth rate of cultured zooxanthellae was a function of incubation irradiance. Zooxanthellae incubated for 10 wk in unsupplemented ES at 5 to 7 E m^-2 s^-1 were capable of growth at this low irradiance, but were also capable of net photosynthetic oxygen production at higher irradiances. This suggests that zooxanthellae can be photoautotrophic or facultatively heterotrophic. An estimate for the duration of mitosis (t _d ) is made on the basis of growth rate of cultured zooxanthellae in log-phase; this estimate of t _d =4.88 h is less than half the estimated t _d for zooxanthellae in situ.     

Nutrient transfer in a marine mutualism: patterns of ammonia excretion by anemonefish and uptake by giant sea anemones

Many symbioses involve multiple partners in complex, multi-level associations, yet little is known concerning patterns of nutrient transfer in multi-level marine mutualisms. We used the anemonefish symbiosis as a model system to create a balance sheet for nitrogen production and transfer within a three-way symbiotic system. We quantified diel patterns in excretion of ammonia by anemonefish and subsequent absorption by host sea anemones and zooxanthellae under laboratory conditions. Rates of ammonia excretion by the anemonefish Amphiprion bicinctus varied from a high of 1.84 μmole g⁻¹ h⁻¹ at 2 h after feeding, to a basal rate of 0.50 μmole g⁻¹ h⁻¹ at 24–36 h since the last meal. Conversely, host sea anemones Entacmaea quadricolor absorbed ammonia at a rate of 0.10 μmole g⁻¹ h⁻¹ during the daytime in ammonia-enriched seawater, but during the night reduced their absorption rate to near zero, indicating that ammonia uptake was driven by zooxanthella photosynthesis. When incubated together, net ammonia excretion was virturally zero, indicating that host anemones absorbed most of the ammonia produced by resident fish. Adult anemonefish weighed about 11 g under laboratory conditions, but on the coral reef may reach up to 64 g, resulting in a maximal potential ammonia load of >200 μmole h⁻¹ produced by two adult fish during daylight hours. In contrast, host sea anemones weighed about 47 g in the laboratory, but under field conditions, large individuals may reach 680 g, so their maximal ammonia clearance rates may reach about 70 μmole h⁻¹ during the daytime. As such, the ammonia load produced by adult anemonefish far exceeds the clearance rate of host anemones and zooxanthellae. Ammonia transfer likely occurs mainly during the daytime, when anemonefish consume zooplankton and excrete rapidly, and in turn the zooxanthellae are photosynthetically active and drive rapid ammonia uptake. We conclude that zooplanktivorous fishes that form mutualisms with coral reef cnidarians may serve as an important link between open water and benthic ecosystems, through the transfer of large quantities of nutrients to zooxanthellate hosts, thus enhancing coral reef productivity.     

Carbon metabolism and strobilation in Cassiopea andromedea (Cnidaria: Scyphozoa): Significance of endosymbiotic dinoflagellates

Scyphopolyps and scyphomedusae of Cassiopea andromeda Forskål (Cnidaria, Scyphozoa) containing dinoflagellate endosymbionts (zooxanthellae) were investigated for rates and pathways of carbon fixation. Photosynthesis by the algae, accounting for 80 and 15 mol C h^-1 on a dry weight basis in medusae and polyps, respectively, by far exceeds dark incorporation of inorganic carbon by the intact association. Photosynthetic carbon fixation is operated via C₃ pathway of carbon reduction. DCMU-treatment (1×10^-6 M and 1×10^-5 M) completely inhibits light-dependent carbon assimilation. Major photosynthates presumably involved in a metabolite flow from algal symbionts to animal tissue are glycerol and glucose. A total of 5–10% net algal photosynthate appears to be seleased in vivo to the host. This is probably less than the energy supply ultimately required for the nutrition of the polyps and medusae. The presence of zooxanthellae proved to be indispensable for strobilation in the scyphopolyps. However, photosynthesis by algal symbionts as well as photosynthate release is obviously not essential for the initiation of ephyrae as is shown by DCMU-treatment, culture in continous darkness, and aposymbiotic controls. It is therefore concluded that strobilation is supported, but not triggered by algal photosynthetic activity. The induction of strobilation thus seems to depend on a more complex system of regulation.     

Nutrient uptake kinetics and growth of zooxanthellae maintained in laboratory culture

Growth characteristics and nutrient uptake kinetics were determined for zooxanthellae (Gymnodinium microadriaticum) in laboratory culture. The maximum specific growth rate (_max) was 0.35 d^-1 at 27 °C, 12 hL:12 hD cycle, 45 E m^-2 s^-1. Anmmonium and nitrate uptake by G. microadriaticum in distinct growth phases exhibited Michaelis-Menten kinetics. Ammonium half-saturation constants (K_s) ranged from 0.4 to 2.0 M; those for nitrate ranged from 0.5 to 0.8 M. Ammonium maximum specific uptake rates (V_max) (0.75 to 1.74 d^-1) exceeded those for nitrate (0.14 to 0.39 d^-1) and were much greater than the maximum specific growth rate (0.35 d^-1), suggesting that ammonium is the more significant N source for cultured zooxanthellae. Ammonium and nitrate V_max values compare with those reported from freshly isolated zooxanthellae. Light enhanced ammonium and nitrate uptake; ammonium inhibited nitrate uptake which was not reported for freshly isolated zooxanthellae, suggesting that physiological differences exist between the two. Knowledge of growth and nutrient uptake kinetics for cultured zooxanthellae can provide insight into the mechanisms whereby nutrients are taken up in coral-zooxanthelae symbioses.Contribution No. 1515 from the University of Maryland Center for Environmental and Estuarine Studies, Chesapeake Biological Laboratory, Solomons, Maryland 20688-0038, USA     

Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam—zooxanthellate symbiosis

The mechanism whereby inorganic carbon (C_i) is acquired by the symbiotic association between the giant clam (Tridacna derasa) and zooxanthellae (Symbiodinium sp.) has been investigated. C_i in the haemolymph of the clam is in equilibrium with the surrounding sea water. The photosynthesis rate exhibited by the intact clam varies as a function of the C_i concentration in the clam haemolymph. The gill tissue contains high carbonic anhydrase activity which may be important in adjusting the C_i equilibrium between haemolymph and sea water. Zooxanthellae (Symbiodinium sp.) isolated from the clam mantle prefer CO₂ to HCO ₃ ^- as a source of inorganic carbon. The zooxanthellae have low levels of carbonic anhydrase on the external surface of the cell; however, mantle extracts display high carbonic anhydrase activity. Carbonic anhydrase is absent from the mantle of aposymbiotic clams (T. gigas), indicating that this enzyme may be essential to the symbiosis. The enzyme is probably associated with the zooxanthellae tubes in the mantle. The results indicate that carbonic anhydrase plays an important role in the supply of carbon dioxide within the clam symbiosis.     

Effects of irradiance and ultraviolet radiation on photoadaptation in the zooxanthellae of Aiptasia pallida: primary production,photoinhibition, and enzymic defenses against oxygen toxicity

Cnidarians which contain symbiotic algae are constantly faced with the challenges of a changing photic regime and a hyperoxic environment. Zooxanthellae (Symbiodinium sp.) from the sea anemone Aiptasia pallida (Verrill), collected and cultured at Bermuda Biological Station in 1986, exhibit a suite of compensatory responses to changes in irradiance, ultraviolet radiation (UV), and to the toxicity resulting from their interaction with photosynthetically produced oxygen. Superoxide dismutase (SOD) and catalase inactivate superoxide radicals (O₂ ^-) and hydrogen peroxide (H₂O₂), which are mediators of oxygen toxicity, show an increase in specific activity with irradiance and in response to UV, both in cultured zooxanthellae (CZ) and freshly isolated zooxanthellae (FIZ) from acclimated anemones. CZ and FIZ exposed to environmentally realistic UV levels show a 30 to 40% increase in SOD activities compared with zooxanthellae exposed to similar irradiances without UV. CZ consistently show higher activities of both SOD and catalase compared to FIZ. Both CZ and FIZ exhibit changes in chlorophyll content and in the relationship between photosynthesis and irradiance which suggest photoadaptive changes in CO₂-fixing enzymes, the photosynthetic-electron transport system, or in photosynthetic unit size (PSU). UV has a greater effect on the photosynthetic capacity (P _max) of FIZ when compared to CZ acclimated at an equivalent irradiance with or without a UV component. UV also enhances the photoinhibition observed at high irradiance in both CZ and FIZ. Differences in enzyme activity between CZ and FIZ suggest an important role for the host in the protection of zooxanthellae against the direct effects of environmentally realistic UV while the photosynthetic performance of zooxanthellae in situ may not be as well protected.     

Daily photosynthesis,respiration, and carbon budgets in a tropical marine jellyfish (Mastigias sp.)

From measured diel photosynthesis and respiration rates, using oxygen electrodes, estimates of carbon flux between symbiotic algae (zooxanthellae) and host animal are presented for the marine scyphomedusan Mastigias sp. from a marine lake in Palau, Western Caroline Islands, during February and March 1982. The carbon budgets calculated for these lake medusae indicate that carbon fixed photosynthetically by zooxanthellae and made available to the host may satisfy up to 100% of the host's daily metabolic carbon demand (CZAR). The stable carbon isotope (¹³C) signature of the mesogleal carbon of lake Mastigias sp. was close to that of the zooxanthellae, supporting the interpretation that while these medusae may feed holozoically, some of their carbon comes from their symbionts. The diel photosynthesis, respiration, and preliminary estimates of carbon budgets of three individuals of another ecotype of Mastigias sp. collected from nearby oceanic lagoons are also given. Photosynthesis of lagoon medusae was generally greater than that for lake medusae of similar size, and lagoon medusae were phototrophic with respect to carbon, with commensurately greater CZAR values. Carbon translocated from the symbiotic algae also may contribute to the growth requirements of both lake and lagoon medusae. From carbon flux data, the lake jellyfish were estimated to contribute about 16% to the total primary productivity of their marine lake habitat.     

Ammonium enhancement of dark carbon fixation and nitrogen limitation in zooxanthellae symbiotic with the reef coralsMadracis mirabilis andMontastrea annularis

The nutrient status (limitation vs sufficiency) of dinoflagellates (zooxanthellae) symbiotic with reef corals in Bermuda was assessed in 1989 and 1990 by measuring the enhancement of dark carbon fixation with 20 M ammonium by isolated symbionts. A colony ofMadracis mirabilis was kept in the laboratory and fed daily or starved for one month. Symbionts from fed portions of the colony had ammonium-enhancement ratios (NH _4dark ⁺ ; SW_dark;SW=seawater without added ammonium) similar to those of the original field population (1.2 to 1.3). Ammonium-enhancement ratios increased with starvation of the host (x1.7) as did values forV _D:V _L [(ammonium dark rate-seawater dark rate): light rate in seawater]. Both parameters indicated decreasing nitrogen sufficiency of the algae when the host was not fed, but starvation appeared to affect these algae less than symbionts of sea anemones. Field samples of zooxanthellae fromM. mirabilis (Three Hill Shoals and Bailey's Bay Flats) yielded results similar to those for fed corals, but those taken from Bailey's Bay Flats in May 1990 yielded exceptionally high values for enhancement (>3) andV _D:V _L indicating pronounced nitrogen limitation at the time of sampling. We sampled zooxanthellae from populations ofMontastrea annularis at 8 m (Three Hill Shoals) and 24 m (Soldier's Point) depths. Enhancement andV _D:V _L values for zooxanthellae from the 8 m corals were density-dependent: symbionts from corals with normal symbiont densities displayed the most nitrogen limitation (enhancement values=1.4 to 2.0), while those from bleached corals with lower density exhibited enhancement andV _D:V _L values typical of nitrogen-sufficient algae. Symbionts isolated from the 25 m corals yielded the highest values, and appeared to exhibit the least nitrogen-sufficiency for this species.     

Role of glutamine synthetase in ammonia assimilation by symbiotic marine dinoflagellates (zooxanthellae)

The dinoflagellate symbionts (zooxanthellae) present in many reef corals aid in the survival of the symbiotic unit in nitrogen deficient tropical waters by providing additional routes of nitrogen uptake and metabolism. The enzymatic pathway of ammonia assimilation from seawater and the re-assimilation of coral ammonium waste by zooxanthellae was studied by examining the affinity of glutamine synthetase for one of its substrates, ammonia. Glutamine synthetase activity was measured in dinoflagellates of the species Symbiodinium microadriaticum found in symbiotic association with various marine coelenterates. Michaelis-Menten kinetics for the substrate ammonia were determined for freshly isolated dinoflagellates from Condylactis gigantea (apparent NH₃ K_m=33 M) and for cultured dinoflagellates from Zoanthus sociatus (apparent NH₃ K_m=60 M). On the basis of the low apparent K_ms for NH₃, it appears that ammonia assimilation by these symbiotic dinoflagellates occurs via the glutamine synthetase/glutamate synthase pathway. Additionally, the uptake of exogenous ammonium by an intact coelenterate-dinoflagellate symbiosis was strongly inhibited by 0.5 mM methionine sulfoximine, and inhibitor of glutamine synthetase.