Primary site and initial products of ammonium assimilation in the symbiotic sea anemone Anemonia viridis

Invertebrates containing endosymbiotic dinoflagellate algae (zooxanthellae) retain excretory nitrogen, and many are able to take up ammonium from the surrounding seawater. However, the site of assimilation and role of nitrogen recycling between symbiont and host remains unclear. In the present study, ammonium uptake by the symbiotic sea anemone Anemonia viridis (Forskål) was examined by following the pathway of assimilation using ¹⁵N-enriched ammonium. Since zooxanthellae became enriched with ¹⁵N from ammonium at up to 17 times the rate of the host, they appear to be the primary site of assimilation. In the light, the rate of zooxanthellae enrichment at 20?M was twice that at 10?M, whereas the rate of host enrichment was not significantly affected by ammonium concentration. When anemones were incubated with [¹⁵N]ammonium in the dark, after 12?h without light the rate of enrichment was lowered in both zooxanthellae and host. However, while the enrichment of the host was significantly reduced when the light level was lowered from 300 to 150?μmol photons m^?2?s^?1, zooxanthellae enrichment was unchanged. Low molecular weight material from the zooxanthellae became enriched at 20 times the rate of that from the host, and enrichment was detected in the amino acids glutamate, glutamine, aspartate, alanine, glycine, phenylalanine, threonine, valine, tyrosine, and leucine from zooxanthellae. In the zooxanthellae, amino acids accounted for 65% of the total enrichment of low molecular weight material. Of the amino acids detected in zooxanthellae, over 90% of the enrichment was accounted for by glutamate, glutamine and aspartate. The enrichment of the amide group of glutamine was greater than that of the amine group of glutamate or glutamine, consistent with the glutamine synthetase/glutamine 2-oxoglutarate amidotransferase cycle as the mechanism of ammonium assimilation. To examine the flux of ¹⁵N from zooxanthellae to host, anemones were pulse-labelled with [¹⁵N]ammonium and then transferred to an unlabelled chase. Over a 2?h period there was no evidence for a flux of nitrogen from zooxanthellae to host. However, during the chase period, the enrichment of low molecular weight material declined and that of high molecular weight material increased in both zooxanthellae and host, indicating that protein was synthesized using ¹⁵N from ammonium in both components of the symbiosis. Again by using a pulse-chase system, it was found that glutamate was metabolised most rapidly by zooxanthellae, followed by (in order of decreasing rate of turnover) aspartate, alanine, glycine and valine (no data are available for glutamine). Unlike these amino acids, nitrogen was transferred to the essential amino acids phenylalanine and threonine, increasing their enrichment during the chase period. While recycled nitrogen is clearly important to this symbiosis, the mechanism by which it is cycled remains to be resolved.     

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.     

Effects of nutritional history on nitrogen assimilation in congeneric temperate and tropical scleractinian corals

The nutritional history of corals is known to affect metabolic processes such as inorganic nutrient uptake and photosynthesis, but little is known about how it affects assimilation efficiency of ingested prey items or the partitioning of prey nitrogen between the host and symbiont. The temperate scleractinian coral Oculina arbuscula and its tropical congener Oculina diffusa were acclimated to three nutritional regimes (fed twice weekly, starved, starved with an inorganic nutrient supplement), then fed Artemia nauplii labeled with the stable isotope tracer ¹⁵N. Fed corals of both species had the lowest assimilation efficiencies (36–51% for O. arbuscula, 38–57% for O. diffusa), but were not statistically different from the other nutritional regimes. Fed and starved corals also had similar NH₄⁺ excretion rates. This is inconsistent with decreased nitrogen excretion and reduced amino acid catabolism predicted by both the nitrogen recycling and conservation paradigms. In coral host tissue, ~90% of the ingested ¹⁵N was in the TCA-insoluble (protein and nucleic acids) and ethanol-soluble (amino acids/low molecular weight compounds) within 4 h of feeding. The TCA-insoluble pool was also the dominant repository of the label in zooxanthellae of both species (40–53% in O. arbuscula, 50–60% in O. diffusa). However, nutritional history had no effect on the distribution of prey ¹⁵N within the biochemical pools of the host or the zooxanthellae for either species. This result is consistent with the nitrogen conservation hypothesis, as preferential carbon metabolism would minimize the effects of starvation on nitrogen-containing biochemical pools.Communicated by P.W. Sammarco, Chauvin     

Symbiotic anemones can grow when starved: nitrogen budget for Anemonia viridis in ammonium-supplemented seawater

The ability of endosymbioses between anthozoans and dinoflagellate algae (zooxanthellae) to retain excretory nitrogen and take up ammonium from seawater has been well documented. However, the quantitative importance of these processes to the nitrogen budget of such symbioses is poorly understood. When starved symbiotic Anemonia viridis were incubated in a flow-through system in seawater supplemented with 20 μM ammonium for 91 d under a light regime of 12 h light at 150 μmol photons m⁻² s⁻¹ and 12 h darkness, they showed a mean net growth of 0.197% of their initial weight per day. Control anemones in unsupplemented seawater with an ammonium concentration of <1 μM lost weight by a mean of 0.263% of their initial weight per day. Attempts to construct a nitrogen budget showed that, over a 14 d period, ≃40% of the ammonium taken up could be accounted for by growth of zooxanthellae. It was assumed that the remainder was translocated from zooxanthellae to host. However, since the budget does not balance, only 60% of the growth of host tissue was accounted for by this translocation. The value for host excretory nitrogen which was recycled to the symbionts equalled that taken in by ammonium uptake from the supplemented seawater, indicating the importance of nitrogen retention to the symbiotic association. Received: 23 December 1997 / Accepted: 12 September 1998     

Growth of zooxanthellae in culture with two nitrogen sources

Physiological characteristics of zooxanthellae were examined under nutrient-saturated conditions created by mixing ammonium (¹⁵NH₄) with nitrate (¹⁵NO₃) to give 0.88 mM total nitrogen. Growth rate varied with the form of nitrogen provided. Ammonium alone resulted in the lowest C:N and C:chl-a ratios. Although zooxanthellae took up nitrate in the absence of ammonium, ammonium assimilation was 1.3 times higher than nitrate assimilation. Ammonium strongly inhibited nitrate assimilation. While high-ammonium treatments resulted in the highest ¹⁴C incorporation into intermediate compounds, high nitrate levels resulted in the highest ¹⁴C incorporation into protein, suggesting that the intermediate compounds are produced prior to the subsequent production of protein when ammonium is the dominant N source. The enhanced production of intermediate compounds at the expense of carbon directed to protein synthesis in the presence of ammonium might be analogous to the “host factor” observed in zooxanthellae–host symbioses, since growth rate is depressed due to low production of protein. Received: 16 March 2000 / Accepted: 26 August 2000     

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.     

Effect of diel photoperiod on nitrogen metabolism of cultured and symbiotic zooxanthellae

Ammonium uptake and assimilation by zooxanthellae (Symbiodinium sp.) cultured with an excess of nitrate was enhanced in light. Uptake was decreased by the same amount when zooxanthellae were incubated in darkness either after 6 h pretreatment in light, or at the end of the dark period of a 12 h light: 12 h dark cycle. This suggested that short-term incubations of zooxanthellae were valid tests for light enhancement of dissolved inorganic nitrogen (DIN) uptake. Assimilation of ammonium into glutamine (Gln) and glutamate (Glu) was also decreased in darkness. During a 12 h light: 12 h dark cycle, free pools of both Gln and Glu fell quickly at the start of the light period, followed by steady increases until the beginning of the next dark period. Of the four other major components of free amino acid pools tested, only the nonprotein amino acid taurine showed diel fluctuations. Gln and Glu pools in zooxanthellae freshly isolated from reef-forming corals also showed differences between day and night, suggesting changes in patterns of DIN assimilation over the diurnal cycle.     

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.     

Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella

Symbiotic Aiptasia pulchella and freshly isolated zooxanthellae were incubated in NaH¹⁴CO₃ and NH₄Cl for 1 to 240 min, and samples were analysed by reverse-phase high-performance liquid chromatography (HPLC) and an online radiochemical detector. NH₄ ⁺ was first assimilated into ¹⁴C-glutamate and ¹⁴C-glutamine in the zooxanthellae residing in A. pulchella. The specific activities (dpm nmol⁻¹) of ¹⁴C-glutamate and ¹⁴C-glutamine in vivo, were far greater in the zooxanthellae than in the host tissue, indicating that NH₄ ⁺ was principally incorporated into the glutamate and glutamine pools of the zooxanthellae. ¹⁴C-α-ketoglutarate was taken up from the medium by intact A. pulchella and assimilated into a small amount of ¹⁴C-glutamate in the host tissue, but no ¹⁴C-glutamine was detected in the host fraction. The ¹⁴C-glutamate that was synthesized was most likely produced from transamination reactions as opposed to the direct assimilation of NH₄ ⁺. The free amino acid composition of the host tissue and zooxanthellae of A. pulchella was also measured. The results presented here demonstrate that NH₄ ⁺ was initially assimilated by the zooxanthellae of A. pulchella. Received: 3 February 1997 / Accepted: 24 October 1997     

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.     

The effects of feeding or addition of dissolved inorganic nutrients in maintaining the symbiosis between dinoflagellates and a tropical marine cnidarian

The availability of solid food (Artemia nauplii) and dissolved inorganic nutrients (ammonium, nitrate, phosphate) to the shallow-water marine hydroid Myrionema amboinense was manipulated for 1-8 days in order to investigate their role in the growth of intracellular symbiotic dinoflagellates (zooxanthellae) of the genus Symbiodinium. Symbionts from hydroids collected from the field or maintained under laboratory conditions (25°C, 12 h:12 h light:dark cycle, 80 µE m^-2 s^-1 fluorescent lighting) always exhibited a single peak in mitotic index (MI) at dawn. Symbionts in freshly collected field animals had an MI peak of about 15%. Symbiotic dinoflagellates in hydroids fed Artemia nauplii twice daily in the laboratory maintained this dawn peak of MI between 10% and 15%, but in the absence of feeding or added inorganic nutrients, this peak declined to less than 1% within 2-4 days. In contrast, when hydroids were placed in solutions containing ammonium (20 µM NH₄Cl), nitrate (10 µM NaNO₃), and a combination of ammonium and phosphate (2 µM Na₂HPO₄) immediately after collection, the algal MI remained between 5% and 15% for 4-7 days; the addition of 2 µM phosphate did not increase MI relative to unfed rates. When unfed animals were placed in dissolved nitrogen or fed Artemia, the symbiont MI increased from <1% to 10-17% within 2-3 days; P alone had no effect. However, the increase resulting from added inorganic nutrients was temporary, lasting only 5-7 days. These observations suggest that algal division in the host is maintained indefinitely in the field or by feeding particulate foods twice daily in the laboratory, but the addition of inorganic nutrients alone (ammonium, nitrate and ammonium/phosphate) appeared to support the completion of a maximum of one additional round of cell division. Nutrients required for continued growth and division of symbiotic dinoflagellates are linked to host feeding and host growth; without external food, neither host nor symbiont continue to grow. The same phenomenon is seen in zooxanthellate anemones, clams and corals, where total numbers of symbionts appear to be linked to changes in host-tissue biomass (protein), achieving relatively stable densities in M. amboinense, corals and other cnidarian symbioses, depending on their local environmental conditions. The results of the present study help explain the cellular responses of algal symbionts in reef-dwelling invertebrates to additions of dissolved inorganic nutrients to coral-reef ecosystems.     

Ammonium, but not nitrate, stimulates an increase in glutamine concentration in the haemolymph of Tridacna gigas

The concentration of glutamine in Tridacna gigas haemolymph increased >35-fold following exposure to sea water supplemented with ammonium (20 μM), but no increase was observed with nitrate (20 μM). Lack of a diel cycle, no decrease in haemolymph glucose levels, the expression patterns of glutamine synthetase in zooxanthellae and host, and the lack of glutamine release in response to nitrate supplementation all support the proposition that the increase in haemolymph glutamine is a product of the host and not the zooxanthellae. Unlike ammonium, nitrate accumulates rapidly in the haemolymph. It has no effect on the concentration of glutamine in the haemolymph, but there is an increase in arginine, histidine and lysine in the haemolymph, suggesting the release of these essential amino acids from zooxanthellae. Glutamine synthetase (GS) activity decreased markedly in the gill and less so in the mantle over a period of 6 d exposure to elevated ammonium (20 μM). In contrast, GS activity in zooxan- thellae doubled. The response of zooxanthellae in situ was confirmed by incubating freshly isolated zooxanthellae for 4 d in ammonium, which resulted in a ten-fold increase in GS activity. Comparison of the in situ response of zooxanthellae with that obtained in vitro indicates that the symbionts are likely to be exposed to ammonium concentrations lower than that found in the haemolymph. Received: 14 November 1997 / Accepted: 28 April 1998     

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.     

Nutrient limitation in the giant clam-zooxanthellae symbiosis: effects of nutrient supplements on growth of the symbiotic partners

The effect of ammonium (5, 10 M N) and phosphate (2, 5, 10 M P) on the growth of the giant clam Tridacna gigas and its symbiotic dinoflagellate Symbiodinium sp. was examined. A 3 mo exposure to these nutritients significantly increased the N or P composition of the soft tissues, as reflected in a corresponding change in C:N:P ratio. Furthermore, exposure to N or N+P markedly increased the amount of soft tissue, but P alone did not, demonstrating that increased availability of inorganic nitrogen enhances tissue growth of the clam host. With addition of N, or N+P, there was a significant increase in the total number of zooxanthellae per clam, with a corresponding decrease in chlorophyll a (chl a) content per zooxanthella. However, only with N+P was there an increase in the zooxanthellae mitotic index. The inverse relationship between zooxanthellae number and chl a per zooxanthella is consistent with phytoplankton studies indicating conditions of nutrient-limitation. Furthermore, the unaffected C:N:P composition of the zooxanthellae and their relatively low specific-growth rates (4 to 10%) also suggest that they are nutrient-limited in vivo. In particular, their high mean C:N:P ratio of 303:52:1 indicates that, relative to C, they are much more depleted in P and less in N than are free-living phytoplankton. Furthermore, polyphosphates (phosphate reserves) were undetectable, and the activity levels of acid phosphatase in the zooxanthellae were relatively high and not influenced by the host's exposure to increased P concentrations in the sea water, implicating the clam host in active regulation of P availability to its symbiotic algae. This is strong evidence that N-limitation of clam zooxanthellae is a function of the availability of ammonium to the symbiosis while, irrespective of nutrient levels in sea water, clam zooxanthellae still show characteristics of P-limitation.     

Host glutamine synthetase activities in the giant clam—zooxanthellae symbiosis: effects of clam size,elevated ammonia and continuous darkness

Host tissues and zooxanthellae of the giant clam Tridacna gigas contained glutamine synthetase, with the highest transferase activities present in the gill, followed by the kidney, mantle, zooxanthellae, foot, heart and adductor muscle, in that order. Synthetase activities of glutamine synthetase in host tissues and zooxanthellae were in a similar order, but the differences were not so marked. Host tissues also contained hexokinase, glucose-6-phosphate dehydrogenase and malate dehydrogenase. Highest hexokinase activities were present in the heart, followed, in order, by the gill, mantle, adductor muscle and foot. Highest glucose-6-phosphate dehydrogenase activities were present in the gill, followed by the mantle, heart, adductor muscle and foot. All tissues assayed contained high malate dehydrogenase activities. There was no detectable glutamate dehydrogenase activity. Glutamine synthetase activity in gill and mantle tissue decreased by 1.6% with every 1 cm increase in clam size. Host glutamine synthetase activity decreased by 80% in gill tissue and by 45% in mantle tissue in clams which were maintained for 8 d in continuous darkness. Similar effects were found when clams were kept in light in the presence of elevated ammonia concentrations. It is suggested that both host and symbionts are nitrogen-deficient in small clams and that host glutamine synthetase plays a role in ammonia assimilation by the intact association.     

Characterization of two glutamate dehydrogenases from the symbiotic microalga Symbiodinium microadriaticum isolated from the coral Acropora formosa

In view of their possible involvement in ammonium assimilation in the coral/algal symbiosis, we have purified two distinct glutamate dehydrogenase isoenzymes from the symbiotic dinoflagellate Symbiodinium microadriaticum (Freudenthal) extracted from the staghorn coral Acropora formosa collected from Magnetic Island, North Queensland, Australia, in 1986–1987. An NADPH-specific glutamate dehydrogenase (GDH) displayed biphasic kinetics with respect to ammonium as the variable substrate; at low substrate concentrations the apparent K _m was below 1 mM, whereas at high substrate concentrations the corresponding value was approximately 200 mM. The NADPH-GDH displayed extremely low activity in the direction of glutamate oxidation; together with the kinetic data this suggests a probable role in ammonium assimilation. A second (NADH-specific) GDH was found to have both amination and deamination activities, and presumably functions in vivo in glutamate oxidation. Kinetic constants are reported for both GDH isoenzymes.     

Some factors influencing selective release of soluble organic material by zooxanthellae from reef corals

Studies were carried out to determine optimum conditions for the investigation of symbiotic zooxanthellae in vitro and to gain insight into factors influencing release of photosynthate by the symbionts. Zooxanthellae isolated from the reef coral Agaricia agaricites and incubated with an homogenate of host tissue release twice as much photosynthate as controls in seawater. The animal homogenate retained its stimulatory activity for 3 h at room temperature (ca. 26°C). Release of photosynthate was markedly influenced by time after isolation of algae from the host, variation in homogenate concentration, and prolonged exposure to homogenate. Release was not influenced by cell concentration, light intensity, or glycerol in the incubation medium. If zooxanthellae are labelled in vitro with glucose ¹⁴C, the principle product released is alanine ¹⁴C. The mechanism of action of homogenate on zooxanthellae in vitro is discussed in terms of its effect on algal cell membrane permeability. A preliminary fractionation of host homogenate is described.     

Tolerance of estuarine benthic diatoms to high concentrations of ammonia,nitrite ion,nitrate ion and orthophosphate

A carbon-14 assimilation method was used to determine action spectra and photosynthesis versus irradiance (P versus I) curves of natural populations of phytoplankton and zooxanthellae from a coral reef fringing Lizard Island in the Australian Barrier Reef. The action spectra were related to the phytoplankton species composition. The curves showed shade adaptation in phytoplankton from deeper waters and in the zooxanthellae. Rates of photosynthesis of zooxanthellae were shown to be highly but variably dependent on their host organisms. Photosynthetic production by zooxanthellae was about 0.9 gC m^-2 day^-1, which is about three times higher than phytoplankton production in the waters close to the reef.     

Lipogenesis in the intact coral Pocillopora capitata and its isolated zooxanthellae: Evidence for a light-driven carbon cycle between symbiont and host

Surface tissue of the reef coral Pocillopora capitata contained approximately 34% lipid on a dry weight basis. Of this, 75% was storage lipid (wax ester and triglyceride) and 25% structural (phospholipid, galactolipid, etc.). Based on chlorophyll a: lipid ratios of intact coral and isolated zooxanthellae, it was determined that over 90% of the storage lipid resided in the host tissue. One half of the structural lipids was found in the host and the other in the symbiotic algae. Gentle fractionation of coral tissue indicated that zooxanthellae possessed less than 14% of the total coral protein. Coral tips and isolated zooxanthellae were incubated with sodium acetate-1-¹⁴C in light and dark to obtain lipogenic rates and proportions of fatty acids and lipid classes synthesized. The rate of lipid synthesis from acetate-1-¹⁴C by intact coral was stimulated three-fold in the light (1200 lux), which indicated that the majority of coral lipogenesis occurred in the zooxanthellae. Intact coral triglycerides contained ca. 68% of the ¹⁴C-activity and wax esters ca. 21%. Zooxanthellae isolated by the Water Pik technique synthesized negligible amounts of wax ester, which implied that wax ester synthesis was a property of the animal tissue. Isolated zooxanthellae and intact coral synthesized identical triglyceride fatty acids from acetate-1-¹⁴C. This study provides evidence for a carbon cycle between host and symbiont whereby the zooxanthellae take up host-derived carbon (probably in the form of acetate), synthesize fatty acids using their photosynthetically derived energy, and return the lipid to the host where it appears as wax ester and triglyceride.     

Glycerol translocation in Condylactis gigantea

Sodium cyanide (NaCN) was used to partially uncouple respiration and photosynthesis in the symbiotic sea anemone Condylactis gigantea. NaCN significantly increased the ratio of gross photosynthesis to respiration in both intact tentacles and isolated zooxanthellae (Symbiodinium microadriaticum), increased carbon translocation from 17.7±3.5% of total fixed in controls to 43.5±5.8%, and doubled the amount of photosynthetically fixed carbon accumulating in the coelenterate host over that in controls. Only 2% of the non-particulate radioactivity recovered in the host tissue was ¹⁴C-glycerol when uninhibited symbiotic tentacles were incubated in ¹⁴C-bicarbonate for 1 h. At 10^-5 M NaCN, approximately 25% of the host nonparticulate radioactivity was recovered as ¹⁴C-glycerol, the absolute concentration of glycerol in the host tissue was three times higher than in controls, and ¹⁴C-glycerol was found in the medium. While glycerol has been proposed to play a major role in the translocation of photosynthetically fixed carbon from zooxanthellae to their coelenterate hosts, its concentration has never been measured in the animal and algal components of the symbiosis. The isolated zooxanthellae contained 3.62±0.33 mM glycerol, 26x the 0.141±0.02 mM found in the anemone. Aposymbiotic anemone tissue contained 0.169±0.06 mM glycerol. The rate of glycerol mineralization was not saturated even when exogenous glycerol levels were 70x internal concentrations. These data show that respiration and photosynthesis in symbiotic associations may be partially uncoupled by NaCN, and that this uncoupling allows the verification of the translocation and rapid catabolism of glycerol within the host.