Effects of nitrogen and phosphorus enrichement on in vivo symbiotic zooxanthellae of Pocillopora damicornis

The reef coral Pocillopora damicornis (Linnaeus) was grown for 8 wk in four nutrient treatments: control, consisting of ambient, unfiltered Kaneohe Bay seawater [dissolved inorganic nitrogen (DIN, 1.0 M) and dissolved inorganic phosphate (DIP, 0.3 M)]; nitrogen enrichment (15 M DIN as ammonium); phosphorus enrichment (1.2 M DIP as inorganic phosphate); and 15 M DIN+1.2 M DIP. Analyses of zooxanthellae for C, N, P and chlorophyll a after the 8 wk experiment indicated that DIN enrichment increased the cellular chlorophyll a and excess nitrogen fraction of the algae, but did not affect C cell^-1. DIP enrichment decreased both C and P cell^-1, but the decrease was proportionally less for C cell^-1. the response of cellular P to both DIN and DIP enrichment appeared to be in the same direction and could not be explained as a primary effect of external nutrient enrichment. The observed response of cellular P might be a consequence of in situ CO₂ limitation. DIN enrichment could increase the CO₂ (aq) demand by increasing the net production per unit area. DIP enrichment could slow down calcification, thus decreasing the availability of CO₂ (aq) in the coral tissue.Hawaii Institute of Marine Biology Contribution No. 920     

Effects of ambient levels of solar ultraviolet radiation on zooxanthellae and photosynthesis of the reef coral Montipora verrucosa

Paired flat plates of the hermatypic coral Montipora verrucosa from Kaneohe Bay, Oahu, Hawaii, were acclimated to photosynthetically active radiation (PAR) only and to full sunlight (PAR+UV) for several weeks in the summer of 1990. After the acclimation period, photosynthesis, both in PAR-only and PAR+UV as well as dark respiration were measured. Levels of the UV-absorbing compounds, S320, density of zooxanthellae, and chlorophyll a concentration were determined. Corals acclimated in PAR+UV had higher levels of the UV-protective compounds and lower areal zooxanthellae densities than corals acclimated in PAR-only. Chlorophyll a per unit volume of coral host and per algal cell did not differ between corals from the two acclimation treatments. Corals acclimated to PAR+UV displayed higher photosynthesis in full sunlight than corals acclimated to PAR-only, but when photosynthesis was measured in the light regime to which the corals had been acclimated, there were no differences in photosynthesis. Dark respiration was the same for corals from the two acclimation treatments regardless of the light quality immediately preceding the dark period.Contribution No. 902 HIMB     

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.     

Effect of daylight variations on the energy budgets of shallow-water corals

Energy budgets were determined for small pieces (nubbins) of the coralsPocillopora damicornis, Montipora verrucosa andPorites lobata living at a water depth of 3 m on the fringing reef of Coconut Island, Kaneohe, Hawaii. The budgets were determined for three different types of day: an ideal day with no cloud and an in situ daily integrated irradiance at 3 m of 14.385 E m^–2 d^–1; a normal day with sporadic cloud cover and daily irradiance of 11.915 E m^–2 d^–1; and an overcast day with daily irradiance of 6.128 E m^–2 d^–1. On the ideal day, the energy fixed in photosynthesis was more than that required for respiration and growth of both zooxanthellae and animal components of the association, and there was a predicted loss of between 19.3 and 32.4% of the energy fixed. On a normal day, the total photosynthetic energy fixation was lower and the excess was between 12.1 and 27.9% of the energy fixed. On the overcast day, however, in bothPocillopora damicornis andPorites lobata energy expenditure exceeded photosynthetic energy fixation and the budget was in deficit. Estimates of rate of mucus secretion on an overcast day were derived and, when incorporated into the energy budget, it was predicted that all three species would have a deficit budget, necessitating the catabolism of lipid reserves. From published values for lipid storage in these species it was calculated that the reserves would last from 28 d inPocillopora damicornis to 114 d inM. verrucosa. A model is suggested in which corals draw upon their extensive lipid stores on days of sub-optimal light, replenishing the reserves again when daily light levels are high, and finally excreting the excess energy fixed, as mucus-lipid when the lipid stores are replete.     

Nutrient enrichment and the ultrastructure of zooxanthellae from the giant clam Tridacna maxima

The separate and combined effects of ammonium (10M) and phosphate (2M) on the ultrastructure of zooxanthellae (Symbiodinium sp.) from giant clams, Tridacna maxima, were examined in the field. Nitrogen addition significantly changed the ultrastructure of the zooxanthellae inhabiting the clams. After 9 mo exposure, the cross-sectional area of zooxanthellae from N-treated clams was significantly lower than that from other treatments [N=39.3 m²; C=47.9 m²; P=43.2m²; N+P=44.5 m²; (P=0.001)]. There was also a significant decrease in the size of starch bodies, especially around the pyrenoid of the zooxanthellae from N and N+P treatments [N=1.2 m²; C=2.0 m²; P=1.8 m²; N+P=1.2 m²; (P=2.08E-11)]. This presumably occurs as a result of the mobilization of organic carbon stores in response to stimulated amino acid synthesis under enriched nutrient conditions. These data strongly suggest that the symbiotic zooxanthellae of clams are limited to some extent by the availability of inorganic nitrogen, and that relatively minor changes to the nutrient loading of the water column can have substantial effects on the biochemistry of symbioses such as that which exists between clams and zooxanthellae.     

The ammonium/methylammonium uptake system of Symbiodinium microadriaticum

The substrate analogue [¹⁴C]-methylammonium was used to study ammonium/methylammonium uptake by Symbiodinium microadriaticum (zooxanthellae). The value of the Michaelis constant (K _m) for the uptake system was approximately 35 M with methylammonium as substrate; ammonium was a competitive inhibitor of methylammonium uptake, and the K _m for ammonium uptake (determined as the inhibition constant, K _i, for methylammonium) was 6.6 M. Methylammonium uptake by zooxanthellae was light-dependent. Methylammonium uptake rates of zooxanthellae which had been freshly isolated from the hermatypic coral Acropora formosa (0.85±0.05x10^-10 mol min^-1 cell^-1) were lower than those of axenic cultures of the zooxanthellae from Montipora verrucosa (Acroporidae) grown under various nitrogen regimes (1.6 to 12x10^-10 mol min^-1 cell^-1). Maximum uptake rates were found for ammonium-starved cultured M. verrucosa zooxanthellae (10.2 to 12x10^-10 mol min^-1 cell^-1); M. verrucosa zooxanthellae growing with ammonium as nitrogen source and zooxanthellae which had been freshly isolated from A. formosa gave similar and considerably lower uptake rates (0.85 to 1.6x10^-1 mol min^-1 cell^-1). These results suggest that either coral tissue contains sufficient ammonium to repress synthesis of the uptake system of the algal symbionts or, alternatively, there are additional barriers to ammonium transport for zooxanthellae in vivo.     

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     

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.     

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.     

A chromatophore system in the hermatypic,deep-water coral Leptoseris fragilis (Anthozoa: Hexacorallia)

Ultrastructural evidence is presented of a chromatophoresystem in the zooxanthellae containing hermatypic, deep-water coral Leptoseris fragilis (Milne Edwards and Haime). It consists of multilobed cells which mainly occupy the intercellular space of the oral gastrodermis. The cellular processes are filled with electron-dense granules up to 1-m-long and 0.5-m-wide. Within the cytoplasm an elaborate system of microtubules is established. The ramifications of the pigment cells, containing the pigment granules, form a dense and nearly continuous layer close to the overlying zooxanthellae. It is speculated that host pigments may transform the violet portion of the incident light into longer wavelengths, thus increasing the photosynthetic efficiency of the zooxanthellae.     

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.     

Light responses of a scleractinian coral (Plerogyra sinuosa)

During daytime Plerogyra sinuosa Dana displays globular expandable tentacles (bubbles) which foster the photosynthetic ability of the coral. Adaptational responses of this coral to different depths (5–25 m) and light conditions were investigated by photosynthetic pigment analysis, insitu measurements of oxygen production, transplantation and shading experiments. Pigment concentrations per unit tissue dry weight were variable, but unrelated to depth. Pigment concentrations per zooxanthellae cell remained constant and bubble size increased with depth. Light intensity at 25 m was 20 to 25% of the 5-m value, but daily integrated rates of photosynthesis were 65% of the 5-m rates, indicating a higher light utilization efficiency in deeper corals. Coral heads transplanted from 25 to 5 m died within 20 d if not protected against UV-radiation, but corals transplanted from 5 to 25 m acclimatized to the new light condition. Photosynthetic oxygen production and bubble size increased in shaded, sun-adapted corals within 60 min and decreased in sun-exposed, shade-adapted corals. The variable bubble size is interpreted as an adaptational mechanism to optimize light exposure of zooxanthellae.     

Uptake of dissolved inorganic nitrogen by the symbiotic clam Tridacna gigas and the coral Acropora sp.

Dissolved inorganic nitrogen flux was studied in the giant clam Tridacna gigas and the corals Acropora sp. and Tubastrea micrantha from the tropical reefs of Belau, Micronesia in 1983. T. micrantha, a nonsymbiotic coral, excreted ammonium. However, Tridacna gigas and Acropora sp., which contain symbiotic dinoflagellates (zooxanthellae) were able to take up both ammonium and nitrate. The requirement for a previous light exposure to sustain uptake by T. gigas is reported. The uptake kinetics of these symbioses are described and include the capacity of the zooxanthellae for surge uptake when given nutrient spikes.Contribution No. 417 of the Allan Hancock Foundation     

Night irradiance and synchronization of lunar release of planula larvae in the reef coral Pocillopora damicornis

Pocillopora damicornis (Linnaeus), which is known to release planula larvae on a monthly cycle, was grown in full daytime solar irradiance, but with four treatments of night irradiance: (1) natural night irradiance, (2) shifted-phase (total darkness during nights of full moon with artificial irradiance at lunar intensity on nights of new moon), (3) constant full moon (full lunar irradiance every night), and (4) constant new moon (total darkness every night). The reproductive cycle of the corals held in the shifted-phase treatment moved out of synchrony with the cycle of corals exposed to a natural lunar cycle of night irradiance. Two previously described types of P. damicornis were tested. The Type Y normally start releasing larvae at full moon, with peak production at third quarter. In the shifted-phase treatment they began releasing planulae at new moon (artificial full moon), with peak production at first quarter. The Type B corals, that normally start releasing planulae at new moon with peak production at first quarter, began to release planulae at full moon (artificial new moon), with peak production at third quarter. Populations of corals grown either in the constant full moon or constant new moon treatment quickly lost synchronization of monthly larva production, although production of planulae continued. Thus spawning is synchronized by night irradiance.Contribution No. 702 of the Hawaii Institute of Marine Biology     

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.     

Laboratory observations on spawning,fecundity and early development of a mesopelagic ostracod,Conchoecia pseudodiscophora,from the Japan Sea

The fate of the polycyclic aromatic hydrocarbon [³H]benzo[a]pyrene (BaP) was examined in two species of scleractinian corals, Favia fragum (Esper) and Montastrea annularis (Ellis and Solander), which were collected in the patch reefs surrounding Alina's Reef (25°23.25N; 80°09.8W) in Biscayne National Park, Florida, USA, in July, 1990. Corals were exposed to initial concentrations of 5 g/l in a simple static system for 25 h. BaP uptake was estimated from the disappearance of BaP from the water. Uptake rates were 6.5±0.7 and 10.8±0.2 g BaP cm^-2h^-1 for F. fragum and M. annularis, respectively, at initial BaP concentrations and were directly proportional to the concentration of BaP in the water. The separation of zooxanthellae from coral tissue revealed that zooxanthellae can accumulate up to 53 and 64% of the total BaP-derived radioactivity present in F. fragum and M. annularis, respectively. Both corals metabolized BaP slowly, as most of the accumulated radioactivity was present as the unmetabolized chemical. However, aqueous and organic-soluble metabolites were found in both the animal and zooxanthellae fractions. Analysis by high-performance liquid chromatography (HPLC) revealed that both species of corals metabolized BaP to various tetrols, triols, dihydrodiols, quinones and phenols, although the pattern of metabolites differed between species. Zooxanthellae contained some of the same Phase I metabolites found in the animal tissue; however, tetrols and triols were absent in extracts from the zooxanthellae. The elimination of BAP from corals was also slow; approximately 38 and 65% of the accumulated radioactivity was still present in F. fragum and M. annularis, respectively, 144 h following the transfer of exposed corals to an uncontaminated flow-through seawater system.     

Effects of light intensity on nitrate and nitrite uptake and excretion by Chaetoceros curvisetus

Michaelis-Menten uptake kinetics were observed at all light intensities. With constant illumination, the V_max and K₁ in nitrate uptake over the natural light intensity range of 0 to 2000 E were 0.343 g-at NO₃–N(g)^-1 at protein-N h^-1 and 26 E, respectively. Nitrate uptake was inhibited at higher light intensities. The K_s for nitrate uptake did not vary as a function of light intensity remaining relatively constant at 0.62 g-at NO₃–N 1^-1. With intermittent illumination, the V_mzx for light intensity in nitrate uptake over a light intensity range of 0 to 5000 E was 0.341 g-at NO₃–N(g)^-1-at protein-N h^-1. No inhibition of nitrate uptake was observed at higher than natural light intensities. Chaetoceros curvisetus will probably never experience light inhibition of nitrate uptake under natural conditions.     

Primary production in Dumbell Bay in the Arctic Ocean

Photosynthesis, chlorophyll, inorganic nutrients, and related factors were measured throughout a productive season in a small coastal inlet of the Arctic Ocean. Significant production was confined to 5 m for a very limited time period beacause of both light and nutrient limitations. Maximum chlorophyll was 8.2 mg Chl a m^-3 and maximum gross and net photosynthesis rates were 830 and 550 mg C m^-2 d^-1. Annual gross and net photosynthesis is estimated at about 12 and 9 g C m^-2 respectively. The effect of light stimulation on assimilation is described, sources of coastal zone nutrient enrichment are considered, and discrepancies between gross and net photosynthesis are discussed with reference to the growth characteristics of the phytoplankton population.Bigelow Laboratory for Ocean Sciences. Contribution No. 76008     

Phosphate acquisition in the giant clam-zooxanthellae symbiosis

The effect of phosphate on the giant clam Tridacna gigas and on its symbiotic dinoflagellate Symbiodinium sp. was compared with that on cultured Symbiodinium sp. originally isolated from the same clarn species. Incubation of whole clams in elevated phosphate (10 M) reduced their capacity for phosphate uptake, but the uptake capacity of the clam's zooxanthellae population was not influenced. In addition, there was no change in the zooxanthellae density and the N:P ratio, of these algae., On the other hand, cultured zooxanthellae were influenced by the phosphate regimen of their culture medium. Compared with controls (0 M P), addition of 10 M phosphate to the culture medium caused an increase of 100% in cell density and decreases of 50% in the N:P ratio, and 80% in the phosphate-uptake capacity of the zooxanthellae. Zooxanthellae freshly isolated from the clams exhibited properties similar to those of zooxanthellae cultured in the absence of phosphate. These results demonstrate that the zooxanthellae population of T. gigas have limited access to the inorganic phosphate in sea water and the phosphate reserves within the animal host.     

The eye and some effects of light on locomotor activity in Nephrops norvegicus

The compound eye of Nephrops norvegicus (L.) is of the superposition type, well-adapted to the low levels of light prevailing at the sea bed during the activity periods of the species. Only the proximal retinal shielding pigment responds to light, the distal retinal shielding pigment being in the dark-adapted position at all times. The response of the proximal pigment appears to vary seasonally. Field observations compared light intensity at the sea bed with the numbers of N. norvegicus caught by trawl at various times of day in the Irish Sea in summer and winter. Laboratory experiments were combined with these field data to indicate that light is an important modulator of locomotor activity in this species.