Nutritional exchange in a tropical tripartite symbiosis: direct evidence for the transfer of nutrients from anemonefish to host anemone and zooxanthellae

The relationship between anemones and anemonefishes is an oft-cited and endearing example of a mutualistic symbiosis. Current research on mutualistic symbioses suggests these relationships are more commonplace and have greater importance at the ecosystem level on nutrient dynamics and evolutionary processes than previously thought. Using stable isotopes ¹⁵N and ¹³C, both field and laboratory experiments were designed to investigate whether nutrient transfer from two species of resident anemonefishes (Amphiprion perideraion and A. clarkii) to host anemones (Heteractis crispa) occurs. Mass spectroscopy indicated that both ¹⁵N and ¹³C were significantly elevated in the tissues of anemonefishes and in both host anemone and zooxanthellae fractions. These experiments provide the first direct empirical evidence of nitrogen and carbon transfer from resident anemonefishes to host anemones and endosymbiotic zooxanthellae. Such transfer of elements within this intriguing tripartite association underscores the central role that nutrient dynamics contributes to the evolutionary processes of these marine symbioses.     

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.     

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.     

Viable algae released by the seastar <Emphasis Type="Italic">Dermasterias</Emphasis><Emphasis Type="Italic">imbricata</Emphasis> feeding on the symbiotic sea anemone <Emphasis Type="Italic">Anthopleura elegantissima</Emphasis>

Echinoderms are major predators of anemones in temperate ecosystems. The fate of two algae, zooxanthellae and zoochlorellae, after their host anemone (Anthopleura elegantissima Brandt) was consumed by the leather star Dermasterias imbricata Grube was determined in experiments conducted in July and August 2004. Productivity, photosynthetic pigments, and mitotic index (percent of cells dividing) were used as indicators of algal health; algae released after leather stars consumed their host were compared with algae freshly isolated from anemones. Two types of waste products contained algae: pellets resulting from extraoral digestion, and feces. Zooxanthellae and zoochlorellae isolated from these waste products were photosynthetic, although to different extents. For algae from feces and pellets, light-saturated photosynthetic rates (P _max) were 85 and 13%, respectively, of P _max of freshly isolated zooxanthellae; and were 20 and 46%, respectively, for zoochlorellae. The photosynthetic pigments and mitotic index (percent of dividing cells) were not altered by the feeding activities of the leather star. These results show that algae released by seastar predation on their hosts remain viable, and are hence available for establishing symbioses in A. elegantissima and other potential hosts.     

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.     

A comparative analysis of the photobiology of zooxanthellae and zoochlorellae symbiotic with the temperate clonal anemone <Emphasis Type="Italic">Anthopleura elegantissima</Emphasis> (Brandt). III. Seasonal effects of natural light and temperature on photosynthesis and respiration

The sea anemone Anthopleura elegantissima hosts two phylogenetically different symbiotic microalgae, a dinoflagellate Symbiodinium (zooxanthellae, ZX) and a chlorophyte (zoochlorellae, ZC). The photosynthetic productivity (P), respiration (R), and contribution of algal carbon translocated to the host (CZAR) in response to a year’s seasonal ambient changes of natural light and temperature are documented for both ZX- and ZC-bearing anemones. Light and temperature both affect photosynthesis, respiration, and CZAR, as well as various algal parameters; while there are evident seasonal differences, for the most part the relative effects on P, R, and CZAR by the two environmental variables cannot be determined. Net photosynthesis (P_n) of both ZX and ZC was significantly higher during spring and summer. During these seasons, the P_n of ZX was always greater than that of ZC. Regardless of algal symbiont, anemone respiration (R) was significantly higher during the spring and summer. The annual net carbon fixation rate of anemones with ZX and ZC was 325 and 276 mg C anemone⁻¹ year⁻¹, respectively, which translates to annual net community productivity rates of 92 and 60 g C m⁻¹ year⁻¹ for anemones with ZX or ZC, respectively. CZAR did not show a clear relationship with season; however the CZAR for ZX was always significantly greater than for ZC. Lower ZX growth rates, coupled with higher photosynthetic rates and higher CZAR estimates, compared to ZC, suggest that if A. elegantissima is simply carbon limited, ZX-bearing anemones should be the dominant symbiont in the field. However ZC-bearing anemones persist in low light and reduced temperature microhabitats, therefore more than the translocation of carbon from ZC must be involved. Given that global climate change will increase water temperatures, the potential for latitudinal range shifts of both ZC and ZX (S. californium and muscatinei) might be used as biological indicators of thermal shifts in the littoral zone of the Pacific Northwest.     

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     

Effects of host feeding and dissolved ammonium on cell division and nitrogen status of zooxanthellae in the hydroid Myrionema amboinense

There is a relationship between host feeding, nitrogen status and mitotic activity of zooxanthellae symbiotic with the marine hydroid Myrionema amboinense. Decreases in the mitotic index of zooxanthellae in starved M. amboinense, and in internal pool sizes of glutamine and glutamate, amino acids involved in ammonium assimilation via the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway, were partially restored by addition of ammonium chloride to seawater in which hydroids were incubated. Levels of glutamine were more sensitive to host starvation than levels of glutamate, resulting in a decrease in the glutamine: glutamate molar ratio to that found in zooxanthellae cultured on nitrate. Hydroids starved for 5 d and then incubated in different concentrations of ammonium chloride showed a positive correlation between ammonium concentration and mitotic index of their symbiotic zooxanthellae. Host starvation caused a decrease in perturbation of levels of glutamine and glutamate during ammonium assimilation, as well as decreases in rates of assimilation of [¹⁴C]-leucine into TCA-insoluble protein, and in photosynthetic incorporation of [¹⁴C]-bicarbonate. These observations suggest that host starvation reduces nitrogen supply to the zooxanthellae, causing nitrogen stress to the symbionts and reduction in metabolic processes associated with nitrogen assimilation and photosynthesis as well as with cell division.     

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.     

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.     

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.     

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.     

Acetate incorporation into the lipids of the anemone Anthopleura elegantissima and its associated zooxanthellae

Anthopleura elegantissima containing zooxanthellae, as well as isolated zooxanthellae, incubated with acetate-1-¹⁴C under both light and dark conditions readily incorporate radioactivity into their total lipid pools. In both cases, the specific activity was greatly increased in the light. Dark-incubated anemones and isolated zooxanthellae incorporate activity predominantly into polar lipid; the remainder being present principally in the triglyceride moiety. Light-incubated organisms, however, show a dramatic redistribution of isotope towards greatly increased triglyceride and was ester incorporation, with a concomitant drop in polar lipid. onder light conditions, 70 to 75% of the radioactivity found in the fatty acids of the total zooxanthellae lipid was present in hexadecanoic (16:0) and octadecenoic (18:1) fatty acids. These are also the two major fatty acids by mass. Octadecanoic acid (18:0) is less than 5% by mass. Isotope incorporation patterns suggest that octadecenoic acids arise by elongation of hexadecenoic acids and that this conversion is blocked in the dark. Isotope incorporation patterns for anemones suggest that fatty acids, primarily in the form of saturated or monoenoic fatty acids, are translocated from algal to animal cells. No activity was found in either octadecadienoic (18:2) or octadecatrienoic (18:3) acids. The significance of these data is discussed.     

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     

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.     

Physiological energetics of the intertidal sea anemone Anthopleura elegantissima

Energy budgets were calculated for individuals of the sea anemone Anthopleura elegantissima (Brandt), collected in 1981 and 1982 from Bodega Harbor, California, USA. Rates of ammonium excretion were measured in high-and low-intertidal, symbiotic and aposymbiotic sea anemones within 24 h of collection. Among symbiotic and aposymbiotic individuals, no differences in excretion rate were found on the basis of intertidal height. However, rates of ammonium excretion in aposymbiotic anemones (2.14 mol NH ₊ ⁴ g^-1 h^-1) were significantly higher than in symbiotic ones (0.288 mol NH ₊ ⁴ g^-1 h^-1). Rates of excretion were used with estimated rates of oxygen uptake to calculate nitrogen quotients (NQ). NQ and RQ values from the literature were used to calculate an oxyenthalpic equivalent [501 kJ (mol O₂)^-1 for R+U], and mass proportions of protein (54%), carbohydrate (44%) and lipid (2%) catabolized during routine metabolism in this species 24 h after feeding. Integrated energy budgets of these experimental anemones were calculated from data on ingestion, absorption and growth, and estimates of translocated energy from the symbiotic algae. Contribution of zooxanthellae to animal respiration based on translocation=90% and RQ=0.97 are 41 and 79% in high-and low-intertidal anemones, respectively. Calculated scope for growth is greater than directly measured growth in both high-and low-intertidal individuals. The deficit, estimated as 30% of assimilated energy in high-intertidal anemones, is attributed to unmeasured costs (specific dynamic effect) or production (mucus). Low-intertidal anemones lost mass during the experiment, implying that the magnitude of the deficit was greater in these anemones than in upper intertidal individuals. Anemones from both shore levels lost zooxanthellae during the experiment, which contributed to energy loss since the contribution of the zooxanthellae is greater in low-intertidal anemones. Scope for growth is preserved in high-intertidal anemones (29% of assimilated energy) because metabolic demands are lower due to aerial exposure, and prey capture rate is higher compared to lowshore anemones. Although possibly underestimated, lower scope for growth in low-shore anemones may result from continuous feeding and digestion processes that are less efficient than those of periodically feeding high-intertidal anemones.     

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     

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.     

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.