Temperate Sea Anemone Research Articles

Comparison of sequence-capture and ddRAD approaches in resolving species and populations in hexacorallian anthozoans

Genome-level sequencing is the next step in understanding species-level relationships within Anthozoa (soft corals, anemones, stony corals, and their kin) as morphological and PCR-directed (single-locus) sequencing methods often fall short of differentiating species. The sea anemone genus Metridium is a common northern temperate sea anemone whose species are difficult to differentiate using morphology alone. Here we use Metridium as a case study to confirm the low level of information available in six loci for species differentiation commonly sequenced for Actiniaria and explore and compare the efficacy of ddRAD and sequence-capture methods in species-level systematics and biogeographic studies. We produce phylogenetic trees from concatenated datasets and perform DAPC and STRUCTURE analyses using SNP data. The six conventional loci are not able to consistently differentiate species within Metridium. The sequence-capture dataset resulted in high support and resolution for both current species and relationships between geographic areas. The ddRAD datasets displayed ambiguity among species, and support between major geographic groupings was not as high as the sequence-capture datasets. The level of resolution and support resulting from the sequence-capture data, combined with the ability to add additional individuals and expand beyond the genus Metridium over time, emphasizes the utility of sequence-capture methods for both systematics and future biogeographic studies within anthozoans. We discuss the strengths and weaknesses of the genomic approaches in light of our findings and suggest potential implications for the biogeography of Metridium based on our sampling.

Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species.

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.

Carbonic anhydrases are influenced by the size and symbiont identity of the aggregating sea anemone Anthopleura elegantissima.

Carbonic anhydrases (CA; EC 4.2.1.1) play a vital role in dissolved inorganic carbon (DIC) transport to photosynthetic microalgae residing in symbiotic cnidarians. The temperate sea anemone Anthopleura elegantissima can occur in three symbiotic states: hosting Breviolum muscatinei (brown), hosting Elliptochloris marina (green) or without algal symbionts (aposymbiotic). This provides a basis for A. elegantissima to be a model for detailed studies of the role of CA in DIC transport. This study investigated the effects of symbiosis, body size and light on CA activity and expression, and suggests that A. elegantissima has a heterotrophy-dominated trophic strategy. We identified putative A. elegantissima CA genes and performed phylogenetic analyses to infer subcellular localization in anemones. We performed experiments on field-collected anemones to compare: (1) CA activity and expression from anemones in different symbiotic states, (2) CA activity in brown anemones as a function of size, and (3) CA activity in anemones of different symbiotic states that were exposed to different light intensities. CA activity in brown anemones was highest, whereas activity in green and aposymbiotic anemones was low. Several CAs had expression patterns that mirrored activity, while another had expression that was inversely correlated with activity, suggesting that symbionts may induce different DIC transport pathways. Finally, CA activity was inversely correlated with anemone size. Our results suggest that the observed CA activity and expression patterns are affected not only by symbiosis, but also by other factors in the host physiology, including trophic strategy as it relates to body size and cellular pH homeostasis.

Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: Lesson learned from Anemonia viridis

The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano. Two versions of fused homing endonuclease gene (HEG) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA, a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function.

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level.

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5 and + 8°C) during 1 and 7days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a + 8°C hyperthermia induced a transient increase of antioxidant defences at 1day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.

Transcriptome sequencing and characterization of Symbiodinium muscatinei and Elliptochloris marina, symbionts found within the aggregating sea anemone Anthopleura elegantissima

There is a growing body of literature using transcriptomic data to study how tropical cnidarians and their photosynthetic endosymbionts respond to environmental stressors and participate in metabolic exchange. Despite these efforts, our understanding of how essential genes function to facilitate symbiosis establishment and maintenance remains limited. The inclusion of taxonomically and ecologically diverse endosymbionts will enhance our understanding of these interactions. Here we characterize the transcriptomes of two very different symbionts found within the temperate sea anemone Anthopleura elegantissima: the chlorophyte Elliptochloris marina and the dinoflagellate Symbiodinium muscatinei. We use a multi-level approach to assess the diversity of genes found across S. muscatinei and E. marina transcriptomes, and compare their overall protein domains with other dinoflagellates and chlorophytes. Our analysis identified several genes that are potentially involved in mitigating stress response (e.g., heat shock proteins pathways for mediating reactive oxygen species) and metabolic exchange (e.g., ion transporters). Finally, we show that S. muscatinei and other Symbiodinium strains are equipped with a high salt peridinin-chl-protein (HSPCP) gene previously identified only in free-living dinoflagellates. The addition of these transcriptomes to the cnidarian-symbiont molecular toolkit will aid in understanding how these vitally important symbiotic relationships are established and maintained across a variety of environmental conditions.

Photophysiology and hydrogen peroxide generation of the dinoflagellate and chlorophyte symbionts of the sea anemone Anthopleura elegantissima

Associating with algal symbionts is considered largely beneficial for cnidarians such as corals and sea anemones, yet there are potential costs of hosting symbionts, such as the production of reactive oxygen species. We compared the photophysiology and H2O2 production rates of Symbiodinium muscatinei and Elliptochloris marina, the dinoflagellate and chlorophyte symbionts, respectively, of the temperate sea anemone Anthopleura elegantissima. Analyses of photosystem II (PSII) function in the two symbionts, including maximum quantum yield and relative electron transport rates, were consistent with prior studies indicating that E. marina has lower photosynthetic performance than S. muscatinei at high temperature and irradiance. The efficiency of PSII in both symbionts was positively affected by the addition of exogenous catalase, suggesting that both symbionts experience H2O2-mediated declines in PSII efficiency. We found that S. muscatinei produced more H2O2 than E. marina across all treatments, with the highest production under high light and temperature. Results were similar in experiments involving both isolated symbionts and symbionts residing within intact tentacles, indicating that the potential stress of symbiont isolation was not a significant factor biasing our results. Despite the lower typical densities of S. muscatinei relative to E. marina, extrapolations of cell-specific H2O2 production rates to the intact symbiosis suggest that S. muscatinei imposes a greater H2O2 burden on A. elegantissima. Even with this burden, however, the considerably higher productivity and fitness of S. muscatinei-bearing anemones documented in prior studies suggests that the net benefit of hosting S. muscatinei exceeds that of E. marina.

Temperature Tolerance Test Exposition with Temperate Sea Anemone Actinia equina, a Climatic and Environmental Changes Simulation

Atlantic and Mediterranean warming-related diseases outbreaks and species shifts recently have been documented. Evaluated tools of short-term effects on the health or organisms resistance are necessary to assess and understand mechanisms affecting marine biodiversity. Until now, climate warming has been studied at the population or community level. Here we offer a better understanding of such phenomena at the individual organism level, using anatomic-morphological approaches to interpret effects of natural physical stressors, according to behavioral patterns. The goal of this work was to evaluate the sea anemones behavior with temperature variance. This study takes a method of behavioral observations (morphological and anatomic parameters, with physiological implications) to identify changes in behavior, after exposure to the physical stressors temperature (10℃, 15℃, 20℃, 25℃ and 30℃) on temperate sea anemone Actinia equina over 96 h of exposure. Other endpoints as condition index and reproduction also assessed. Behavioral patterns analysis placed the differentially ecological functions in a wide range of categories including tentacle flexion, tentacle retraction, column cavitation, peristome depression and oral disc flexion. These parameters suggest that the “early stress response” (before result on individual death) to elevated temperature involves essentially all aspects of same chemical reactions. In this case we observed receptors functioning and the frequency of open-close oral sea anemones, tentacles and columns anatomic alterations to detect earlier the effects of physical stress induction. The superiority of results tested was that the key species reacted to different temperature ranges in order to demonstrate that species from different climatic zones could have the same behavioral pattern but have intrinsic adaptations on each climatic zone. Also some collections of parameters such as: 1) water nutrients availability, 2) reproductions rate (number of polyps), 3) survival (condition index) and 4) temperature variations were significant on behavioral answers.

Resilience to ocean acidification: decreased carbonic anhydrase activity in sea anemones under high pCO2 conditions

Non-calcifying photosynthetic anthozoans have emerged as a group that may thrive under high carbon dioxide partial pressure (pCO(2)) conditions via increased productivity. However, the physiological mechanisms underlying this potential success are unclear. Here we investigated the impact of high pCO(2) on the dissolved inorganic carbon (DIC) use in the temperate sea anemone Anemonia viridis. We assessed the impacts of long-term exposure to high pCO(2), i.e. sampling in situ natural CO2 vents (Vulcano, Italy), and short-term exposure, i.e. during a 3 wk controlled laboratory experiment. We focused on photo-physiological parameters (net photosynthesis rates, chlorophyll a content and Symbiodinium density) and on carbonic anhydrase (CA) activity, an enzyme involved in the energy-demanding process of DIC absorption. Long-term exposure to high pCO(2) had no impact on Symbiodinium density and chlorophyll a content. In contrst, short-term exposure to high pCO(2) induced a significant reduction in Symbiodinium density, which together with unchanged net photosynthesis resulted in the increase of Symbiodinium productivity per cell. Finally, in both in situ long-term and laboratory short-term exposure to high pCO(2), we observed a significant decrease in the CA activity of sea anemones, suggesting a change in DIC use (i.e. from an HCO3- to a CO2 user). This change could enable a shift in the energy budget that may increase the ability of non-calcifying photosynthetic anthozoans to cope with ocean acidification.

Diet of the sea anemone Anthopleura nigrescens: composition and variation between daytime and nighttime high tides

The diverse feeding habits of sea anemones have led them to be classified as opportunistic polyphagous predators. However, most studies have focused on the diet of temperate sea anemones, and little is known about the trophic ecology of tropical species and their role in tropical ecosystems. We studied the diet of the intertidal sea anemone Anthopleura nigrescens by examining the gut contents of specimens collected at two sites on Costa Rica’s Pacific coast, testing for differences in prey items captured between daytime and nighttime high tides. We found 25 prey items including: mollusks, crustaceans, annelids, insects and eggs. Juvenile bivalves, barnacle cyprids, and copepods were the main prey items. The anemones at Playa Dominical captured significantly more juvenile bivalves during nighttime high tides, and those at Mata de Limon captured more cyprids during daytime high tides. This study shows that A. nigrescens is an opportunistic polyphagous predator, like most of its temperate counterparts.

Relationships Between Host and Symbiont Cell Cycles in Sea Anemones and Their Symbiotic Dinoflagellates

The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbiotic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastrodermal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across species and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

The effects of symbiotic state on heterotrophic feeding in the temperate sea anemone Anthopleura elegantissima

The temperate sea anemone Anthopleura elegantissima is facultatively symbiotic with unicellular algae. Symbiotic A. elegantissima can supplement heterotrophic feeding with excess photosynthate from their algal partners, while asymbiotic individuals must rely solely on heterotrophy. A. elegantissima individuals were collected from Swirl Rocks, Washington (48°25′6″ N, 122°50′58″ W) in July 2010, and prey capture and feeding characteristics were measured to determine whether asymbiotic individuals are more efficient predators. Feeding abilities were then measured again after a 3-week exposure to full sunlight or shaded conditions. Freshly collected asymbiotic anemones had larger nematocysts, but symbiotic individuals showed greater nematocyte sensitivity. Sunlight enhanced digestion and reduced cnida density in all anemones regardless of symbiotic state. Results suggest that the phototropic potential of A. elegantissima, as influenced by symbiotic condition, has little effect on heterotrophic capacity. The anemones appear to maximize heterotrophic energy input independent of the presence or identity of their algal symbionts.

Response of the symbiotic cnidarian Anthopleura elegantissima transcriptome to temperature and UV increase

Elevated temperature and solar radiation, including ultraviolet radiation, are now recognized as the primary environmental stresses that lead to mass cnidarian bleaching. This study takes a functional genomics approach to identifying genes that change expression soon after exposure to these stressors in the temperate sea anemone Anthopleura elegantissima that harbors Symbiodinium, the same genus of symbionts found in reef-building corals. Symbiotic anemones were subjected to elevated temperature or UV over a 24 h period. cDNA from these animals was hybridized to a 10,000-feature cDNA microarray of A. elegantissima. Overall 2.7% of the 10,000 features were found to be differentially expressed as a function of temperature or UV stress. Of the 86 features sequenced, 45% displayed significant homology to sequences in GenBank. There are 27 features that were differentially expressed in both stress conditions. Gene ontology analysis placed the differentially expressed genes in a wide range of categories including cytoskeleton organization and biogenesis, protein biosynthesis, cell proliferation, apoptosis and transport. This suggests that the early stress response to elevated temperature and UV involves essentially all aspects of host cellular regulation and machinery and that downstream cnidarian bleaching is a complex cellular response in host tissues.

Symbiont distribution along a light gradient within an intertidal cave

The potentially lethal bleaching of invertebrates through loss of their algal symbionts is a major global conservation issue; cnidarians in such interactions could potentially withstand environmental change by having multiple symbiotic partners whose physiologies vary with critical field parameters such as temperature and light. Spatial variation in symbiont distributions in the field is a strong indicator of such potential. We show that, in an association between an abundant temperate sea anemone and its two endosymbionts, the dinophyte Symbiodinium muscatinei (zooxanthellae) and an unidentified green alga (zoochlorellae), densities and ratios of anemones’ symbionts change along a light gradient generated by intertidal caves in Washington state. In the laboratory, the photosynthetic performance of zooxanthellate anemones exceeded that of zoochlorellate anemones under high irradiance levels (.100 mmol quanta m 22 s 21 ). Further, field sampling revealed that caves are divided into three distinct regions based on anemones’ algal complements: a brown region of zooxanthellate anemones near the mouth of the cave, a green region of zoochlorellate anemones in the middle of the cave, and a white region of algae-free anemones near the back of the cave. These data, together with field reciprocal transplant experiments showing algae-free anemones’ ability to regrow algae consistent with local physical conditions, suggest that algal performance (vs. host selection) determines observed variation in these symbioses. Our results support the idea that temperate, as well as tropical, host‐symbiont associations can respond plastically to environmental change. Our data also suggest that intact symbioses in adult hosts are surprisingly stable; bleaching may be required in rearranging host‐symbiont associations.

Differential accumulation of heavy metals in the sea anemone Anthopleura elegantissima as a function of symbiotic state

The accumulation of metals by the North American Pacific Coast temperate sea anemone Anthopleura elegantissima, and its dinoflagellate-algal symbiont Symbiodinium muscatinei was examined following laboratory metal exposures. Both, naturally occurring symbiotic and symbiont-free (aposymbiotic) anemones were used in this study to investigate differences in metal uptake due to the symbiotic state of the animal. The effects of metal exposures on the anemone-algal symbiosis were determined using measures of algal cell density and mitotic index (MI). Anemones were exposed to either cadmium, copper, nickel or zinc chloride (0, 10, 100 μg l −1 for Cd, Cu and Ni; 0, 100, 1000 μg l −1 for Zn) for 42 days followed by a 42-day recovery period in ambient seawater. Anemones were analyzed for metal content using inductively coupled plasma mass spectroscopy (ICP-MS) at various time points during the study. Symbiotic anemones accumulated Cd, Ni and Zn to a greater extent than aposymbiotic anemones. A dramatically different pattern of Cu accumulation was observed, with aposymbiotic anemones accumulating higher levels than symbiotic anemones. Following recovery in ambient seawater, all tissue metal levels were reduced to near pre-exposure control levels in most cases. No changes in algal cell density or MI were observed in symbiotic anemone tentacle clips at any dose or time point in the Cd and Cu exposures. However, significant reductions in algal cell densities were observed in the Ni-exposed and some Zn-exposed animals, although levels returned to control values following recovery. There were no changes in mitotic index (MI) following Ni or Zn exposures. These results demonstrate that the extent of heavy metal accumulation depends upon cnidarian symbiotic state and the heavy metal in question.

Differential accumulation of cadmium and changes in glutathione levels as a function of symbiotic state in the sea anemone Anthopleura elegantissima

Coastal ecosystems are increasingly impacted by anthropogenic activities and contaminant inputs, including heavy metals. This paper investigates the responses to laboratory cadmium exposures in the North American Pacific coast temperate sea anemone Anthopleura elegantissima. Anemones were exposed to cadmium chloride (control, 20 and 100 μg l −1 Cd) for 14 days (sampled at days 0, 2, 7 and 14) and analyzed for Cd content using inductively coupled plasma mass spectroscopy (ICP-MS). Higher levels of Cd were demonstrated in symbiotic anemones compared to symbiont-free (aposymbiotic) anemones (e.g. 100 μg l −1 Cd exposure at day 14; 12.4±2.4 and 4.5±1.9 ng Cd mg −1 dry weight, respectively; t-test; p<0.001). These higher levels in symbiotic anemones were not due to increasing uptake of Cd by the algal symbionts (levels in algal-containing pellet fractions were constant over dose and time; t-test; p>0.05) but rather to increased accumulation by both partners. No changes in algal cell density were observed in symbiotic anemone tentacle clips at any dose or time point (ANOVA; p>0.05). Changes in glutathione (GSH) levels have been proposed as a useful biomarker in other marine invertebrates in response to heavy metal stress, but GSH studies have not been reported for this species. We demonstrate the presence of GSH in A. elegantissima and its depletion (58.2% of control values) following 1-week exposure to buthionine sulfoximine (BSO), a nontoxic chemical known to deplete GSH. Levels of GSH were higher in symbiotic compared to aposymbiotic anemones (control averages, 563±81 and 485±58 nmol GSH mg −1 wet weight, respectively; t-test; p<0.001). Following Cd exposure, significant reductions ( t-test; p<0.05) in GSH levels were observed only in the aposymbiotic anemones (exposure to 100 μg l −1 Cd resulted in a 27% reduction from control levels at day 14). Our results demonstrate that the accumulation of Cd depends upon symbiotic state and provides the first identification of a GSH response in anemones and indeed in any cnidarian in response to heavy metals.

Development of symbiosis-specific genes as biomarkers for the early detection of cnidarian–algal symbiosis breakdown

Coral bleaching, i.e. the loss of dinoflagellate symbionts from cnidarian hosts, is occurring globally at increasing rates, scales, and severity. The significance of these bleaching events to the health of coral reef ecosystems is extreme, as bleached corals exhibit high mortality, reduced fecundity and productivity and increased susceptibility to disease. This decreased coral fitness leads to reef degradation and ultimately to the breakdown of the coral reef ecosystem. To date there has been little work describing the application of biomarkers to assess coral health. The most commonly applied biomarker is, in fact, the bleaching event itself. We are interested in developing early warning biomarkers that can detect coral stress before bleaching occurs. Recently, several genes that are likely to function in regulating interactions between cnidarians and their symbionts have been characterized, using the temperate sea anemone Anthopleura elegantissima as a model species. One “symbiosis gene” identified from the host genome, sym32, is expressed as a function of anemone symbiotic-state, where sym32 expression is higher in symbiotic cf. aposymbiotic (symbiont-free) anemones. Real-time quantitative RT-PCR suggested that the level of sym32 expression was correlated with the abundance of algae in the host. Furthermore, laboratory exposures of anemones to low levels of cadmium (0, 20, 100 μg−1 CdCl2; 14 days), which caused no change in algal cell numbers, resulted in a down-regulation of sym32 compared to controls, indicating that sym32 expression may serve as a new sensitive early warning biomarker of cnidarian–algal symbiosis breakdown.

Feeding behavior and acquisition of zooxanthellae by planula larvae of the sea anemone Anthopleura elegantissima

Symbiotic associations between cnidarians and photosynthetic dinoflagellates (i.e., zooxanthellae) are common in the marine environment. Many symbiotic cnidarians produce offspring that are initially nonsymbiotic. These new hosts must acquire symbiotic algae from environmental sources. We examined zooxanthella acquisition by laboratory-reared planula larvae of the temperate sea anemone Anthopleura elegantissima. Larvae ingested zooxanthellae while they were feeding. However, the signal that prompted larval feeding behavior did not originate from the symbiotic algae; the addition of algal cells to larval cultures never elicited a feeding response. In contrast, the addition of macerated animal tissue from several sources invariably generated a strong feeding response, which resulted in the larvae indiscriminately ingesting any particulate matter that was present, including zooxanthellae or other unicellular algae. Ingested zooxanthellae were incorporated into endodermal cells, where they remained undigested, while all other ingested material was digested or expelled within 24 h. Our results provide evidence that one source of zooxanthellae likely to serve as a route of infection in the natural environment is zooxanthella-laden mucus egested by anemones. This egested material fulfilled both of the criteria necessary for successful infection: it prompted larvae to begin feeding and provided an abundant supply of zooxanthellae that were ingested and taken up into endodermal cells of the new host.

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis.

The temperate sea anemone Anemonia viridis (Forskal) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 μM ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 μM ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.

Temperature induction of viruses in symbiotic dinoflagellates

Bleaching manifests itself as a loss of symbiotic dinoflagellates (zooxanthellae) and/or chlorophyll from a variety of symbiotic hosts, including corals and sea anemones. Bleaching is known to result from a range of environmental stresses, the most significant of which is elevated tempera- ture; how these stresses elicit a bleaching response is cur- rently the focus of intense research. One consequence of environmental stress that has yet to be considered is viral attack. Here, we have isolated a transferable infectious agent believed to be a virus, from zooxanthellae of the temperate sea anemone Anemonia viridis. The infectious agent is induced by elevated temperature. Once induced, the filter- able agent can be further propagated without heat induction, thus fulfilling Koch's postulates. We propose that zooxanthel- lae harbor a latent viral infection that is induced by exposure to elevated temperatures. If such a mechanism also operates in the zooxanthellae harbored by reef corals, and these viruses kill the symbionts, then this could contribute to tem- perature-induced bleaching.