Tropical Sea Anemone Aiptasia Research Articles

Host and Symbiont Cell Cycle Coordination Is Mediated by Symbiotic State, Nutrition, and Partner Identity in a Model Cnidarian-Dinoflagellate Symbiosis.

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G2/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

Development and Symbiosis Establishment in the Cnidarian Endosymbiosis Model Aiptasia sp.

Symbiosis between photosynthetic algae and heterotrophic organisms is widespread. One prominent example of high ecological relevance is the endosymbiosis between dinoflagellate algae of the genus Symbiodinium and reef-building corals, which typically acquire symbionts anew each generation during larval stages. The tropical sea anemone Aiptasia sp. is a laboratory model system for this endosymbiosis and, similar to corals, produces non-symbiotic larvae that establish symbiosis by phagocytosing Symbiodinium from the environment into the endoderm. Here we generate the first overview of Aiptasia embryogenesis and larval development and establish in situ hybridization to analyze expression patterns of key early developmental regulators. Next, we quantify morphological changes in developing larvae and find a substantial enlargement of the gastric cavity over time. Symbiont acquisition starts soon after mouth formation and symbionts occupy a major portion of the host cell in which they reside. During the first 14 days of development, infection efficiency remains constant while in contrast, localization of phagocytosed symbionts changes, indicating that the occurrence of functional phagocytosing cells may be developmentally regulated. Taken together, here we provide the essential framework to further develop Aiptasia as a model system for the analysis of symbiosis establishment in cnidarian larvae at the molecular level.

Induction of Gametogenesis in the Cnidarian Endosymbiosis Model Aiptasia sp.

Endosymbiosis is widespread among cnidarians and is of high ecological relevance. The tropical sea anemone Aiptasia sp. is a laboratory model system for endosymbiosis between reef-building corals and photosynthetic dinoflagellate algae of the genus Symbiodinium. Here we identify the key environmental cues to induce reproducible spawning in Aiptasia under controlled laboratory conditions. We find that simulating a lunar cycle with blue-wavelength light is necessary to promote abundant gamete production and synchronous release in well-fed animals. Sexual reproduction rates are genetically determined and differ among clonal lines under similar conditions. We also find the inverse difference in rates of asexual reproduction. This study provides the requisite basis for further development of the Aiptasia model system, allowing analysis of basic cellular and molecular mechanisms in the laboratory as well as investigations of broad questions of ecological and evolutionary relevance.

Elevated temperature inhibits recruitment of transferrin-positive vesicles and induces iron-deficiency genes expression in Aiptasia pulchella host-harbored Symbiodinium.

Coral bleaching is the consequence of disruption of the mutualistic Cnidaria-dinoflagellate association. Elevated seawater temperatures have been proposed as the most likely cause of coral bleaching whose severity is enhanced by a limitation in the bioavailability of iron. Iron is required by numerous organisms including the zooxanthellae residing inside the symbiosome of cnidarian cells. However, the knowledge of how symbiotic zooxanthellae obtain iron from the host cells and how elevated water temperature affects the association is very limited. Since cellular iron acquisition is known to be mediated through transferrin receptor-mediated endocytosis, a vesicular trafficking pathway specifically regulated by Rab4 and Rab5, we set out to examine the roles of these key proteins in the iron acquisition by the symbiotic Symbiodinium. Thus, we hypothesized that the iron recruitments into symbiotic zooxanthellae-housed symbiosomes may be dependent on rab4/rab5-mediated fusion with vesicles containing iron-bound transferrins and will be retarded under elevated temperature. In this study, we cloned a novel monolobal transferrin (ApTF) gene from the tropical sea anemone Aiptasia pulchella and confirmed that the association of ApTF with A. pulchella Rab4 (ApRab4) or A. pulchella Rab5 (ApRab5) vesicles is inhibited by elevated temperature through immunofluorescence analysis. We confirmed the iron-deficient phenomenon by demonstrating the induced overexpression of iron-deficiency-responsive genes, flavodoxin and high-affinity iron permease 1, and reduced intracellular iron concentration in zooxanthellae under desferrioxamine B (iron chelator) and high temperature treatment. In conclusion, our data are consistent with algal iron deficiency being a contributing factor for the thermal stress-induced bleaching of symbiotic cnidarians.

Effects of Cd, Co, Cu, Ni and Zn on asexual reproduction and early development of the tropical sea anemone Aiptasia pulchella.

Currently few studies present sub-lethal toxicity data for tropical marine species, and there are no routine toxicity tests using marine cnidarians. The symbiotic sea anemone Aiptasia pulchella has been identified as a useful species for ecotoxicological risk assessment, and would provide a tropical marine cnidarian representative. Chronic sub-lethal toxicity tests assessing the effects of 28-day trace metal exposure on asexual reproduction in A. pulchella were investigated, and concentration-dependant reductions in the number of offspring that were produced were evident for all metal exposures. Metal concentration estimates causing 50% reductions in the numbers of asexually-reproduced juveniles after 28-day exposures (28-day effect concentrations 50%: EC50s) were 14 µg/L for copper, 63 µg/L for zinc, 107 µg/L for cobalt, 145 µg/L for cadmium, and 369 µg/L for nickel. Slightly higher 28-day EC50s of 16 µg/L for copper, 192 µg/L for zinc, 172 µg/L for cobalt, 185 µg/L for cadmium, and 404 µg/L for nickel exposures and were estimated based on reductions in the total number of live developed and undeveloped offspring. These sensitive and chronic sub-lethal toxicity estimates help fill the knowledge gap related to metal effects on cnidarians over longer exposure periods, and this newly-developed bioassay may provide a much needed tool for ecotoxicological risk assessment relevant to tropical marine environments.

Interactions between the tropical sea anemone Aiptasia pallida and Serratia marcescens, an opportunistic pathogen of corals.

Coral reefs are under increasing stress caused by global and local environmental changes, which are thought to increase the susceptibility of corals to opportunistic pathogens. In the absence of an easily culturable model animal, the understanding of the mechanisms of disease progression in corals remains fairly limited. In the present study, we tested the susceptibility of the tropical sea anemone Aiptasia pallida to an opportunistic coral pathogen (Serratia marcescens). A. pallida was susceptible to S. marcescens PDL100 and responded to this opportunistic coral pathogen with darkening of the tissues and retraction of tentacles, followed by complete disintegration of polyp tissues. Histological observations revealed loss of zooxanthellae and structural changes in eosinophilic granular cells in response to pathogen infection. A screen of S. marcescens mutants identified a motility and tetrathionate reductase mutants as defective in virulence in the A. pallida infection model. In co-infections with the wild-type strain, the tetrathionate reductase mutant was less fit within the surface mucopolysaccharide layer of the host coral Acropora palmata.

Molecular assessment of heterotrophy and prey digestion in zooxanthellate cnidarians

Zooxanthellate cnidarians are trophically complex, relying on both autotrophy and heterotrophy. Although several aspects of heterotrophy have been studied in these organisms, information linking prey capture with digestion is still missing. We used prey-specific PCR-based tools to assess feeding and prey digestion of two zooxanthellate cnidarians - the tropical sea anemone Aiptasia sp. and the scleractinian coral Oculina arbuscula. Prey DNA disappeared rapidly for the initial 1-3days, whereas complete digestion of prey DNA required up to 10days in O.arbuscula and 5 or 6days in Aiptasia sp. depending on prey species. These digestion times are considerably longer than previously reported from microscopy-based examination of zooxanthellate cnidarians and prey DNA breakdown in other marine invertebrates, but similar to prey DNA breakdown reported from terrestrial invertebrates such as heteroptera and spiders. Deprivation of external prey induced increased digestion rates during the first days after feeding in O.arbuscula, but after 6days of digestion, there were no differences in the remaining prey levels in fed and unfed corals. This study indicates that prey digestion by symbiotic corals may be slower than previously reported and varies with the type of prey, the cnidarian species and its feeding history. These observations have important implications for bioenergetic and trophodynamic studies on zooxanthellate cnidarians.

Cellular mechanisms underlying temperature-induced bleaching in the tropical sea anemone Aiptasia pulchella.

Temperature-induced bleaching in symbiotic cnidarians is a result of the detachment and loss of host cells containing symbiotic algae. We tested the hypothesis that host cell detachment is evoked through a membrane thermotropic event causing an increase in intracellular calcium concentration, [Ca(2+)](i), which could then cause collapse of the cytoskeleton and perturb cell adhesion. Electron paramagnetic resonance measurements of plasma membranes from the tropical sea anemone Aiptasia pulchella and the Hawaiian coral Pocillopora damicornis labeled with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) revealed no membrane thermotropic event. In addition, intracellular imaging using Fura-2AM as well as labeling anemones with (45)Ca revealed no significant change in [Ca(2+)](i). However, bleaching could be evoked at ambient temperature with 25 mmol l(-1) caffeine without affecting [Ca(2+)](i). [Ca(2+)](i) could be altered with ionomycin in isolated host cells, but ionomycin could not induce bleaching in A. pulchella. As caffeine can affect levels of intracellular protein phosphorylation, the ability of other agents that alter intracellular levels of protein phosphorylation to evoke bleaching was investigated. The protein phosphatase inhibitor vanadate could induce bleaching in A. pulchella. Two-dimensional gels of (32)P-labeled proteins from cold-shocked, caffeine-treated and control anemones show that both temperature shock and caffeine alter the array of phosphorylated host soluble proteins. We conclude that cnidarian bleaching is linked to a temperature-induced alteration in protein phosphorylation.

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [ 3H]arginine to [ 3H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N G-methyl- l-arginine ( l-NMA), but not the arginase inhibitors l-valine and l-ornithine. NOS activity was predominantly cytosolic, and was characterised by a K m for arginine of 19.05 μM and a V max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.

The influence of an anthozoan “host factor” on the physiology of a symbiotic dinoflagellate

Anthozoan host factors are components of host animal homogenates which elicit the release of newly fixed carbon by symbiotic dinoflagellates in vitro. The host factor active component of an homogenate of the reef coral Pocillopora damicornis has recently been isolated and identified as a mixture of free amino acids. A synthetic host factor (SHF) based on the measured free amino acid pool of P. damicornis elicits an identical response in symbiotic dinoflagellates in vitro as does the crude host factor. SHF was used as a tool with which to investigate the impact of anthozoan host factor on other aspects of symbiotic dinoflagellate metabolism. Here we demonstrate that dinoflagellates isolated from the tropical sea anemone Aiptasia pulchella (Carlgren) incubated in SHF, in addition to releasing fixed carbon, also exhibit higher photosynthetic carbon fixation, heterotrophic carbon fixation, photosynthetic oxygen production, respiration rates, and cell specific concentrations of chlorophyll a as compared to dinoflagellates incubated in sea water in vitro. Thus, the performance of symbiotic dinoflagellates in vitro appears to be sustained in an amino acid rich environment, but compromised in sea water. This observation raises the possibility that in nature symbiotic dinoflagellates released from anthozoan hosts may survive in amino acid rich environments such as fish guts and “intermediate hosts”.

Temperature Stress Causes Host Cell Detachment in Symbiotic Cnidarians: Implications for Coral Bleaching.

During the past decade, acute and chronic bleaching of tropical reef corals has occurred with increasing frequency and scale. Bleaching, i.e., the loss of pigment and the decrease in population density of symbiotic dinoflagellates (zooxanthellae), is often correlated with an increase or decrease in sea surface temperature. Because little is known of the cellular events concomitant with thermal bleaching, we have investigated the mechanism of release of zooxanthellae by the tropical sea anemone Aiptasia pulchella and the reef coral Pocillopora damicornis in response to cold and heat stress. Both species released intact host endoderm cells containing zooxanthellae. The majority of the released host cells were viable, but they soon disintegrated in the seawater leaving behind isolated zooxanthellae. The detachment and release of intact host cells suggests that thermal stress causes host cell adhesion dysfunction in these cnidarians. Knowledge of the cellular entity released by the host during bleaching provides insight into both the underlying release mechanism and the way in which natural environmental stresses evoke a bleaching response.

The Induction of Carbonic Anhydrase in the Symbiotic Sea Anemone Aiptasia pulchella.

The activity and nature of carbonic anhydrase (CA, EC 4.2.1.1 .) was measured and described in the tropical sea anemone Aiptasia pulchella. The hypothesis that high CA activity in animal tissue is induced by the presence of symbiotic algae was tested. CA activity was positively correlated with the number of symbiotic dinoflagellates (zooxanthellae) present. CA activity in aposymbiotic anemone tissue was 2.5 times lower than that in control symbiotic animals or in aposymbiotic animals repopulated with algae. Polyclonal antisera against human CA were used to probe for the presence of CA in both symbiotic and aposymbiotic anemone tissue, and in freshly isolated and cultured zooxanthellae. The resulting immunoblots showed one band with a molecular weight of 30 kDa in symbiotic animal tissue and control mammalian CA lanes, no bands in the aposymbiotic animal lanes, and one band at a molecular weight of 22.5 kDa in freshly isolated and cultured zooxanthellae lanes. Because no 22.5 kDa band was detected in the symbiotic animal tissue lanes, the high CA activity found in symbiotic animal tissue is considered to be due to the induction of animal enzyme by the presence of algae. The lack of any band in the aposymbiotic lanes further supports the hypothesis that CA activity in A. pulchella is induced by the presence of algae.

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 μE m-2 s-1 (below compensation irradiance) grew heterotrophically when supplied with 100 μM glycerol, glycolate, acetate, malate, or propionate, and grew in darkness on 100 μM 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 μE 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 (td) is made on the basis of growth rate of cultured zooxanthellae in log-phase; this estimate of td=4.88 h is less than half the estimated td for zooxanthellae in situ.