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.

Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment.

The purpose of this study was to determine whether the addition of iron alone or in combination with nitrate affects growth and photosynthesis of the scleractinian coral, Stylophora pistillata, and its symbiotic dinoflagellates. For this purpose, we used three series of two tanks for a 3-week enrichment with iron (Fe), nitrate (N) and nitrate+iron (NFe). Two other tanks were kept as a control (C). Stock solutions of FeCl(3) and NaNO(3) were diluted to final concentrations of 6 nM Fe and 2 &mgr;M N and continuously pumped from batch tanks into the experimental tanks with a peristaltic pump. Results obtained showed that iron addition induced a significant increase in the areal density of zooxanthellae (ANOVA, p=0.0013; change from 6.3+/-0.7x10(5) in the control to 8.5+/-0.6x10(5) with iron). Maximal gross photosynthetic rates normalized per surface area also significantly increased following iron enrichment (ANOVA, p=0.02; change from 1.23+/-0.08 for the control colonies to 1.81+/-0.24 &mgr;mol O(2) cm(-2) h(-1) for the iron-enriched colonies). There was, however, no significant difference in the photosynthesis normalized on a per cell basis. Nitrate enrichment alone (2 &mgr;M) did not significantly change the zooxanthellae density or the rates of photosynthesis. Nutrient addition (both iron and nitrogen) increased the cell-specific density of the algae (CSD) compared to the control (G-test, p=0.3x10(-9)), with an increase in the number of doublets and triplets. CSD was equal to 1.70+/-0.04 in the Fe-enriched colonies, 1.54+/-0.12 in the N- and NFe-enriched colonies and 1.37+/-0.02 in the control. Growth rates measured after 3 weeks in colonies enriched with Fe, N and NFe were 23%, 34% and 40% lower than those obtained in control colonies (ANOVA, p=0.011).

Preferential expulsion of dividing algal cells as a mechanism for regulating algal-cnidarian symbiosis.

A wide range of both intrinsic and environmental factors can influence the population dynamics of algae in symbiosis with marine cnidarians. The present study shows that loss of algae by expulsion from cnidarian hosts is one of the primary regulators of symbiont population density. Because there is a significant linear correlation between the rate of algal expulsion and the rate of algal division, factors that increase division rates (e.g., elevated temperature) also increase expulsion rates. Additionally, 3H-thymidine is taken up to a greater extent by algae destined to be expelled than by algae retained in the host cnidarians. Taken together, data for rates of expulsion, rates of division at different temperatures, and uptake of 3H-thymidine suggest that dividing algal cells are preferentially expelled from their hosts. The preferential expulsion of dividing cells may be a mechanism for regulation of algal population density, where the rate of expulsion of algae may be an inverse function of the ability of host cells to accommodate new algal daughter cells. This kind of regulation is present in some cnidarian species (e.g., Aiptasia pulchella, Pocillopora damicornis), but not in all (e.g., Montipora verrucosa, Porites compressa, and Fungia scutaria).

Oxidative Stress in the Symbiotic Sea Anemone Aiptasia pulchella (Carlgren, 1943): Contribution of the Animal to Superoxide Ion Production at Elevated Temperature.

Production of superoxide ions within tissues of the symbiotic sea anemone Aiptasia pulchella was detected using SOD-inhibitable cytochrome c reduction and quantified by SOD-inhibitable reduction of nitro blue tetrazolium (NBT). Intact aposymbiotic and symbiotic specimens of A. pulchella produced superoxide in response to acute, sublethal thermal stress. Neither light nor inhibition of symbiont photosynthesis by (3,4-di-chlorophenyl) -1, 1-dimethylurea (DCMU) affected superoxide production. The time course of superoxide ion production strongly resembled the time course of increased dark respiration by intact anemones, suggesting that the effect of elevated temperature on host mitochondria may account for increased superoxide production. Interestingly, freshly isolated algae (FIZ) did not release superoxide ions in response to elevated temperature, and net oxygen production decreased greatly in both intact symbiotic anemones and in FIZ within 20 minutes after temperature elevation. These results show that oxidative stress in A. pulchella is primarily an animal response, and suggest that the presence of symbiotic algae, although sufficient to cause hyperoxia, is not necessary for the appearance of oxidative stress in these anemones at elevated temperature.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.

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 cell cycle of symbiotic Chlorella. IV. DNA content of algae slowly increases during host starvation of green hydra.

The distribution of DNA content of symbiotic Chlorella algae freshly isolated from green hydra was compared with that of cultured Chlorella of the NC64A strain, using flow cytometry. In nonlogarithmic cultures of NC64A most cells had accumulated in G1 phase, while in logarithmic cultures a peak containing cells in S phase and mitosis could be distinguished from the larger G1 peak. However, symbiotic algae showed a single broad peak in which there was no clear distinction between G1 and S phase/mitosis. When hydra were starved for a prolonged period, inhibiting host cell and algal division, the DNA content of the symbiotic algae slowly increased, and the number of daughter cells produced after a single feeding increased with the length of the preceding period of starvation. This suggests that symbiotic algae are able to cycle slowly through S phase, but unless the host is fed they cannot traverse into mitosis and complete the cell division cycle. No significant difference in cell size was found between algae producing either four or eight daughter cells after 1-day- or 22-day-starved hydra were fed, suggesting that algal cell size did not determine the number of daughter cells produced. Instead, this may be dependent upon the length of time the cell had spent in S phase prior to receiving the, as yet unknown, stimulus to enter into mitosis.

DAILY BUDGETS OF PHOTOSYNTHETICALLY FIXED CARBON IN SYMBIOTIC ZOANTHIDS.

We tested the hypothesis that some zoanthids are able to meet a portion of their daily respiratory carbon requirement with photosynthetic carbon from symbiotic algal cells (= zooxanthellae). A daily budget was constructed for carbon (C) photosynthetically fixed by zooxanthellae of the Bermuda zoanthids Zoanthus sociatus and Palythoa variabilis. Zooxanthellae have an average net photosynthetic C fixation of 7.48 and 15.56 µgC·polyp-1·day-1 for Z. sociatus and P. variabilis respectively. The C-specific growth rate (µc) was 0.215·day-1 for Z. sociatus and 0.152·day-1 for P. variabilis. The specific growth rate (µ) of zooxanthellae in the zoanthids was measured to be 0.011 and 0.017·day-1 for Z. sociatus and P. variabilis zooxanthellae respectively. Z. sociatus zooxanthellae translocated 95.1% of the C assimilated in photosynthesis, while P. variabilis zooxanthellae translocated 88.8% of their fixed C. As the animal tissue of a polyp of Z. sociatus required 14.75 µgC·day-1 for respiration, and one of P. variabiis required 105.54 µgC·day-1, the contribution of zooxanthellae to animal respiration (CZAR) was 48.2% for Z. sociatus and 13.1% for P. variabilis.

Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis.

Certain species of Chlorella live within the digestive cells of the fresh water cnidarian Hydra viridis. When introduced into the hydra gut, these symbiotic algae are phagocytized by digestive cells but avoid host digestion and persist at relatively constant numbers within host cells. In contrast, heat-killed symbionts are rapidly degraded after phagocytosis. Live symbionts appear to persist because host lysosomes fail to fuse with phagosomes containing live symbionts. Neither acid phosphatase nor ferritin was delivered via lysosomes into phagosomes containing live symbionts, whereas these lysosomal markers were found in 50% of the vacuoles containing heat-killed symbionts 1 h after phagocytosis. Treatment of symbiotic algae before phagocytosis with polycationic polypeptides abolishes algal persistence and perturbs the ability of these algae to control the release of photosynthate in vitro. Similarly, inhibition of photosynthesis and hence of the release of photosynthetic products as a result of prolonged darkness and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) treatment also abolishes persistence. Symbiotic algae are not only protected from host digestive attack but are also selectively transported within host cells, moving from the apical site of phagocytosis to a basal position of permanent residence. This process too is disrupted by polycationic polypeptides, DCMU and darkness. Both algal persistence and transport may, therefore, be a function of the release of products from living, photosynthesizing symbionts. Vinblastine treatment of host animals blocked the movement of algae within host cells but did not perturb algal persistence: algal persistence and the transport of algae may be initiated by the same signal, but they are not interdependent processes.

Transmission of symbiotic algae to eggs of green hydra.

Evidence from light and electron microscopy shows that symbiotic algae occur in a large percentage of fertilized eggs of two strains of green hydra. Transmission of algae to eggs may occur by insertion of algae-laden endodermal cell processes across the mesogloea into the ooplasm of the ectodermally-derived egg when oocyte maturation is well under way. This transmission mechanism is contrasted with mechanisms postulated for other strains of green hydra.

Regulation of numbers of intracellular algae.

Members of three classes of unicellular algae have exploited an intracellular habitat and occur as endosymbionts in aquatic invertebrates, including Protozoa. Such associations manifest a range of host--symbiont cellular interactions and achieve stability through the regulation of symbiont numbers. The mechanism of regulation is poorly understood. Steady-state algae:host cell ratios might be achieved by expulsion, digestion, or inhibition of growth of algal symbionts. Digestion and expulsion have been observed directly in some associations but their role in regulating numbers is circumstantial. Inhibition of growth as a result of nutrient limitation or inhibitor secretion is an attractive, but inadequately tested, hypothesis. The relation between the host cell mitosis and algal proliferation is a potential focal point for further study.

Endocytic mechanisms of the digestive cells of Hydra viridis. 1. Morphological aspects.

The endocytic mechanisms of the digestive cells of Hydra viridis were examined by transmission electron microscopy. Algae which form a stable intracellular symbiosis are phagocytosed by uncoated plasmalemma, as are large (greater than 0.5 micron) food particles. Discoidal coated vesicles apparently effect the endocytosis of smaller particles, including macromolecules. Competition experiments indicate that the uptake of algae and larger food particles utilize similar endocytic membrane.