Impact of symbiotic algae on sea anemone metabolism: analysis by in vivo 31P nuclear magnetic resonance spectroscopy

High-field pulsed Fourier-transform nuclear magnetic resonance spectroscopy (NMR) was used to quantify the adenylate levels of sea anemones (Aiptasia pulchella) with and without symbiotic dinoflagellates (Symbiodinium sp.). Animals were fed to repletion, then starved in darkness for up to six days before collection of in vivo NMR spectra. The host adenylate ratio of ATP: (ATP + ADP) declined significantly with increasing periods of starvation in both symbiotic and aposymbiotic hosts (P less than 0.05). However, the decline in the animal adenylate ratio was significantly more rapid in animals bearing symbiotic algae (P less than 0.05). This suggests that symbiotic algae in darkness cause more rapid depletion of host energy reserves, possibly by drawing on host pools of organic substrates. In vivo NMR spectroscopy was also used to evaluate the effect on A. pulchella of photosynthesis by zooxanthellae. Symbiotic anemones were fed to repletion, then starved under high irradiance (300 to 320 mu Ein m-2 s-1) or low irradiance (70 to 80 mu Ein m-2 s-1) conditions for up to five days. The host adenylate ratio declined significantly (P less than 0.01) with starvation under both treatments, but no significant difference was detected between treatments (P greater than 0.35). Blotted wet weight of anemones under high and low irradiance declined by 50% over eight days of starvation, but there was no significant difference in the rate of weight loss by anemones in the two treatments. There results suggest that translocation of photosynthate from symbiotic zooxanthellae does not significantly affect host adenylate ratio or have a sparing effect on host biomass during starvation in this symbiotic sea anemone.     

Nutrient availability for zooxanthellae derived from physiological activities of Condylactus spp

Differences in phosphate metabolism of symbiotic and aposymbiotic Condylactus suggest that the host animal makes available quantities of phosphate to support growth of zooxanthellae. Nitrite may serve as a nitrogen source for symbionts as indicated by host removal of nitrite from sea water.The presence of zooxanthellae is responsible for removal of phosphate from sea water in the dark whereas there is excretion during light periods. There is a greater uptake of nitrite from sea water in the light compared with the dark in symbiotic animals.Since nitrate is removed from sea water by aposymbiotic animals, the presence of nitrate reducing bacteria is proposed.     

The Symbiotic Anthozoan: A Physiological Chimera between Alga and Animal

The symbiotic life style involves mutual ecological, physiological,structural, and molecular adaptations between the partners.In the symbiotic association between anthozoans and photosyntheticdinoflagellates (Symbiodinium spp., also called zooxanthellae),the presence of the endosymbiont in the animal cells has constrainedthe host in several ways. It adopts behaviors that optimizephotosynthesis of the zooxanthellae. The animal partner hashad to evolve the ability to absorb and concentrate dissolvedinorganic carbon from seawater in order to supply the symbiont'sphotosynthesis. Exposing itself to sunlight to illuminate itssymbionts sufficiently also subjects the host to damaging solarultraviolet radiation. Protection against this is provided bybiochemical sunscreens, including mycosporine-like amino acids,themselves produced by the symbiont and translocated to thehost. Moreover, to protect itself against oxygen produced duringalgal photosynthesis, the cnidarian host has developed certainantioxidant defenses that are unique among animals. Finally,living in nutrient-poor waters, the animal partner has developedseveral mechanisms for nitrogen assimilation and conservationsuch as the ability to absorb inorganic nitrogen, highly unusualfor a metazoan. These facts suggest a parallel evolution ofsymbiotic cnidarians and plants, in which the animal host hasadopted characteristics usually associated with phototrophicorganisms.     

珊瑚共生体中“细菌-虫黄藻-宿主”三角关系的通讯交流

“细菌-虫黄藻-珊瑚”是生态系统中一对经典的三角关系,其中包含着复杂的物质流、信息流和能量流,三者的平衡与稳定是维护珊瑚礁生态系统健康的重要保障。过去20年里针对共生体交互关系进行了大量研究,并取得了一些重要成果,明确了“细菌-虫黄藻-宿主”三者之间的物质代谢、营养交换以及与环境的交互关系。然而,基于共生系统的复杂性,一些现象背后的机制仍然未被充分揭示,尤其是共生体之间的通讯交流。信号分子介导的相互作用是珊瑚共生体稳态维持和高效运转的内在驱动力。本文以珊瑚共生体系中化学信号为重点,尝试梳理最新的研究进展,包括细菌与细菌、细菌与珊瑚、细菌与虫黄藻以及虫黄藻与珊瑚之间的通讯方式,重点关注了群体感应信号(QS)、二甲基巯基丙酸盐(DMSP)、糖类信号、脂类信号以及非编码RNA。选择性例举了QS信号介导的微生物协作和竞争、DMSP调节下的细菌和宿主的相互作用,以及环境胁迫下珊瑚和虫黄藻对非编码RNA的响应过程,强调了它们在共生体中的作用模式和生态意义。并对今后的研究重点和可能方向进行了提炼,包括研究维度的扩充、新技术-新方法的应用以及生态模型的构建等,旨在提升对三角关系互作方式的认识,增进对珊瑚共生体的理解,探索基于通讯语言的操纵方式为珊瑚礁生态系统的恢复和保护提供新思路。     

Application of chlorophyll fluorescence technique in the study of coral symbiotic zooxanthellae micro-ecology

Mutualistic relationship between coral polyps and their symbiotic zooxanthellae living within their tissues are the most essential features of a coral reef ecosystem. In this symbiotic system, the coral polyps provide a protected habitat, carbon dioxide and nutrients needed for photosynthesis to zooxanthellae; in turn, the symbiotic zooxanthellae provide food as products of photosynthesis to coral polyps. The Photosynthesis of zooxanthellae is therefore an important process of this symbiotic system as well as the development of the whole coral reef ecosystem. The recent application of chlorophyll fluorescence technique in the study of the zooxanthellae’s photosynthesis has greatly improved our understanding on the micro-ecology of corals and the symbiotic zooxanthellae. This paper summarizes the recent progress as the following aspects: (1) The ecological characteristics of the photosynthesis of symbiotic zooxanthellae, such as the diurnal and seasonal changes in the photochemical efficiency of the zooxanthellae, and the relationship between zooxanthellae photosynthesis and the world-wide coral bleaching. (2) The mechanism of corals acclimating to the changes of irradiance via spatial and temporal photoacclimations, including the corals’ photobiology; zooxanthella size, pigmentation, location and clade, and the relationship between light extremes and the corals’ metabolism and calcification. (3) The understanding of the response of zooxanthellae to various environmental stresses, such as long-term changes in the chlorophyll fluorescence of bleached and recovering corals; the tolerance of corals to thermal bleaching; the changes to photosystem II of symbiotic zooxanthellae after heat stress and bleaching. Due to the above findings, the chlorophyll fluorescence values of those coral species sensitive to environmental changes have been utilized as indicators of coral health as well as the status of coral reef ecosystems. In summary, the chlorophyll fluorescence technique has great potential in the understanding, monitoring, protecting and managing coral reefs.     

Trophic status and population density of zooxanthellae in hermatypic corals

This paper discusses experimental data and theoretical concepts characterizing the trophic status of symbiotic zooxanthellae and the mechanisms regulating their density in hermatypic corals. Under natural conditions, the growth of zooxanthellae is not limited by the deficiency of nitrogen. The main factor regulating the density of zooxanthellae is their ingestion by the animal host.     

Catalase characterization and implication in bleaching of a symbiotic sea anemone

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.     

The adaptive bleaching hypothesis: experimental tests of critical assumptions

Coral bleaching, the loss of color due to loss of symbiotic zooxanthellae or their pigment, appears to be increasing in intensity and geographic extent, perhaps related to increasing sea surface temperatures. The adaptive bleaching hypothesis (ABH) posits that when environmental circumstances change, the loss of one or more kinds of zooxanthellae is rapidly, sometimes unnoticeably, followed by formation of a new symbiotic consortium with different zooxanthellae that are more suited to the new conditions in the host's habitat. Fundamental assumptions of the ABH include (1) different types of zooxanthellae respond differently to environmental conditions, specifically temperature, and (2) bleached adults can secondarily acquire zooxanthellae from the environment. We present simple tests of these assumptions and show that (1) genetically different strains of zooxanthellae exhibit different responses to elevated temperature, (2) bleached adult hosts can acquire algal symbionts with an apparently dose-dependent relationship between the concentration of zooxanthellae and the rate of establishment of the symbiosis, (3) and finally, bleached adult hosts can acquire symbionts from the water column.     

The taxonomy and biology of further aeolidacean and arminacean nudibranch molluscs with symbiotic zooxanthellae

The occurrence of symbiotic zooxanthellae in further aeolid and arminacean nudibranch molluscs is described for the first time. The aeolid Aeolidiopsis ransoni Pruvot-Fol is redescribed, and a new species of Aeolidiopsis , also feeding on the colonial zoantharian Palythoa , is described. Both have symbiotic zooxanthellae. The taxonomy of the family Aeolidiidae is discussed and the genus Berghia Trinchese, 1877 is considered a synonym of Spurilla Bergh, 1864. Spurilla major (Eliot, 1903) and a new species of Spurilla are reported to have zooxanthellae while another new species of Spurilla is without zooxanthellae. The glaucid aeolid Pteraeolidia ianthina (Angas) is shown to have symbiotic zooxanthellae, as is the arminacean Doridomorpha gardineri Eliot, which is reported to feed on the alcyonarian blue coral, Heliopora. In all cases, the morphological adaptations developed for this symbiosis are described. Further notes on the Porites-fetding arminacean Pinufius rebus Marcus & Marcus and the aeolid Phestilla lugubris (Bergh) are included and a facultative symbiosis with zooxanthellae is suggested for the latter. The tion of symbiosis with zooxanthellae within nudibranchs is discussed and it is suggested that the relationship has evolved independently on several occasions.     

Experimental infection of aposymbiotic gorgonian polyps with zooxanthellae

The planula larvae and young polyp stages of the West Indian gorgonian Pseudopterogorgia bipinnata (Verrill) do not have symbiotic zooxanthellae. Planulae were allowed to settle in water free of any algae and presented with a number of strains of dinoflagellates. Motile forms of these algae were attracted to the polyps and in some cases were seen to swim into the polyp's open mouth. Successful infection was obtained only with Gymnocdinium (probably microadriaticum (Freudenthal)). Algae of the genus Amphidinium were apparently not able to infect polyps. The differences in transmission of zooxanthellae in symbiotic animals are discussed.     

Repopulation of a sea anemone with symbiotic dinoflagellates: Analysis by in vivo fluorescence

Individuals in a population of aposymbiotic Aiptasia pulchella Carlgren were each inoculated with homologous zooxanthellae. The rate of repopulation of the anemones (i.e. the in situ growth rate of the zooxanthellae) was determined non-destructively from the mean in vivo fluorescence per anemone over 19 days. As zooxanthellae cell density increased, chlorophyll a per cell increased, but fluorescence per cell decreased, probably as a result of self-shading. The emergent relationship between in vivo fluorescence and number of zooxanthellae was linear over the range of cell densities investigated. The-specific growth rate during exponential growth was 0.4·day⁻¹ between days 7 and 15. As repopulation approached saturation (ca. 0.5 × 10⁶ cells per mg animal soluble protein) at about 19 days, the growth rate decreased and approached the steady state growth rate of about 0.02 · day⁻¹ of normal symbiotic anemones. Rates of repopulation of A. pulchella by freshly isolated and cultured homologous zooxanthellae were virtually identical.     

动物的共生菌固氮

介绍了共生菌固氮涉及的动物和微生物类群、动物共生菌固氮的性质和机理。应用乙炔还原法和固氮酶基因检测等研究表明,所涉及的动物有7门13纲23目50科99属174种。动物肠道具有丰富的微生境,供不同生理需求的固氮菌生长发育,所蕴含的共生固氮菌类群也十分丰富,涵盖植物共生固氮菌、植物内生固氮菌、植物根际固氮菌、自生固氮菌等生态类型。一般认为动物共生固氮菌来源于环境,其性质属于联合共生固氮。动物共生固氮菌一般与其他共生生物形成复合体,以满足固氮过程中对电子和质子供体、能量供给、固氮酶活性保护以及氨阻遏解除等方面的需求。动物共生菌固氮产物氨的同化也需要多种共生物的协同作用,可能通过谷氨酰胺合成酶/谷氨酸合成酶等途径。总体上,食物氮、非蛋白氮和共生菌固氮相互协调,形成营养和解毒的代谢网络,共同维持动物体内氮素营养的动态平衡,并对未来研究提出展望。     

Productivity and organic consumption in Cassiopea and Condylactus

Oxygen production and consumption was determined for Cassiopea and Condylactus containing symbiotic algae (zooxanthellae). 88 % of both animals produce more organic material than they consumed during a 12-h light period.It is suggested that zooxanthellae contribute a significant nutritional supplementation and a greater ecological efficiency to the host animals.     

Nitrogen and photosynthetic function of hermatypic corals. Oxygen exchange of Stylophora pistillata coral under artificial feeding

The change of Stylophora pistillata coral photosynthetic function (oxygen exchange and biomass of symbionts) under starvation and food enrichment was studied to understand the role of heterotrophy in nitrogen supplements of zooxanthellae. The starvation caused the decrease of frequency of zooxanthellae cells division in 7-10 times. The number of degraded algae cells increased in same proportion and, as a result, the density of zooxanthellae in corals decreased about two times during one-two weeks. Under starvation corals kept their photosynthetic capacity at the level of corals in situ by means of enhancing the zooxanthellae gross photosynthesis. The respiration rate of coral had tendency to increase and the dry mass of polyp tissue to decrease. Under artificial feeding which was following starvation the zooxanthellae density increased in 1.5-2 times, and particular food caused more intensive accumulation of zooxanthellae comparing to dissolved inorganic ammonium. The feeding regime did not affect dry mass of polyp tissue and chlorophyll content as well as respiration and gross productivity of the corals. The conclusion about high effectiveness of particular feeding for supplying symbiotic algae with nitrogen was made and trophic status of zooxanthellae in hospite was determined as unlimited by nitrogen.     

The effects of feeding frequency and symbiosis with zooxanthellae on the biochemical composition of Astrangia Danae Milne Edwards & Haime 1849

The coral Astrangia danae Milne Edwards & Haime 1849 occurs naturally with and without symbiotic algae and thus may have two sources of nourishment: (1) particles captured by the coral polyps, and (2) photosynthetic products translocated from their zooxanthellae. Symbiotic colonies may have both sources, and nonsymbiotic ones certainly have only the former. The relative importance of these two food sources was studied in the laboratory by examining the tissues of corals fed with frozen brine shrimp. Stock corals were fed once per week. Two to three weeks prior to each experiment, selected corals were placed on one of three feeding schedules: starved (S), fed once per week (1/wk), and fed three times per week (3/wk). The coral tissues were analyzed for protein, lipid, carbohydrate, and zooxanthellae content. Increased feeding frequency (1/wk → 3/wk) resulted in an increased tissue biomass and lipid to protein (L/P) ratio; starvation (1/wk → S) caused a decrease in these parameters. Symbiosis with zooxanthellae had an effect similar to increased feeding frequency in that the S and 1/wk symbiotic corals had a higher L/P ratio than comparable nonsymbiotic ones. There were no significant differences in L/P ratios between the 3/wk symbiotic and nonsymbiotic corals. Freshly collected colonies had a tissue composition most similar to the laboratory animals fed 3/wk. This result is consistent with the hypothesis that ingestion of solid food is the major nutritional source for A. danae in Narragansett Bay, Rhode Island, but our experiments suggest that the algae can have an important effect on tissue L/P ratios during times of food scarcity.     

Localization and quantification of carbonic anhydrase activity in the symbiotic Scyphozoan Cassiopea xamachana

The relationship between density and location of zooxanthellae and levels of carbonic anhydrase (CA) activity was examined in Cassiopea xamachana. In freshly collected symbiotic animals, high densities of zooxanthellae corresponded with high levels of CA activity in host bell and oral arm tissues. Bleaching resulted in a significant loss of zooxanthellae and CA activity. Recolonization resulted in full restoration of zooxanthellar densities but only partial restoration of CA activity. High levels of CA activity were also seen in structures with inherently higher zooxanthellar densities, such as oral arm tissues. Similarly, the oral epidermal layer of bell tissue had significantly higher zooxanthellar densities and levels of CA activity than did aboral bell tissues. Fluorescent labeling, using 5-dimethylaminonapthalene-1-sulfonamide (DNSA) also reflected this tight-knit relationship between the presence and density of zooxanthellae, as DNSA-CA fluorescence intensity was greatest in host oral epithelial cells directly overlying zooxanthellae. However, the presence and density of zooxanthellae did not always correspond with enzyme activity levels. A transect of bell tissue from the margin to the manubrium revealed a gradient of CA activity, with the highest values at the bell margin and the lowest at the manubrium, despite an even distribution of zooxanthellae. Thus, abiotic factors may also influence the distribution of CA and the levels of CA activity.     

Dimethylsulfoniopropionate in giant clams (Tridacnidae)

The tridacnid clams maintain symbiotic associations with certain dinoflagellates (termed zooxanthellae). Tridacnids are thus candidates to have high tissue concentrations of dimethylsulfoniopropionate (DMSP), a tertiary sulfonium compound that is not synthesized by animals but is commonly produced by dinoflagellates. This study establishes that DMSP is about an order of magnitude more concentrated in the light-exposed and shaded mantle and gills of Tridacna maxima and T. squamosa than in any other known animal tissues. The DMSP concentration in the light-exposed, siphonal mantle--the location of most zooxanthellae--is an inverse function of body size, paralleling an inverse relation between apparent density of zooxanthellae (measured as pheophytin concentration) and body size. The shaded mantle and gills are high in DMSP despite having low densities of zooxanthellae, indicating that high DMSP concentrations occur in molluscan tissue, not just in algal cells. DMSP is almost an order of magnitude less concentrated in the adductor muscle than in other tissues. The high DMSP concentrations found in tridacnids, by providing abundant substrate for formation of volatile dimethylsulfide, probably explain the peculiar tendency of tridacnids to rapidly develop offensive odors and tastes after death: a serious problem for their exploitation as food. Tridacnids are the one group of animals in which DMSP concentrations are high enough in some tissues to be in the range capable of perturbing enzyme function at high physiological temperatures. Thus, tridacnids may require enzyme forms adapted to DMSP.     

Comparative gene expression in the symbiotic and aposymbiotic Aiptasia pulchella by expressed sequence tag analysis

Intracellular symbiotic relationships are prevalent between cnidarians, such as corals and sea anemones, and the photosynthetic dinoflagellate symbionts. However, there is little understanding about how the genes express when the symbiotic relationship is set up. To characterize genes involved in this association, the endosymbiosis between sea anemone, Aiptasia pulchella, and dinoflagellate zooxanthellae, Symbiodinium spp., was employed as a model. Two complementary DNA (cDNA) libraries were constructed from RNA isolated from symbiotic and aposymbiotic A. pulchella. Using single-pass sequencing of cDNA clones, a total of 870 expressed sequence tags (ESTs) clones were generated from the two libraries: 474 from symbiotic animal and 396 from aposymbiotic animal. The initial ESTs consisted of 143 clusters and 231 singletons. A BLASTX search revealed that 147 unique genes had similarities with protein sequences available from databases; 120 of these clones were categorized according to their putative function. However, many ESTs could not assign functionally. The putative roles of some of the identified genes relative to endosymbiosis were discussed. This is the first report of the use of EST analysis to examine the gene expression in symbiotic and aposymbiotic states of the cnidarians. The systematic analysis of EST from this study provides a useful database for future investigations of the molecular mechanisms involved in algal-cnidarian symbiosis.     

Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral

Summary. Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C₅-DMB-SM). Incubation of symbiotic endoderm cells with C₅-DMB-SM–defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research. Correspondence and reprints: National Museum of Marine Biology and Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan, R.O.C.