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.     

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.     

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.     

The impact of bleaching on the metabolic contribution of dinoflagellate symbionts to their giant clam host

Bleaching (loss of symbiotic dinoflagellates) is known to significantly decrease the fitness of symbiotic marine invertebrates resulting in reduced growth, fecundity and survival. This report is the first to quantify the effects of bleaching on inorganic carbon (C_i) and ammonium flux, fixation and export of photosynthate to the host, in this case the giant clam Tridacna gigas. The 1998 bleaching event was found to decrease the zooxanthellae population 30‐fold when comparing bleached to non‐bleached clams. This resulted in significant increases in haemolymph C_i and decreases in haemolymph pH and glucose concentration, the predominant photosynthate exported from zooxanthellae in this symbiosis. There was also a decrease in the expression levels of host carbonic anhydrase, an enzyme involved in C_i transport to the zooxanthellae, and although host glutamine synthase levels were unaffected, the clams ability to assimilate ammonium was eliminated in bleached individuals, suggesting that photosynthate from the zooxanthellae is required for ammonium assimilation. In an artificial bleaching experiment haemolymph C_i (r² = 0.97), pH (r² = 0.94) and glucose levels (r² = 0.95) were correlated to zooxanthellae numbers during both bleaching and recovery. Recovery of the zooxanthellae population, was enhanced four‐fold by the addition of organic and inorganic nutrients, as were related haemolymph characteristics. These results highlight the profound physiological changes that occur in symbiotic organisms during and after a bleaching event.     

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.     

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.     

The Energy Budget of Coral Polyps

We analyzed the recent data on the distribution of the photosynthetic energy of zooxanthellae in corals and the models of interactions between the plant and animal cells in the course of matter and energy exchange developed based on this information. The models of energy exchange of animals symbiotic with zooxanthellae are characterized by the following features: (1) A flow chart of carbon or energy fluxes is the main form of representation of the energetics in native symbiotic organisms. (2) The relations between the symbionts are relatively adequately revealed and correspond to the modern notions; however, the intensities of the energy fluxes ascribed to these relations are dependent on the experimental and design methods used by the authors. (3) The inputs into the energy budget consist of the autotrophic production of zooxanthellae and the heterotrophy of the polyp. The energy expenditures comprise excretion, respiration, development, and growth of the animal and algae. (4) The differences between the species, genera, and phyla of animals that develop symbiotic relations with zooxanthellae are confined to the absolute values of energy fluxes in the organism.     

不同发育阶段的黄癣蜂巢珊瑚共生虫黄藻多样性

【背景】黄癣蜂巢珊瑚是我国南海主要的造礁石珊瑚之一,但至今未见对其共生虫黄藻的相关研究报道。【目的】研究黄癣蜂巢珊瑚不同发育阶段的共生虫黄藻多样性,探讨其获取途径。【方法】用荧光显微镜观察黄癣蜂巢珊瑚卵子、浮浪幼虫以及珊瑚成体中的共生虫黄藻。基于ITS2序列的克隆文库技术分析珊瑚卵子、浮浪幼虫、成体中共生虫黄藻的多样性。【结果】荧光显微镜观察结果显示,黄癣蜂巢卵子及发育至第4天的浮浪幼虫中均无共生虫黄藻。没有在黄癣蜂巢珊瑚卵子及4 d浮浪幼虫中检测到虫黄藻ITS2序列,但是在黄癣蜂巢珊瑚成体中检测到共生虫黄藻序列,系统发育分析提示为C1型虫黄藻。【结论】成体黄癣蜂巢珊瑚中发现了共生虫黄藻,而卵子、浮浪幼虫阶段均未发现虫黄藻,提示黄癣蜂巢珊瑚中的虫黄藻可能是后期从海水中水平转移而来,而不是由母体垂直遗传的。     

Computational Biology Approaches to Plant Metabolism and Photosynthesis: Applications for Corals in Times of Climate Change and Environmental Stress

Knowledge of factors that are important in reef resilience helps us to understand how reef ecosystems react following major an-thropogenic and environmental disturbances. The symbiotic rela-tionship between the photosynthetic zooxanthellae algal cells and corals is that the zooxanthellae provide the coral with carbon, while the coral provides protection and access to enough light for the zooxanthellae to photosynthesise. This article reviews some recent advances in computational biology relevant to photosynthetic or-ganisms, including Beyesian approaches to kinetics, computational methods for flux balances in metabolic processes, and determina-tion of clades of zooxanthallae. Application of these systems will be important in the conservation of coral reefs in times of climate change and environmental stress.     

Acclimation of the Hermatypic Coral Stylophora pistillata to Bright Light

Photoacclimation dynamics to bright light was studied in the symbiotic reef-building coral Stylophora pistillata. Coral colonies were collected from shallow shaded sites (2 m, 40–20% PAR_s) from a fringing reef at Sesoko Island, Okinawa, Japan. Outer branches were broken off from the colonies and placed in an outdoor aquarium until the start of the experiments. After maintenance of the branches in an aquarium under a light intensity of 30% PAR_s for 30 days (experiment 1) or for 90 days (experiment 2), the samples were exposed to 95% PAR_s for 120 days in the same aquarium. The population density of zooxanthellae, chlorophyll concentration, locations of zooxanthellae, proliferating zooxanthellae frequency (PZF), and degrading zooxanthellae frequency (DZF) were examined. It was shown that after acclimation of coral branches to bright light, the population density of zooxanthellae, chlorophyll concentration calculated per 1000 polyps, and chlorophyll concentration in zooxanthellae decreased. The size of zooxanthellae significantly decreased. A decrease in the population density of zooxanthellae was detected by the eighth day of acclimation, and stabilization in the density of the symbionts occurred in the period from the 40th to the 60th day of the experiment. The chlorophyll concentration in zooxanthellae significantly decreased by the second day of exposure to bright light and stabilized by the fourth day. The PZF level sharply dropped on the second day, while the DZF level sharply increased and was higher than the PZF level for 40 days of exposure to bright light. We conclude, therefore, that the population density of zooxanthellae is regulated by the rates of two processes: cell division and the cell degradation.     

Light intensity as a factor in the regulation of the density of symbiotic zooxanthellae in Aiptasia tagetes (Coelenterata, Anthozoa)

Experiments designed to investigate the effect of different levels of illumination on the density of symbiotic zooxanthellae in the anemone Aiptasia tagetes (Duch. & Mich.) are described.
The anemones are found to regulate the densities of their zooxanthella populations to fixed levels which are dependent upon ambient light intensity.
Regulation is continuous and results from the interaction of increase in algal numbers, growth of host tissues, population of new host tissues by symbionts and extrusion of zooxanthellae by the host.
The nature of the process by which zooxanthellae are selected for extrusion is discussed and a scheme is outlined indicating the pathways through which the level of the algal population in Aiptasia , and other coelenterates, is believed to be controlled.     

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.     

Strain-Specific Differences in Surface Antigens of Symbiotic Algae

Intracellular symbiotic dinoflagellates (zooxanthellae) isolated from different invertebrate hosts are morphologically similar but could be differentiated by the fluorescent-antibody technique.     

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.     

Yellow band and dark spot syndromes in Caribbean corals: distribution,rate of spread,cytology, and effects on abundance and division rate of zooxanthellae

Yellow band and dark spot syndromes have been frequently observed to affect coral species throughout the Caribbean within the last 10 years. These syndromes significantly impair at least three important reef-building species. Yellow band (also known as yellow blotch) appears as rings or blotches on Montastrea annularis throughout the Caribbean. The coral tissue necrosis occurs at a rate of approximately 0.6 cm/month. Transect measurements at various locations indicated that as many as 90% of M. annularis were affected by yellow band during 1997–98. Tissue samples reveal a 41–96.9% decrease in zooxanthellae/sample compared to healthy specimens, depending on distance from healthy tissue. Mitotic indices (MI) of zooxanthellae (symbiotic algae appearing as doublets) for M. annularis are 2.5%. MI in yellow band samples directly bordering healthy tissue are less than 0.9%, and zooxanthellae directly within the band bordering exposed skeleton had a mitotic index of 0.0%. This indicates impairment of zooxanthellae cell division in yellow band specimens. Zooxanthellae are not expelled and appear vacuolated and devoid of organelles. Dark spot, characterized by tissue necrosis as well as a depression of the colony surface, affects Stephanocoenia michelinii and Siderastrea siderea throughout the Caribbean. Transects showed that as many as 56% of S. michelinii and S. siderea showed signs of dark spot during 1997–98. Affected tissues of S. siderea died at a rate of 4.0 cm/month. In dark spot samples from S. siderea, the total number of zooxanthellae was 56% of that in healthy tissue; dark spot-affected specimens of S. michelinii showed a 14% decrease in the number of zooxanthellae compared to healthy tissue samples. Mitotic indices of zooxanthellae from healthy specimens of S. sidereawere 1.20% compared to 0.40% in dark spot samples. Mitotic indices of healthy S. michelinii were 1.54% compared to 0.23% in dark spot samples, also indicating a decrease in algal cell division. Zooxanthellae from dark spot tissue are swollen and darker in pigment. Due to the changes that are evident in the symbiotic algae, we suggest that both syndromes act primarily on the zooxanthellae symbiont, and secondarily on the cnidarian host.     

Expulsion of zooxanthellae by symbiotic cnidarians from the Red Sea

The expulsion of zooxanthellae by octocorals (Xenia macrospiculata and Heteroxenia fuscescens), the scleractinian coral Stylophora pistillata, and the hydrocoral Millepora dichotoma, was measured in the field. The numbers expelled did not exceed 0.1% of the total standing stock of symbiotic algae per day, the rate of expulsion was less than 4% of the rate at which cells are added to symbiotic populations, and the carbon lost represented 0.01% of the total carbon fixed on a daily basis. Expulsion of zooxanthellae is therefore not a significant sink for fixed carbon in these symbiotic associations. In contrast to field populations, expulsion by X. macrospiculata increased 5-fold or more in the laboratory, suggesting that laboratory conditions may introduce stress.     

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.     

Copper accumulation and oxidative stress in the sea anemone, Aiptasia pallida, after waterborne copper exposure

Copper is a common marine pollutant yet its effects on symbiotic cnidarians are largely understudied. To further understand the impact of elevated copper concentrations on marine symbiotic organisms, toxicity tests were conducted using the model sea anemone, Aiptasia pallida, with and without its zooxanthellae symbiont. Symbiotic and aposymbiotic A. pallida were exposed to sublethal copper concentrations (0, 5, 15, and 50 µg/L) for 7 d and copper accumulation, behavior, and the activity of the oxidative stress enzymes, superoxide dismutase (SOD), and catalase (CAT) were measured. Additionally, acute 96-h toxicity tests were conducted to determine LC₅₀ values of the organisms after copper exposure. Both symbiotic and aposymbiotic A. pallida rapidly accumulated copper in a time and dose dependent manner. However, higher copper concentrations accumulated in the aposymbiotic as compared to the symbiotic A. pallida. In response to the highest two copper exposures (15 and 50 µg/L) symbiotic A. pallida upregulated CAT activity to combat the damaging effects of hydrogen peroxide. Contrary to these results, SOD activity significantly decreased during the highest copper exposure, when compared to controls. CAT activity was not detected and SOD was substantially (> 10 fold) reduced in aposymbiotic A. pallida, suggesting that the zooxanthellae are associated with the oxidative stress response. Copper exposure as low as 5 µg/L caused tentacle retraction and increased mucus production in both symbiotic and aposymbiotic anemones. The LC₅₀ values for symbiotic and aposymbiotic A. pallida exposed to copper for 96 h were 148 µg/L (95% confidence interval = 126.4, 173.8) and 206 µg/L (95% confidence interval = 175.2, 242.2), respectively. Understanding the varying responses of symbiotic and aposymbiotic A. pallida to copper stress may advance our comprehension of the functional roles of zooxanthellae and host. Although the mechanism of copper toxicity has not been fully elucidated, it is clear that A. pallida accumulate copper and are sensitive, as effects were detected at environmentally relevant copper concentrations. Likewise, A. pallida may be useful in biomonitoring copper polluted environments.     

卫星遥感珊瑚礁白化概述

珊瑚礁白化是由于珊瑚失去体内共生的虫黄藻或者共生的虫黄藻失去体内色素而导致五彩缤纷的珊瑚礁变白的现象,严重的白化可以带来珊瑚礁的死亡.国内外研究表明海水温度升高和珊瑚礁白化关系最为紧密.卫星遥感能够提供大范围、同步与连续的海洋数据,如海水表层温度和海色数据,从而能够及时监测和预测珊瑚礁的白化.基于AVHRR (Advanced Very High Resolution Radiometer),NOAA(National Oceanic and Atmospheric Administration,US)开发了全球监测珊瑚礁白化的方法,热点(HotSpot)和周热度(DHW)两种主要指数.目前,我国珊瑚礁白化现象的监测和研究明显滞后于国际动态,迫切需要发展和利用卫星遥感的方法监测南海珊瑚礁白化状况.     

Biogeography and microhabitat variation in temperate algal-invertebrate symbioses: zooxanthellae and zoochlorellae in two Pacific intertidal sea anemones, Anthopleura elegantissima and A. xanthogrammica

Abstract. Temperate sea anemones in the genus Anthopleura are unique among cnidarians in harboring two phylogenetically distinct symbiotic algae, zooxanthellae (golden-brown dinophytes, Symbiodinium ) and zoochlorellae (green chlorophytes). To determine whether their physiological differences generate patterns in anemone habitat and biogeographic distribution, we sampled symbiotic algae in the small clonal A. elegantissima and the large solitary A. xanthogrammica at 8 field sites (and the other large solitary Anthopleura species at one site) spanning 18° of latitude along 2500 km of the Pacific coast of North America. We found that zoochlorellae predominate in low intertidal habitats and northerly latitudes and in A. xanthogrammica , while zooxanthellae constitute the majority of symbionts in high intertidal habitats and more southerly latitudes and in A. elegantissima. These data are consistent with published predictions based on photosynthetic efficiency of the two algae under varied temperature and light regimes in the laboratory. This anemone-algal system provides a potential biological signal of benthic intertidal communities' responses to El Niño events and long-term climate changes in the Pacific.