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.     

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.     

共生珊瑚异养营养研究进展

共生珊瑚的异养营养指有虫黄藻共生的珊瑚在同化作用过程中,除通过虫黄藻光合作用获取营养之外,还可以从外界环境中直接摄取现成的有机物,经消化吸收后转变为自身的组成物质或储存为能量.国内外异地养殖或繁育虫黄藻共生珊瑚的研究多集中在光照、水流、水质等条件对珊瑚生长的影响,对共生珊瑚异养营养需求与供应方面的关注较少.本文从共生珊瑚异养营养来源、影响共生珊瑚异养营养供应的因素以及研究手段等方面,对国内外研究进展进行了综述,探讨异养营养供应对共生珊瑚的意义.总的来说,目前共生珊瑚异养营养的研究尚处于起步阶段,不论是研究方法或者珊瑚选择摄食的内在机制,都需要深入探究.     

Symbiont diversity in scleractinian corals from tropical reefs and subtropical non-reef communities in Taiwan

We examined zooxanthellae diversity in scleractinian corals from southern Taiwan and the Penghu Archipelago, a tropical coral reef and a subtropical non-reefal community, respectively. Zooxanthellae diversity was investigated in 52 species of scleractinian corals from 26 genera and 13 families, using restriction fragment length polymorphism (RFLP), and phylogenetic analyses of the nuclear small-subunit ribosomal DNA (nssrDNA) and large-subunit ribosomal DNA (nlsrDNA). RFLP and phylogenetic analyses of nuclear-encoded ribosomal RNA genes showed that Symbiodinium clade C was the dominant zooxanthellae in scleractinian corals in the seas around Taiwan; Symbiodinium clade D was also found in some species. Both Symbiodinium clade C and D were found in colonies of seven species of scleractinian corals. Symbiodinium clade D was associated with corals that inhabit either shallow water or the reef edge in deep water, supporting the hypothesis that Symbiodinium clade D is a relatively stress-tolerant zooxanthellae found in marginal habitats.Communicated by Biological Editor H.R. Lasker     

Carbonic anhydrases in anthozoan corals—A review

Coral reefs are among the most biologically diverse and economically important ecosystems on the planet. The deposition of massive calcium carbonate skeletons (biomineralization or calcification) by scleractinian corals forms the coral reef framework/architecture that serves as habitat for a large diversity of organisms. This process would not be possible without the intimate symbiosis between corals and photosynthetic dinoflagellates, commonly called zooxanthellae. Carbonic anhydrases play major roles in those two essential processes of coral’s physiology: they are involved in the carbon supply for calcium carbonate precipitation as well as in carbon-concentrating mechanisms for symbiont photosynthesis. Here, we review the current understanding of diversity and function of carbonic anhydrases in corals and discuss the perspective of theses enzymes as a key to understanding impacts of environmental changes on coral reefs.     

Symbiosis regulation in a facultatively symbiotic temperate coral: zooxanthellae division and expulsion

Zooxanthellae mitotic index (MI) and expulsion rates were measured in the facultatively symbiotic scleractinian Astrangia poculata during winter and summer off the southern New England coast, USA. While MI was significantly higher in summer than in winter, mean expulsion rates were comparable between seasons. Corals therefore appear to allow increases in symbiont density when symbiosis is advantageous during the warm season, followed by a net reduction during the cold season when zooxanthellae may draw resources from the coral. Given previous reports that photosynthesis in A. poculata symbionts does not occur below approximately 6°C, considerable zooxanthellae division at 3°C and in darkness suggests that zooxanthellae are heterotrophic at low seasonal temperatures. Finally, examination of expulsion as a function of zooxanthellae density revealed that corals with very low zooxanthellae densities export a significantly greater proportion of their symbionts, apparently allowing them to persist in a stable azooxanthellate state.     

Conservation genetics and the resilience of reef-building corals

Coral reefs have suffered long-term decline due to a range of anthropogenic disturbances and are now also under threat from climate change. For appropriate management of these vulnerable and valuable ecosystems it is important to understand the factors and processes that determine their resilience and that of the organisms inhabiting them, as well as those that have led to existing patterns of coral reef biodiversity. The scleractinian (stony) corals deposit the structural framework that supports and promotes the maintenance of biological diversity and complexity of coral reefs, and as such, are major components of these ecosystems. The success of reef-building corals is related to their obligate symbiotic association with dinoflagellates of the genus Symbiodinium. These one-celled algal symbionts (zooxanthellae) live in the endodermal tissues of their coral host, provide most of the host's energy budget and promote rapid calcification. Furthermore, zooxanthellae are the main primary producers on coral reefs due to the oligotrophic nature of the surrounding waters. In this review paper, we summarize and critically evaluate studies that have employed genetics and/or molecular biology in examining questions relating to the evolution and ecology of reef-building corals and their algal endosymbionts, and that bear relevance to coral reef conservation. We discuss how these studies can focus future efforts, and examine how these approaches enhance our understanding of the resilience of reef-building corals.     

Laboratory models for the study of coral pathologies

Worldwide, the scleractinian corals that characterize contemporary coral reef communities are exhibiting a variety of pathological conditions. These conditions range from diseases linked with specific pathogens to the syndrome known as bleaching. The latter phenomenon involves the loss or reduction of the symbiotic zooxanthellae on which the corals depend. Bleaching appears to be a generalized stress syndrome, but in some cases it may be due to pathogenic infections. A full understanding of coral pathologies requires the development of laboratory models. We have developed two complementary protocols that will facilitate the study of coral pathologies at a number of levels. The first method involves the induction of bleaching by exposing the coral to an acute period of reduced temperature. The second protocol allows the dissociation of coral polyps into a number of cell types that can be maintained long-term in primary culture. Among these are multicellular endothelial isolates (MEI) that contain zooxanthellae and show a high rate of motility. The bleaching protocol will enable investigators to study the processes by which corals recover from bleaching, and it will offer a standard that can be compared to other conditions that lead to bleaching. The cell culture technique will enable the study of mechanisms underlying pathological conditions at the cellular level, and permit studies of how pathological conditions disrupt the relationship between corals and their zooxanthellae.     

造礁石珊瑚对低温的耐受能力及响应模式

通过实验室生态模拟,研究了低温胁迫下三亚湾5种造礁石珊瑚（十字牡丹珊瑚、佳丽鹿角珊瑚、花鹿角珊瑚、强壮鹿角珊瑚、澄黄滨珊瑚）的耐受性,分析了造礁石珊瑚对低温的响应模式.结果表明：造礁石珊瑚耐受低温能力与其骨骼类型有关,枝状珊瑚最先死亡,块状珊瑚的耐受能力明显高于枝状珊瑚;14 ℃持续3 d是三亚湾枝状造礁石珊瑚的致死低温;14 ℃持续3 d为块状澄黄滨珊瑚的致白化低温;12 ℃持续10 d为叶片状十字牡丹珊瑚的致死温度;块状澄黄滨珊瑚受到低温胁迫时表面形成粘膜,阻止了珊瑚进一步排出共生虫黄藻. 耐高温的珊瑚对低温也表现出较强的耐受能力,珊瑚对低温胁迫的响应模式与对高温的响应模式基本一致, 即珊瑚首先不伸展触手,紧接着不断释放粘液并排出共生藻,最后白化、死亡.     

A Comparison of the Composition of Wax Ester Molecular Species of Different Coral Groups (Subclasses Hexacorallia and Octocorallia)

Wax esters, which are esters of fatty alcohols and fatty acids (FAs), are one of the main classes of reserve lipids in all coral species. The chemical structures and the content of wax ester molecular species were determined for the first time in nine coral species from three taxonomic groups: symbiotic reef-building corals, (Hexacorallia subclasses), symbiotic soft coral alcyonarians, and asymbiotic soft coral gorgonians (Octocorallia subclasses) collected in the South China Sea (Vietnam). Our comparison of these groups showed that the absence of symbiotic microalgae (zooxanthellae) and the exoskeleton affects the profile of molecular species of wax esters considerably. The main components of wax esters of all corals were cetyl palmitate (16:0-16:0) and other saturated wax esters containing 30, 34, and 36 carbon atoms. The content of unsaturated molecular species 6:0–16:1, 16:0–18:1, and 16:0–20:1 in wax esters of symbiotic soft corals (alcyonarians) was greater than that in wax esters of reef-building corals. In contrast to symbiotic coral species, wax esters of asymbiotic soft corals, namely azooxanthellate gorgonians, contained a considerable amount of long-chain molecular species (C₃₇-C₄₁) with an odd number of carbon atoms. The presence of such molecular species indicates that asymbiotic gorgonians may use bacterial FAs in biosynthesis of their own wax esters. This observation confirms our hypothesis that bacterial community is important for maintaining the energy balance of azooxanthellate corals.     

Coral photobiology: new light on old views

The relationship between reef-building corals and light-harvesting pigments of zooxanthellae (Symbiodinium sp.) has been acknowledged for decades. The photosynthetic activity of the algal endocellular symbionts may provide up to 90% of the energy needed for the coral holobiont. This relationship limits the bathymetric distribution of coral reefs to the upper 100 m of tropical shorelines. However, even corals growing under high light intensities have to supplement the photosynthates translocated from the algae by predation on nutrient-rich zooplankton. New information has revealed how the fate of carbon acquired through photosynthesis differs from that secured by predation, whose rates are controlled by light-induced tentacular extension. The Goreau paradigm of “light-enhanced calcification” is being reevaluated, based on evidence that blue light stimulates coral calcification independently from photosynthesis rates. Furthermore, under dim light, calcification rates were stoichiometrically uncoupled from photosynthesis. The rates of photosynthesis of the zooxanthellae exhibit a clear endogenous rhythmicity maintained by light patterns. This daily pattern is concomitant with a periodicity of all the antioxidant protective mechanisms that wax and wane to meet the concomitant fluctuation in oxygen evolution. The phases of the moon are involved in the triggering of coral reproduction and control the spectacular annual mass-spawning events taking place in several reefs. The intensity and directionality of the underwater light field affect the architecture of coral colonies, leading to an optimization of the exposure of the zooxanthellae to light. We present a summary of major gaps in our understanding of the relationship between light and corals as a roadmap for future research.     

Diversity of symbiotic algae of the genus Symbiodinium in scleractinian corals of the Xisha Islands in the South China Sea

Symbiotic algae （Symbiodinium sp.） in scleractinian corals are important in understanding how coral reefs will respond to global climate change. The present paper reports on the diversity of Symbiodinium sp. in 48 scleractinian coral species from 25 genera and 10 families sampled from the Xisha Islands in the South China Sea, which were identified with the use of restriction fragment length polymorphism （RFLP） of the nuclear ribosomal DNA large subunit gene （rDNA）. The results showed that： （i） Symbiodinium Clade C was the dominant zooxanthellae in scleractinian corals in the Xisha Islands; （ii） Symbiodinium Clade D was found in the corals Montipora aequituberculata, Galaxea fascicularis, and Plerogyra sinuosa; and （iii） both Symbiodinium Clades C and D were found simultaneously in Montipora digitata, Psammocora contigua, and Galaxeafascicularis. A poor capacity for symbiosis polymorphism, as uncovered by RFLP, in the Xisha Islands indicates that the scleractinian corals have low adaptability to environmental changes. Further studies are needed to investigate zooxanthellae diversity using other molecular markers.     

Calcification and associated physiological parameters during a stress event in the scleractinian coral Stylophora pistillata

High calcification rates observed in reef coral organisms are due to the symbiotic relationship established between scleractinian corals and their photosynthetic dinoflagellates, commonly called zooxanthellae. Zooxanthellae are known to enhance calcification in the light, a process referred as "light-enhanced calcification". The disruption of the relationship between corals and their zooxanthellae leads to bleaching. Bleaching is one of the major causes of the present decline of coral reefs related to climate change and anthropogenic activities. In our aquaria, corals experienced a chemical pollution leading to bleaching and ending with the death of corals. During the time course of this bleaching event, we measured multiple parameters and could evidence four major consecutive steps: 1) at month 1 (January 2005), the stress affected primarily the photosystem II machinery of zooxanthellae resulting in an immediate decrease of photosystem II efficiency, 2) at month 2, the stress affected the photosynthetic production of O2 by zooxanthellae and the rate of light calcification, 3) at month 3, there was a decrease in both light and dark calcification rates, the appearance of the first oxidative damage in the zooxanthellae, the disruption of symbiosis, 4) and finally the death of corals at month 6.     

Feeding sustains photosynthetic quantum yield of a scleractinian coral during thermal stress

Thermal resistance of the coral-zooxanthellae symbiosis has been associated with chronic photoinhibition, increased antioxidant activity and protein repair involving high demands of nitrogen and energy. While the relative importance of heterotrophy as a source of nutrients and energy for cnidarian hosts, and as a means of nitrogen acquisition for their zooxanthellae, is well documented, the effect of feeding on the thermal sensitivity of the symbiotic association has been so far overlooked. Here we examine the effect of zooplankton feeding versus starvation on the bleaching susceptibility and photosynthetic activity of photosystem II (PSII) of zooxanthellae in the scleractinian coral Stylophora pistillata in response to thermal stress (daily temperature rises of 2-3 degrees C) over 10 days, employing pulse-amplitude-modulated chlorophyll fluorometry. Fed and starved corals displayed a decrease in daily maximum potential quantum yield (F (v)/F (m)) of PSII, effective quantum yield (F/F (m)') and relative electron transport rates over the course of 10 days. However after 10 days of exposure to elevated temperature, F (v)/F (m) of fed corals was still 50-70% higher than F (v)/F (m) of starved corals. Starved corals showed strong signs of chronic photoinhibition, which was reflected in a significant decline in nocturnal recovery rates of PSII relative to fed corals. This was paralleled by the progressive inability to dissipate excess excitation energy via non-photochemical quenching (NPQ). After 10 days, NPQ of starved corals had decreased by about 80% relative to fed corals. Feeding treatment had no significant effect on chlorophyll a and c (2) concentrations and zooxanthellae densities, but the mitotic indices were significantly lower in starved than in fed corals. Collectively the results indicate that exogenous food may reduce the photophysiological damage of zooxanthellae that typically leads to bleaching and could therefore play an important role in mediating the thermal resistance of some corals.     

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.     

Sediment as a possible source of food for corals

Reef building corals are generally believed to require clear, nutrient-poor water. Yet, paradoxically, coral reefs are among the most productive marine ecosystems in the world. This paradox is commonly explained by the coral's ability to utilize a number of food resources, including zooplankton, phytoplankton, dissolved organic matter and photosynthetic products derived from their endosymbiotic algae, the zooxanthellae. Sedimentation is a known stressor for corals, inhibiting most feeding modes in various ways. However, evidence for enhanced sediment deposition on certain corals, induced by their morphologies, as well as for sediment ingestion by some corals, has led us to examine the use of sediment as a possible source of food for corals in addition to the other, known food sources. Our experiments with fluorescently labelled sediment show transfer of labelled organic matter from the sediment into the cells of the solitary coral Fungia horrida Dana 1846. The results provide the first direct evidence for the ability of a coral to digest the sediment's organic fraction. These results may indicate a positive role of sediment, which up until now was considered to have only deleterious effects on corals.     

Effect of water currents on organic matter release by two scleractinian corals

Organic matter release by scleractinian corals fulfils an important ecological role as energy carrier and particle trap in reef ecosystems, but the hypothetically stimulating impact of water currents, an essential and ubiquitous environmental factor in coral reefs, on this process has not been investigated yet. This study therefore quantifies organic matter release by two species of scleractinian corals subjected to ambient water current velocities ranging from 4 to 16?cm?s^?1 using closed-system flow-through chambers. Findings revealed that particulate organic matter (POM) concentration was significantly increased in the flow-through chambers in all investigated coral species compared to still water conditions, while no effect on dissolved organic carbon (DOC) concentration could be observed. These results suggest that POM release by corals may be controlled by hydro-mechanical impacts, while DOC fluxes are rather influenced by the physiological condition of the corals. Hence, this study indicates that previous POM release quantification results are conservative estimates and may have underestimated in situ POM release through corals in reef environments. The contribution of coral-derived POM to biogeochemical cycles in reef ecosystems, therefore, may be more pronounced than already assumed.     

Antagonistic interactions among coral-associated bacteria

Reef-building corals are comprised of close associations between the coral animal, symbiotic zooxanthellae, and a diversity of associated microbes (including Bacteria, Archaea and Fungi). Together, these comprise the coral holobiont – a paradigm that emphasizes the potential contributions of each component to the overall function and health of the coral. Little is known about the ecology of the coral-associated microbial community and its hypothesized role in coral health. We explored bacteria–bacteria antagonism among 67 bacterial isolates from the scleractinian coral Montastrea annularis at two temperatures using Burkholder agar diffusion assays. A majority of isolates exhibited inhibitory activity (69.6% of isolates at 25°C, 52.2% at 31°C), with members of the γ-proteobacteria ( Vibrionales and Alteromonadales ) being especially antagonistic. Elevated temperatures generally reduced levels of antagonism, although the effects were complex. Several potential pathogens were observed in the microbial community of apparently healthy corals, and 11.6% of isolates were able to inhibit the growth of the coral pathogen Vibrio shiloi at 25°C. Overall, this study demonstrates that antagonism could be a structuring force in coral-associated microbial communities and may contribute to pathogenesis as well as disease resistance.     

Specificity is rarely absolute in coral-algal symbiosis: implications for coral response to climate change

Some reef-building corals have been shown to respond to environmental change by shifting the composition of their algal symbiont (genus Symbiodinium) communities. These shifts have been proposed as a potential mechanism by which corals might survive climate stressors, such as increased temperatures. Conventional molecular methods suggest this adaptive capacity may not be widespread because few (～25%) coral species have been found to associate with multiple Symbiodinium clades. However, these methods can fail to detect low abundance symbionts (typically less than 10-20% of the total algal symbiont community). To determine whether additional Symbiodinium clades are present, but are not detected using conventional techniques, we applied a high-resolution, real-time PCR assay to survey Symbiodinium (in clades A-D) from 39 species of phylogenetically and geographically diverse scleractinian corals. This survey included 26 coral species thought to be restricted to hosting a single Symbiodinium clade ('symbiotic specialists'). We detected at least two Symbiodinium clades (C and D) in at least one sample of all 39 coral species tested; all four Symbiodinium clades were detected in over half (54%) of the 26 symbiotic specialist coral species. Furthermore, on average, 68 per cent of all sampled colonies within a given coral species hosted two or more symbiont clades. We conclude that the ability to associate with multiple symbiont clades is common in scleractinian (stony) corals, and that, in coral-algal symbiosis, 'specificity' and 'flexibility' are relative terms: specificity is rarely absolute. The potential for reef corals to adapt or acclimatize to environmental change via symbiont community shifts may therefore be more phylogenetically widespread than has previously been assumed.     

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.