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.     

Dispersal of Symbiodinium by the stoplight parrotfish Sparisoma viride

Environmental reservoirs of zooxanthellae are essential for coral larvae settlement; understanding where they occur and how they are maintained is important for coral reef ecology. This study investigated the dispersal of Symbiodinium spp. by the stoplight parrotfish Sparisoma viride, which had high mean densities of viable and cultivable Symbiodinium (3207-8900 cells ml(-1)) in faeces. Clades A, B and G were detected using amplified chloroplast ribosomal sequences (cp23S-HVR), and corresponded with diet preferences of fish and the environmental Symbiodinium diversity of the region. Cells are constantly dispersed in the water column and deposited in the substrate at a local level (86 ± 17.8 m(2)), demonstrating that parrotfishes are vectors for short-distance dispersal of zooxanthellae. Such dispersal could constitute a key role in the maintenance of environmental Symbiodinium reservoirs.     

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.     

Identity and diversity of coral endosymbionts (zooxanthellae) from three Palauan reefs with contrasting bleaching, temperature and shading histories

The potential of corals to associate with more temperature-tolerant strains of algae (zooxanthellae, Symbiodinium) can have important implications for the future of coral reefs in an era of global climate change. In this study, the genetic identity and diversity of zooxanthellae was investigated at three reefs with contrasting histories of bleaching mortality, water temperature and shading, in the Republic of Palau (Micronesia). Single-stranded conformation polymorphism and sequence analysis of the ribosomal DNA internal transcribed spacer (ITS)1 region was used for genotyping. A chronically warm but partly shaded coral reef in a marine lake that is hydrographically well connected to the surrounding waters harboured only two single-stranded conformation polymorphism profiles (i.e. zooxanthella communities). It consisted only of Symbiodinium D in all 13 nonporitid species and two Porites species investigated, with the remaining five Porites harbouring C*. Despite the high temperature in this lake (> 0.5 degrees above ambient), this reef did not suffer coral mortality during the (1998) bleaching event, however, no bleaching-sensitive coral families and genera occur in the coral community. This setting contrasts strongly with two other reefs with generally lower temperatures, in which 10 and 12 zooxanthella communities with moderate to low proportions of clade D zooxanthellae were found. The data indicate that whole coral assemblages, when growing in elevated seawater temperatures and at reduced irradiance, can be composed of colonies associated with the more thermo-tolerant clade D zooxanthellae. Future increases in seawater temperature might, therefore, result in an increasing prevalence of Symbiodinium phylotype D in scleractinian corals, possibly associated with a loss of diversity in both zooxanthellae and corals.     

Restriction Fragment Length Polymorphism Analysis of Large Subunit rDNA of Symbiotic Dinoflagellates from Scleractinian Corals in the Zhubi Coral Reef of the Nansha Islands

Zooxanthellae are very important for the coral reef ecosystem. The diversity of coral hosts Is high in the South China Sea, but the diversity of zooxanthellae has not yet been investigated. We chose the Zhubi Coral Reef of the Nansha Islands as the region to be surveyed in the present study because it represents a typical tropical coral reef of the South China Sea and we investigated zooxanthellae diversity In 10 host scleractinlan coral species using polymerase chain reaction （PCR） of the large subunit rRNA and restriction fragment length polymorphlsm （RFLP） patterns. Poclllopora verrucosa, Acropora pefifera, Acropora mlllepora, Fungla fungltes, Galaxea fasclcularls, and Acropora pruinosa harbor Clade C, Goniastrea aspera harbors Clade D, and Acropora formosa harbors Clades D and C. Therefore, the Clade C is the dominant type in the Zhubi Coral Reef of the Nansha Islands. Furthermore, the results of the present also disprove what has been widely accepted, namely that one coral host harbors only one algal symblont. The coral-algal symbiosis Is flexible, which may be an Important mechanism for surviving coral bleaching. Meanwhile, on the basis of the results of the present study, we think that Symblodlnium Clade D may be more tolerant to stress than Symbiodlnlum Clade C.     

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。     

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.     

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.     

Coral reef bleaching: ecological perspectives

Coral reef bleaching, the whitening of diverse invertebrate taxa, results from the loss of symbiotic zooxanthellae and/or a reduction in photosynthetic pigment concentrations in zooxanthellae residing within the gastrodermal tissues of host animals. Of particular concern are the consequences of bleaching of large numbers of reef-building scleractinian corals and hydrocorals. Published records of coral reef bleaching events from 1870 to the present suggest that the frequency (60 major events from 1979 to 1990), scale (co-occurrence in many coral reef regions and often over the bathymetric depth range of corals) and severity (>95% mortality in some areas) of recent bleaching disturbances are unprecedented in the scientific literature. The causes of small scale, isolated bleaching events can often be explained by particular stressors (e.g., temperature, salinity, light, sedimentation, aerial exposure and pollutants), but attempts to explain large scale bleaching events in terms of possible global change (e.g., greenhouse warming, increased UV radiation flux, deteriorating ecosystem health, or some combination of the above) have not been convincing. Attempts to relate the severity and extent of large scale coral reef bleaching events to particular causes have been hampered by a lack of (a) standardized methods to assess bleaching and (b) continuous, long-term data bases of environmental conditions over the periods of interest. An effort must be made to understand the impact of bleaching on the remainder of the reef community and the long-term effects on competition, predation, symbioses, bioerosion and substrate condition, all factors that can influence coral recruitment and reef recovery. If projected rates of sea warming are realized by mid to late AD 2000, i.e. a 2°C increase in high latitude coral seas, the upper thermal tolerance limits of many reef-building corals could be exceeded. Present evidence suggests that many corals would be unable to adapt physiologically or genetically to such marked and rapid temperature increases.     

珊瑚及共生藻在白化过程中的适应机制研究进展

珊瑚礁生态系统具有非常重要的生态学功能。但是随着全球气候变暖和CO2浓度的升高,珊瑚白化事件越来越频繁,珊瑚礁生态系统面临严重的危机。影响珊瑚白化的重要因子主要有海水温度的异常（过高或过低）,太阳辐射与紫外线辐射,海水盐度的偏离,珊瑚疾病,海洋污染,长棘海星的爆发,人类的过度捕鱼和全球CO2浓度升高等。其中,海洋表面水体温度（SST）的异常升高为珊瑚白化的主要因素。珊瑚主要是通过珊瑚与共生藻的生理适应机制以及更换共生藻基因型机制两种方式来适应环境胁迫的。生理适应机制主要通过叶黄素循环、珊瑚色素荧光（热）、活性氧清除系统（自由基）、分泌紫外线吸收物质MAAs（紫外光）、产生热休克蛋白HspS（热）来实现的。珊瑚共生藻基因型更换适应机制是指珊瑚的适应性白化假说。珊瑚的适应性白化假说还有很多争议,还需要更多的实验证据提供支持。未来的研究重点将在珊瑚白化过程中共生藻-珊瑚共生功能体作为整体性的研究,尤其是珊瑚宿主在白化过程中对共生功能体作出贡献的研究。     

Bleaching of Hermatypic Corals

In this paper, I review data on the magnitude and extent of reef coral bleaching events and consider modern hypotheses on the mechanisms of this natural phenomenon and experimental data lying at their basis. Four possible mechanisms of color loss by hermatypic corals have been confirmed experimentally: bacterial infection, change of zooxanthellae type in the polyps to improve the heat resistance of the photosynthetic function of coral to elevated seawater temperature, intoxication of zooxanthellae by animal metabolic wastes at high temperature and light levels, and thermal and photodestruction of the animal and algal cells. The heating effect of photosynthetic active radiation on the zooxanthellar cells in coral polyps was verified theoretically. The calculations showed that in the natural environment, the additional light-induced heating of zooxanthellae is not above 0.01°C and that it cannot cause disruption of the animal and zooxanthellae symbiosis.     

Coral bleaching: causes and consequences

It has been over 10 years since the phenomenon of extensive coral bleaching was first described. In most cases bleaching has been attributed to elevated temperature, but other instances involving high solar irradiance, and sometimes disease, have also been documented. It is timely, in view of our concern about worldwide reef condition, to review knowledge of physical and biological factors involved in bleaching, the mechanisms of zooxanthellae and pigment loss, and the ecological consequences for coral communities. Here we evaluate recently acquired data on temperature and irradiance-induced bleaching, including long-term data sets which suggest that repeated bleaching events may be the consequence of a steadily rising background sea temperature that will in the future expose corals to an increasingly hostile environment. Cellular mechanisms of bleaching involve a variety of processes that include the degeneration of zooxanthellae in situ, release of zooxanthellae from mesenterial filaments and release of algae within host cells which become detached from the endoderm. Photo-protective defences (particularly carotenoid pigments) in zooxanthellae are likely to play an important role in limiting the bleaching response which is probably elicited by a combination of elevated temperature and irradiance in the field. The ability of corals to respond adaptively to recurrent bleaching episodes is not known, but preliminary evidence suggests that phenotypic responses of both corals and zooxanthellae may be significant.     

Geographic and habitat partitioning of genetically distinct zooxanthellae (Symbiodinium) in Acropora corals on the Great Barrier Reef

Intra- and intercolony diversity and distribution of zooxanthellae in acroporid corals is largely uncharted. In this study, two molecular methods were applied to determine the distribution of zooxanthellae in the branching corals Acropora tenuis and A. valida at several reef locations in the central section of the Great Barrier Reef. Sun-exposed and shaded parts of all colonies were examined. Single-stranded conformational polymorphism analysis showed that individual colonies of A. tenuis at two locations harbour two strains of Symbiodinium belonging to clade C (C1 and C2), whereas conspecific colonies at two other reefs harboured a single zooxanthella strain. A. valida was found to simultaneously harbour strains belonging to two distinct phylogenetic clades (C and D) at all locations sampled. A novel method with improved sensitivity (quantitative polymerase chain reaction using Taqman fluorogenic probes) was used to map the relative abundance distribution of the two zooxanthella clades. At two of the five sampling locations both coral species were collected. At these two locations, composition of the zooxanthella communities showed the same pattern in both coral species, i.e. correlation with ambient light in Pioneer Bay and an absence thereof in Nelly Bay. The results show that the distribution of genetically distinct zooxanthellae is correlated with light regime and possibly temperature in some (but not all) colonies of A. tenuis and A. valida and at some reef locations, which we interpret as acclimation to local environmental conditions.     

Interspecific comparison of the symbiotic relationship in corals with high and low rates of bleaching-induced mortality

Some coral species are more resistant than others to environmental factors that cause bleaching and bleaching-related mortality. This study compared aspects of the coral/zooxanthellae symbiosis in species and genera that suffered either high or low mortality during a bleaching event. These characteristics were assessed in Okinawa between March and June 1999, 5-10 months after the bleaching event there in August-September 1998. Species with low mortality rates generally had higher densities of zooxanthellae per square centimeter and a very low rate of release of degraded zooxanthellae. Low-mortality species also had more total coral tissue per square centimeter of coral surface area. The size of zooxanthellae varied little among species. The differences in these characteristics among coral species suggest that the symbiotic relationship operates very differently among coral species.     

Review of coral reef ecosystem remote sensing

Coral reef communities face unprecedented pressures at local, regional and global scales as a consequence of climate change and anthropogenic disturbance. Remote sensing, from satellites or aircraft, is possibly the only means to measure the effects of such stresses at appropriately large spatial scales. In the past 30 years, remote sensing of coral reefs has made rapid progress. However, the current technology is still not mature enough to monitor complicated coral reef ecosystems. Compared with foreign research in this field, our work lags far behind. There are still deficiencies in many aspects, such as basic data collection, theoretical research and platform construction. In our nation, it is even unclear how coral reefs disperse and where they may be unhealthy. In this paper, general characteristics of coral reef ecosystems and spectral features of different reef benthos have been summarized, based initially on a review of relevant literature in recent years. Based on the spectral separability of different reef types or benthos, remote sensing can be used to monitor two aspects of coral reefs: (1) Measurement of the ecological properties of reefs. (2) Health assessment of the coral reef ecosystem. In the first part, optical remote sensing methods are widely used to map reef geomorphology and habitats or biotopes. The investigation of geomorphologic zonation has proven to be one of the most successful applications, as different geomorphologic zones are associated with characteristic benthic community structures and occur at spatial scales of tens to hundreds of meters, they are amenable to remote detection by moderate to high resolution sensors. With more and more attention on the ecological problems of coral reefs, a number of studies have used high resolution sensors to map reef communities. The number of classes distinguishable depends on many factors, including the platforms, resolution (spectral, spatial and temporal resolution) and environmental conditions (water depth, water clarity, surface roughness, etc.). Compared with deep water color remote sensing, or terrestrial remote sensing, three techniques for the measurement of reef ecological properties are examined in this paper: (1) Coral reef classification system using remote sensing. (2) Techniques of sea surface correction and water column correction. (3) Techniques of coral reef information extraction from images. In terms of the complexity of coral reef ecosystems, the current techniques still need further improvement or optimization. In the health assessment of coral reef ecosystems, there are two ways to carry out the monitoring using remote sensing: (1) Monitoring the pigment or symbiotic zooxanthellae contents in corals. (2) Measuring the environmental properties of reefs. The first way is theoretically feasible, but difficult to achieve in practice. Currently, most reef health assessments are carried out by measuring environmental parameters, including sea surface temperature, solar radiation, ultraviolet radiation, water color, wind speed and direction, rainfall, ocean acidification, sea level, etc., of which sea surface temperature has been routinely measured by NOAA to monitor coral bleaching. In addition to the contents above, this article puts forward five main prospects for development in the future: (1) Establishment of a coral reef classification system using remote sensing. (2) Satellite launch for monitoring coral reefs. (3) Theoretical and methodological development. (4) Establishment of a spectral database for different reef benthos. (5) Integrated application of multi-source remote sensing data. It is hoped that the information provided here will be a reference for subsequent similar studies.     

Is photoinhibition of zooxanthellae photosynthesis the primary cause of thermal bleaching in corals?

The bleaching of corals in response to increases in temperature has resulted in significant coral reef degradation in many tropical marine ecosystems. This bleaching has frequently been attributed to photoinhibition of photosynthetic electron transport and the consequent photodamage to photosystem II (PSII) and the production of damaging reactive oxygen species (ROS) in the zooxanthellae (Symbiodinium spp.). However, these events may be because of perturbations of other processes occurring within the zooxanthellae or the host cells, and consequently constitute only secondary responses to temperature increase. The processes involved with the onset of photoinhibition of electron transport, photodamage to PSII and pigment bleaching in coral zooxanthellae are reviewed. Consideration is given to how increases in temperature might lead to perturbations of metabolic processes in the zooxanthellae and/or their host cells, which could trigger events leading to bleaching. It is concluded that production of ROS by the thylakoid photosynthetic apparatus in the zooxanthellae plays a major role in the onset of bleaching resulting from photoinhibition of photosynthesis, although it is not clear which particular ROS are involved. It is suggested that hydrogen peroxide generated in the zooxanthellae may have a signalling role in triggering the mechanisms that result in expulsion of zooxanthellae from corals.     

Host population genetic structure and zooxanthellae diversity of two reef-building coral species along the Florida Reef Tract and wider Caribbean

In preparation for a large-scale coral restoration project, we surveyed host population genetic structure and symbiont diversity of two reef-building corals in four reef zones along the Florida reef tract (FRT). There was no evidence for coral population subdivision along the FRT in Acropora cervicornis or Montastraea faveolata based on microsatellite markers. However, in A. cervicornis, significant genetic differentiation was apparent when extending the analysis to broader scales (Caribbean). Clade diversity of the zooxanthellae differed along the FRT. A. cervicornis harbored mostly clade A with clade D zooxanthellae being prominent in colonies growing inshore and in the mid-channel zones that experience greater temperature fluctuations and receive significant nutrient and sediment input. M. faveolata harbored a more diverse array of symbionts, and variation in symbiont diversity among four habitat zones was more subtle but still significant. Implications of these results are discussed for ongoing restoration and conservation work.     

Zooxanthellar symbionts shape host sponge trophic status through translocation of carbon

Sponges belonging to the genus Cliona are common inhabitants of many coral reefs, and as bioeroders, they play an important role in the carbonate cycle of the reef. Several Cliona species maintain intracellular populations of dinoflagellate zooxanthellae (i.e., Symbiodinium spp.), which also form symbioses with a variety of other invertebrates and protists (e.g., corals, molluscs, foraminifera). Unlike the case of coral symbioses, however, almost nothing is known of the metabolic interaction between sponges and their zooxanthella symbionts. To assess this interaction, we performed a tracer experiment to follow C and N in the system, performed a reciprocal transplant experiment, and measured the stable carbon isotope ratio of Cliona spp. with and without zooxanthellae to study the influence of environment on the interaction. We found strong evidence of a transfer of C from zooxanthellae to their sponge hosts but no evidence of a transfer of N from sponge to zooxanthellae. We also saw significant influences of the environment on the metabolism of the sponges. Finally, we observed significant differences in carbon metabolism of sponge species with and without symbionts. These data strongly support hypotheses of metabolic integration between zooxanthellae and their sponge host and extend our understanding of basic aspects of benthic-pelagic coupling in shallow-water marine environments.     

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.     

Bleaching response of Symbiodinium (zooxanthellae): Determination by flow cytometry

Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and β-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 38°C) over 24 h, with ambient temperature (29°C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. ? 2012 International Society for Advancement of Cytometry.