Metronidazole (2-methyl-5-nitroimidazole- 1-ethanol) is shown to be effective for the selective enrichment of mutants of Chiamydomonas reinhardtii that possess impaired photosynthetic electron transport. More than 99.9% of wild-type cells are killed when incubated in the presence of 6-10 mM metronidazole for 24 hr under illumination of 7500 lux. Survival of wild-type cells in darkness and of mutants that are blocked at different steps in photosynthetic electron transport is nearly 100% when incubated in the presence of the drug under iden- tical conditions. The toxicity of metronidazole is demonstrated to depend upon its reduction by photosynthetic electron transport. Light-de- pendent oxygen uptake mediated by metronidazole is shown to require active photosystem I in vitro and in vivo. Ferredoxin is necessary for metronidazole reduction by thylakoid mem- brane fractions enriched in photosystem I activity. We propose that the toxicity of metronidazole is due to the formation of le- thal derivatives of the drug or to the accumulation of hydrogen peroxide, which could occur upon autooxidation of metron- idazole reduced by one electron. The results indicate that mu- tants of C. reinhardtii, and probably other photosynthetic or- ganisms, with any lesion in photosynthetic electron transport from the oxidizing side of photosystem II to ferredoxin can be isolated by metronidazole treatment of mutagenized cul- tures.

Recently, the Caribbean reef-building coral Orbicella faveolata was listed as “threatened” under the U.S. Endangered Species Act. Despite attention to this species’ conservation, the extent of geographic variation within O. faveolata warrants further investigation. O. faveolata is unusual in that it can simultaneously harbor multiple genetically distinct and co-dominant species of endosymbiotic dinoflagellates in the genus Symbiodinium. Here, we investigate the geographic and within-colony complexity of Symbiodinium - O. faveolata associations from Florida Keys, USA; Exuma Cays, Bahamas; Puerto Morelos, Mexico; and Carrie Bow Cay, Belize. We collected coral samples along intracolony axes, and Symbiodinium within O. faveolata samples was analyzed using the nuclear ITS2 region and chloroplast 23S rDNA genotyping. O. faveolata associated with species of Symbiodinium in clades A (type A3), B (B1 and B17), C (C3, C7, and C7a), and D (D1a/ Symbiodinium trenchii ). Within-colony distributions of Symbiodinium species correlated with light availability, cardinal direction, and depth, resulting in distinct zonation patterns of endosymbionts within a host. Symbiodinium species from clades A and B occurred predominantly in the light-exposed tops, while species of clade C generally occurred in the shaded sides of colonies or in deeper-water habitats. Furthermore, geographic comparisons of host–symbiont associations revealed regional differences in Symbiodinium associations. Symbiodinium A3 was detected in Mesoamerican coral colonies, but not in colonies from the Florida Keys or Bahamas. Likewise, Symbiodinium B17 was unique to Mesoamerican O. faveolata , whereas Symbiodinium B1 was found at all localities sampled. However, using cp23S genotyping paired with ITS2 analysis revealed geographically endemic haplotypes among Symbiodinium clades A, B, and C. Since Symbiodinium spatial heterogeneity among this coral species is greater than most corals, a question arises as to whether all western Atlantic populations of O. faveolata should be considered equally “threatened”? Alternatively, geographically and spatially distinct coral–symbiont associations may benefit from specialized management protocols.

Community dynamics and physiology of Symbiodinium associated with Orbicella ( = Montastraea) faveolata were examined before, during, and after a thermally induced coral bleaching event in Puerto Morelos, Mexico. We combined microsampling molecular genotyping with in situ pulse‐amplitude modulated fluorometry to correlate colony variability of Symbiodinium population identities and the phenomena of partial coral bleaching. Pigmented nonbleached portions of O. ( = M.) faveolata were compared with bleached portions of the same colony. During bleaching, maximum quantum yield of photosystem II (PSII; Fv : Fm) was significantly lower and highly variable (range 0.110 to 0.680) compared with previous summers in which coral bleaching was absent (range 0.516 to 0.661) and recovery (range 0.480 to 0.716). Differential susceptibility to environmental perturbation of Fv : Fm corresponded to distinct genetic identities of Symbiodinium. Analysis of ribosomal deoxyribonucleic acid (rDNA) internal transcribed spacer 2 (ITS2) revealed regions of the coral colonies that had phylotype A3 prior to bleaching were more resistant to the bleaching perturbation than adjacent bleaching‐prone patches that harbored phylotypes B17 and C7. During environmental perturbation, regions of the colonies containing predominantly Symbiodinium phylotypes A3 or D1a retained significantly higher Fv : Fm values than adjacent regions with phylotypes B17 and C7. Following bleaching, rapid recovery of symbiotic algal densities greatly exceeded normal seasonal oscillations. During recovery we document shifts in Symbiodinium populations and increase prevalence of Symbiodinium types A3 and D1a, phylotypes known to have enhanced thermal tolerances. Thermal tolerance of Symbiodinium spp. influences the changes of coral—Symbiodinium communities during disturbance events and the dynamics of coral—Symbiodinium repopulation.

The symbiosis between hermatypic corals and their dinoflagellate endosymbionts, genus Symbiodinium , is based on carbon exchange. This symbiosis is disrupted by thermally induced coral bleaching, a stress response in which the coral host expels its algal symbionts as they become physiologically impaired. The disruption of the dissolved inorganic carbon (DIC) supply or the thermal inactivation of Rubisco have been proposed as sites of initial thermal damage that leads to the bleaching response. Symbiodinium possesses a highly unusual Form II ribulose bisphosphate carboxylase/oxygenase (Rubisco), which exhibits a lower CO 2 :O 2 specificity and may be more thermally unstable than the Form I Rubiscos of other algae and land plants. Components of the CO 2 concentrating mechanism (CCM), which supplies inorganic carbon for photosynthesis, may also be temperature sensitive. Here, we examine the ability of four cultured Symbiodinium strains to acquire and fix DIC across a temperature gradient. Surprisingly, the half-saturation constant of photosynthesis with respect to DIC concentration ( K P ), an index of CCM function, declined with increasing temperature in three of the four strains, indicating a greater potential for photosynthetic carbon acquisition at elevated temperatures. In the fourth strain, there was no effect of temperature on K P . Finding no evidence for thermal inhibition of the CCM, we conclude that CCM components are not likely to be the primary sites of thermal damage. Reduced photosynthetic quantum yields, a hallmark of thermal bleaching, were observed at low DIC concentrations, leaving open the possibility that reduced inorganic carbon availability is involved in bleaching.

A terminal electron acceptor alternative to mitochondrial cytochrome c oxidase (COX), mitochondrial alternative oxidase (AOX), is ubiquitous in higher plants and represented in nearly every algal taxon but is poorly documented in dinoflagellates. AOX competes for electrons with the conventional COX and has been hypothesized to function as a means of reducing oxidative stress in mitochondria, as well as a potential mechanism for ameliorating thermal and other physiological stressors. Here, the presence of an active AOX in cultured Symbiodinium was assayed by the response of oxygen consumption to the AOX inhibitor salicylhydroxamic acid (SHAM) and the COX inhibitor cyanide (CN). CN-insensitive, SHAM-sensitive oxygen consumption was found to account for a large portion (26 %) of Symbiodinium dark respiration and is consistent with high levels of AOX activity. This experimental evidence of the existence of a previously unreported terminal oxidase was further corroborated by analysis of publicly available Symbiodinium transcriptome data. The potential for enhanced AOX expression to play a compensatory role in mediating thermal stress was supported by inhibitor assays of cultured Symbiodinium at low (18 °C), moderate (26 °C), and high (32 °C) temperature conditions. Maximum capacity of the putative AOX pathway as a proportion of total dark oxygen consumption was found to increase from 26 % at 26 °C to 45 % and 53 % at 18 °C and 32 °C, respectively, when cells were acclimated to the treatment temperatures. Cells assayed at 18 and 32 °C without acclimation exhibited either the same or lower AOX capacity as controls, suggesting that the AOX protein is upregulated under temperature stress. The physiological implications for the presence of AOX in the coral/algal symbiosis and its potential role in response to many forms of biotic and abiotic stress, particularly oxidative stress, are discussed.

Conventional means of assessing net photosynthetic rates of microalgae have largely relied upon the use of oxygen electrodes or carbon isotope radiolabeling. These methods are simple but inadequately resolve simultaneous fluxes of the gaseous substrates and products of photosynthesis, CO2, and O2. Fluorometric methods allow for assessment of the photosynthetic efficiency of excitation energy capture by the light‐harvesting complexes and electron transport chain, but do not directly measure photosynthetic rates, because they do not resolve the fate of excitation energy downstream of electron transport chain. Here we describe and validate the accuracy of a compact system for the simultaneous, real‐time, and comprehensive measurement of photosynthetic CO2 and O2 flux as well as photosynthetic electron transport in microalgae, from which net and gross rates of carbon fixation and oxygen evolution can be derived. Essential components include an infrared gas analyzer, gas‐phase galvanic oxygen electrode, and pulse‐amplitude modulated fluorometer.

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994-2007), eleven years in the Exuma Cays, Bahamas (1995-2006), and four years in Puerto Morelos, Mexico (2003-2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1-4 m) compared to deeper-dwelling conspecifics (12-15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels.

Coral reefs of the Florida Keys typically experience seasonal temperatures of 20-31 degrees C. Deviation outside this range causes physiological impairment of reef-building corals, potentially leading to coral colony death. In January and February 2010, two closely spaced cold fronts, possibly driven by an unusually extreme Arctic Oscillation, caused sudden and severe seawater temperature declines in the Florida Keys. Inshore coral reefs [e. g., Admiral Reef (ADM)] experienced lower sustained temperatures (i.e., < 12 degrees C) than those further offshore [e. g., Little Grecian Reef (LG), minimum temperature = 17.2 degrees C]. During February and March 2010, we surveyed ADM and observed a mass die-off of reef-building corals, whereas 12 km away LG did not exhibit coral mortality. We subsequently measured the physiological effects of low-temperature stress on three common reef-building corals (i.e., Montastraea faveolata, Porites astreoides, and Siderastrea siderea) over a range of temperatures that replicated the inshore cold-water anomaly (i.e., from 20 to 16 to 12 degrees C and back to 20 degrees C). Throughout the temperature modulations, coral respiration as well as endosymbiont gross photosynthesis and maximum quantum efficiency of photosystem II were measured. In addition, Symbiodinium genotypic identity, cell densities, and chlorophyll a content were determined at the beginning and conclusion of the experiment. All corals were significantly affected at 12 degrees C, but species-specific physiological responses were found indicating different coral and/or Symbiodinium cold tolerances. Montastraea faveolata and P. astreoides appeared to be most negatively impacted because, upon return to 20 degrees C, significant reductions in gross photosynthesis and dark respiration persisted. Siderastrea siderea, however, readily recovered to pre-treatment rates of dark respiration and gross photosynthesis. Visual surveys of inshore reefs corroborated these results, with S. siderea being minimally affected by the cold-water anomaly, whereas M. faveolata and P. astreoides exhibited nearly 100% mortality. This study highlights the importance of understanding the physiological attributes of genotypically distinct coral-Symbiodinium symbioses that contribute to tolerance, recovery, and consequences to an environmental perturbation. These data also document effects of a rarely studied environmental stressor, possibly initiated by remote global climate events, on coral-Symbiodinium symbioses and coral reef communities.

The reticulated chloroplasts of zooxanthellae (Symbiodinium) and differences in chlorophyll localization among life cycle stages

Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR–DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

There are at least two photosynthetic cyclic electron transport (CET) pathways in most C3 plants: the NAD(P)H dehydrogenase (NDH)-dependent pathway and a pathway dependent upon putative ferredoxin:plastoquinone oxidoreductase (FQR) activity. While the NDH complex has been identified, and shown to play a role in photosynthesis, especially under stress conditions, less is known about the machinery of FQR-dependent CET. Recent studies indicate that FQR-dependent CET is dependent upon PGR5, a small protein of unknown function. In a previous study we found that overexpression of PGR5 causes alterations in growth and development associated with decreased chloroplast development and a transient increase in nonphotochemical quenching (NPQ) after the shift from dark to light. In the current study we examine the spatiotemporal expression pattern of PGR5, and the effects of overexpression of PGR5 in Arabidopsis under a host of light and stress conditions. To investigate the conserved function of PGR5, we cloned PGR5 from a species which apparently lacks NDH, loblolly pine, and overexpressed it in Arabidopsis. Although greening of cotyledons was severely delayed in overexpressing lines under low light, mature plants survived exposure to high light and drought stress better than wild-type. In addition, PSI was more resistant to high light in the PGR5 overexpressors than in wild-type plants, while PSII was more sensitive to this stress. These complex responses corresponded to alterations in linear and cyclic electron transfer, suggesting that over-accumulation of PGR5 induces pleiotropic effects, probably via elevated CET. We conclude that PGR5 has a developmentally-regulated, conserved role in mediating CET.

The Gulf of California (Sea of Cortez) acts as a refuge for zooxanthellate coral communities at high northern latitudes in the eastern Pacific. The diversity of dinoflagellate endosymbionts living with cnidarians (9 genera, 16 species) was examined using denaturing gradient gel electrophoresis (PCR‐DGGE) fingerprint analysis of the rDNA Internal Transcribed Spacers (ITS) 1 and 2. Although geographically isolated from the rest of the Pacific, coral‐algal associations in the Gulf of California were similar in cladal composition (dominated by clade C Symbiodiniu m). However, levels of host‐symbiont specificity appeared to be greater than in the western Pacific, where host diversity is considerably higher and environmental conditions are relatively stable. As a result, the cnidarian community lacked a host‐generalist symbiont. Most colonies of the ecologically dominant genus Pocillopora contained uniform populations of either Symbiodinium D1 (in ~70% of colonies) or C1b‐c (~30%). The proportions of D versus C colonies (i.e., D and C holobionts) remained stable between the years 2004 and 2006. The specificity, stability, and prevalence of Symbiodinium D1 found among individuals of Pocillopora spp. in a region minimally impacted by coral bleaching indicate that these symbiotic combinations, exhibiting specific variability, appear to be products of long‐term ecological and evolutionary processes.

Many corals form obligate symbioses with photosynthetic dinoflagellates of the genus Symbiodinium Freudenthal (1962). These symbionts vary genotypically, with their geographical distribution and abundance dependent upon host specificity and tolerance to temperature and light variation. Despite the importance of these mutualistic relationships, the physiology and ecology of Symbiodinium spp. remain poorly characterized. Here, we report that rDNA internal transcribed spacer region 2 (ITS2) defined Symbiodinium type B2 associates with the cnidarian hosts Astrangia poculata and Oculina arbuscula from northerly habitats of the western Atlantic. Using pulse‐amplitude‐modulated (PAM) fluorometry, we compared maximum photochemical efficiency of PSII of type B2 to that of common tropical Symbiodinium lineages (types A3, B1, and C2) under cold‐stress conditions. Symbiont cultures were gradually cooled from 26°C to 10°C to simulate seasonal temperature declines. Cold stress decreased the maximum photochemical efficiency of PSII and likely the photosynthetic potential for all Symbiodinium clades tested. Cultures were then maintained at 10°C for a 2‐week period and gradually returned to initial conditions. Subsequent to low temperature stress, only type B2 displayed rapid and full recovery of PSII photochemical efficiency, whereas other symbiont phylotypes remained nonfunctional. These findings indicate that the distribution and abundance of Symbiodinium spp., and by extension their cnidarian hosts, in temperate climates correspond significantly with the photosynthetic cold tolerance of these symbiotic algae.

Photoinhibition, exacerbated by elevated temperatures, underlies coral bleaching, but sensitivity to photosynthetic loss differs among various phylotypes of Symbiodinium , their dinoflagellate symbionts. Symbiodinium is a common symbiont in many cnidarian species including corals, jellyfish, anemones, and giant clams. Here, we provide evidence that most members of clade A Symbiodinium , but not clades B–D or F, exhibit enhanced capabilities for alternative photosynthetic electron-transport pathways including cyclic electron transport (CET). Unlike other clades, clade A Symbiodinium also undergo pronounced light-induced dissociation of antenna complexes from photosystem II (PSII) reaction centers. We propose these attributes promote survival of most cnidarians with clade A symbionts at high light intensities and confer resistance to bleaching conditions that conspicuously impact deeper dwelling corals that harbor non-clade A Symbiodinium .

Genotypic characterization of Symbiodinium symbionts in hard corals has routinely involved coring, or the removal of branches or a piece of the coral colony. These methods can potentially underestimate the complexity of the Symbiodinium community structure and may produce lesions. This study demonstrates that microscale sampling of individual coral polyps provided sufficient DNA for identifying zooxanthellae clades by RFLP analyses, and subclades through the use of PCR amplification of the ITS-2 region of rDNA and denaturing-gradient gel electrophoresis. Using this technique it was possible to detect distinct ITS-2 types of Symbiodinium from two or three adjacent coral polyps. These methods can be used to intensely sample coral-symbiont population/communities while causing minimal damage. The effectiveness and fine scale capabilities of these methods were demonstrated by sampling and identifying phylotypes of Symbiodinium clades A, B, and C that co-reside within a single Montastraea faveolata colony.

The reproductive mode of corals largely determines how zooxanthellae (Symbiodinium spp.) are acquired. Typically, broadcast spawning corals obtain symbionts from the surrounding environment, whereas most brooders transfer symbionts from maternal parent to offspring. Brooding corals are therefore predicted to harbor stable communities of Symbiodinium. This study documents the associations between Symbiodinium spp. and brooding corals in response to seasonal environmental fluctuations. Between March 2002 and December 2005, endosymbiont identity was determined seasonally from replicate colonies (n = 6) of three brooding species, Agaricia agaricites, Porites astreoides and Siderastrea radians, from shallow environments (1–4 m) of the Florida Keys and Bahamas. Symbionts were identified via denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer 2 (ITS2) region. No change was detected in the Symbiodinium communities harbored within these brooding colonies. Additionally, no change in symbiosis was observed through a moderate bleaching event, thereby demonstrating that some bleached corals recover without changing symbionts.

The ability to acquire different types of the symbiotic dinoflagellate Symbiodinium (zooxanthellae) from the environment was investigated using aposymbiotic scyphistomae of the jellyfish Cassiopea xamachana. Non-symbiotic scyphistomae were placed on an offshore Florida patch reef and in Florida Bay during 3- and 5-day periods in March, and 5-day exposures in May, August and December of 2003. Scyphistomae were maintained in culture for several months, after which members of clades A, B, C and D Symbiodinium were detected in these hosts by denaturing gradient gel electrophoresis (DGGE) analyses. These findings contrast with naturally collected C. xamachana medusa from Florida Bay where all specimens possessed only Symbiodinium type A1. Furthermore, the polyps did not acquire the symbionts found in nearby cnidarian colonies, suggesting that a diverse pool of symbiont lineages exists in the environment. These results support previous laboratory studies where aposymbiotic hosts were initially non-selective and capable of acquiring many kinds of Symbiodinium. The specificity seen in adult hosts is likely a result of post-infection processes due to competitive exclusion or other mechanisms. A higher percentage of polyps became infected after 5 days of exposure, compared to 3 days, and no infections were observed in laboratory controls held in filtered seawater. Infections were lowest (50% at both sites) in March of 2003, when seawater temperatures were at their annual minima. Infection was 100% in scyphistomae exposed for 5 days during the months of May, August and December of 2003. These findings suggest that this host system, in addition to addressing questions of host-symbiont selectivity, can be employed to monitor and define the abundance and distribution of natural pools of Symbiodinium. (c) 2006 Elsevier B.V. All rights reserved.

This report documents the extent to which coral colonies show fluctuations in their associations with different endosymbiotic dinoflagellates. The genetic identity of Symbiodinium from six coral species [Acropora palmata (Lamarck), A. cervicornis (Lamarck), Siderastrea siderea (Ellis and Solander), Montastrea faveolata (Ellis and Solander), M. annularis (Ellis and Solander), and M. franksi (Gregory)] was examined seasonally over five years (1998 and 2000–2004) in the Bahamas and Florida Keys at shallow (1 to 4 m) fore-reef/patch reef sites and at deeper fore-reef (12–15 m) locations. Symbionts were identified genetically using denaturing gradient gel electrophoresis (DGGE) fingerprinting of the internal transcribed spacer region 2 (ITS2) of ribosomal RNA gene loci. Repetitive sampling from most labeled colonies from the Bahamas and the Florida Keys showed little to no change in their dominant symbiont. In contrast, certain colonies of M. annularis and M. franksi from the Florida Keys exhibited shifts in their associations attributed to recovery from the stresses of the 1997–1998 El Niño southern oscillation (ENSO) event. Over several years, a putatively stress-tolerant clade D type of Symbiodinium was progressively replaced in these colonies by symbionts typically found in M. annularis and M. franksi in Florida and at other Caribbean locations. Greater environmental fluctuations in Florida may explain the observed changes among some of the symbioses. Furthermore, symbiotic associations were more heterogeneous at shallow sites, relative to deep sites. The exposure to greater environmental variability near the surface may explain the higher symbiont diversity found within and between host colonies.