The production, purification and crystallization of a pocilloporin pigment from a reef-forming coral

Reef-building corals contain fluorescent pigments termed pocilloporins that function by regulating the light environment of coral and acting as a photoprotectant in excessive sunlight. These pocilloporins are related to the monomeric green fluorescent protein and the tetrameric DsRed fluorescent proteins, which have widespread use as biotechnological tools. An intensely blue-coloured pocilloporin, termed Rtms5, was expressed in Escherichia coli, purified and crystallized. Rtms5 was shown to be tetrameric, with deep blue crystals that diffract to 2.2 Angstrom resolution and belong to space group I4(1)22. The colour of this pocilloporin was observed to be sensitive to pH and a yellow (pH 3.5) and a red form (pH 4.5) of Rtms5 were also crystallized. These crystals belong to space group P4(2)22 and diffract to 2.4 Angstrom resolution or better.

Nitrogen ecophysiology of Heron Island, a subtropical coral cay of the Great Barrier Reef, Australia

Coral cays form part of the Australian Great Barrier Reef. Coral cays with high densities of seabirds are areas of extreme nitrogen (N) enrichment with deposition rates of up to 1000 kg N ha(-1) y(-1). The ways in which N sources are utilised by coral cay plants, N is distributed within the cay, and whether or not seabird-derived N moves from cay to surrounding marine environments were investigated. We used N metabolite analysis, N-15 labelling and N-15 natural abundance (delta(15)N) techniques. Deposited guano-derived uric acid is hydrolysed to ammonium (NH4+) and gaseous ammonia (NH3). Ammonium undergoes nitrification, and nitrate (NO3-) and NH4+ were the main forms of soluble N in the soil. Plants from seabird rookeries have a high capacity to take up and assimilate NH4+, are able to metabolise uric acid, but have low rates of NO3- uptake and assimilation. We concluded that NH4+ is the principal source of N for plants growing at seabird rookeries, and that the presence of NH4+ in soil and gaseous NH3 in the atmosphere inhibits assimilation of NO3-, although NO3- is taken up and stored. Seabird guano, Pisonia forest soil and vegetation were similarly enriched in N-15 suggesting that the isotopic enrichment of guano (delta(15)N 9.9parts per thousand) carries through the forest ecosystem. Soil and plants from woodland and beach environments had lower delta(15)N (average 6.5parts per thousand) indicating a lower contribution of bird-derived N to the N nutrition of plants at these sites. The aquifer under the cay receives seabird-derived N leached from the cay and has high concentrations of N-15-enriched NO3- (delta(15)N 7.9parts per thousand). Macroalgae from reefs with and without seabirds had similar delta(15)N values of 2.0-3.9parts per thousand suggesting that reef macroalgae do not utilise N-15-enriched seabird-derived N as a main source of N. At a site beyond the Heron Reef Crest, macroalgae had elevated delta(15)N of 5.2parts per thousand, possibly indicating that there are locations where macroalgae access isotopically enriched aquifer-derived N. Nitrogen relations of Heron Island vegetation are compared with other reef islands and a conceptual model is presented.

Benthic microalgae in coral reef sediments of the southern Great Barrier Reef, Australia

The abundance and productivity of benthic microalgae in coral reef sediments are poorly known compared with other, more conspicuous (e.g. coral zooxanthellae, macroalgae) primary producers of coral reef habitats. A survey of the distribution, biomass, and productivity of benthic microalgae on a platform reef flat and in a cross-shelf transect in the southern Great Barrier Reef indicated that benthic microalgae are ubiquitous, abundant (up to 995.0 mg chlorophyll (chl) a m(-2)), and productive (up to 110 mg O-2 m(-2) h(-1)) components of the reef ecosystem. Concentrations of benthic microalgae, expressed as chlorophyll a per surface area, were approximately 100-fold greater than the integrated water column concentrations of microalgae throughout the region. Benthic microalgal biomass was greater on the shallow water platform reef than in the deeper waters of the cross-shelf transect. In both areas the benthic microalgal communities had a similar composition, dominated by pennate diatoms, dinoflagellates, and cyanobacteria. Benthic microalgal populations were potentially nutrient-limited, based on responses to nitrogen and phosphorus enrichments in short-term (7-day) microcosm experiments. Benthic microalgal productivity, measured by O-2 evolution, indicated productive communities responsive to light and nutrient availability. The benthic microalgal concentrations observed (92-995 mg chl a m(-2)) were high relative to other reports, particularly compared with temperate regions. This abundance of productive plants in both reef and shelf sediments in the southern Great Barrier Reef suggests that benthic microalgae are key components of coral reef ecosystems.

Metamorphosis of a scleractinian coral in response to microbial biofilms

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.

Coral reefs in a century of rapid environmental change

Coral reefs are the most diverse marine ecosystem and embrace possibly millions of plant, animal and protist species. Mutualistic symbioses are a fundamental feature of coral reefs that have been used to explain their structure, biodiversity and existence. Complex inter-relationships between hosts, habitats and symbionts belie closely coupled nutrient and community dynamics that create the circumstances for something from nothing (or the oasis in a nutrient desert). The flip side of these dynamics is a close dependency between species, which results in a series of non-linear relationships as conditions change. These responses are being highlighted as anthropogenic influences increase across the world's tropical and subtropical coastlines. Caribbean as well as Indo-Pacific coral populations are now in a serious decline in many parts of the world. This has resulted in a significant reorganization of how coral reef ecosystems function. Among the spectra of changes brought about by humans is rapid climate change. Mass coral bleaching - the loss of the dinoflagellate symbionts from reef-building corals - and mortality has affected the world's coral reefs with increasing frequency and intensity since the late 1970s. Mass bleaching events, which often cover thousands of square kilometres of coral reefs, are triggered by small increases (+1-3degreesC) in water temperature. These increases in sea temperature are often seen during warm phase weather conditions (e.g. ENSO) and are increasing in size and magnitude. The loss of living coral cover (e.g. 16% globally in 1998, an exceptionally warm year) is resulting in an as yet unspecified reduction in the abundance of a myriad of other species. Projections from general circulation models (GCM) used to project changes in global temperature indicate that conditions even under the mildest greenhouse gas emission scenarios may exceed the thermal tolerances of most reef-building coral communities. Research must now explore key issues such as the extent to which the thermal tolerances of corals and their symbionts are dynamic if bleaching and disease are linked; how the loss of high densities of reef-building coral will affect other dependent species; and, how the loss of coral populations will affect the millions of people globally who depend on coral reefs for their daily survival.

Cell death and degeneration in the symbiotic dinoflagellates of the coral Stylophora pistillata during bleaching

Rising sea temperatures are increasing the incidences of mass coral bleaching (the dissociation of the coral-algal symbiosis) and coral mortality. In this study, the effects of bleaching (induced by elevated light and temperature) on the condition of symbiotic dinoflagellates (Symbiodinium sp.) within the tissue of the hard coral Stylophora pistillata (Esper) were assessed using a suite of techniques. Bleaching of S. pistillata was accompanied by declines in the maximum potential quantum yield of photosynthesis (F-v/F-m, measured using pulse amplitude modulated [PAM] fluorometry), an increase in the number of Sytox-green-stained algae (indicating compromised algal membrane integrity and cell death), an increase in 2',7'-dichlorodihydrofluroscein diacetate (H(2)DCFDA)stained algae (indicating increased oxidative stress), as well as ultrastructural changes (vacuolisation, losses of chlorophyll, and an increase in accumulation bodies). Algae expelled from S. pistillata exhibited a complete disorganisation of cellular contents; expelled cells contained only amorphous material. In situ samples taken during a natural mass coral bleaching event on the Great Barrier Reef in February 2002 also revealed a high number of Sytox-labelled algae cells in symbio. Dinoflagellate degeneration during bleaching seems to be similar to the changes resulting from senescence-phase cell death in cultured algae. These data support a role for oxidative stress in the mechanism of coral bleaching and highlight the importance of algal degeneration during the bleaching of a reef coral.

Effect of early marine diagenesis on coral reconstructions of surface-ocean 13C/12C and carbonate saturation state

Recent research suggests that future decreases in the carbonate saturation state of surface seawater associated with the projected build-up of atmospheric CO2 could cause a global decline in coral reef-building capacity. Whether significant reductions in coral calcification are underway is a matter of considerable debate. Multicentury records of skeletal calcification extracted from massive corals have the potential to reconstruct the progressive effect of anthropogenic changes in carbonate saturation on coral reefs. However, early marine aragonite cements are commonly precipitated from pore waters in the basal portions of massive coral skeletons and, if undetected, could result in apparent nonlinear reductions in coral calcification toward the present. To address this issue, we present records of coral skeletal density, extension rate, calcification rate, δ13C, and δ18O for well preserved and diagenetically altered coral cores spanning ∼1830-1994 A.D. at Ningaloo Reef Marine Park, Western Australia. The record for the pristine coral shows no significant decrease in skeletal density or δ13C indicative of anthropogenic changes in carbonate saturation state or δ13C of surface seawater (oceanic Suess effect). In contrast, progressive addition of early marine inorganic aragonite toward the base of the altered coral produces an apparent ∼25% decrease in skeletal density toward the present, which misleadingly matches the nonlinear twentieth century decrease in coral calcification predicted by recent modeling and experimental studies. In addition, the diagenetic aragonite is enriched in 13C, relative to coral aragonite, resulting in a nonlinear decrease in δ13C toward the present that mimics the decrease in δ13C expected from the oceanic Suess effect. Taken together, these diagenetic changes in skeletal density and δ13C could be misinterpreted to reflect changes in surface-ocean carbonate saturation state driven by the twentieth century build-up of atmospheric CO2. Copyright 2004 by the American Geophysical Union.

The ecological significance of the combtoothed blenny in a coral reef ecosystem

At Heron Island reef, Great Barrier Reef Australia, biomass densities and mean wet mass of Ward's damselfish Pomacentrus wardi and the jewelled blenny Salarias fasciatus were not significantly different at 2-37 v. 2-95 g m(-2) and 8-7 v. 7-9 g, respectively. Whereas S. fasciatus significantly exceeded P. wardi in (1) total number of bites per day (3427 v. 1155), (2) the mass of epilithic algal community consumed per bite (2-19 1,. 0-14mg) and (3) total organic carbon consumed per day (487-31 v. 35-46 mg C m(-2) day(-1)). Territorial behaviour differed also between the two species. Pomacentrus wardi chased from their territories a smaller proportion of blennies than roving grazers (i.e. scarids, acanthurids, siganids and pomacentrids) relative to S. fasciatus. Salarias fasciatus chased c. 90% of other blennies from their territories, while chasing only c. 20% of all damsels that entered. Both P. wardi and S. fasciatus rarely chased non-grazers. The chasing behaviour of S. fascialus was size dependent, with resident fish chasing only individuals of its own family (i.e. Blenniidae) that were the same or smaller size. Pomacentrus wardi may have tolerated S. fasciatus grazing within its territory, as it contributes to territory defence from other blennies. The possibility that the interaction between the two species is facilitative, rather than competitive, is discussed. It was concluded that salariine blennies play an important, and previously underestimated role in coral reef trophodynamics. (C) 2004 The Fisheries Society of the British Isles.

Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.

High-frequency winter cooling and reef coral mortality during the Holocene climatic optimum

A detailed ecological, micro-structural and skeletal Sr/Ca study of a 3.42 m thick Goniopora reef profile from an emerged Holocene reef terrace at the northern South China Sea reveals at least nine abrupt massive Goniopora stress and mortality events occurred in winter during the 7.0-7.5 thousand calendar years before present (cal. ka BP) (within the Holocene climatic optimum). Whilst calculated Sr/Ca-SST (sea surface temperature) maxima during this period are comparable to those in the 1990s, Sr/Ca-SST minima are significantly lower, probably due to stronger winter monsoons. Such generally cooler winters, superimposed by further exceptional winter cooling on inter-annual to decadal scales, may have caused stress and mortality of the corals about every 50 years. Sea level rose by similar to 3.42 m during this period, with present sea-level reached at similar to 7.3 ka BP and a sea-level highstand of at least similar to 1.8 m occurred at similar to 7.0 ka. The results show that it took about 20-25 years for a killed Goniopora coral reef to recover. (C) 2004 Elsevier B.V. All rights reserved.

Endolithic algae photoacclimate to increased irradiance during coral bleaching

The photoacclimation of endolithic algae ( of the genus Ostreobium) inhabiting the skeleton of the Mediterranean coral Oculina patagonica during a bleaching event was examined. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques in situ were used to assess the photosynthetic efficiency of endolithic algae in the coral skeleton and the symbiotic dinoflagellates (zooxanthellae) in the coral tissue. Relative photosynthetic electron transport rates (ETRs) of the endolithic algae under bleached areas of the colony were significantly higher than those of endolithic algae from a healthy section of the colony and those of zooxanthellae isolated from the same section. Endolithic algae under healthy parts of the colony demonstrated an ETRmax of 16.5% that of zooxanthellae from tissue in the same section whereas endolithic algae under bleached sections showed ETRmax values that were 39% of those found for healthy zooxanthellae. The study demonstrates that endolithic algae undergo photoacclimation with increased irradiance reaching the skeleton. As PAM fluorometry has become a major tool for assessing levels of stress and bleaching in corals, the importance of considering the contribution of the endolithic algae to the overall chlorophyll fluorescence measured is highlighted.

Dynamics of a temperature-related coral disease outbreak

During the austral summer of 2001/2002, a coral epizootic occurred almost simultaneously with a bleaching event on the fringing reefs of Magnetic Island (Great Barrier Reef region), Australia. This resulted in a 3- to 4-fold increase in the mean percentage of partial mortality rate in a population of the hard coral Montipora aequituberculata. The putative disease state, ‘atramentous necrosis’, was observed on both bleached and normally-pigmented M. aequituberculata, and presented blackened lesions that spread within days across the colony surface and throughout the population. Diseased portions of the corals were only visible for 3 to 4 wk, with diseased tissues becoming covered in sediment and algae, which rapidly obscured evidence of the outbreak. Diseased colonies were again observed in the summer of 2002/2003 after being absent over the 2002 winter. Analysis of when diseased and bleached corals were first observed, and when and where the mortality occurred on individual colonies, indicated virtually all the mortality over the summer could be attributed to the disease and not to the bleaching. Fluorescence in situ hybridisation (FISH) techniques and cloning, and analysis of the 16S rRNA genes from diseased coral tissue, identified a mixed microbial assemblage in the diseased tissues particularly within the Alphaproteobacteria, Firmicutes and Bacteroidetes. While it is not possible in this study to distinguish between a disease-causing microbial community versus secondary invaders, the bacterial 16S rDNA sequences identified within the blackened lesions demonstrated high similarity to sequences from black band disease and white plague infected corals, suggesting either common aetiological agents or development of a bacterial community that is specific to degrading coral tissues. Temperature-induced coral disease outbreaks, with the potential for elevated levels of mortality, may represent an added problem for corals during the warmer summer months and an added dimension to predicted increases in water temperature from climate change.

Use of X-radiographs to distinguish members of the Montastraea annularis reef-coral species complex

Recent work suggests the Montastraea annularis species complex consists of at least three species, which can be distinguished qualitatively in the field using features related to colony growth (e.g. overall growth form. bumpiness, growth along the colony edge). However, when whole colonies are not available and surfaces are eroded, identification becomes problematic when relying on such characteristics. Characters based on internal skeletal structures are less prone to loss due to taphonomic processes. Previous work has shown that internal corallite architectural features measured in transverse thin sections can be used to distinguish species. To determine whether internal colony-level features measured on X-radiographs can be used. eight characters related to corallite budding and accretionary growth were measured on specimens representing three modern members of the M. annularis species complex (M. annularis, M. flaveolata and M. franksi), as well as two fossil forms (columnar and organ-pipe). All eight characters showed significant differences among species. Discriminant function analysis using seven of these characters resulted in distinct species groupings In canonical scores plots and a 100% classification success for specimens from Panama. These results suggest that measurements made on X-radiographs provide a useful tool for quantitatively distinguishing members of the M. annularis complex as well as between other massive reef corals.

Overlapping species boundaries and hybridization within the Monastraea "annularis" reef coral complex in the Pleistocene of the Bahama Islands

Recent molecular analyses indicate that many reef coral species belong to hybridizing species complexes or "syngameons." Such complexes consist of numerous genetically distinct-species or lineages, which periodically split and/or fuse as they extend through time. During splitting and fusion, morphologic intermediates form and species overlap. Here we focus on processes associated with lineage fusion, specifically introgressive hybridization, and the recognition of such hybridization in the fossil record. Our approach involves comparing patterns of ecologic and morphologic overlap in genetically characterized modern species with fossil representatives of the same or closely related species. We similarly consider the long-term consequences of past hybridization on the structure of modern-day species boundaries. Our study involves the species complex Montastraea annularis s.l. and is based in the Bahamas, where, unlike other Caribbean locations, two of the three members of the complex today are not genetically distinct. We measured and collected colonies along linear transects across Pleistocene reef terraces of last interglacial age (approximately 125 Ka) on the islands of San Salvador, Andros, and Great Inagua. We performed quantitative ecologic and morphologic analyses of the fossil data, and compared patterns of overlap among species with data from modern localities where species are and are not genetically distinct. Ecologic and morphologic analyses reveal "moderate" overlap (>10%, but statistically significant differences) and sometimes "high" overlap (no statistically significant differences) among Pleistocene growth forms (= "species"). Ecologic analyses show that three species (massive, column, organ-pipe) co-occurred. Although organ-pipes had higher abundances in patch reef environments, columnar and massive species exhibited broad, completely overlapping distributions and had abundances that were not related to reef environment. For morphometric analyses, we used multivariate discriminant analysis on landmark data and linear measurements. The results show that columnar species overlap "moderately" with organ-pipe and massive species. Comparisons with genetically characterized colonies from Panama show that the Pleistocene Bahamas species have intermediate morphologies, and that the observed "moderate" overlap differs from the morphologic separation among the three modern species. In contrast, massive and columnar species from the Pleistocene of the Dominican Republic comprise distinct morphologic clusters, similar to the modern species; organ-pipe species exhibit "low" overlap (