Highly repeated DNA sequences in the scleractinian coral genusAcropora: Evaluation of cloned repeats as taxonomic probes

A plasmid clone containing highly repeated DNA sequence fromAcropora formosa was used to probe slot blots of genomic DNA and Southern blots of Taq I digests from other Acroporidae. Homologous repeated DNA sequences were detected in 12 otherAcropora species, but not in three other species of Acroporidae (Astreopora sp.,Montipora digitata andM. aequituberculata). Slot-blot experiments indicated that the repeated sequences inAcropora latistella, A. tenuis, A. longicyathus andA. nobilis were distantly homologous with the clonedA. formosa repeat, whereas those inA. pulchra, A. millepora, A. valida, A. hyacinthus andA. microphthalma were highly homologous with this probe and those inA. digitifera were intermediate. Relatedness of the highhomology group toA. formosa was assessed by comparison of Taq I-digestion patterns. The predominant repeats inA. pulchra andA. hyacinthus had two Taq I sites per repeat unit (as didA. formosa), whereas repeats inA. digitifera, A. valida andA. microphthalma had one Taq I site per repeat; the pattern given byA. millepora implied that its highly homologous repeat units contained either one or two Taq I sites. Within the high-homology group, decreasing number of Taq I sites implies increasing taxonomic distance fromA. formosa. The relatedness series implied by these data differs from that based on morphological criteria.     

Nucleotide sequencing of highly repetitive DNA from seven species in the coral genusAcropora (Cnidaria: Scleractinia) implies a division contrary to morphological criteria

Nucleotide sequences have been determined for 31 homologous 118 base-pair highly repeated DNA sequences from seven species ofAcropora. A matrix was constructed from the sequence data and subjected to phylogenetic analysis using heuristic search routines in the PAUP (phylogenetic analysis using parsimony) program, Version 3.0L. These analyses confirm a close relationship between two species of one subgeneric group (A. pulchra andA. millepora), but identify a division in a group of six species which is contrary to taxonomic groupings based on morphological criteria.     

Simulating light-saturation curves for photosynthesis and calcification by reef-building corals

Light-saturation curves for photosynthesis by reef-building corals have previously been simulated by three functions: the right rectangular hyperbola, a simple exponential function, and the hyperbolic tangent function. Studies of photosynthesis by other organisms have also frequently considered the application of a rectilinear function. This communication analyzes lightsaturation curves for photosynthesis by the Atlantic rose coral Manicina aerolata, the Atlantic staghorn coral Acropora cervicornis, and the Pacific staghorn coral A. formosa. It also analyzes light-saturation curves for calcification by A. cervicornis and A. formosa. This communication demonstrates that the two most accurate functions (as measured by coefficients of determination) are the simple exponential function and the hyperbolic tangent function. The hyperbolic tangent function is preferred because parameter estimates obtained with this function have narrower confidence intervals than those obtained through the application of the simple exponential function. The hyperbolic tangent function can also be used successfully to simulate light-saturation curves for light-enhanced calcification.     

NADP+-dependent glutamate dehydrogenase from Acropora formosa: purification and properties

As an initial step in our study of nitrogen metabolism in the coral/algal symbiosis we have purified glutamate dehydrogenase (EC 1.4.1.4) to homogeneity from polyp tissue of the staghorn coral Acropora formosa collected from Magnetic Island (North Queensland) in 1985–1986. The purified enzyme had a specific activity of 78 U mg^-1. The native enzyme had a relative molecular weight, M _r, of 360 000 (±20 000), and appears to be a hexamer with subunits of M _r=56000 (±3 000). Like the enzyme from other coelenterates, the coral glutamate dehydrogenase (GDH) was absolutely specific with respect to the coenzyme substrate (NADP⁺/NADPH), and was insensitive to allosteric regulation by nucleotides; unlike other coelenterate GDHs, the coral enzyme was absorlutely specific for ammonium as amino group donor in the reductive amination reaction, and major differences in kinetic properties were apparent. Linear Michaelis-Menten kinetics were observed for the substrates a-ketoglutarate, NADPH and NADP⁺, the K _m values being 0.93, 0.11 and 0.03 mM, respectively. However glutamate dehydrogenase displayed biphasic kinetics with respect to l-glutamate and ammonium, indicating two apparent K _m values (18 and 81 mM for l-glutamate and 9.2 and 416 mM for ammonium). The enzyme also exhibits Scatchard plots, Hill coefficients and cooperativity indices characteristic of enzymes displaying negative cooperativity.     

Structural investigations on the mucus from six species of coral

The chemical composition of the mucus from three hard corals (Acropora formosa, Pachyseris speciosa and Fungia fungites) and three soft corals (Sarcophyton sp., Lemnalia sp., and Cespitularia sp.) collected on the Great Barrier Reef (1982–1985) was determined. Significant variation exists in the composition and structure of the six mucus samples, indicating the absence of a common structure for coral mucus. In all cases protein and/or carbohydrate polymers are the major components of the mucus, and lipids are present only in small amounts. The glycose composition varied between species, with fucose (F. fungites and Lemnalia sp.), arabinose (A. formosa), galactose (P. speciosa) and N-acetyl glucosamine (Sarcophyton sp.) being present in high concentrations. With the exception of Sarcophyton sp. and Lemnalia sp., all mucus samples were acidic and contained significant sulphate but no uronic or sialic acids. The amino acid composition of the mucus samples was not unusual, apart from A. formosa, which contained a high percentage of serine and threonine, and F. fungites, which had high levels of glutamic acid present.     

Characterization of two glutamate dehydrogenases from the symbiotic microalga Symbiodinium microadriaticum isolated from the coral Acropora formosa

In view of their possible involvement in ammonium assimilation in the coral/algal symbiosis, we have purified two distinct glutamate dehydrogenase isoenzymes from the symbiotic dinoflagellate Symbiodinium microadriaticum (Freudenthal) extracted from the staghorn coral Acropora formosa collected from Magnetic Island, North Queensland, Australia, in 1986–1987. An NADPH-specific glutamate dehydrogenase (GDH) displayed biphasic kinetics with respect to ammonium as the variable substrate; at low substrate concentrations the apparent K _m was below 1 mM, whereas at high substrate concentrations the corresponding value was approximately 200 mM. The NADPH-GDH displayed extremely low activity in the direction of glutamate oxidation; together with the kinetic data this suggests a probable role in ammonium assimilation. A second (NADH-specific) GDH was found to have both amination and deamination activities, and presumably functions in vivo in glutamate oxidation. Kinetic constants are reported for both GDH isoenzymes.     

Why does the white tip of stony coral grow so fast without zooxanthellae?

The photosynthesis of zooxanthellae in a coral polyp greatly enchances the calcification rate of a coral. However, the white tip of a coral branch is free of zooxanthellae yet still has a very high calcification rate. Furthermore, the reason for the difference is not clear. In this study, the amount of photopigment, total protein (TP), total organic carbon (TOC), ATP, and lipid in polyps from the white tip and brown stalk of a branch of stony coral were measured. Samples of Acropora hyacinthus and A. formosa were collected from southern Taiwan between 1985 and 1987. The results showed that the ATP concentration in polyps of the white tip was much higher than that in polyps of the brown stalk. Conversely, the amount of TP, TOC and measured lipids in polyps of the brown stalk were all higher than those of the white tip. It was the high concentration of ATP in cells that gave these polyp tips the vitality to sustain the energy requirements of such a rapid calification rate. Facilitated diffusion, due to the high metabolite gradient created by cell activity, could be the major driving force for the transport of photosynthetic product from stalk to tip.     

Predominance of clade D <Emphasis Type="Italic">Symbiodinium</Emphasis> in shallow-water reef-building corals off Kish and Larak Islands (Persian Gulf,Iran)

Scleractinian coral species harbour communities of photosynthetic taxa of the genus Symbiodinium. As many as eight genetic clades (A, B, C, D, E, F, G and H) of Symbiodinium have been discovered using molecular biology. These clades may differ from each other in their physiology, and thus influence the ecological distribution and resilience of their host corals to environmental stresses. Corals of the Persian Gulf are normally subject to extreme environmental conditions including high salinity and seasonal variation in temperature. This study is the first to use molecular techniques to identify the Symbiodinium of the Iranian coral reefs to the level of phylogenetic clades. Samples of eight coral species were collected at two different depths from the eastern part of Kish Island in the northern Persian Gulf, and Larak Island in the Strait of Hormuz. Partial 28S nuclear ribosomal (nr) DNA of Symbiodinium (D1/D2 domains) were amplified by polymerase chain reaction (PCR). PCR products were analyzed using single stranded conformational polymorphism and phylogenetic analyses of the LSU DNA sequences from a subset of the samples. The results showed that Symbiodinium populations were generally uniform among and within the populations of eight coral species studied, and there are at least two clades of Symbiodinium from Kish and Larak islands. Clade D was detected from eight of the coral species while clade C was found in two of species only (one species hosted two clades simultaneously). The dominance of clade D might be explained by high temperatures or the extreme temperature variation, typical of the Persian Gulf. Publication of this article was held up owing to technical problems. The publisher apologizes sincerely for this lengthy delay.     

Assessing stress response of Stylophora pistillata towards oil and phosphate pollution in the Gulf of Aqaba,using molecular and biochemical markers

Crude oil (from oil terminal) and raw phosphate (from phosphate port) pollution are responsible for the lowered health conditions of coral reefs at their vicinity in the Jordanian coast of the Gulf of Aqaba. Both in situ incubations and ex situ laboratory exposure experiments were used to study the effects of those pollutants on corals, by using molecular and biochemical biomarkers in the coral Stylophora pistillata. For ex situ part of the experiment, crude oil and raw phosphate were added to a final concentration of 500?ppm for both pollutants. The DNA damage was assessed by Comet assay, while biochemical stress markers were reassessed by lipid peroxidation (LPO) test. Although the corals looked healthy from outside, the use of stress biomarkers indicated that they are under high pressure at the cellular level. The corals incubated with oil and phosphate had more DNA damage and LPO in comparison with the control samples. The results obtained suggest that the use of stress biomarkers can be used as important prognostic tools for examining the sub-lethal stress on corals before their death.     

Genetic structure of the subtidal red alga Delisea pulchra

We used random amplified polymorphic DNA (RAPDs) to examine small-scale spatial genetic structure in the red alga Delisea pulchra (Greville) Montagne at two locations near Sydney, Australia. We examined genetic structure among plants at four spatial scales ranging from 2 km apart down to <50 cm apart between locations, among sites within locations, among quadrats within sites, and among plants within quadrats. Haploid stages of D. pulchra were absent from the populations studied, suggesting that they are maintained through asexual reproduction of diploid plants. Consistent with this, we found that 19 RAPD phenotypes scored in this study had multiple individuals, indicating the presence of clones in these populations. However, there were no RAPD phenotypes common to two locations separated by only 2 km. Analysis of molecular variance revealed that strong genetic differences occurred between plants from these two locations, with 46.3% of the total genetic variation occurring at this scale, most probably reflecting limited gene flow. Within each location, <25% of the genetic variation was attributable to differences among sites or quadrats, indicating gene flow at those smaller scales. Most of the variation within each location occurred at the smallest spatial scale, among plants within 0.25 m² quadrats. Nonetheless, some pairwise genetic distances (φST) between sites or quadrats within locations were large, indicating some genetic divergence on smaller scales. Genetic distance was independent of spatial distance within both locations, suggesting that fine-scale differences within locations were most probably caused by variation in fine-scale patterns of water movement or fine-scale natural selection. We assessed the impact of one potential selective agent, grazing sea urchins, on the fine-scale genetic structure of D. pulchra. There was no evidence that grazing by sea urchins affected the genetic structure of D. pulchra. In combination with demographic data, our results indicated that local populations of D. pulchra within locations were relatively open and that fine-scale genetic structure was probably constrained by gene flow. At the larger scale however, strong genetic differentiation indicated little gene flow between locations and restricted dispersal of spores. Received: 22 April 1999 / Accepted: 29 November 1999     

Growth of the staghorn coral Acropora formosa at Houtman Abrolhos, Western Australia

The growth (extension rate, number of radial branches, skeletal mass, branch diameter) of the␣staghorn coral Acropora formosa (Dana, 1846) was examined at four sites on the Beacon Island platform at Houtman Abrolhos, in subtropical Western Australia (28°S). Sites were at depths of 7 to 11 m, with variable exposure to weather and swell conditions. Two sites on the western reef slope were partly exposed to the oceanic swell, and two sites in the lagoon were largely protected from wave action. Linear extension rate between 1994 and 1995 varied significantly between sites, with greater linear extension at the more protected lagoonal sites. However, accumulation of skeletal mass per branch and number of newly initiated radial branches did not vary significantly between the sites. Carbonate was deposited in similar amounts, but either as porous, rapidly extending branches, or as denser branches which extended more slowly. Branch extension rate over 11.5 mo ranged from a mean of 50.3 mm (range=13 to 93 mm) at a reef slope site to a mean of 76.0 mm (range=31 to 115 mm) at a sheltered lagoonal site. Mean extension rates were almost twice that previously reported for this species in Houtman Abrolhos (37 to 43 mm yr⁻¹) from a shallower site where environmental conditions were apparently sub-optimal. Growth was within the range reported for A. formosa from tropical sites, which is consistent with the relatively high calcification and reef-accretion rates recorded for Houtman Abrolhos in geological and metabolic studies. The role of reduced coral growth-rate in limiting coral reef formation at high latitudes remains equivocal. Received: 19 November 1997 / Accepted: 5 May 1998     

DNA damage induced by ultraviolet radiation in coral-reef microbial communities

Ultraviolet radiation (UVR) has been implicated in coral-bleaching processes and UVR may create stress to marine organisms by damage to DNA. Absorption of energy from UVB (280 to 320 nm) induces direct damage to DNA via cyclobutane pyrimidine dimer photoproduct-formation. We examined the extent of DNA damage created by UVR in coral reef microbial communities and whether the coral-surface microlayer (CSM) provides protection from UVR to the microorganisms found there. Diel patterns and depth profiles of UVR effects were examined in coral mucus (coral-surface microlayer, CSM) from Montastraea faveolata and Colpophyllia natans, and water-column samples of similar depths. UV-induced photodamage was determined using a radioimmunoassay specific for cyclobutane pyrimidine dimers (thymine dimers). Significant photodamage was detected in water-column and CSM samples, although the level of damage in CSM samples was consistently lower than in water-column samples collected from the same depth, suggesting the presence of photoprotective mechanisms within the CSM. Diel patterns of photodamage were detected in both water-column and CSM samples, but peak damage occurred earlier in the day for the CSM samples, suggesting differences in damage and repair kinetics between the water column and CSM. The results suggest that microorganisms within the CSM are afforded some protection from UVR stress and that changes in the amount of DNA damage in these organisms may be an indicator of changing UVR stress to corals. Received: 10 January 1997 / Accepted: 15 September 1997     

Photosynthetic carbon dioxide fixation in zooxanthellae

The carbon-fixation patterns of freshly isolated zooxanthellae from the hermatypic coral Acropora formosa were examined during a 15 min exposure to sodium mosa were examined during a 15 min exposure to sodium [¹⁴C]bicarbonate. The labelling pattern during the first 60 s exposure showed that the C₃ carbon-fixation pathway is the major route for photosynthetic carbon fixation in Symbiodinium sp. 3-Phosphoglyceric acid, which constituted >50% of the label after 5 s, steadily decreased over the first 60 s. Hexose phosphates, aspartate, malate and glucose were the other main products during the first 60 s. Over longer periods, significant amounts of the organic acids succinate, aspartate and glutamate were found in the extract along with glucose; but no glycerol.     

Molecular genetic evidence for probable reticulate speciation in the coral genus Madracis from a Caribbean fringing reef slope

For many corals, the existence of morphologically distinct yet sympatric populations/species implies reproductive isolation. Conversely, the presence of many intermediate and overlapping morphologies combined with synchronous, mass spawning suggests incomplete reproductive isolation. In Madracis (Scleractinia: Astrocoeniina: Pocilloporidae), high levels of morphological plasticity among the five most commonly recognized species (M. mirabilis, M. senaria, M. decactis, M. formosa and M. pharensis) on Caribbean reefs led us to question species boundaries. Phylogenetic relationships were investigated at the intra-individual, inter-individual and inter-specific levels using the ITS1-5.8S-ITS2 region (ca. 613 bp) of the ribosomal DNA cistron. Inter-specific divergence was ca. 6%, while intra-individual and intra-specific divergences ranged from 0% to 4.9% and 3.3% to 3.5%, respectively. M. senaria and M. mirabilis formed monophyletic groups. M. formosa, M. decactis and M. pharensis formed a paraphyletic complex. High levels of intra-individual and intra-specific ITS polymorphism in the decactis-formosa-pharensis cluster may be the result of very recent speciation within the clade (i.e. maintenance of ancestral polymorphism and incomplete lineage sorting), or the result of repeated introgressive hybridization among the three taxa. Polymorphism parsimony of 89 sites, including nine that showed additivity, revealed a phylogenetic topology more consistent with inter-taxal hybridization. Results are discussed in terms of weak reproductive barriers, and phylogenetic fission and fusion under Veron's model of reticulate speciation in corals. Ecological studies involving Madracis should consider M. decactis, M. formosa and M. pharensis as a complex.     

The reef environment and competitive success in the Corallimorpharia

Competitive success within coral reef communities is controlled by various factors. In addition to competitive abilities in direct interactions with a contestant, external influences such as disturbance caused by nutrient input may determine the outcome of antagonistic interactions. We examined the competitive success of corallimorpharians on coral reefs by investigating their distribution patterns within reefs and how well they perform in interference competition with staghorn corals in different environments. Substrate composition and corallimorpharian growth were examined on three reefs in Tanzania under different disturbance regimes using the line-intercept transect and point techniques. A transplant experiment was conducted in which staghorn corals (Acropora formosa) were exposed to the polyps of Rhodactis rhodostoma to establish how competition between corals and corallimorpharians affects their respective distributions. Within reefs corallimorpharians seemed to be more competitive in shallow waters. This could be due to both environmental factors as well as varied competitive abilities depending on surrounding benthos that changed with depth. Reef environment also seemed to influence corallimorpharian growth among reefs as they had the highest densities in the areas with the highest nutrient loads. The transplant experiment revealed that the corallimorpharians had a competitive advantage over the corals, and in comparisons of reefs influenced by different degrees of disturbance, corallimorpharians were most competitive in the area with the highest nutrient content. Hence, stress on coral reefs in the form of raised nutrient loads may favour the competitive success of corallimorpharians.Communicated by P.W. Sammarco, Chauvin     

Isolation and cloning of DNA from somatic tissue of soft corals (Cnidaria: Octocorallia)

A method is described for isolating high molecular weight DNA from somatic tissue of soft corals. The method avoids problems associated with the presence of nucleases, pigments and other secondary metabolites in soft corals. Tissue is frozen in liquid nitrogen, pulverized with a pestle and mortar, and the powder extracted with buffered sodium dodecyl sulphate at 4°C in the presence of phenol/chloroform. This technique has been applied toAlcyonium, Sinularia, Sarcophyton andLobophytum species. Repeated sequences have been cloned fromAlcyonium sp., and used to probe slot-blots of genomic DNA and Southern blots of restriction digests. Homologous repeated sequences were detected in threeAlcyonium sp., but not in threeSinularia sp., despite these genera being closely related.     

Phylogenetic relationships of coral-associated gobies (Teleostei,Gobiidae) from the Red Sea based on mitochondrial DNA data

Bryaninops, Gobiodon, Paragobiodon and Pleurosicya are the most abundant genera of coral-associated gobies. These genera are adapted to live among coral, while other small reef gobies (e.g., the genus Eviota) show no obligate association with this living substrate. Thirteen coral-associated species and two Eviota species were sampled from different regions of the Red Sea, along with four populations/species of Gobiodon from the Indian and western Pacific Oceans. A molecular phylogenetic analysis was performed using partial sequences of 12S rRNA, 16S rRNA and cytochrome b mitochondrial genes, 1,199 base pairs in total. Several clades were consistently resolved in neighbor joining-, maximum parsimony-, maximum likelihood and Bayesian analyses. While each of the four genera Gobiodon, Paragobiodon, Bryaninops and Pleurosicya proved to be monophyletic, their relative position in the phylogeny did not support an emergence of coral-associated gobiids as a monophyletic assemblage. Instead, two separate monophyletic sub-groups were discovered, the first comprising Gobiodon and Paragobiodon, and the second Bryaninops and Pleurosicya. Our molecular phylogenetic examinations also revealed one unassigned species of Gobiodon from the Maldives as a distinct species and confirmed three putative and yet unassigned species from the Red Sea. Moreover, the uniformly black colored species of Gobiodon are not monophyletic but have evolved independently within two distinct species groups. Genetic distances were large in particular within Pleurosicya and Eviota. Estimated divergence times suggest that coral-associated gobies have diversified in parallel to their preferred host corals. In particular, divergence times of Gobiodon species closely match those estimated for their typical host coral genus Acropora.     

Phylogenetic analyses of potentially free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> spp. isolated from coral reef sand in Okinawa,Japan

The existence of “free-living” Symbiodinium that can form symbioses with hosts is implied by the presence of hosts that produce Symbiodinium-free gametes and expulsion and/or expelled symbiotic algae from host. However, it is still unclear if potentially symbiotic Symbiodinium are found “free-living” in the coral reef environment. Sixteen Symbiodinium strains were established from samples taken from three sampling locations of coral reef sand in Okinawa, Japan. Phylogenetic analyses of the partial large subunit ribosomal DNA (28S-rDNA) and the internal transcribed spacer of ribosomal DNA (ITS-rDNA) conclusively showed that all 16 isolates belonged to Symbiodinium clade A sensu Rowan and Powers (1991). The lack of other Symbiodinium clades besides clade A in this study may be due to other clades not being readily culturable under culture conditions used here. The new isolates could be phylogenetically divided into four groups, though no sequences were identical to previously reported Symbiodinium. Two of the four groups were closely related to symbiotic Symbiodinium clade A isolated from a variety of host species. One isolate group formed a highly supported monophyly with Symbiodinium types that have previously been characterized as “free-living”. The remaining isolate group, although within clade A, was quite divergent from other clade A Symbiodinium. These results indicate that novel diversity of free-living Symbiodinium exists in coral sand.     

Growth of Acropora pulchra

Transplantation experiments were carried out on the branching hermatypic coral Acropora pulchra (Brook) from October 1980 to March 1982 in the northern Philippines. Primary objectives were to determine the effects on the coral of the stresses associated with transplantation, relocation, and the use of different artificial substrates such as flagstones and tires, and to determine possible effects of sedimentation on survival and growth of the transslants. Results of transplantation at different times of the year were also assessed. Comparisons of the responses of transplants with those of undisturbed, control colonies indicated possible significant effects of transplantation tending to retard growth. Sedimentation likewise appeared to suppress growth of the transplants. Growth and survival of the transplants may have been further inhibited by transplantation during the warmer times of the year, and by setting them in prone positions on tires. Effects of relocation at varying distances from source colonies appeared to be negligible.     

DNA damage in mouse organs and in human sperm cells by bisphenol A

Abstract

The in vivo genotoxic potential of bisphenol A using the comet assay in mice and in human sperm cells in vitro without metabolizing enzymes was studied. Male mice were exposed by oral gavage to the following doses of bisphenol A (0 125, 250 and 500?mg/kg body weight). DNA damage was investigated in liver, kidney, testes, urinary bladder, colon and lungs cells. In testicular cells, a significant increase in DNA strand breaks was observed in the lowest, but not in the medium or highest dose groups. Histopathological investigation of the testicular samples did not show any treatment dose-related effects. No DNA strand breaks were observed in any of the other investigated tissues. In human sperm cells in vitro, bisphenol A did not induce DNA strand breaks.