Preparation of high molecular weight DNA from hermatypic corals and its use for DNA hybridization and cloning

Previous attempts to extract high molecular weight DNA from hermatypic corals have failed because the starting material was mature tissue. The following paper describes the isolation of DNA from coral sperm which is of high molecular weight, and is suitable for DNA hybridization, digestion with restriction endonucleases and cloning. Gross DNA hybridization and hybridization with cloned repetitive DNA was used to investigate relatedness amongst members of the scleractinian coral family Acroporidae. Acropora formosa and A. pulchra were found to share a common (highly homologous) DNA sequence repeat; a related but less homologous sequence was present in A. latistella, and no homologous sequence was detected in Montipora digitata. These results imply a close taxonomic relationship between A. formosa and A. pulchra, and suggest that molecular criteria of this sort will be extremely useful in coral taxonomy.     

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.     

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.     

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.     

Trade-offs associated with dietary specialization in corallivorous butterflyfishes (Chaetodontidae: <Emphasis Type="Italic">Chaetodon</Emphasis>)

Increasing dietary specialization is an inherently risky strategy because it increases a species’ vulnerability to resource depletion. However, risks associated with dietary specialization may be offset by increased performance when feeding on preferred prey. Although rarely demonstrated, highly specialized species are expected to outperform generalists when feeding on their preferred prey, whereas generalists are predicted to have more similar performance across a range of different prey. To test this theory, we compared the growth rates of two obligate coral-feeding butterflyfishes (Chaetodon trifascialis and Chaetodon plebeius) maintained on exclusive diets of preferred vs nonpreferred prey. In the field, C. trifascialis was the most specialized species, feeding almost exclusively on just one coral species, Acropora hyacinthus. C. plebeius meanwhile, was much less specialized, but fed predominantly on Pocillopora damicornis. During growth experiments, C. trifascialis grew fastest when feeding on A. hyacinthus and did not grow at all when feeding on less preferred prey (P. damicornis and Porites cylindrica). C. plebeius performed equally well on both A. hyacinthus and P. damicornis (its preferred prey), but performed poorly when feeding on P. cylindrica. Both butterflyfishes select coral species that maximize juvenile growth, but contrary to expectations, the more specialized species (C. trifascialis) did not outperform the generalist species (C. plebeius) when both consumed their preferred prey. Increased dietary specialization, therefore, appears to be a questionable strategy, as there was no evidence of any increased benefits to offset increases in susceptibility to disturbance.     

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.     

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.     

Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island,northern Great Barrier Reef

This study documented the range of corals, and other prey types, consumed by 20 species of butterflyfishes, which co-occur at Lizard Island, northern Great Barrier Reef, Australia. Six species (Chaetodon aureofasciatus, C. baronessa, C. lunulatus, C. plebius, C. rainfordi and C. trifascialis) fed almost exclusively on scleractinian corals, and a further eight species (C. citrinellus, C. kleinii, C. lunula, C. melannotus, C. rafflesi, C. speculum, C. ulietensis, and C. unimaculatus) took a significant proportion of their bites from corals. The other six species (C. auriga, C. ephippium, C. lineolatus, C. semeion, C. vagabundus, and Chelmon rostratus) rarely consumed coral, but fed on small discrete prey items from non-coral substrates. Coral-feeding butterflyfishes consumed a wide range of corals. Chaetodon lunulatus, for example, consumed 51 coral species from 24 different genera. However, there was up to 72% dietary overlap between coral-feeding butterflyfishes, with 11/14 species feeding predominantly on Acropora hyacinthus or Pocillopora damicornis. The most specialised corallivore, C. trifascialis, took 88% of bites from A. hyacinthus. Chaetodon trifascialis defend territories encompassing one or more colonies of A. hyacinthus, and may have prevented other species such as C. lunulatus from feeding even more extensively on this coral. This study has shown that coexistence of coral-feeding butterflyfishes occurs despite an apparent lack of partitioning of prey resources. While different coral-feeding butterflyfishes were more or less selective in their use of different coral prey, virtually all species fed predominantly on A. hyacinthus or P. damicornis.Electronic Supplementary Material Supplementary material is available for this article at     

Changes of respiration and biomass of spider crab (Hyas araneus) larvae during starvation

The influence of starvation on respiration (R), dry weight (W), carbon (C), nitrogen (N), hydrogen (H), and energy content (E; calculated fromC) of spider crab (Hyas araneus L.) larvae was studied in the laboratory. In all larval stages (zoea I and II, megalopa)W increased during postmoult, independent of food, and decreased subsequently. The final reduction inW after continued starvation increased from stage to stage (9, 13, and 20% respectively), but it was always much lower than the decrease inC (44 to 52%),N (42 to 46%),H (50 to 58%), andE (53 to 62%). Individual (R) and weight-specific respiration rates (QO ₂) were reduced by 83 to 88%. The time-dependence of these reductions in metabolism and biomass as well as the rates of change in all parameters studied were described with non-linear regression models and differential equations, respectively. Rates and total amounts of energy lost during starvation were independently calculated fromC andR values, and similar results were mostly obtained. Only in the megalopa stage was there a conspicuous difference between the two estimates: higher losses were calculated fromR. This shows that further (biochemical) data are required for a more complete understanding of the energetics of this stage. Estimates of total protein (fromN) and lipid (fromC) suggest that both constituents serve as metabolic substrates during starvation, but most of the energy originates from the breakdown of protein.Supported by the Deutsche Forschungsgemeinschaft (An 145/1-1)     

The short-term partitioning of carbon-14 assimilate between zooxanthellae and polyp tissue in Acropora formosa

The usefulness of Fluorinert for the extraction of Acropora formosa polyp tissue and zooxanthellae was demonstrated. The latter remain intact, with no leakage of metabolites, and the polyp tissue can be extracted in a minimal volume. Intact A. formosa and its isolated zooxanthellae were incubated in the light with sodium [¹⁴C]bicarbonate for 5 s to 15 min and the kinetics of carbon-14 fixation was determined. The isolated zooxanthellae showed a linear response for carbon fixation, whilst the zooxanthellae in the intact association showed a lag period of 1 to 2 min, containing only 12% of the total fixed carbon in the first 1 min. After 10 min, the distribution of fixed carbon between the symbiotic partners was approximately even and the total carbon fixed was in a range similar to that fixed by the isolated zooxanthellae. A pulse-chase experiment showed rapid movement of fixed carbon from the polyp tissue to the zooxanthellae after the 30 s pulse. The paper discusses two possible explanations for the observed results.     

The effects of antifoulant-paint-contaminated sediments on coral recruits and branchlets

The 184-m cargo ship "Bunga Teratai Satu" ran aground on Sudbury Reef, within the Great Barrier Reef Marine Park, on 2 November 2000. Although no cargo or fuel was lost, the ship remained aground for 12 days and a large quantity of antifoulant paint containing tributyltin (TBT), zinc, and copper was scraped from the hull during the grounding and subsequent refloating operation. This resulted in extensive contamination of the reef sediments for up to 250 m surrounding the grounding site. Two laboratory-based experiments assessed the impact of contaminated sediments on the survival of both newly settled corals of Acropora microphthalma and branchlets of A. formosa. Newly settled corals exposed to sediments containing 8.0 mg kg^–1 TBT, 72 mg kg^–1 Cu, and 92 mg kg^–1 Zn or greater suffered significantly higher mortality after 72 h, compared to control or low-concentration treatments. Coral recruits exposed to 40 mg kg^–1 TBT (Sn), 306 mg kg^–1 Cu, and 403 mg kg^–1 Zn were all killed within 38 h. Branchlets from adult corals exposed to sediments with a high concentration of contaminants (TBT 160 mg kg^–1, Cu 1,180 mg kg^–1, and Zn 1,570 mg kg^–1) suffered significant mortality (38%), whereas branchlets placed in treatments with lower levels of contaminants suffered no mortality. Visual bleaching of the branchlets was observed at high contaminant levels, but an overall reduction in the symbiotic zooxanthellae populations was not observed in surviving corals. The photosynthetic yields of light-adapted zooxanthellae remained constant in live branchlets, indicating that the TBT-contaminated sediment may be more toxic to the host than the symbiont. Our results show that antifoulant contamination at ship-grounding sites has the potential to cause major mortality of resident coral communities and can have a negative impact on the recovery of adult populations.Communicated by P.W. Sammarco, Chauvin     

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.     

Adaptive host choice and avoidance of superparasitism in the spawning decisions of bitterling (Rhodeus sericeus)

Choice of a site for oviposition can have fitness consequences. We investigated the consequences of female oviposition decisions for offspring survival using the bitterling, Rhodeus sericeus, a freshwater fish that spawns inside living unionid mussels. A field survey of nine bitterling populations in the Czech Republic revealed a significantly lower rate of release of juvenile bitterling from Anodonta cygnea compared to three other mussel species. A field experiment demonstrated that female bitterling show highly significant preferences for spawning in A. anatina, Unio pictorum, and U. tumidus. Within a species, female bitterling avoided mussels containing high numbers of bitterling embryos. Mortality rates of bitterling embryos in mussels were strongly density dependent and the strength of density dependence varied significantly among mussel species. Female preferences for mussels matched survival rates of embryos within mussels and females distributed their eggs among mussels such that embryo mortalities conformed to the predictions of an ideal free distribution model. Thus, female oviposition choice is adaptive and minimizes individual embryo mortality. Received: 6 October 1999 / Received in revised form: 7 January 2000 / Accepted: 13 March 2000     

Comparative electrophysiological analysis of plant odor perception in females of threePapilio species

Summary Antennae of femalePapilio butterflies perceive many volatile plant constituents with widely differing, constituent-specific sensitivities. We compared the responses of threePapilio species to volatiles from host and non-host plants to assess species-specificity and the degree of evolutionary conservatism in olfactory responses.Since previous studies had demonstrated that the polar constituents in odor fromDaucus carota stimulate oviposition behavior inPapilio polyxenes, we collected headspace volatiles fromD. carota, Pastinaca sativa (both Apiaceae) andArtemisia dracunculus (Asteraceae) and separated the polar fraction of these volatiles by gas chromatography. GC-coupled electroantennograms (GC-EAG) were recorded from the speciesPapilio polyxenes, P. machaon hippocrates andP. troilus. In addition, the responses of the three species to five compounds known as generally occurring constituents of plant odor were recorded. The relative sensitivities for these compounds were nearly identical in all threePapilio species. The response spectra to the separated plant volatiles also showed considerable similarities among the species.From the limited set of GC peaks evoking a response in one of the species, 64% (D. carota), 44% (P. sativa) and 29% (A. dracunculus) also evoked a response in both of the other species. The responses of the two closely related Apiaceae feeders (P. polyxenes, P. m. hippocrates) to volatiles fromD. carota were more similar to each other than was either to the response ofP. troilus, which feeds on Lauraceae. However, this was not true for the responses to volatiles fromP. sativa. The least congruence among the three species was found in the responses to volatiles fromA. dracunculus, a non-host for all of them. The differences and similarities found in the response profiles of the threePapilio species are discussed with respect to evolutionary adaptation to host odor versus evolutionary conservatism in adaptation of olfactory receptors.     

Morphological and DNA sequence variation in the kelp Costaria costata (Phaeophyta)

Kelp species show substantial intraspecific morphological variation. The annual kelp, Costaria costata (C. Agardh) Saunders, is found in the lower intertidal and subtidal regions of shores which vary in exposure from strongly wave-exposed to sheltered. Phenotypic variation was studied in this monotypic genus. Individuals of C. costata were collected from two morphologically-distinct populations (one wave-exposed and the other wave-sheltered) at Cape Beale and Stanley Park, Vancouver, Canada, in May 1985. Analysis of morphological variation, using multivariate statistical techniques, indicated significant differentiation between plants from the two sites. Blade widths and thicknesses were the major discriminating variables. Restriction fragment length polymorphism (RFLP) analysis of nuclear DNA with 42 anonymous probes demonstrated one polymorphism. Most of the probes are hypothesized to encode highly repeated, dispersed sequences. These repeat sequence probes comprised 80.9% of all cloned fragments. Primary sequence analysis of 1595 base pairs of small-subunit ribosomal DNA and 204 base pairs of upstream sequence (probe pCc 18) failed to show any divergence between plants from the two sites.     

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.     

Biochemical composition of cephalopods with different life strategies,with special reference to a giant squid,<Emphasis Type="Italic"> Architeuthis</Emphasis> sp.

The present study is a comprehensive comparison of the biochemical composition (protein, lipid, glycogen and cholesterol contents, and amino acid and fatty acid profiles) of 14 species of cephalopods with different life strategies (benthic, nektobenthic, benthopelagic and pelagic) in distinct habitats (neritic, demersal, oceanic and deep sea), with special emphasis placed on a male Architeuthis sp. The giant squid showed a significantly lower protein and total amino acid content in the gonad and digestive gland. The major essential amino acids were leucine, lysine and arginine. The major non-essential amino acids were glutamic acid, aspartic acid and proline. The benthopelagic cirrate octopus Opisthoteuthis sp. showed a lower content of these nitrogen compounds in the muscle. In respect to lipid and fatty acid contents, the giant squid showed the highest values in the gonad and digestive gland and the lowest in the muscle. Most of saturated fatty acid content was presented as 16:0; monounsaturated fatty acid content, as 18:1 and 20:1; and polyunsaturated fatty acid content, as 20:4n-6, 20:5n-3 and 22:6n-3. The highest cholesterol content in the gonad was detected in Opisthoteuthis sp. and Architeuthis sp.; in the digestive gland. in Todarodes sagittatus; and in the muscle, in Sepia elegans. The highest glycogen value in the gonad was detected in Octopus vulgaris; in the digestive gland and muscle, the highest values were attained in Opisthoteuthis sp. The relationships between life strategies and biochemical composition were investigated and principal component analysis (PCA) was performed. The different cephalopod life strategies could be well separated on the basis of the first two principal components. The nektobenthic and pelagic strategies were clearly separated from the benthic, suggesting that these groups of species are characterized by lower lipid content and higher protein content in the gonad. A rather similar life-strategy distinction was obtained for the digestive gland. The benthopelagic strategy was also well separated from benthic and pelagic strategies and from Architeuthis sp. In the muscle, the results indicated lower nitrogen and carbon compound contents in Architeuthis sp. and Opisthoteuthis sp. The environmental conditions that possibly explain the Architeuthis sp. stranding, the relationships between biochemical compositions and the life strategies of the different cephalopod species studied are discussed.Communicated by S.A. Poulet, Roscoff     

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.     

Non-random mating and population genetic subdivision of two broadcasting corals at Ningaloo Reef,Western Australia

Allozyme electrophoresis of two corals was used to assess whether populations at Ningaloo Reef, Western Australia are primarily self-seeding or whether recruitment is from a broader geographic pool. Significant genetic subdivision across a range of spatial scales (between 6.5 km and 155 km) was found for both Acropora digitifera and A. aspera, with mean F _ST values of 0.010 and 0.067 respectively. Large departures from Hardy-Weinberg expectations were found for both species. Without exception these were due to deficits of heterozygotes; mean D values were –0.341 for A. digitifera and –0.455 for A. aspera. The magnitude of the deficits was consistent both across loci for all sites and across all sites for each locus. Some loci were found to be in linkage disequilibrium but no consistent pattern was observed. Also, multi-locus genotypic diversity values were generally high (between 0.83 and 1.00) and so departures from equilibria cannot be attributed to asexual reproduction. The most plausible explanation for the patterns observed is restricted gene flow at both the planktonic and gametic stages, with mating between close relatives.Communicated by G.F. Humphrey, Sydney     

Illumination and the coral community beneath tabular Acropora species

Light profiles beneath the tabular coral species Acropora cytherea and A. hyacinthus were examined at Rib and Broadhurst Reefs, central Great Barrier Reef, in November 1980. They show a strongly decreasing illumination gradient towards the central stem. Beneath tables at 10m deep, which receive about 7.0 mW cm^–2 at solar noon, substrate irradiance falls to a minimum of about 5% ambient or to about 0.4 mW cm^–2. Thus, from previously reported compensation values, most sub-table substrate may receive above-compensation irradiance. Illumination beneath tables screened by foil is significantly lower. The coral communities beneath tables of these two species plus those of A. subulata and A. clathrata support an average 26 colonies m^–2, at a cover of 40%, nearly identical to adjacent, unshaded quadrats. Species richness is likewise very similar. Species richness and colony density beneath the tables increase inwards from the perimeter of the shaded areas, decreasing only near the central stems. Dark adaptation and reduced competitive and grazing pressure are suggested explanations for the latter. It is concluded that shading does not provide a significant competitive advantage for Acropora spp. tables at 10 m deep.