Coral-seeding devices with fish-exclusion features reduce mortality on the Great Barrier Reef.

Restoration methods that seed juvenile corals show promise as scalable interventions to promote population persistence through anthropogenic warming. However, challenges including predation by fishes can threaten coral survival. Coral-seeding devices with refugia from fishes offer potential solutions to limit predation-driven mortality. In an 8-month field study, we assessed the efficacy of such devices for increasing the survival of captive-reared Acropora digitifera (spat and microfragments) over control devices (featureless and caged). Devices with fish-exclusion features demonstrated a twofold increase in coral survival, while most corals seeded without protection suffered mortality within 48 h. Overall, spat faced more grazing and higher mortality compared to microfragments, and upward-facing corals were more vulnerable than side-facing corals. Grazing-induced mortality varied by site, with lower activity in locations abundant in mat-forming cyanobacteria or Scleractinian corals. Many scraping parrotfish were found feeding on or near the seeded corals; however, bites by Scarus globiceps explained the most site-related variation in grazing. Cyanobacteria may be preferred over corals as a nutritional resource for scraping parrotfish-advancing our understanding of their foraging ecology. Incorporating side-facing refugia in seeding devices and deploying to sites with nutrient-rich food sources for fish are potential strategies to enhance coral survival in restoration programs.

Hierarchical settlement behaviours of coral larvae to common coralline algae.

Natural regeneration of degraded reefs relies on the recruitment of larvae to restore populations. Intervention strategies are being developed to enhance this process through aquaculture production of coral larvae and their deployment as spat. Larval settlement relies on cues associated with crustose coralline algae (CCA) that are known to induce attachment and metamorphosis. To understand processes underpinning recruitment, we tested larval settlement responses of 15 coral species, to 15 species of CCA from the Great Barrier Reef (GBR). CCA in the family Lithophyllaceae were overall the best inducer across most coral species, with Titanoderma cf. tessellatum being the most effective species that induced at least 50% settlement in 14 of the coral species (mean 81%). Taxonomic level associations were found, with species of Porolithon inducing high settlement in the genus Acropora; while a previously understudied CCA, Sporolithon sp., was a strong inducer for the Lobophyllidae. Habitat-specific associations were detected, with CCA collected from similar light environment as the coral inducing higher levels of settlement. This study revealed the intimate relationships between coral larvae and CCA and provides optimal coral-algal species pairings that could be utilized to increase the success of larval settlement to generate healthy spat for reef restoration.

Effects of suspended coal particles on gill structure and oxygen consumption rates in a coral reef fish.

Large quantities of coal are transported through tropical regions; however, little is known about the sub-lethal effects of coal contamination on tropical marine organisms, including fish. Here, we measured aerobic metabolism and gill morphology in a planktivorous coral reef damselfish, Acanthochromis polyacanthus to elucidate the sub-lethal effects of suspended coal particles over a range of coal concentrations and exposure durations. Differences in the standard oxygen consumption rates (MO2) between control fish and fish exposed to coal particles (38 and 73 mg L-1) were minimal and generally not dose dependent; however, the MO2 of fish exposed to 38 mg coal L-1 (21 days) and 73 mg coal L-1 (31 days) were both significantly higher than the MO2 of control fish. Chronic coal exposure (31 days) altered gill structure in the higher coal treatments (73 and 275 mg L-1), with fish exposed to 275 mg L-1 exhibiting significant reductions in gill mucous and thinning of lamellar and filament epithelium. These findings contribute to our limited understanding of the potential impacts of coal on tropical reef species; however, most of the observed effects occurred at high coal concentrations that are unlikely under most coal spill scenarios. Future studies should investigate other contamination scenarios such as the impacts of chronic exposures to lower concentrations of coal.

Comparative toxicity of five dispersants to coral larvae.

Oil spill responders require information on the absolute and relative toxicities of chemical dispersants to relevant receptor species to assess their use in spill response. However, little toxicity data are available for tropical marine species including reef-building corals. In this study, we experimentally assessed the sub-lethal toxicity of five dispersants to larvae of the coral Acropora millepora over three short exposure periods (2, 6 and 24 h) reflecting real-world spill response scenario durations. Inhibition of larval settlement increased rapidly between 2 and 6 h, and was highest at 24 h: EC50 Corexit EC9500A = 4.0 mg l-1; Ardrox 6120 = 4.0 mg l-1; Slickgone LTSW = 2.6 mg L-1; Slickgone NS = 11.1 mg L-1 and Finasol OSR52 = 3.4 mg L-1. Coral larvae were more sensitive to dispersants than most other coral life stages and marine taxa, but the toxic thresholds (EC10s) exceeded most realistic environmental dispersant concentrations. Estimating toxic threshold values for effects of dispersants on coral should benefit the decision-making of oil spill responders by contributing to the development of species sensitivity distributions (SSDs) for dispersant toxicity, and by informing net environmental benefit assessment (NEBA) for dispersant use.

Host-associated coral reef microbes respond to the cumulative pressures of ocean warming and ocean acidification.

Key calcifying reef taxa are currently threatened by thermal stress associated with elevated sea surface temperatures (SST) and reduced calcification linked to ocean acidification (OA). Here we undertook an 8 week experimental exposure to near-future climate change conditions and explored the microbiome response of the corals Acropora millepora and Seriatopora hystrix, the crustose coralline algae Hydrolithon onkodes, the foraminifera Marginopora vertebralis and Heterostegina depressa and the sea urchin Echinometra sp. Microbial communities of all taxa were tolerant of elevated pCO2/reduced pH, exhibiting stable microbial communities between pH 8.1 (pCO2 479-499 µatm) and pH 7.9 (pCO2 738-835 µatm). In contrast, microbial communities of the CCA and foraminifera were sensitive to elevated seawater temperature, with a significant microbial shift involving loss of specific taxa and appearance of novel microbial groups occurring between 28 and 31 °C. An interactive effect between stressors was also identified, with distinct communities developing under different pCO2 conditions only evident at 31 °C. Microbiome analysis of key calcifying coral reef species under near-future climate conditions highlights the importance of assessing impacts from both increased SST and OA, as combinations of these global stressors can amplify microbial shifts which may have concomitant impacts for coral reef structure and function.

Effects of sediments on the reproductive cycle of corals.

Dredging, river plumes and natural resuspension events can release sediments into the water column where they exert a range of effects on underlying communities. In this review we examine possible cause-effect pathways whereby light reduction, elevated suspended sediments and sediment deposition could affect the reproductive cycle and early life histories of corals. The majority of reported or likely effects (30+) were negative, including a suite of previously unrecognized effects on gametes. The length of each phase of the life-cycle was also examined together with analysis of water quality conditions that can occur during a dredging project over equivalent durations, providing a range of environmentally relevant exposure scenarios for future testing. The review emphasizes the need to: (a) accurately quantify exposure conditions, (b) identify the mechanism of any effects in future studies, and (c) recognize the close interlinking of proximate factors which could confound interpretation of studies.

Chemical mediation of coral larval settlement by crustose coralline algae.

The majority of marine invertebrates produce dispersive larvae which, in order to complete their life cycles, must attach and metamorphose into benthic forms. This process, collectively referred to as settlement, is often guided by habitat-specific cues. While the sources of such cues are well known, the links between their biological activity, chemical identity, presence and quantification in situ are largely missing. Previous work on coral larval settlement in vitro has shown widespread induction by crustose coralline algae (CCA) and in particular their associated bacteria. However, we found that bacterial biofilms on CCA did not initiate ecologically realistic settlement responses in larvae of 11 hard coral species from Australia, Guam, Singapore and Japan. We instead found that algal chemical cues induce identical behavioral responses of larvae as per live CCA. We identified two classes of CCA cell wall-associated compounds--glycoglycerolipids and polysaccharides--as the main constituents of settlement inducing fractions. These algae-derived fractions induce settlement and metamorphosis at equivalent concentrations as present in CCA, both in small scale laboratory assays and under flow-through conditions, suggesting their ability to act in an ecologically relevant fashion to steer larval settlement of corals. Both compound classes were readily detected in natural samples.

Combined thermal and herbicide stress in functionally diverse coral symbionts.

Most reef building corals rely on symbiotic microalgae (genus Symbiodinium) to supply a substantial proportion of their energy requirements. Functional diversity of different Symbiodinium genotypes, endorsing the host with physiological advantages, has been widely reported. Yet, the influence of genotypic specificity on the symbiont's susceptibility to contaminants or cumulative stressors is unknown. Cultured Symbiodinium of presumed thermal-tolerant clade D tested especially vulnerable to the widespread herbicide diuron, suggesting important free-living populations may be at risk in areas subjected to terrestrial runoff. Co-exposure experiments where cultured Symbiodinium were exposed to diuron over a thermal stress gradient demonstrated how fast-growing clade C1 better maintained photosynthetic capability than clade D. The mixture toxicity model of Independent Action, considering combined thermal stress and herbicide contamination, revealed response additivity for inhibition of photosynthetic yield in both tested cultures, emphasizing the need to account for cumulative stressor impacts in ecological risk assessment and resource management.

Near-future ocean acidification causes differences in microbial associations within diverse coral reef taxa.

Microorganisms form symbiotic partnerships with a diverse range of marine organisms and can be critical to the health and survival of their hosts. Despite the importance of these relationships, the sensitivity of symbiotic microbes to ocean acidification (OA) is largely unknown and this needs to be redressed to adequately predict marine ecosystem resilience in a changing climate. We adopted a profiling approach to explore the sensitivity of microbes associated with coral reef biofilms and representatives of three ecologically important calcifying invertebrate phyla [corals, foraminifera and crustose coralline algae (CCA)] to OA. The experimental design for this study comprised four pHs consistent with current IPCC predictions for the next few centuries (pHNIST 8.1, 7.9, 7.7, 7.5); these pH/pCO2 conditions were produced in flow-through aquaria using CO2 bubbling. All reduced pH/increased pCO2 treatments caused clear differences in the microbial communities associated with coral, foraminifera, CCA and reef biofilms over 6 weeks, while no visible signs of host stress were detected over this period. The microbial communities of coral, foraminifera, CCA and biofilms were significantly different between pH 8.1 (pCO2 = 464 µatm) and pH 7.9 (pCO2 = 822 µatm), a concentration likely to be exceeded by the end of the present century. This trend continued at lower pHs/higher pCO2. 16S rRNA gene sequencing revealed variable and species-specific changes in the microbial communities with no microbial taxa consistently present or absent from specific pH treatments. The high sensitivity of coral, foraminifera, CCA and biofilm microbes to OA conditions projected to occur by 2100 is a concern for reef ecosystems and highlights the need for urgent research to assess the implications of microbial shifts for host health and coral reef processes.

Turbulence, cleavage, and the naked embryo: a case for coral clones.

After mass spawning events, coral embryos, lacking the protective capsule of other metazoans, are directly exposed to the environment at the ocean surface. Here, we present evidence that modest turbulence disrupts the integrity of these embryos, which fragment into totipotent cells that develop into proportionately smaller functional larvae. The level of turbulence required to fragment coral embryos can be generated from small wind-generated waves, which occur frequently during coral spawning on the Great Barrier Reef. The formation of planktonic coral clones, through natural embryo fragmentation of broadcast spawn, is a previously unknown mode of reproduction in the animal kingdom.

Effects of chlorpyrifos on cholinesterase activity and stress markers in the tropical reef fish Acanthochromis polyacanthus.

Tropical coastal ecosystems, including the Great Barrier Reef (GBR) of Australia are increasingly threatened by pollution; yet few studies have investigated the sensitivity of GBR species to these pollutants. Here we exposed juveniles of the tropical reef fish Acanthochromis polyacanthus (spiny damselfish) to three concentrations of the insecticide chlorpyrifos (CPF) and measured (i) muscle cholinesterase (ChE) activity; (ii) hepatic glutathione-S-transferase (GST) activity; and (iii) coenzyme Q (CoQ) redox balance, after 6h and 96h of exposure. After 96h, muscle ChE activity was significantly inhibited by 26%, 49% and 53% when fish were exposed to 1, 10 or 100µg/L CPF, respectively. Muscle ChE characterization revealed three types of ChEs, including two atypical forms. Hepatic CoQ antioxidant form significantly increased at 10µg/L after 6h of exposure, potentially demonstrating an early response to CPF-induced oxidative stress in liver. Hepatic GST was not affected by CPF exposure.

The biology of coral metamorphosis: molecular responses of larvae to inducers of settlement and metamorphosis.

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring >8000 Acropora unigenes were used to follow gene expression changes during the 12h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common-an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.

Effects of alumina refinery wastewater and signature metal constituents at the upper thermal tolerance of: 1. The tropical diatom Nitzschia closterium.

Ecotoxicological studies, using the tropical marine diatom, Nitzschia closterium (72-h growth rate), were undertaken to assess potential issues relating to the discharge from an alumina refinery in northern Australia. The studies assessed: (i) the species' upper thermal tolerance; (ii) the effects of three signature metals, aluminium (Al), vanadium (V) and gallium (Ga) (at 32°C); and (iii) the effects of wastewater (at 27 and 32°C). The critical thermal maximum and median inhibition temperature for N. closterium were 32.7°C and 33.1°C, respectively. Single metal toxicity tests found that N. closterium was more sensitive to Al compared to Ga and V, with IC(50)s (95% confidence limits) of 190 (140-280), 19,640 (11,600-25,200) and 42,000 (32,770-56,000) µg L(-1), respectively. The undiluted wastewater samples were of low toxicity to N. closterium (IC(50)s>100% wastewater). Environmental chemistry data suggested that the key metals and discharge are a very low risk to this species.

TBT contamination identified in Antarctic marine sediments.

We report for the first time butyltin contamination of near-shore sediments at six sites in the Ross Sea, Antarctica. A very high concentration of 2290 microg Sn kg(-1) sediment was recorded in one sample. The most likely source is abrasion of antifouling paint from the hulls of ice-breakers, but this pattern of contamination is also possible following ship groundings. Antifoulant biocides, such as TBT, have not been considered or detected in Antarctica previously and represent a new challenge to environmental managers and custodians.

Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneus.

After ingestion of a specimen of the crab Zosimus aeneus (Xanthidae), an East Timorese adult male died within several hours. Xanthid crabs are known to harbour paralytic shellfish toxins (PSTs), tetrodotoxin and palytoxin. A post-mortem examination did not find any obvious pathological abnormalities. This absence of pathologies is more often associated with PSTs and tetrodotoxin intoxication. A second, yet uneaten specimen of Z. aeneus from the same meal, contained a significant amount of PSTs and these same toxins were identified in the gut contents, blood, liver and urine of the victim. Metabolism of the PSTs occurred with the ingested crab harbouring gonyautoxin 2, gonyautoxin 3 and saxitoxin (STX) whereas neoSTX, decarbamoylSTX and STX dominated the PSTs in the victim's urine. The PST composition in the gut contents, in both their identity and proportion, was intermediate between the eaten crab and the urine suggesting that toxin conversion commenced in the victim's gut. The dose consumed by the victim was calculated to be between 1 and 2 microg STX equivalents/kg based upon the concentration in the remains of the cooked crab. The victim's meal did not consist solely of the toxic crab eaten and the possibility of other food items acting in a synergistic manner with the consumed PSTs cannot be discounted.

Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis.

Toxic freshwater cyanobacteria can contaminate water supplies and adversely effect humans, agricultural livestock, and wildlife. Toxicity is strain-specific so morphological observations alone cannot predict the hazard level. Two microtiter plate based bioassays have emerged for measuring saxitoxin (STX) and its derivatives, commonly found in the freshwater cyanobacteria Anabaena and Aphanizomenon. They use radioactively labeled STX binding by sodium channels, STX's pharmacological target, or an unrelated protein, saxiphilin. These bioassays were challenged with extracts of toxic and nontoxic strains of Anabaena circinalis, and the results were compared with HPLC analysis. Both radioreceptor assays had detection limits of 2 microg STX equivalents (STXeq)/L, which is belowthe concentration proposed for a health alert, namely 3 microg STXeq/L. In all cases, statistically significant correlations existed between all toxicity measurements of the same extracts with the methods used herein. Sodium channel and saxiphilin assays however predicted less toxicity relative to HPLC analysis. The only exception to this was the equivalency observed between saxiphilin measurement and HPLC quantitation corrected for mammalian toxicity. Saxiphilin assay predicted toxicity in one strain was 3 orders of magnitude more than by sodium channel assay, and no STX was detected by HPLC. Lack of acetylcholinesterase inhibition showed this bioactivity was not anatoxin-a(S), a toxin also produced by this A. circinalis with some resemblance to the region of STX bound by saxiphilin. Presence of anatoxin-a(S) was predicted for another strain by this same acetylcholinesterase assay that, if confirmed by chemical analysis, would be the first report of anatoxin-a(S) in an Australian cyanobacterium.

The effects of copper on the microbial community of a coral reef sponge.

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators for sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha-proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 microg l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 microg l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 microg l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction in the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 microg l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morpho-type actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 microg l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 microg l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 microg l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 microg l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.

Inhibition of coral fertilisation and larval metamorphosis by tributyltin and copper.

Fertilisation and larval metamorphosis of reef-building corals are important life history events leading to recruitment of juvenile corals to reef populations. Little is known of the sensitivity of these early life phases to pollution, or their relative susceptibility to certain toxicants compared with established coral colonies. Inhibition of fertilisation and larval metamorphosis of the coral Acropora millepora (Ehrenberg, 1834) was assessed in response to solutions of the antifoulants tributyltin (TBT) and copper (Cu) using laboratory-based bioassays. Nominal concentrations that inhibited 50% fertilisation and metamorphosis (IC50) were calculated from 4 h fertilisation and 24 h metamorphosis assays and were based on introduced dose. Cu was most potent towards fertilisation with an IC50 of 17.4 micrograms/l. TBT however, proved more toxic to larval metamorphosis having an IC50 of 2.0 micrograms/l. Inert surfaces coated with either Cu- or TBT-based antifouling paint also inhibited fertilisation and metamorphosis. The degree of inhibition was correlated with surface area of the paint coating. These results indicate fertilisation and metamorphosis of coral can be sensitive to active components of antifouling paints.

A high-throughput, microtiter plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin.

An isoform of the paralytic shellfish poison (PSP)-specific receptor saxiphilin, from the tropical centipede Ethmostigmus rubripes, was used as the basis for a radiometric, high-throughput, microtiter plate assay for this group of toxins. Characterization of the assay revealed that it was able to detect several representatives from the various structural PSP subgroups and yet was insensitive toward tetrodotoxin. To test the utility of the assay as a seafood toxin-monitoring tool, the assay was subjected to a variety of marine organism extracts, some of which were known to contain PSPs, and whole extract toxicity expressed as STX equivalents (STXeq) was measured by two methods: First, by comparison of values from a screening assay with a standard STX inhibition curve and, second, for highly active extracts, by calculation using the IC50 from a full inhibition curve of the extract. For extracts which could be quantified by both methods, there was almost 100% correlation between the derived values. STXeq derived by both methods from the bioassay highly correlated with absolute toxin quantities from HPLC analysis.

Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.

An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 micrograms/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.