Marine organisms as sources of C4-weed-specific herbicides

Imagine that you are a plant propagule looking for a home. You find a nice warm sunny place where the nutrients wash over you and think that you have arrived in paradise. And, what is more, there are few other plants with which to compete. However there are many animals close by in the form of corals and sponges and a variety of animals which make up the Great Barrier Reef. If you were on land, where plants occupy much of the available surface area over which animals wander, you would seem less out of place. Yet on healthy and pristine coral reefs found on the outer shelf of the Great Barrier Reef off the north east coast of Australia, only 20–28% of the available surface area comprises plants, i.e. algae (Sweatman et al., 1998) (Figure 1). Sea grass and kelp beds are more reminiscent of the terrestrial situation. Why is this so? Maybe coral reefs are, in fact, not a good place for plants to grow. But if this were the case, why then would the space be dominated by animals reliant for much of their nutrition upon symbiotic relationships with plants such as are found in corals and their symbiotic unicellular plants, zooxanthellae. What then, keeps the number of freegrowing plants low on coral reefs relative to the land? Does chemical warfare by the animals play some role? And if so, can these chemicals be developed for use as herbicide in the terrestrial environment

Purification and Characterization of a Collagenolytic Enzyme from a Pathogen of the Great Barrier Reef Sponge, Rhopaloeides odorabile

BACKGROUND:In recent years there has been a global increase in reports of disease affecting marine sponges. While disease outbreaks have the potential to seriously impact on the survival of sponge populations, the ecology of the marine environment and the health of associated invertebrates, our understanding of sponge disease is extremely limited. METHODOLOGY/PRINCIPAL FINDINGS:A collagenolytic enzyme suspected to enhance pathogenicity of bacterial strain NW4327 against the sponge Rhopaloeides odorabile was purified using combinations of size exclusion and anion exchange chromatography. After achieving a 77-fold increase in specific activity, continued purification decreased the yield to 21-fold with 7.2% recovery (specific activity 2575 collagen degrading units mg(-1)protein) possibly due to removal of co-factors. SDS-PAGE of the partially pure enzyme showed two proteins weighing approximately 116 and 45 kDa with the heavier band being similar to reported molecular weights of collagenases from Clostridium and marine Vibrios. The enzyme degraded tissue fibres of several sponge genera suggesting that NW4327 could be deleterious to other sponge species. Activity towards casein and bird feather keratin indicates that the partially purified collagenase is either a non-selective protease able to digest collagen or is contaminated with non-specific proteases. Enzyme activity was highest at pH 5 (the internal pH of R. odorabile) and 30 degrees C (the average ambient seawater temperature). Activity under partially anaerobic conditions also supports the role of this enzyme in the degradation of the spongin tissue. Cultivation of NW4327 in the presence of collagen increased production of collagenase by 30%. Enhanced enzyme activity when NW4327 was cultivated in media formulated in sterile natural seawater indicates the presence of other factors that influence enzyme synthesis. CONCLUSIONS/SIGNIFICANCE:Several aspects of the sponge disease etiology were revealed, particularly the strong correlation with the internal tissue chemistry and environmental temperature. This research provides a platform for further investigations into the virulence mechanisms of sponge pathogens

Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species

Pharmacokinetic modelling of multi-decadal luminescence time series in coral skeletons

As corals grow, they incorporate chemical indicators of seawater conditions into their aragonite skeleton after they have traversed an outer living tissue layer. Long-lived, massive coral skeletons can record decade-and century-long time series of seawater status. One such environmental clue is luminescence intensity which can correspond to river flow patterns and has been attributed to humic acid incorporation. Seawater humic acid levels are linked to river flow as rainfall extracts them from catchment soils to then flow into rivers and coastal seas. However, discrepancies exist when validating coral luminescence records against river flow data with intense luminescence sometimes occurring in the absence of increased flows. This contributes to uncertainty when reconstructing pre-instrumental river flows and rainfall from coral luminescence. Here we demonstrate that a major portion of coral core luminescence time series can be explained using a single-compartment, pharmacokinetic model that incorporates river flow measurements as the equivalent of drug dose. The model was robust for luminescence series in corals from near-shore reefs regularly influenced by river flow. The model implies that after floods, a proportion of subsequent luminescence peaks can be derived from the initial flood. This explains why some luminescence peaks after floods often do not correspond to additional significant river flows. This provides the first mechanism-based explanation for temporal changes in coral skeleton luminescence that incorporates a mathematical link between two independent time series making this proxy even more robust for reconstructing river flow and rainfall

Synthesis of bifunctional saxitoxin analogues by biotinylation

Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety

Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts

Several radioreceptor assays using tritiated saxitoxin ([3H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [3H]STX within the range of 1-90 μg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600 mM NaCl and 500 mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5

Robust cross-validation of linear regression QSAR models

A quantitative structure−activity relationship (QSAR) model is typically developed to predict the biochemical activity of untested compounds from the compounds' molecular structures. "The gold standard" of model validation is the blindfold prediction when the model's predictive power is assessed from how well the model predicts the activity values of compounds that were not considered in any way during the model development/calibration. However, during the development of a QSAR model, it is necessary to obtain some indication of the model's predictive power. This is often done by some form of cross-validation (CV). In this study, the concepts of the predictive power and fitting ability of a multiple linear regression (MLR) QSAR model were examined in the CV context allowing for the presence of outliers. Commonly used predictive power and fitting ability statistics were assessed via Monte Carlo cross-validation when applied to percent human intestinal absorption, blood-brain partition coefficient, and toxicity values of saxitoxin QSAR data sets, as well as three known benchmark data sets with known outlier contamination. It was found that (1) a robust version of MLR should always be preferred over the ordinary-least-squares MLR, regardless of the degree of outlier contamination and that (2) the model's predictive power should only be assessed via robust statistics. The Matlab and java source code used in this study is freely available from the QSAR-BENCH section of www.dmitrykonovalov.org for academic use. The Web site also contains the java-based QSAR-BENCH program, which could be run online via java's Web Start technology (supporting Windows, Mac OSX, Linux/Unix) to reproduce most of the reported results or apply the reported procedures to other data sets

HPLC chromatogram obtained with analytical molecular size exclusion chromatography using Superdex 200 HR.

<p>The chromatograms depicted show the absorbance of the eluted material at 254 nm (lower trace) and at 280 nm (upper trace). Note that the absorbance values at 280 nm have been offset by 7.5 mAU to distinguish it from the values at 254 nm.</p

The toxicity of skin secretions from coral-dwelling gobies and their potential role as a predator deterrent

Coral-dwelling gobies in the genus Gobiodon (family Gobiidae) posses toxic skin secretions. We used bioassays to investigate interspecific variation in the toxicity of skin secretions from six species of Gobiodon from Lizard Island on the Great Barrier Reef. We then used feeding experiments with two common species of predatory fish to test if skin secretions might act as a chemical defence against predation. The skin secretions of all species were toxic to the bioassay species, Apogon fragilis, but there were marked differences in toxicity among Gobiodon species. Feeding experiments showed that both small- and large-gaped predators avoided food items to which goby skin secretions, or a whole goby, had been added. These experiments indicate that skin toxins could function as a predator deterrent in coral-dwelling gobies