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.

The Pathogen of the Great Barrier Reef Sponge Rhopaloeides odorabile Is a New Strain of Pseudoalteromonas agarivorans Containing Abundant and Diverse Virulence-Related Genes.

Sponge diseases have increased dramatically, yet the causative agents of disease outbreaks have eluded identification. We undertook a polyphasic taxonomic analysis of the only confirmed sponge pathogen and identified it as a novel strain of Pseudoalteromonas agarivorans. 16S ribosomal RNA (rRNA) and gyraseB (gyrB) gene sequences along with phenotypic characteristics demonstrated that strain NW4327 was most closely related to P. agarivorans. DNA-DNA hybridization and in silico genome comparisons established NW4327 as a novel strain of P. agarivorans. Genes associated with type IV pili, mannose-sensitive hemagglutinin pili, and curli formation were identified in NW4327. One gene cluster encoding ATP-binding cassette (ABC) transporter, HlyD and TolC, and two clusters related to the general secretion pathway indicated the presence of type I secretion system (T1SS) and type II secretion system (T2SS), respectively. A contiguous gene cluster of at least 19 genes related to type VI secretion system (T6SS) which included all 13 core genes was found. The absence of T1SS and T6SS in nonpathogenic P. agarivorans S816 established NW4327 as the virulent strain. Serine proteases and metalloproteases of the classes S8, S9, M4, M6, M48, and U32 were identified in NW4327, many of which can degrade collagen. Collagenase activity in NW4327 and its absence in the nonpathogenic P. agarivorans KMM 255(T) reinforced the invasiveness of NW4327. This is the first report unambiguously identifying a sponge pathogen and providing the first insights into the virulence genes present in any pathogenic Pseudoalteromonas genome. The investigation supports a theoretical study predicting high abundance of terrestrial virulence gene homologues in marine bacteria.

Draft Genome Sequence of Pseudoalteromonas sp. Strain NW 4327 (MTCC 11073, DSM 25418), a Pathogen of the Great Barrier Reef Sponge Rhopaloeides odorabile.

To date, only one marine sponge pathogen (Pseudoalteromonas sp. strain NW 4327) has fulfilled Koch's postulates. We report the 4.48-Mbp draft genome sequence of this strain, which is pathogenic to the Great Barrier Reef sponge Rhopaloeides odorabile. The sequence provides valuable information on sponge-pathogen interactions, including the mode of transmission and associated virulence factors.

Phylogeny drives large scale patterns in Australian marine bioactivity and provides a new chemical ecology rationale for future biodiscovery.

Twenty-five years of Australian marine bioresources collecting and research by the Australian Institute of Marine Science (AIMS) has explored the breadth of latitudinally and longitudinally diverse marine habitats that comprise Australia's ocean territory. The resulting AIMS Bioresources Library and associated relational database integrate biodiversity with bioactivity data, and these resources were mined to retrospectively assess biogeographic, taxonomic and phylogenetic patterns in cytotoxic, antimicrobial, and central nervous system (CNS)-protective bioactivity. While the bioassays used were originally chosen to be indicative of pharmaceutically relevant bioactivity, the results have qualified ecological relevance regarding secondary metabolism. In general, metazoan phyla along the deuterostome phylogenetic pathway (eg to Chordata) and their ancestors (eg Porifera and Cnidaria) had higher percentages of bioactive samples in the assays examined. While taxonomy at the phylum level and higher-order phylogeny groupings helped account for observed trends, taxonomy to genus did not resolve the trends any further. In addition, the results did not identify any biogeographic bioactivity hotspots that correlated with biodiversity hotspots. We conclude with a hypothesis that high-level phylogeny, and therefore the metabolic machinery available to an organism, is a major determinant of bioactivity, while habitat diversity and ecological circumstance are possible drivers in the activation of this machinery and bioactive secondary metabolism. This study supports the strategy of targeting phyla from the deuterostome lineage (including ancestral phyla) from biodiverse marine habitats and ecological niches, in future biodiscovery, at least that which is focused on vertebrate (including human) health.

Characterisation of nitric oxide synthase in three cnidarian-dinoflagellate symbioses.

BACKGROUND: Nitric oxide synthase (NOS) is an enzyme catalysing the conversion of L-arginine to L-citrulline and nitric oxide (NO), the latter being an essential messenger molecule for a range of biological processes. Whilst its role in higher vertebrates is well understood little is known about the role of this enzyme in early metazoan groups. For instance, NOS-mediated signalling has been associated with Cnidaria-algal symbioses, however controversy remains about the contribution of enzyme activities by the individual partners of these mutualistic relationships. METHODOLOGY/PRINCIPAL FINDINGS: Using a modified citrulline assay we successfully measured NOS activity in three cnidarian-algal symbioses: the sea anemone Aiptasia pallida, the hard coral Acropora millepora, and the soft coral Lobophytum pauciflorum, so demonstrating a wide distribution of this enzyme in the phylum Cnidaria. Further biochemical (citrulline assay) and histochemical (NADPH-diaphorase) investigations of NOS in the host tissue of L. pauciflorum revealed the cytosolic and calcium dependent nature of this enzyme and its in situ localisation within the coral's gastrodermal tissue, the innermost layer of the body wall bearing the symbiotic algae. Interestingly, enzyme activity could not be detected in symbionts freshly isolated from the cnidarians, or in cultured algal symbionts. CONCLUSIONS/SIGNIFICANCE: These results suggest that NOS-mediated NO release may be host-derived, a finding that has the potential to further refine our understanding of signalling events in cnidarian-algal symbioses.

Revisiting the association between sea surface temperature and the epidemiology of fish poisoning in the South Pacific: reassessing the link between ciguatera and climate change.

The most detailed dataset of ciguatera intensity is that produced by the South Pacific Epidemiological and Health Information Service (SPEHIS) of the Secretariat of the Pacific Community. The SPEHIS fish poisoning database has been previously analysed yielding statistically significant correlations between the Southern Oscillation Index (SOI) and ciguatera case numbers in several countries raising concerns this affliction will increase as oceans warm. Mapping of the SPEHIS records and other data hints at ciguatera not only being restricted to warm waters but that the Indo-Pacific Warm Pool, a body of water that remains hot throughout much of the year, may inhibit ciguatera prevalence. A qualitative assessment of ciguatera intensity and sea surface temperature (SST) behaviour within the EEZ of selected South Pacific nations supported the notion that ciguatera intensity was highest when SST was between an upper and lower limit. Many more climate and SST indices beyond the SOI are now available, including some that measure the abovementioned phenomenon of oceanic warm pools. Statistically significant, positive and negative cross-correlations were obtained between time series of annual ciguatera case rates from the SPEHIS dataset and the Pacific Warm Pool Index and several ENSO related indices which had been lagged for up to 2 years before the ciguatera time series. This further supports the possibility that when considering the impact of climate change on ciguatera, one has to consider two thresholds, namely waters that remain warm enough for a long enough period can lead to ciguatera and that extended periods where the water remains too hot may depress ciguatera case rates. Such a model would complicate projections of the effects of climate change upon ciguatera beyond that of a simple relationship where increased SST may cause more ciguatera.

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.

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.

Eusynstyelamides A, B, and C, nNOS inhibitors, from the ascidian Eusynstyela latericius.

Eusynstyelamides A-C (1-3) were isolated from the Great Barrier Reef ascidian Eusynstyela latericius, together with the known metabolites homarine and trigonelline. The structures of 1-3, with relative configurations, were elucidated by interpretation of their spectroscopic data (NMR, MS, UV, IR, and CD). The NMR data of 1 were found to be virtually identical to that reported for eusynstyelamide (4), isolated from E. misakiensis, indicating that a revision of the structure of 4 is needed. Eusynstyelamides A-C exhibited inhibitory activity against neuronal nitric oxide synthase (nNOS), with IC(50) values of 41.7, 4.3, and 5.8 microM, respectively, whereas they were found to be nontoxic toward the three human tumor cell lines MCF-7 (breast), SF-268 (CNS), and H-460 (lung). Compounds 1 and 2 displayed mild inhibitory activity toward Staphylococcus aureus (IC(50) 5.6 and 6.5 mM, respectively) and mild inhibitory activity toward the C(4) plant regulatory enzyme pyruvate phosphate dikinase (PPDK) (IC(50) values of 19 and 20 mM, respectively).

Sodium channel inhibiting marine toxins.

Saxitoxin (STX), tetrodotoxin (TTX) and their many chemical relatives are part of our daily lives. From killing people who eat seafood containing these toxins, to being valuable research tools unveiling the invisible structures of their pharmacological receptor, their global impact is beyond measure. The pharmacological receptor for these toxins is the voltage-gated sodium channel which transports Na ions between the exterior to the interior of cells. The two structurally divergent families of STX and TTX analogues bind at the same location on these Na channels to stop the flow of ions. This can affect nerves, muscles and biological senses of most animals. It is through these and other toxins that we have developed much of our fundamental understanding of the Na channel and its part in generating action potentials in excitable cells.

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.

Disulfide-containing peptides from the glandular skin secretions of froglets of the genus Crinia: structure, activity and evolutionary trends.

The skin secretions of Crinia signifera, C. riparia and C. deserticola contain bioactive disulfide-containing peptides. Signiferin 1 (RLCIPYIIPC-OH) from C. signifera and C. deserticola) contracts smooth muscle at a concentration of 10(-9) M, and effects proliferation of lymphocytes at 10(-6) M. In contrast, riparin 1.1 (RLCIPVIFC-OH) and riparin 1.2 (FLPPCAYKGTC-OH) from C. riparia show lymphocyte activity but do not contract smooth muscle. The lymphocyte and smooth muscle activities involve CCK2R. 3D structures of signiferin 1 and riparin 1.1 have been established using 2D NMR methods: these studies show significant differences in the shapes of the disulfide rings and with the orientations of the N-terminal residues. cDNA cloning establishes that the pre sections of the precursor pre-pro-riparin 1.4-1.6 peptides are different from the conserved pre regions of disulfide-containing antimicrobial peptides from species of the genus Rana found in the northern hemisphere and caerin antimicrobial peptides isolated from Australian tree frogs of the genus Litoria. This suggests that (i) either that riparins 1 have converged to similar structure and function to the ranid and hyloid prepropeptides which were lost initially from the myobatrachid lineage, or (ii) the prepropeptides in all three groups were derived from a single ancestral form that has remained relatively conserved in the hyloid and ranoid lineages but has undergone substantial divergent evolution in the myobatrachids.

A tropical marine microbial natural products geobibliography as an example of desktop exploration of current research using web visualisation tools.

Microbial marine biodiscovery is a recent scientific endeavour developing at a time when information and other technologies are also undergoing great technical strides. Global visualisation of datasets is now becoming available to the world through powerful and readily available software such as Worldwind, ArcGIS Explorer and Google Earth. Overlaying custom information upon these tools is within the hands of every scientist and more and more scientific organisations are making data available that can also be integrated into these global visualisation tools. The integrated global view that these tools enable provides a powerful desktop exploration tool. Here we demonstrate the value of this approach to marine microbial biodiscovery by developing a geobibliography that incorporates citations on tropical and near-tropical marine microbial natural products research with Google Earth and additional ancillary global data sets. The tools and software used are all readily available and the reader is able to use and install the material described in this article.

Comparison of the biological properties of several marine sponge-derived sesquiterpenoid quinones.

Eight naturally occurring marine-sponge derived sesquiterpenoid quinones were evaluated as potential inhibitors of pyruvate phosphate dikinase (PPDK), a C4 plant regulatory enzyme. Of these, the hydroxyquinones ilimaquinone, ethylsmenoquinone and smenoquinone inhibited PPDK activity with IC50's (reported with 95% confidence intervals) of 285.4 (256.4-317.7), 316.2 (279.2-358.1) and 556.0 (505.9-611.0) microM, respectively, as well as being phytotoxic to the C4 plant Digitaria ciliaris. The potential anti-inflammatory activity of these compounds, using bee venom phospholipase A2 (PLA2), was also evaluated. Ethylsmenoquinone, smenospongiarine, smenospongidine and ilimaquinone inhibited PLA2 activity (% inhibition of 73.2 +/- 4.8 at 269 microM, 61.5 +/- 6.1 at 242 microM, 41.0 +/- 0.6 at 224 microM and 36.4 +/- 8.2 at 279 microM, respectively). SAR analyses indicate that a hydroxyquinone functionality and a short, hydroxide/alkoxide side-chain atC-20 is preferred for inhibition of PPDK activity, and that a larger amine side-chain at C-20 is tolerated for PLA2 inhibitory activity.

Predictive toxinology: an initial foray using calculated molecular descriptors to describe toxicity using saxitoxins as a model.

Molecular descriptors and their mathematical combination have been used for predictive toxicology and risk assessments of environmental pollutants and pharmaceutical leads. However, this approach has not yet been used for natural toxins and may contribute to health and environmental risk assessments of newly discovered toxins without having to undertake whole animal toxicology. To explore this approach, over 3000 descriptors were calculated for each of the 30 saxitoxins for which mouse toxicities have been reported. This dataset was reduced to only 87 descriptors by firstly eliminating descriptors that were the same for all toxins or could not be calculated for all 30 toxins, and then removing those descriptors that did not have a statistically significant linear relationship with toxicity values. From the remaining 87 descriptors, a subset of seven descriptors was identified upon which various mathematical approaches were assessed for their ability to fit the dataset both with and without leave-one-out cross-validation. K-nearest neighbours and support vector machine regression along with various combinations of these seven descriptors fit the toxicity data almost perfectly and also achieved high predictability as measured by leave-one-out cross-validation. Of these seven descriptors, five incorporated weighting by estimates of atomic polarizability and electronic states. Predicted toxicities of several saxitoxins of unknown toxicity bore similarities to the pattern of known potencies of these toxins on various isoforms of the voltage-gated sodium channel. Some of these predicted toxicity values however would not be expected if there was a direct relationship between mammalian sodium channel affinity of the saxitoxins and whole animal toxicity.

Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase.

Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.

Screening marine fungi for inhibitors of the C4 plant enzyme pyruvate phosphate dikinase: unguinol as a potential novel herbicide candidate.

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C(4) plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 +/- 0.8 muM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C(4) plant but no effect on a model C(3) plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.

Host-defence peptide profiles of the skin secretions of interspecific hybrid tree frogs and their parents, female Litoria splendida and male Litoria caerulea.

Five healthy adult female first-generation hybrid tree frogs were produced by interspecific breeding of closely related tree frogs Litoria splendida and L. caerulea in a cage containing large numbers of males and females of both species. Phylogenetic analysis of mitochondrial DNA sequences established the female parent to be L. splendida. The peptide profile of the hybrid frogs included the neuropeptide caerulein, four antibiotics of the caerin 1 family and several neuronal nitric oxide synthase inhibitors of the caerin 1 and 2 classes of peptides. The skin secretions of the hybrids contained some peptides common to only one parent, some produced by both parental species, and four peptides expressed by the hybrids but not the parental species.

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

Several radioreceptor assays using tritiated saxitoxin ([(3)H]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 [(3)H]STX within the range of 1-90microg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5.

The behavior of mixtures of paralytic shellfish toxins in competitive binding assays.

Organisms that contain paralytic shellfish toxins (PSTs) may contain many members of this toxin family. PSTs block voltage-gated sodium channels (Na channel) and elicit neurotoxicity. Animals, including humans, may encounter PST mixtures via consumption of tainted seafood, contaminated water, or the microalgae that produce the toxins. PST binding by the Na channel as well as other proteins such as antibodies and saxiphilin have been used to develop biomolecular assays for PSTs. An equation that predicts the combined effects of binary and ternary PST mixtures has been experimentally validated for two unrelated STX-binding proteins, the rat brain Na channel and a saxiphilin from the xanthid crab Liomera tristis. It was found that the most potent toxin or toxins in any mixture profoundly affect the cumulative potency of the mixture, overwhelming weaker toxins with the transition from strong to weak toxicity and changing in a curvilinear manner. Less active PSTs must be several orders of magnitude more concentrated than stronger toxins for the mixture to reflect their potency. This behavior is important in understanding how toxin mixtures may act at the Na channel receptor via which PSTs exert their neurotoxicity and that the presence of weaker toxins does not dilute the effect of stronger toxins in a linear fashion. This strong dominance of a mixture by the most potent toxins also has implications for measurement of toxic test samples and for standards that may contain low levels of highly potent bioactive impurities. This equation has been extended to mixtures of PSTs containing more than three toxins and may be applicable to other natural contaminants and any competitive binding assays used to detect their presence and measure their concentration.