Lyndon E. Llewellyn

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance.

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i). recognize particular populations of anurans of the same species and to differentiate between species, and (ii). investigate evolutionary aspects of peptide formation.

Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum

Gymnodinium catenatum is one of several dinoflagellates that produce a suite of neurotoxins called the paralytic shellfish toxins (PST), responsible for outbreaks of paralytic shellfish poisoning in temperate and tropical waters. Previous research suggested that the bacteria associated with the surface of the sexual resting stages (cyst) were important to the production of PST by G. catenatum. This study sought to characterise the cultivable bacterial diversity of seven different strains of G. catenatum that produce both high and abnormally low amounts of PST, with the long-term aim of understanding the role the bacterial flora has in bloom development and toxicity of this alga. Sixty-one bacterial isolates were cultured and phylogenetically identified as belonging to the Proteobacteria (70%), Bacteroidetes (26%) or Actinobacteria (3%). The Alphaproteobacteria were the most numerous both in terms of the number of isolates cultured (49%) and were also the most abundant type of bacteria in each G. catenatum culture. Two phenotypic (functional) traits inferred from the phylogenetic data were shown to be a common feature of the bacteria present in each G. catenatum culture: firstly, Alphaproteobacteria capable of aerobic anoxygenic photosynthesis, and secondly, Gammaproteobacteria capable of hydrocarbon utilisation and oligotrophic growth. In relation to reports of autonomous production of PST by dinoflagellate-associated bacteria, PST production by bacterial isolates was investigated, but none were shown to produce any PST-like toxins. Overall, this study has identified a number of emergent trends in the bacterial community of G. catenatum which are mirrored in the bacterial flora of other dinoflagellates, and that are likely to be of especial relevance to the population dynamics of natural and harmful algal blooms.

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 victims 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 victims 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 victims 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.

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.

Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia

The increased frequency and distribution of red tides requires the development of high-throughput detection methods for paralytic shellfish toxins (PST). Community ethics also requires that there be a reduced reliance upon the standard mouse bioassay. A biomolecular assay such as the sodium channel 3H-saxitoxin binding assay can satisfy both of these requirements but may be compromised by cross-reactivity with the structurally unrelated tetrodotoxins (TTX). This study utilised the sodium channel assay but also an alternative 3H-saxitoxin binding assay based upon a saxiphilin isoform from the centipede Ethmostigmus rubripes to screen for PSTs. Saxiphilin is a novel transferrin which binds saxitoxin (STX) but differs from the sodium channel in not having any measurable affinity for TTX. A detailed analysis of toxin composition was achieved by high performance liquid chromatography (HPLC). Various crustaceans and molluscs accumulate PSTs and TTX, thus proving useful biomarkers for these toxins in their immediate environment and an ideal challenge to the detection and analysis of PSTs in this presumptive screening program. Also, there has been little investigation of PSTs in invertebrates from the Indian Ocean so this region was selected to extend our knowledge of the distribution of these toxins. 190 crabs and shellfish encompassing 31 species were collected from reefs along the North-West Australian coast and tested for PSTs and TTX by sodium channel and saxiphilin bioassays as well as HPLC. PSTs were detected in 18 species of crabs and shellfish of the 31 species tested. Eight of these species have not been previously described as toxic, these being the crabs Euzanthus exsculptus, Lophozozymus octodentatus, Metopograpsus frontalis, Pilumnus pulcher, Platypodia pseudogranulosa and Portunus pelagicus, and the molluscs Tectus fenestratus and Trochus hanleyanus. By HPLC, only one or both of STX and decarbamoyl-STX was detected in any extract. Some extracts markedly inhibited 3H-saxitoxin binding by the sodium channel but not by saxiphilin. The close agreement between toxin quantification by the PST specific methods of HPLC and the saxiphilin bioassay is indicative that the additional toxicity detected by the sodium channel assay is TTX.

PARALYTIC SHELLFISH TOXINS IN TROPICAL OCEANS

The tropics possess some of the worlds richest marine environments, most notably coral reefs. Concealed within these ecosystems are a group of potent neurotoxins called the paralytic shellfish toxins (PSTs), the most famous of which is saxitoxin. Thirty years ago, PSTs were recognized as a major danger to seafood consumers in the tropics. The tropical dinoflagellate Pyrodinium bahamense biosynthesizes PSTs and its contamination of seafood has caused more illnesses and deaths than any other PST-producing microalga. Apart from this and other dinoflagellates, PSTs have been confirmed in tropical benthic algae, molluscs, echinoderms, crustacea, and other arthropods. Some of these organisms are unique in that, to date, they have only been found to be toxic in tropical oceans. For example, species of grazing and predatory gastropods, crabs, and more recently cephalopods have been discovered to contain PSTs in a number of intertidal tropical locations. These animals are thought to accumulate the toxins from benthic sources rather than toxic dinoflagellates as happens with filter-feeding bivalve molluscs such as clams and oysters. Here we evaluate the current understanding of PST transmission through tropical food webs. Finally, we consider the prevalence of PST intoxications in tropical regions and their social and economic costs.

Effects of the bloom-forming alga Trichodesmium erythraeum on the pearl oyster Pinctada maxima

Farmed pearl oysters (Pinctada maxima) suffered high mortality in the Dampier Archipelago of Western Australia in 1996. The mortality event affected all oyster sizes and coincided with extensive blooms of the cyanobacterium Trichodesmium erythraeum. The potent neurotoxin saxitoxin was detected in small amounts in some of the affected adult oysters but was not detectable in T. erythraeum. Vibrio species were isolated from some of the affected oysters but not in patterns consistent with a primary disease and no virus-like particles were observed. Juvenile oysters were exposed to medium and high concentrations of T. erythraeum in experimental aquaria for 7 days. No mortality of juvenile oysters occurred but individuals exposed to T. erythraeum at 105 cells/ml were less healthy than those fed upon the diatom Chaetoceros calcitrans. Histopathology of adult oysters from the affected farm and juvenile oysters exposed to T. erythraeum in the aquarium experiments were similar and included dilation of digestive gland lumens, sloughing of epithelial cells and granulocytes under the epithelial layer. These symptoms suggest that the T. erythraeum blooms in the archipelago were not a suitable food source for the oysters and may have contributed to the observed mortalities.

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).

Paralytic shellfish toxins are restricted to few species among Australia's taxonomic diversity of cultured microalgae

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.

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−1protein) 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°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.