Molecular responses of sponge larvae exposed to partially weathered condensate oil.

Anthropogenic inputs of petroleum hydrocarbons into the marine environment can have long lasting impacts on benthic communities. Sponges form an abundant and diverse component of benthic habitats, contributing a variety of important functional roles; however, their responses to petroleum hydrocarbons are largely unknown. This study combined a traditional ecotoxicological experimental design and endpoint with global gene expression profiling and microbial indicator species analysis to examine the effects of a water accommodated fraction (WAF) of condensate oil on a common Indo-Pacific sponge, Phyllospongia foliascens. A no significant effect concentration (N(S)EC) of 2.1 % WAF was obtained for larval settlement, while gene-specific (N(S)EC) thresholds ranged from 3.4 % to 8.8 % WAF. Significant shifts in global gene expression were identified at WAF treatments ≥20 %, with larvae exposed to 100 % WAF most responsive. Results from this study provide an example on the incorporation of non-conventional molecular and microbiological responses into ecotoxicological studies on petroleum hydrocarbons.

Effects of aromatic hydrocarbons and evaluation of oil toxicity modelling for larvae of a tropical coral.

Application of oil toxicity modelling for assessing the risk of spills to coral reefs remains uncertain due to a lack of data for key tropical species and environmental conditions. In this study, larvae of the coral Acropora millepora were exposed to six aromatic hydrocarbons individually to generate critical target lipid body burdens (CTLBBs). Larval metamorphosis was inhibited by all six aromatic hydrocarbons, while larval survival was only affected at concentrations >2000 µg L-1. The derived metamorphosis CTLBB of 9.7 µmol g-1 octanol indicates larvae are more sensitive than adult corals, and places A. millepora larvae among the most sensitive organisms in the target lipid model (TLM) databases. Larvae were also more sensitive to anthracene and pyrene when co-exposed to ecologically relevant levels of ultraviolet radiation. The results suggest that the application of the phototoxic TLM would be protective of A. millepora larvae, provided adequate chemical and light data are available.

Sensitivity of the Indo-Pacific coral Acropora millepora to aromatic hydrocarbons.

The risks posed by petroleum spills to coral reefs are poorly understood and quantifying acute toxicity thresholds for aromatic hydrocarbons to reef-building corals is required to assess their sensitivity relative to other taxa. In this study, we exposed Acropora millepora to toluene, naphthalene and 1-methylnaphthalene (1-MN) in a flow-through system and assessed survivorship and sublethal responses including growth, colour and the photosynthetic performance of symbionts. Median 50% lethal concentrations (LC50s) decreased over the 7-d exposure period, reaching asymptotic values of 22,921, 5,268, 1167 µg L-1 for toluene, naphthalene and 1-MN, respectively. Corresponding toxicokinetic parameters (εLC50) defining the time progression of toxicity were 0.830, 0.692, and 0.256 d-1, respectively. Latent effects after an additional 7-d recovery in uncontaminated seawater were not observed. Effect concentrations (EC50s) for 50% growth inhibition were 1.9- to 3.6-fold lower than the LC50s for each aromatic hydrocarbon. There were no observed effects of aromatic hydrocarbon exposure on colour score (a proxy for bleaching) or photosynthetic efficiency. Acute and chronic critical target lipid body burdens (CTLBBs) of 70.3 ± 16.3 and 13.6 ± 18.4 µmol g-1 octanol (± standard error) were calculated for survival and growth inhibition based on 7-d LC50 and EC10 values, respectively. These species-specific constants indicate adult A. millepora is more sensitive than other corals reported so far but is of average sensitivity in comparison with other aquatic taxa in the target lipid model database. These results advance our understanding of acute hazards of petroleum contaminants to key habitat-building tropical coral reef species.

Coral recruits are highly sensitive to heavy fuel oil exposure both in the presence and absence of UV light.

Oil pollution remains a prominent local hazard to coral reefs, but the sensitivity of some coral life stages to oil exposure remains unstudied. Exposure to ultraviolet radiation (UVR), ubiquitous on coral reefs, may significantly increase oil toxicity towards these critical habitat-forming taxa. Here we present the first data on the sensitivity of two distinct post-settlement life stages of the model coral species Acropora millepora to a heavy fuel oil (HFO) water accommodated fraction (WAF) in the absence and presence of UVR. Assessment of lethal and sublethal endpoints indicates that both 1-week-old and 2-month-old recruits (1-wo and 2-mo) were negatively affected by chronic exposures to HFO (7 and 14 days, respectively). Relative growth (1-wo and 2-mo recruits) and survival (1-wo recruits) at end of exposure were the most sensitive endpoints in the absence of UVR, with no effect concentrations (NEC) of 34.3, 5.7 and 29.3 µg L-1 total aromatic hydrocarbons (TAH; ∑39 monocyclic- and polycyclic aromatic hydrocarbons), respectively. On average, UVR increased the negative effects by 10% for affected endpoints, and latent effects of exposure were evident for relative growth and symbiont uptake of recruits. Other sublethal endpoints, including maximum quantum yield and tissue colour score, were unaffected by chronic HFO exposure. A comparison of putative species-specific sensitivity constants for these ecologically relevant endpoints, indicates A. millepora recruits may be as sensitive as the most sensitive species currently included in oil toxicity databases. While the low intensity UVR only significantly increased the negative effects of the oil for one endpoint, the majority of endpoints showed trends towards increased toxicity in the presence of UVR. Therefore, the data presented here further support the standard incorporation of UVR in oil toxicity testing for tropical corals.

Food-chain length determines the level of phenanthrene bioaccumulation in corals.

Exposure from the dissolved-phase and through food-chains contributes to bioaccumulation of polycyclic aromatic hydrocarbons (PAHs) in organisms such as fishes and copepods. However, very few studies have investigated the accumulation of PAHs in corals. Information on dietary uptake contribution to PAHs accumulation in corals is especially limited. Here, we used Cavity-Ring-Down Spectroscopy (CRDS) to investigate the uptake rates and accumulation of a 13C-labeled PAH, phenanthrene, in Acropora millepora corals over 14 days. Our experiment involved three treatments representing exposure levels of increasing food-chain length. In Level W, corals were exposed to 13C-phenanthrene directly dissolved in seawater. In Level 1 representing herbivory, Dunaliella salina microalgal culture pre-exposed to 13C-phenanthrene for 48 h was added to the coral treatment jars. In Level 2 representing predation, corals were provided a diet of copepod (Parvocalanus crassirostris) nauplii fed on D. salina pre-exposed to 13C-phenanthrene. Bioconcentration factors (BCF) and bioaccumulation factors (BAF) were calculated as appropriate for all organisms, and biomagnification factors (BMF) were calculated for A. millepora. We found that while phenanthrene uptake rates were not significantly different for the treatments, the accumulated concentration in corals was significantly higher in Level W (33.5 ± 2.83 mg kg-1) than in Level 1 (27.55 ± 2.77 mg kg-1) and Level 2 (29.36 ± 3.84 mg kg-1). Coral log BAF values increased with food-chain length; Level 2 log BAF (6.45) was higher than Level W log BCF (4.18) and Level 1 log BAF (4.5). Coral BMF was also higher for Level 2 than for Level 1. Exposure to dissolved or diet-bound phenanthrene had no significant effect on the coral symbionts' photosynthetic efficiency (Fv/Fm) as monitored by pulse-amplitude-modulation (PAM) fluorometry, indicating the PAH can be accumulated without toxic effects to their Photosystem II. Our study highlights the critical role of dietary exposure for pollutant accumulation in corals.

Biomass pre-treatments of the N2-fixing cyanobacterium Tolypothrix for co-production of methane.

Tolypothrix, a self-flocculating, fast growing, CO2 and nitrogen-fixing cyanobacterium, can be cultivated in nutrient-poor ash dam waters of coal-fired power stations, converting CO2 emissions into organic biomass. Therefore, the biomass of Tolypothrix sp. is a promising source for bio-fertiliser production, providing micro- and macronutrients. Energy requirements for production could potentially be offset via anaerobic digestion (AD) of the produced biomass, which may further improve the efficiency of the resulting biofertilizer. The aim of this study was to evaluate the effectiveness of pre-treatment conditions and subsequent methane (CH4) production of Tolypothrix under out-door cultivation conditions. Pre-treatments on biogas and methane production for Tolypothrix sp. biomass investigated were: (1) thermal at 95 °C for 10 h, (2) hydrothermal by autoclave at 121 °C at 1013.25 hPa for 20 min, using a standard moisture-heat procedure, (3) microwave at an output power of 900 W and an exposure time of 3 min, (4) sonication at an output power of 10 W for 3.5 h at 10 min intervals with 20 s breaks and (5) freeze-thaw cycles at -80 °C for 24 h followed by thawing at room temperature. Thermal, hydrothermal and sonication pre-treatments supported high solubilization of organic compounds up to 24.40 g L-1. However, higher specific CH4 production of 0.012 and 0.01 L CH4 g-1 volatile solidsadded. was achieved for thermal and sonic pre-treatments, respectively. High N- and low C-content of the Tolypothrix biomass affected CH4 recovery, while pre-treatment accelerated production of volatile acids (15.90 g L-1) and ammonia-N-accumulation (1.41 g L-1), leading to poor CH4 yields. Calculated theoretical CH4 yields based on the elemental composition of the biomass were ~55% higher than actual yields. This highlights the complexity of interactions during AD which are not adequately represented by elemental composition.

Derivation of toxicity thresholds for gas condensate oils protective of tropical species using experimental and modelling approaches.

Toxicity thresholds for dissolved oil applied in tropical ocean risk assessments are largely based on the sensitivities of temperate and/or freshwater species. To explore the suitability of these thresholds for tropical habitats we experimentally determined toxicity thresholds for eight tropical species for a partially weathered gas condensate, applied the target lipid model (TLM) to predict toxicity of fresh and weathered condensates and compared sensitivities of the tropical species with model predictions. The experimental condensate-specific hazard concentration (HC5) was 167 µg L-1 total aromatic hydrocarbons (TAH), with the TLM-modelled HC5 (78 µg L-1 TAH) being more conservative, supporting TLM-modelled thresholds for tropical application. Putative species-specific critical target lipid body burdens (CTLBBs) indicated that several of the species tested were among the more sensitive species in the TLM database ranging from 5.1 (coral larvae) to 97 (sponge larvae) µmol g-1 octanol and can be applied in modelling risk for tropical marine ecosystems.

Bioproduct Potential of Outdoor Cultures of Tolypothrix sp.: Effect of Carbon Dioxide and Metal-Rich Wastewater.

Rising CO2 levels, associated climatic instability, freshwater scarcity and diminishing arable land exacerbate the challenge to maintain food security for the fast growing human population. Although coal-fired power plants generate large amounts of CO2 emissions and wastewater, containing environmentally unsafe concentrations of metals, they ensure energy security. Nitrogen (N2)-fixation by cyanobacteria eliminate nitrogen fertilization costs, making them promising candidates for remediation of waste CO2 and metals from macronutrient-poor ash dam water and the biomass is suitable for phycocyanin and biofertilizer product development. Here, the effects of CO2 and metal mixtures on growth, bioproduct and metal removal potential were investigated for the self-flocculating, N2-fixing freshwater cyanobacterium Tolypothrix sp. Tolypothrix sp. was grown outdoors in simulated ash dam wastewater (SADW) in 500 L vertical bag suspension cultures and as biofilms in modified algal-turf scrubbers. The cultivation systems were aerated with air containing either 15% CO2 (v/v) or not. CO2-fertilization resulted in ∼1.25- and 1.45-fold higher biomass productivities and ∼40 and 27% increased phycocyanin and phycoerythrin contents for biofilm and suspension cultures, respectively. CO2 had no effect on removal of Al, As, Cu, Fe, Sr, and Zn, while Mo removal increased by 37% in both systems. In contrast, Ni removal was reduced in biofilm systems, while Se removal increased by 73% in suspension cultures. Based on biomass yields and biochemical data obtained, net present value (NPV) and sensitivities analyses used four bioproduct scenarios: (1) phycocyanin sole product, (2) biofertilizer sole product, (3) 50% phycocyanin and 50% biofertilizer, and (4) 100% phycocyanin and 100% biofertilizer (residual biomass) for power station co-located and not co-located 10 ha facilities over a 20-year period. Economic feasibility for the production of food-grade phycocyanin either as a sole product or with co-production of biofertilizer was demonstrated for CO2-enriched vertical and raceway suspension cultures raised without nitrogen-fertilization and co-location with power stations significantly increased profit margins.

Sources, presence and potential effects of contaminants of emerging concern in the marine environments of the Great Barrier Reef and Torres Strait, Australia.

Current policy and management for marine water quality in the Great Barrier Reef (GBR) in north-eastern Australia primarily focusses on sediment, nutrients and pesticides derived from diffuse source pollution related to agricultural land uses. In addition, contaminants of emerging concern (CECs) are known to be present in the marine environments of the GBR and the adjacent Torres Strait (TS). Current and projected agricultural, urban and industrial developments are likely to increase the sources and diversity of CECs being released into these marine ecosystems. In this review, we evaluate the sources, presence and potential effects of six different categories of CECs known to be present, or likely to be present, in the GBR and TS marine ecosystems. Specifically, we summarize available monitoring, source and effect information for antifouling paints; coal dust and particles; heavy/trace metals and metalloids; marine debris and microplastics; pharmaceuticals and personal care products (PPCPs); and petroleum hydrocarbons. Our study highlights the lack of (available) monitoring data for most of these CECs, and recommends: (i) the inclusion of all relevant environmental data into integrated databases for building marine baselines for the GBR and TS regions, and (ii) the implementation of local, targeted monitoring programs informed by predictive methods for risk prioritization. Further, our spatial representation of the known and likely sources of these CECs will contribute to future ecological risk assessments of CECs to the GBR and TS marine environments, including risks relative to those identified for sediment, nutrients and pesticides.

Effect of CO2 and metal-rich waste water on bioproduct potential of the diazotrophic freshwater cyanobacterium, Tolypothrix sp.

Continued economic growth is reliant on stable, affordable energy, requiring at present fossil fuel-derived energy production. Coal-fired power stations produce metal-rich but macro-nutrient-poor waste waters and emit flue gas, containing ∼10% CO2. Algae and cyanobacteria remediate metals and CO2, but use of N2-fixing (diazotrophic) cyanobacteria can reduce nitrogen-fertilization costs. The resulting biomass represents a promising source for biofuel and bio-product development. This study investigated the effect of CO2- and trace metals on growth performance, biochemical profiles and metal content of the freshwater diazotrophic cyanobacterium Tolypothrix sp. to assess bioproduct potential. Aerated 2 L batch cultures were grown in simulated ash-dam water (SADW) and BG11 without nitrogen (BG11(-N) controls). Supplied air was supplemented with either 15% CO2 or not (non-CO2 controls). CO2 supplementation resulted in 2.4 and 3.3-fold higher biomass productivities and 1.3 and 1.2-fold higher phycocyanin and phycoerythrin contents, whilst metals (media) had no effect. Al, Cu, Ni and V were more efficiently removed (50-90%) with CO2-addition, while As, Mo, Se and Sr removal was higher (30-87%) for non-CO2 controls. No significant effect on Zn and Fe removal was evident. Calculated biomass metal concentrations, at quantities required to meet N-requirements of wheat, suggests no metal toxicity when applied as a mineral-nitrogen biofertilizer. With a carbohydrate content of 50%, the biomass is also suitable for bioethanol production. In summary, Tolypothrix sp. raised in ash dam waste water supplemented with flue gas CO2 could yield high-value phycobiliproteins, bioethanol or biogas, and mineral-rich nitrogen fertilizer which would offset remediation costs and improve agricultural productivity.

Phototoxic effects of two common marine fuels on the settlement success of the coral Acropora tenuis.

Coral reefs are at risk of exposure to petroleum hydrocarbons from shipping spills and uncontrolled discharges during extraction. The toxicity of petroleum hydrocarbons can substantially increase in the presence of ultraviolet radiation (UVR), therefore spills in shallow coral reef environments may be particularly hazardous to reef species. Here we investigated the sensitivity of coral larvae (Acropora tenuis) to dissolved hydrocarbons from heavy fuel oil (HFO) and diesel in the absence and presence of UVR. Larval settlement success decreased with increasing concentrations of dissolved HFO, and co-exposure to UVR doubled the toxicity: 50% effect concentrations (EC50) decreased from 96 (-UVR) to 51 (+UVR) total petroleum aromatic hydrocarbons (TPAH). Toxic thresholds for HFO were similar to concentrations reported during marine spills: EC10s of 24 (-UVR) and 15 (+UVR) µg l-1. While less toxic, diesel also reduced settlement and exhibited phototoxicity: EC10s of 122 (+UVR) and 302 (-UVR) µg l-1. This study demonstrates that the presence of UVR increases the hazard posed by oil pollution to tropical, shallow-water coral reefs. Further research on the effects of oils in the presence of UVR is needed to improve the environmental relevance of risk assessments and ensure appropriate protection for shallow reef environments against oil pollution.

Phototoxic effects of PAH and UVA exposure on molecular responses and developmental success in coral larvae.

Exposure to polycyclic aromatic carbons (PAHs) poses a growing risk to coral reefs due to increasing shipping and petroleum extraction in tropical waters. Damaging effects of specific PAHs can be further enhanced by the presence of ultraviolet radiation, known as phototoxicity. We tested phototoxic effects of the PAHs anthracene and phenanthrene on larvae of the scleractinian coral Acropora tenuis in the presence and absence of UVA (320-400 nm). Activity of superoxide dismutase (SOD) enzyme was reduced by anthracene while phenanthrene and UVA exposure did not have any effect. Gene expression of MnSod remained constant across all treatments. The genes Catalase, Hsp70 and Hsp90 showed increased expression levels in larvae exposed to anthracene, but not phenanthrene. Gene expression of p53 was upregulated in the presence of UVA, but downregulated when exposed to PAHs. The influence on stress-related biochemical pathways and gene expresson in A. tenuis larvae was considerably greater for anthracene than phenanthrene, and UVA-induced phototoxicity was only evident for anthracene. The combined effects of UVA and PAH exposure on larval survival and metamorphosis paralleled the sub-lethal stress responses, clearly highlighting the interaction of UVA on anthracene toxicity and ultimately the coral's development.

Effects of coal contamination on early life history processes of a reef-building coral, Acropora tenuis.

Successful reproduction and larval dispersal are important for the persistence of marine invertebrate populations, and these early life history processes can be sensitive to marine pollution. Coal is emerging as a contaminant of interest due to the proximity of ports and shipping lanes to coral reefs. To assess the potential hazard of this contaminant, gametes, newly developed embryos, larvae and juveniles of the coral Acropora tenuis were exposed to a range of coal leachate, suspended coal, and coal smothering treatments. Fertilisation was the most sensitive reproductive process tested. Embryo survivorship decreased with increasing suspended coal concentrations and exposure duration, effects on larval settlement varied between treatments, while effects on juvenile survivorship were minimal. Leachate exposures had negligible effects on fertilisation and larval settlement. These results indicate that coral recruitment could be affected by spills that produce plumes of suspended coal particles which interact with gametes and embryos soon after spawning.

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa).

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms.

Acute ecotoxicology of natural oil and gas condensate to coral reef larvae.

Risks posed by oil spills to coral reefs are difficult to evaluate, partially due to the absence of studies that adequately assess toxicity to relevant coral reef species. Here we experimentally tested the acute toxicity of condensate, representing a fraction of light crude oil, to coral (Acropora tenuis) and sponge (Rhopaloeides odorabile) larvae. The metamorphosis of coral larvae was inhibited at total petroleum aromatic hydrocarbon (TPAH) concentrations of water accommodated fractions (WAF) as low as 103 µg l(-1), similar to concentrations detected in seawater following large spills. The sensitivity of coral larvae increased by 40% when co-exposed to UV light that they might encounter in shallow reefal systems. Condensate WAF was more toxic to coral larvae than predicted by summing the toxicity of its main components (benzene, toluene, p-xylene and napthalene). In contrast, the sensitivity of sponge larvae to condensate WAF (>10,000 µg l(-1) TPAH) was far less than coral in the presence and absence of UV, but similar to that of other marine invertebrates. While these results highlight the relative sensitivity of coral larvae to oil, further research is needed to better understand and predict the impacts and risks posed by hydrocarbons to tropical reef systems.

Comparative study of the toxic effects of Chrysaora quinquecirrha (Cnidaria: Scyphozoa) and Chironex fleckeri (Cnidaria: Cubozoa) venoms using cell-based assays.

The venoms of jellyfish cause toxic effects in diverse biological systems that can trigger local and systemic reactions. In this study, the cytotoxic and cytolytic effects of Chrysaora quinquecirrha and Chironex fleckeri venoms were assessed and compared using three in vitro assays. Venoms from both species were cytotoxic to fish gill cells and rat cardiomyocytes, and cytolytic in sheep erythrocytes. Both venoms decreased cell viability in a concentration-dependent manner; however, the greatest difference in venom potencies was observed in the fish gill cell line, wherein C. fleckeri was 12.2- (P = 0.0005) and 35.7-fold (P < 0.0001) more potently cytotoxic than C. quinquecirrha venom with 30 min and 120 min cell exposure periods, respectively. Gill cells and rat cardiomyocytes exposed to venoms showed morphological changes characterised by cell shrinkage, clumping and detachment. The cytotoxic effects of venoms may be caused by a group of toxic proteins that have been previously identified in C. fleckeri and other cubozoan jellyfish species. In this study, proteins homologous to CfTX-1 and CfTX-2 toxins from C. fleckeri and CqTX-A toxin from Chironex yamaguchii were identified in C. quinquecirrha venom using tandem mass spectrometry. The presence and relative abundance of these proteins may explain the differences in venom potency between cubozoan and scyphozoan jellyfish and may reflect their importance in the action of venoms.

Transcriptome and venom proteome of the box jellyfish Chironex fleckeri.

BACKGROUND: The box jellyfish, Chironex fleckeri, is the largest and most dangerous cubozoan jellyfish to humans. It produces potent and rapid-acting venom and its sting causes severe localized and systemic effects that are potentially life-threatening. In this study, a combined transcriptomic and proteomic approach was used to identify C. fleckeri proteins that elicit toxic effects in envenoming. RESULTS: More than 40,000,000 Illumina reads were used to de novo assemble ∼ 34,000 contiguous cDNA sequences and ∼ 20,000 proteins were predicted based on homology searches, protein motifs, gene ontology and biological pathway mapping. More than 170 potential toxin proteins were identified from the transcriptome on the basis of homology to known toxins in publicly available sequence databases. MS/MS analysis of C. fleckeri venom identified over 250 proteins, including a subset of the toxins predicted from analysis of the transcriptome. Potential toxins identified using MS/MS included metalloproteinases, an alpha-macroglobulin domain containing protein, two CRISP proteins and a turripeptide-like protease inhibitor. Nine novel examples of a taxonomically restricted family of potent cnidarian pore-forming toxins were also identified. Members of this toxin family are potently haemolytic and cause pain, inflammation, dermonecrosis, cardiovascular collapse and death in experimental animals, suggesting that these toxins are responsible for many of the symptoms of C. fleckeri envenomation. CONCLUSIONS: This study provides the first overview of a box jellyfish transcriptome which, coupled with venom proteomics data, enhances our current understanding of box jellyfish venom composition and the molecular structure and function of cnidarian toxins. The generated data represent a useful resource to guide future comparative studies, novel protein/peptide discovery and the development of more effective treatments for jellyfish stings in humans. (Length: 300).

Chironex fleckeri (box jellyfish) venom proteins: expansion of a cnidarian toxin family that elicits variable cytolytic and cardiovascular effects.

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 µg kg(-1)) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml(-1)) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.

Venom proteome of the box jellyfish Chironex fleckeri.

The nematocyst is a complex intracellular structure unique to Cnidaria. When triggered to discharge, the nematocyst explosively releases a long spiny, tubule that delivers an often highly venomous mixture of components. The box jellyfish, Chironex fleckeri, produces exceptionally potent and rapid-acting venom and its stings to humans cause severe localized and systemic effects that are potentially life-threatening. In an effort to identify toxins that could be responsible for the serious health effects caused by C. fleckeri and related species, we used a proteomic approach to profile the protein components of C. fleckeri venom. Collectively, 61 proteins were identified, including toxins and proteins important for nematocyte development and nematocyst formation (nematogenesis). The most abundant toxins identified were isoforms of a taxonomically restricted family of potent cnidarian proteins. These toxins are associated with cytolytic, nociceptive, inflammatory, dermonecrotic and lethal properties and expansion of this important protein family goes some way to explaining the destructive and potentially fatal effects of C. fleckeri venom. Venom proteins and their post-translational modifications (PTMs) were further characterized using toxin-specific antibodies and phosphoprotein/glycoprotein-specific stains. Results indicated that glycosylation is a common PTM of the toxin family while a lack of cross-reactivity by toxin-specific antibodies infers there is significant divergence in structure and possibly function among family members. This study provides insight into the depth and diversity of protein toxins produced by harmful box jellyfish and represents the first description of a cubozoan jellyfish venom proteome.

Biochemical and molecular characterisation of cubozoan protein toxins.

Class Cubozoa includes several species of box jellyfish that are harmful to humans. The venoms of box jellyfish are stored and discharged by nematocysts and contain a variety of bioactive proteins that are cytolytic, cytotoxic, inflammatory or lethal. Although cubozoan venoms generally share similar biological activities, the diverse range and severity of effects caused by different species indicate that their venoms vary in protein composition, activity and potency. To date, few individual venom proteins have been thoroughly characterised, however, accumulating evidence suggests that cubozoan jellyfish produce at least one group of homologous bioactive proteins that are labile, basic, haemolytic and similar in molecular mass (42-46 kDa). The novel box jellyfish toxins are also potentially lethal and the cause of cutaneous pain, inflammation and necrosis, similar to that observed in envenomed humans. Secondary structure analysis and remote protein homology predictions suggest that the box jellyfish toxins may act as alpha-pore-forming toxins. However, more research is required to elucidate their structures and investigate their mechanism(s) of action. The biological, biochemical and molecular characteristics of cubozoan venoms and their bioactive protein components are reviewed, with particular focus on cubozoan cytolysins and the newly emerging family of box jellyfish toxins.