Selective Binding of Small Molecules to Vibrio cholerae DsbA Offers a Starting Point for the Design of Novel Antibacterials.

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446±10 µM. The same benzimidazole compound has ∼8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD >3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

Pre-pyrolysis metal and base addition catalyzes pore development and improves organic micropollutant adsorption to pine biochar.

Biochars were produced from pine feedstock pretreated with aqueous base, NaOH, at pH 9 and 11, and alkali and alkaline earth metals (AAEMs) Na, K, Ca, and Mg at 10-3 and 1 M. The effects of base and AAEM feedstock pretreatment on biochar surface area, pore size distribution, and adsorption capacity of two organic micropollutants (OMPs), 2,4-dichlorophenoxyacetic acid and sulfamethoxazole, from surface water with background dissolved organic matter (DOM) were evaluated. Base pretreatment significantly increased surface area within micropores (<2 nm diameter). AAEM pretreatment caused pore widening, increasing surface area within pores >2 nm in diameter. The catalytic activity of AAEMs, assessed by generation of non-micropore surface area, decreased in the following order: Ca > K > Na > Mg. All pretreated biochars outperformed untreated biochar for OMP adsorption. Biochar pretreated by aqueous base at pH 11 showed over an order of magnitude increase in OMP adsorption, nearly matching the performance of commercial activated carbon. OMP adsorption from surface water was positively correlated with biochar micropore surface area and negatively correlated with non-micropore surface area, which was linked to higher levels of DOM competition. Base and AAEM pretreatment of biochar feedstocks can increase OMP adsorption for water treatment applications by tuning pore structure and surface area.

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiLX).

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.

Assessing the impact of diclofenac, ibuprofen and sildenafil citrate (Viagra®) on the fertilisation biology of broadcast spawning marine invertebrates.

Exposure to synthetic chemicals is a key environmental challenge faced by aquatic organisms. The time and dose effects of the pharmaceuticals diclofenac, ibuprofen, and sildenafil citrate on sperm motility and successful fertilisation are studied using the echinoderms, Asterias rubens and Psammechinus miliaris, and the polychaete worm Arenicola marina, all important components of the marine benthos. Motility was reduced for all species when exposed to diclofenac concentrations ≥0.1 µg/L. Exposure to ≥1.0 µg/L of ibuprofen affected only P. miliaris gametes and fertilisation success of A. marina. A. rubens and P. miliaris sperm increased in both percentage motility and swimming velocity when exposed to sildenafil citrate at concentrations ≥18 and ≥ 50 ng/L, respectively. Pre-incubation of sperm with sildenafil citrate significantly increased fertilisation success in A. rubens and P. miliaris but not in A. marina. Pre-incubated A. rubens oocytes fertilised successfully in ibuprofen. According to EU Directive 93/67/EEC, diclofenac is classified as a very toxic substance to gametes of A. rubens, P. miliaris, and A. marina (EC50 = 100-1000 µg/L) while ibuprofen is classified as very toxic to gametes of P. miliaris but non-toxic to gametes of A. marina (EC50 > 10,000 µg/L). The present study indicates that diclofenac exposure may have negative impacts on invertebrate reproductive success, whereas ibuprofen potentially may compromise P. miliaris reproduction. This study provides a valuable insight into the mechanisms that allow marine invertebrates to survive and reproduce in contaminated and changing habitats.

Response of copepods to elevated pCO2 and environmental copper as co-stressors--a multigenerational study.

We examined the impacts of ocean acidification and copper as co-stressors on the reproduction and population level responses of the benthic copepod Tisbe battagliai across two generations. Naupliar production, growth, and cuticle elemental composition were determined for four pH values: 8.06 (control); 7.95; 7.82; 7.67, with copper addition to concentrations equivalent to those in benthic pore waters. An additive synergistic effect was observed; the decline in naupliar production was greater with added copper at decreasing pH than for decreasing pH alone. Naupliar production modelled for the two generations revealed a negative synergistic impact between ocean acidification and environmentally relevant copper concentrations. Conversely, copper addition enhanced copepod growth, with larger copepods produced at each pH compared to the impact of pH alone. Copepod digests revealed significantly reduced cuticle concentrations of sulphur, phosphorus and calcium under decreasing pH; further, copper uptake increased to toxic levels that lead to reduced naupliar production. These data suggest that ocean acidification will enhance copper bioavailability, resulting in larger, but less fecund individuals that may have an overall detrimental outcome for copepod populations.

The relevance of marine chemical ecology to plankton and ecosystem function: an emerging field.

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.

Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex.

The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.

Exposure to copper and a cytotoxic polyunsaturated aldehyde induces reproductive failure in the marine polychaete Nereis virens (Sars).

A number of metabolites from microalgae, including polyunsaturated aldehydes (PUAs), have been implicated as inducers of reproductive failure in aquatic invertebrates. Current work describes the impacts of the model PUA 2E, 4E-decadienal and copper sulphate applied in isolation and combination on the reproductive performance of the infaunal polychaete, Nereis virens (Sars). The reproductive and life cycle parameters investigated were; fertilisation success, larval survival, sperm motility (percent motility and curvilinear velocity) and sperm DNA damage. Exposure to decadienal and copper sulphate in isolation resulted in dose- and time-dependent reductions for each evaluated endpoint. Fertilisation success was heavily impacted at concentrations of up to 10µM for both compounds. Copper sulphate was more toxic in larval survival assays. Sperm motility impacts, although variable, exhibited rapid onset with pronounced reductions in sperm swimming performance observed within 3min of exposure. The extent of DNA damage was dose-dependent, and in the case of decadienal, rapid in onset. Dual compound exposures resulted in enhanced overall toxicity in all assays. Logistic regression analysis of fertilisation and larval survival assays showed significant synergistic interactions between decadienal and copper sulphate; an increase in concentration of either compound resulted in enhanced toxicity of the other. Longer exposure durations during larval survival assays demonstrated a further increase in both toxicity and synergism. The results indicate that the effects of additional environmental stressors must be considered when attempting to extrapolate laboratory-derived single compound exposures to field situations.

Neurovascular anatomy of the embryonic quail hindlimb.

Blood vessel and nerve development in the vertebrate embryo possess certain similarities in pattern and molecular guidance cues. To study the specific influence of shared guidance molecules on nervous and vascular development, an understanding of the normal neurovascular anatomy must be in place. The present study documents the pattern of nervous and vascular development in the Japanese quail hindlimb using immunohistochemistry and fluorescently labeled intravital injection combined with confocal and epifluorescent microscopy. The developmental patterns of major nerves and blood vessels of embryonic hindlimbs between stages E2.75 (HH18) and E6.0 (HH29) are described. By E2.75, the dorsal aortae have begun to fuse into a single vessel at the level of the hindlimb, and have completely fused by E3 (HH20). The posterior cardinal vein is formed at the level of the hindlimb by E3, as is the main artery of the early hindlimb, the ischiadic artery, as an offshoot of the dorsal aorta. Our data suggest that eight spinal segments, versus seven as reported by others (Tanaka and Landmesser,1986a; Tyrrell et al.,1990), contribute to innervation of the quail hindlimb. Lumbosacral neurites reach the plexus region by E3.5 (HH21 & 22), pause for approximately 24 hr, and then enter the hindlimb along with the ischiadic and crural arteries through shared foramina in the pelvic anlage. The degree of anterior-posterior spatial congruency between major nerves and blood vessels of the quail hindlimb was found to be highest medial to the pelvic girdle precursor, versus in the hindlimb proper.

Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform.

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.

Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism.

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.

Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus.

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the beta6/beta7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca(2+)-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k(cat)/K(m), indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.

Effects of 4-nonylphenol on the endocrine system of the shore crab, Carcinus maenas.

There is a considerable body of evidence to suggest that many anthropogenic chemicals, most notably xeno-estrogens, are able to disrupt the endocrine system of vertebrates. There have been few comparable studies on the effects of exposure to these chemicals that may serve as biomarkers of endocrine disruption in aquatic invertebrate species. In addition, the evidence available is complex, conflicting, and far from conclusive. The present study aimed to investigate the impact of the xeno-estrogen 4-nonylphenol (4-NP, nominal concentrations 10-100 microg L(-1)) on the regulation and functioning of the endocrine system of the shore crab Carcinus maenas. It also set out to establish whether 4-NP are causing the effects (i.e., changes of exoskeletons including secondary sexual characteristics, pheromonally mediated behavior and ecdysone levels, and the presence of vt in the male hepatopancreas) found recently in wild shore crabs (Lye et al.,2005). The study utilizes morphological (e.g., gonadosomatic and hepatosomatic indices) and hormonal (ecdysteroid moulting hormone levels and the induction of female specific proteins, vitellins) biomarkers using radioimmunoassay and an indirect enzyme linked immunosorbent assay applied to the soluble protein fraction of adult male hepatopancreatic homogenates. Exposure of C. maenas to an effective concentration as low as 1.5 microg L(-1) 4-NP resulted in a reduced testis weight, increased liver weight, and altered levels of ecdysone equivalents compared to controls. Induction of vitellin-like proteins was absent in all samples tested. The ecological implications and the possible mechanisms for the action of 4-NP on the response of the shore crab to xeno-estrogen exposure are discussed.

Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA.

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.

Engineering the substrate specificity of Staphylococcus aureus Sortase A. The beta6/beta7 loop from SrtB confers NPQTN recognition to SrtA.

The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the beta6/beta7 loop in SrtA with the corresponding domain from SrtB. The chimeric beta6/beta7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a k(cat)/K(m)(app) of 0.0062 +/- 0.003 m(-1) s(-1). This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (k(cat)/K(m)(app)(NPQTN)/k(cat)/K(m)(app)(LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the beta6/beta7 loop is an important site for substrate recognition in sortases.

Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC-ssaV-) M01ZH09, with a defined mutation in the Salmonella pathogenicity island 2, as a live, oral typhoid vaccine in human volunteers.

Salmonella enterica serovar Typhi strains with mutations in the Salmonella pathogenicity island-2 (SPI-2) may represent an effective strategy for human vaccine development, and a vectoring system for heterologous antigens. S. Typhi (Ty2 aroC-ssaV-) M01ZH09 is an attenuated, live, oral typhoid vaccine harboring defined deletion mutations in ssaV, which encodes an integral component in the SPI-2 type III secretion system (TTSS), as well as a mutation in an aromatic biosynthetic pathway needed for bacterial growth in vivo (aroC). SPI-2 mutant vaccines have yet to be evaluated in a large, randomized human trial. A simplified or single-oral dose oral typhoid vaccine using the SPI-2 strategy would offer significant advantages over the currently licensed typhoid vaccines. We performed a double-blinded, placebo-controlled, dose-escalating clinical trial in 60 healthy adult volunteers to determine the tolerability and immunogenicity of a single dose of M01ZH09. Three groups of 20 healthy adult volunteers were enrolled; 16 in each group received a single oral dose of the freeze-dried vaccine at 5 x 10(7), 5 x 10(8) or 5 x 10(9)CFU in a bicarbonate buffer. Four volunteers in each cohort received placebo in the same buffer. Adverse events were infrequent and not statistically different between vaccine and placebo recipients, although two subjects in the mid-range dose and three subjects in the highest dose had temperature measurements >37.5 degrees C. No blood or urine cultures were positive for M01ZH09, and fecal shedding was brief. The immune response was dose-related; the highest vaccine dose (5 x 10(9)CFU) was the most immunogenic. All tested subjects receiving the highest dose had a significant ASC response (mean 118 spots/10(6) cells). A >or=4-fold increase in antibody titer for S. Typhi LPS or flagellin was detected in 75% of volunteers in the highest-dose cohort by day 28. The SPI-2 mutant vaccine, M01ZH09, is a promising typhoid vaccine candidate and deserves further study as a vectoring system for heterologous vaccine antigens.

Comparison of the antibodies in lymphocyte supernatant and antibody-secreting cell assays for measuring intestinal mucosal immune response to a novel oral typhoid vaccine (M01ZH09).

Antibody-secreting cell (ASC) and antibodies in lymphocyte supernatant (ALS) assays are used to assess intestinal mucosal responses to enteric infections and vaccines. The ALS assay, performed on cell supernatants, may represent a convenient alternative to the more established ASC assay. The two methods, measuring immunoglobulin A to Salmonella enterica serovar Typhi lipopolysaccharide, were compared in volunteers vaccinated with a live-attenuated typhoid vaccine M01ZH09. The specificity of the ALS assay compared to the ASC assay was excellent (100%), as was sensitivity (82%). The ALS assay was less sensitive than the ASC assay at

The novel oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in 2 vaccine presentations.

BACKGROUND: M01ZH09 (Salmonella enterica serovar Typhi [Ty2 aroC(-) ssaV(-)] ZH9) is a live oral-dose typhoid vaccine candidate. M01ZH09 was rationally modified with 2 independently attenuating mutations, including a novel mutation in Salmonella pathogenicity island (SPI)-2. We demonstrate that M01ZH09, in a single oral dose, is well tolerated and prompts broad immune responses, regardless of whether prevaccination with a bicarbonate buffer is given. METHODS: Thirty-two healthy adult subjects were randomized and given 5x109 cfu of M01ZH09, with (presentation 1) or without (presentation 2) prevaccination with a bicarbonate buffer. Immunogenicity data included Salmonella Typhi lipopolysaccharide (LPS)-specific immunoglobulin (Ig) A antibody-secreting cells (enzyme-linked immunospot [ELISPOT] assay), IgG serologic responses to Salmonella Typhi LPS, lymphocyte proliferation, and interferon (IFN)- gamma production. RESULTS: The vaccine was well tolerated; adverse events after vaccination were mild. No fever or prolonged vaccine shedding occurred. Immunogenicity data demonstrated that 88% and 93% of subjects who received presentation 1 and presentation 2, respectively, had a positive response by ELISPOT assay; 81% of subjects in both groups underwent IgG seroconversion on day 14. Both groups had similar cellular immune responses to presentation 1 and presentation 2; lymphocyte proliferation to Salmonella Typhi flagellin occurred in 63% and 67% of subjects, respectively, and 69% and 73% of subjects, respectively, had an increase in IFN- gamma production. CONCLUSION: The oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in a single oral dose, with and without prevaccination with a bicarbonate buffer. Field studies to demonstrate protective efficacy are planned.

Toxicity of algal-derived aldehydes to two invertebrate species: do heavy metal pollutants have a synergistic effect?

The recent discovery of the production of anti-proliferative aldehydes in a variety of microalgal species has lead to considerable investigation into the effects of these toxins on aquatic invertebrates. Studies have, however, rarely considered the impact pollutants may have on grazer responses to algal toxins. In this study, the acute toxicities of five aldehydes to the rotifer Brachionus plicatilis and nauplii of the brine shrimp Artemia salina are examined using immersion assays. In addition, the effect of a representative of these aldehydes in the presence of sub-lethal levels of heavy metals was examined. B. plicatilis generally showed greater sensitivity to the aldehydes than A. salina. The polyunsaturated 2-trans,4-trans-decadienal was the most toxic to both species having 24h LD(50) values of 7 and 20 microM for B. plicatilis and A. salina, respectively. The remaining aldehydes had different orders of toxicity for the two species with a stronger relationship observed between mortality and aldehyde carbon-chain length for A. salina whereas B. plicatilis mortality showed a stronger dependence on the presence of carbon-carbon double bonds in the aldehydes. The presence of 1 microM of copper sulphate in solutions of decadienal resulted in the reduction of the 24h LD(50) of decadienal by approximately a third for both species. 1 microM of copper chloride in solutions of decadienal reduced the 24h LD(50) of decadienal to A. salina nauplii by approximately 11% and 1 microM zinc sulphate caused a reduction of only 3%. Pre-exposure of the organisms to 1 microM copper sulphate had no significant impact on their subsequent mortality in decadienal. The ecological implications and the possible mechanisms for the action of copper sulphate on the response of organisms to decadienal are discussed.

Exposure to 2,4-decadienal negatively impacts upon marine invertebrate larval fitness.

Diatoms liberate volatile, biologically active unsaturated aldehydes following cell damage, which negatively impact upon invertebrate reproductive processes such as fertilization, embryogenesis and larval survival. 2,4-Decadienal is frequently identified among the aldehydes produced and is one of the more biologically active. The majority of studies which have examined the toxic effects of diatom aldehydes to invertebrate reproduction have scored egg production and/or hatching success as indicators of biological impacts. There are very few studies which have dealt specifically with the impacts of diatom-derived aldehydes on larval fitness. Larval stages of the polychaetes Arenicola marina and Nereis virens and the echinoderms Asterias rubens and Psammechinus miliaris exposed to 2,4-decadienal at sub 1 microg ml(-1) concentrations suffered reduced survival over the incubation period (day 1-8 post fertilization) with detectable differences for the polychates at a concentration of 0.005 and 0.01-0.1 microg ml(-1) for the echinoderms. Susceptibility of larval N. virens was investigated using stage specific 24 h exposures at 2,4-decadienal concentrations up to 1.5 microg ml(-1). A clear stage specific effect was found, with earlier larval stages most vulnerable. Nectochaete larvae (9-10 d) showed no reduction in survival at the concentrations assayed. Fluctuating asymmetry (FA), defined as random deviations from perfect bilateral symmetry, was used to analyse fitness of larval P. miliaris exposed to 2,4-decadienal at concentrations of 0.1, 0.5 and 1 microg ml(-1). The degree and frequency of asymmetrical development increased with increasing 2,4-decadienal concentration. Equally, as FA increased larval survival decreased. These results provide further support for the teratogenic nature of 2,4-decadienal and its negative impact on invertebrate larval fitness.