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Giovanni Appendino, Mark Brönstrup and Julia Kubanek introduce the Natural Product Reports themed issue on ‘Olfaction, taste and chemoreception’.

As global warming problem is becoming serious in recent decades, the global sea level is continuously rising. This will cause damages to the coastal deltas with the characteristics of low-lying land, dense population, and developed economy. Continuously reclamation costal intertidal and wetland areas are making Shanghai, the mega city of Yangtze River Delta, more vulnerable to sea level rise. I

The world s population density in flood-prone coastal zones and megacities is expected to grow up to 25% by 2050. Global sea-levels have risen during the 20th century, and they will rise by up to ~60 cm by 2100. Non-climate-related anthropogenic processes (such as ground subsidence due to groundwater extraction,ground settlements due to large scaleland-reclamation,and fast and non-linear subsidence phenomena of artificial sea wall), as well as frequently encountered natural hazards (such as storms and storm-surge) will exacerbate the risk to coastal zones and megacities and amplify local vulnerability.Making the situation worse is the combination of sea-level rise resulting from climate change, local sinking of land resulting from anthropogenic and natural hazards.The coastal vulnerability of Yangtze River Delta (YRD) and Pearl River Delta (PRD) is currently being amplified by the compounding effects of the time-dependent ground subsidence, the accelerated rate of sea level rise, and natural hazards.The provided examples of delta regions affected by the combination of sea-level rise, significant modifications over time, and natural hazards make clear the need of extended analyses for the understanding of the mechanisms at the base of the surface modifications of coastal areas, estimating of future regional sea level change, and evaluating the potential submerged land area. In this project, the use of well-established remote sensing technologies, based on the joint exploitation of multi-spectral information gathered at different spectral wavelengths, the advanced Differential Interferometric Synthetic Aperture (DInSAR) techniques, GPS/leveling campaigns aiming to perform sound and extended geophysical analyses,satellite altimeter data and tide gauge data, and the CoupledModelInter-comparisonProject Phase 5 (CMIP5) climate model projections will be employed for these purposes.The results obtained in this project represents an asset for the planning of present and future scientific activities devoted to the monitoring of such fragile environments.

The bloom-forming dinoflagellate Karenia brevis produces a suite of allelopathic compounds that inhibit the growth of several phytoplankton competitors in laboratory experiments. However, it is less clear how allelopathy affects competition in the field, including whether allelopathic compounds impact K. brevis bloom dynamics. We investigated the extent to which phytoplankton species typically found offshore in the Gulf of Mexico, where K. brevis blooms initiate, are sensitive to K. brevis allelopathy. Natural assemblages of offshore phytoplankton dominated by diatoms were largely resistant to K. brevis allelopathy, even experiencing slight stimulation of growth from exposure to K. brevis exudates. When tested in pair-wise laboratory experiments, four diatom species often found offshore in the Gulf of Mexico exhibited varying degrees of resistance to K. brevis similar to that observed with field assemblages, although some competitors displayed a trend toward growth inhibition. In concurrent pair-wise laboratory experiments, four diatom species whose nearshore habitats frequently experience dense K. brevis blooms also demonstrated a range of responses to K. brevis allelopathy, from strong sensitivity to resistance. Overall, Gulf of Mexico phytoplankton that co-occur with K. brevis blooms in both nearshore and offshore environments respond variably to K. brevis allelopathy.

Summary Resource quality can have conflicting effects on the spread of disease. High-quality resources could hinder disease spread by promoting host immune function. Alternatively, high-quality food might enhance the spread of disease through other traits of hosts or parasites. Thus, to assess how resource quality shapes epidemics, we need to delineate mechanisms by which food quality affects key epidemiological traits. Here, we disentangle effects of food quality on ‘transmission potential’ – a key component of parasite fitness that combines transmission rate and parasite production – using a zooplankton host and fungal parasite. We estimated the components of transmission potential (i.e. parasite encounter rate, susceptibility and yield of parasite propagules) for hosts fed a high-quality green alga and a low-quality cyanobacterium. A focal experiment was designed to disentangle food quality effects on various components of transmission potential. The low-quality resource decreased transmission potential by stunting host growth and altering foraging behaviour. Hosts reared on low-quality food were smaller and had lower size-corrected feeding rates. Due to their slower grazing, they encountered fewer parasite spores in the water. Smaller hosts also had lower risk of an ingested spore causing infection (i.e. lower susceptibility) and yielded fewer parasite propagules. Hosts switched from high- to low-quality food during spore exposure also had low transmission potential – despite their large size – because the poor quality resource strongly depressed foraging. A follow-up experiment investigated traits of the low-quality resource that might have driven those results. Cyanobacterial compounds that can inhibit digestive proteases of a related grazer likely did not cause the observed reductions in transmission potential. Our study highlights the value of using mechanistic models to pinpoint how resource quality can change transmission potential. Overall, our results show that low-quality resources could inhibit the spread of disease through effects on multiple components of transmission potential. They also provide insight into how disease outbreaks in wildlife may respond to shifts in resource quality caused by eutrophication or climate change.

A suite of pharmacokinetic and pharmacological studies show that bromophycolide A (1), an inhibitor of drug-sensitive and drug-resistant Plasmodium falciparum, displays a typical small molecule profile with low toxicity and good bioavailability. Despite susceptibility to liver metabolism and a short in vivo half-life, 1 significantly decreased parasitemia in a malaria mouse model. Combining these data with prior structure–activity relationship analyses, we demonstrate the potential for future development of 1 and its bioactive ester analogues.

This review examines the state of the field for chemically mediated interactions involving marine angiosperms (seagrasses, mangroves, and salt marsh angiosperms). Small-scale interactions among these plants and their herbivores, pathogens, fouling organisms, and competitors are explored, as are community-level effects of plant secondary metabolites. At larger spatial scales, secondary metabolites from marine angiosperms function as reliable cues for larval settlement, molting, or habitat selection by fish and invertebrates, and can influence community structure and ecosystem function. Several recent studies illustrate the importance of chemical defenses from these plants that deter feeding by herbivores and infection by pathogens, but the extent to which allelopathic compounds kill or inhibit the growth of competitors is less clear. While some phenolic compounds such as ferulic acid and caffeic acid act as critical defenses against herbivores and pathogens, we find that a high total concentration of phenolic compounds within bulk plant tissues is not a strong predictor of defense. Residual chemical defenses prevent shredding or degradation of plant detritus by detritivores and microbes, delaying the time before plant matter can enter the microbial loop. Mangroves, marsh plants, and seagrasses remain plentiful sources of new natural products, but ecological functions are known for only a small proportion of these compounds. As new analytical techniques are incorporated into ecological studies, opportunities are emerging for chemical ecologists to test how subtle environmental cues affect the production and release of marine angiosperm chemical defenses or signaling molecules. Throughout this review, we point to areas for future study, highlighting opportunities for new directions in chemical ecology that will advance our understanding of ecological interactions in these valuable ecosystems.

Prey responses to predator cues are graded in intensity in accordance with the degree of threat presented by the predator. In systems in which prey gather information on predators by using chemicals, prey often respond more to the odor of predators that have consumed conspecifics, as opposed to heterospecifics. We investigated the response of a prey species, the mud crab, Panopeus herbstii, to urine of blue crab, Callinectes sapidus, fed mud crabs or oysters. Behavioral analysis was combined with metabolomics to characterize bioactive deterrents in the urine of predators fed different diets. Urine from blue crabs fed oysters or mud crabs depressed mud crab foraging when presented singly, with the urine derived from a mud crab diet being more potent. The magnitude of foraging depression increased with urine concentration. When urine from blue crabs fed oysters or mud crabs was combined, response to the urine mixture was no different from that to urine derived only from a mud crab diet. Metabolomics analysis indicated diet-dependent differences were related to a set of shared spectral features that differed in concentration in the respective urines, likely consisting of aromatic compounds, amino acids, and lipids. Taken together, these results suggest mud crabs distinguish diet of, and therefore the risk imposed by, predators through detection of a suite of compounds that together represent what the predator has recently consumed.

Coral reefs are in global decline, with seaweeds increasing as corals decrease. Although seaweeds inhibit coral growth, recruitment, and survivorship, the mechanism of these interactions is poorly understood. Here, we used field experiments to show that contact with four common seaweeds induces bleaching on natural colonies of Porites rus. Controls in contact with inert, plastic mimics of seaweeds did not bleach, suggesting seaweed effects resulted from allelopathy rather than shading, abrasion, or physical contact. Bioassay-guided fractionation of the hydrophobic extract from the red alga Phacelocarpus neurymenioides revealed a previously characterized antibacterial metabolite, neurymenolide A, as the main allelopathic agent. For allelopathy of lipid-soluble metabolites to be effective, the compounds would need to be deployed on algal surfaces where they could transfer to corals on contact. We used desorption electrospray ionization mass spectrometry (DESI-MS) to visualize and quantify neurymenolide A on the surface of P. neurymenioides, and we found the molecule on all surfaces analyzed, with highest concentrations on basal portions of blades.

Bioassay-guided fractionation of extracts from a Fijian red alga in the genus Callophycus resulted in the isolation of five new compounds of the diterpene-benzoate class. Bromophycoic acids A-E (1-5) were characterized by NMR and mass spectroscopic analyses and represent two novel carbon skeletons, one with an unusual proposed biosynthesis. These compounds display a range of activities against human tumor cell lines, malarial parasites, and bacterial pathogens including low micromolar suppression of MRSA and VREF.

The bistatic acquisition mode of the German TanDEM-X radar satellite mission provides a reliable source for measuring morphological changes associated with volcanic activity. We present the use of this system to measure key lava flow parameters including thickness, volume, runout, and flow extent by using two TanDEM-X data pairs to generate digital elevation models (DEMs) prior to and immediately following the 2012–2013 eruption of Tolbachik Volcano, Kamchatka. Morphometric parameters and areal distribution of the new lava flow field are determined using a cell-by-cell elevation difference between the two DEMs. A total flow volume of 0.53 ± 0.07 km3, a mean flow thickness of 14.5 m, and a modal thickness of 7.8 m are calculated. We use these calculated flow parameters as input to a volume-limited lava flow emplacement model. Model simulations are able to reproduce the SW portion of the 2012–2013 Tolbachik lava flow using a 75-m Shuttle Radar Topography Mission (SRTM) DEM and the 15-m TanDEM-X derived DEM, with goodness-of-fit measures of 56.3 and 59.6 %, respectively, based on the Jaccard similarity coefficient. The flow simulation done using SRTM data underestimates the observed 14.4 km flow runout by over 3 km, while the simulation with TanDEM-X data overestimates flow runout by about 1.5 km. Performance of the lava flow modeling algorithm is highly dependent on the modal lava thickness, highlighting the importance of using TanDEM-X DEMs to provide precise lava flow measurements in order to constrain input parameters for numerical modeling of lava flows.

The frequent occurrence of devastating blooms of the harmful dinoflagellate Karenia brevis in the Gulf of Mexico has motivated research into bloom dynamics and potential mitigation strategies. The use of competing phytoplankton to lower waterborne concentrations of the most abundant and toxic brevetoxins produced during these blooms has been proposed. However the ecological impacts of using such biocontrol agents have not been addressed. This study investigated the impact on marine invertebrates of lowered brevetoxin concentrations due to the presence of competing phytoplankton. Even at low brevetoxin concentrations, the presence of the common diatom Skeletonema grethae ameliorated harmful toxic effects of brevetoxins upon the brine shrimp, Artemia salina , and reduced the incidence of negative physiological and morphological responses of the sea anemone Aiptasia pallida . In addition, brevetoxin biotransformation products formed by competing phytoplankton appear to be non-toxic or do not trigger the same physiological responses as brevetoxins in the model organisms used. These findings may impact the interpretation of ecotoxicological data gathered during bloom events, since the presence of phytoplankton competitors in Karenia blooms is likely to reduce the harmful effects seen on many marine invertebrates.

During recent decades, many tropical reefs have transitioned from coral to macroalgal dominance. These community shifts increase the frequency of algal–coral interactions and may suppress coral recovery following both anthropogenic and natural disturbance. However, the extent to which macroalgae damage corals directly, the mechanisms involved, and the species specificity of algal–coral interactions remain uncertain. Here, we conducted field experiments demonstrating that numerous macroalgae directly damage corals by transfer of hydrophobic allelochemicals present on algal surfaces. These hydrophobic compounds caused bleaching, decreased photosynthesis, and occasionally death of corals in 79% of the 24 interactions assayed (three corals and eight algae). Coral damage generally was limited to sites of algal contact, but algae were unaffected by contact with corals. Artificial mimics for shading and abrasion produced no impact on corals, and effects of hydrophobic surface extracts from macroalgae paralleled effects of whole algae; both findings suggest that local effects are generated by allelochemical rather than physical mechanisms. Rankings of macroalgae from most to least allelopathic were similar across the three coral genera tested. However, corals varied markedly in susceptibility to allelopathic algae, with globally declining corals such as Acropora more strongly affected. Bioassay-guided fractionation of extracts from two allelopathic algae led to identification of two loliolide derivatives from the red alga Galaxaura filamentosa and two acetylated diterpenes from the green alga Chlorodesmis fastigiata as potent allelochemicals. Our results highlight a newly demonstrated but potentially widespread competitive mechanism to help explain the lack of coral recovery on many present-day reefs.

Tydemania expeditionis Weber-van Bosse (Udoteaceae) is a weakly calcified green alga. In the present paper, liquid chromatography coupled with photodiode array detection and electrospray mass spectrometry was developed to identify the fingerprint components. A total of four triterpenoid sulfates and three hydroxy fatty acids in the ethyl acetate fraction of the crude extract were structurally characterized on the basis of retention time, online UV spectrum, and mass fragmentation pattern. Furthermore, a detailed liquid chromatography-mass spectrometry analysis revealed two new hydroxy fatty acids, which were then prepared and characterized by extensive nuclear magnetic resonance (NMR) analyses. The proposed method provides a scientific and technical platform for the rapid identification of triterpenoid sulfates and hydroxy fatty acids in similar marine algae and terrestrial plants.

Plasmodium falciparum, the most deadly human malaria parasite, poses a major threat to human health worldwide, with over 500 million clinical cases and between one and two million deaths annually.[1] Natural products and their synthetic derivatives have provided a significant number of successful antimalarial treatments to date, representing approximately 65 % of current drugs.[2] Quinine, discovered from cinchona tree bark, has been used to treat malaria for centuries and was the primary antimalarial drug until it was replaced by chloroquine, a synthetic derivative, in the 1940s.[3] Chloroquine became the mainstay antimalarial until resistant strains began to appear nearly a decade after its introduction. Artemisinin, isolated from the plant Artemisia annua used in traditional Chinese medicine, ushered in a new wave of antimalarials and became the most potent and rapid-acting drug available.[4] Several synthetic artemisinin derivatives have since been developed, and artemisinin-based combination therapies are currently in use throughout the world to treat this parasitic disease. However, artemisinin-resistant strains have recently been reported,[5] and new antiparasitic drugs are urgently needed to combat these strains.

Blooms and cultures of the red tide dinoflagellate Karenia brevis exude compounds that inhibit the growth of competing phytoplankton, but the identities of these compounds are unknown. We characterized allelopathic compounds from K. brevis using a variety of chromatographic and spectroscopic techniques. K. brevis produces multiple compounds that are inhibitory towards the Gulf of Mexico diatom Asterionellopsis glacialis. However, brevetoxins, potent neurotoxins responsible for massive fish kills and neurotoxic shellfish poisoning in humans, had no effect on A. glacialis growth. We determined that most allelopathic compounds produced by K. brevis are unstable, polar, organic molecules produced at low concentrations, which are either neutral or positively charged. K. brevis also produces a suite of less polar, but more stable compounds that are moderately allelopathic towards A. glacialis. Given that K. brevis encounters a variety of competitor species in the field, the production of multiple allelopathic compounds could provide a broader chemical arsenal to successfully inhibit these competitors. The allelopathic compounds characterized in more detail had molecular weights between 500 and 1000 Da and possessed aromatic functional groups. Metabolic fingerprinting of K. brevis extracts has not yet led to identification of specific compound(s) responsible for allelopathy. Our results indicate that the ability of K. brevis to outcompete co-occurring phytoplankton may be facilitated by the production of multiple allelopathic compounds.

The increasing frequency of devastating blooms of the harmful dinoflagellate Karenia brevis has motivated investigations into understanding bloom dynamics and the potential for mitigation. Previous findings indicate that waterborne concentrations of the most abundant brevetoxin (brevetoxin B or PbTx-2) associated with these blooms decrease in the presence of other phytoplankton species. The current study explores the mechanism of brevetoxin removal from seawater upon exposure to phytoplankton competitors. Live phytoplankton removed waterborne brevetoxins more rapidly than lysates, but phytoplankton did not need to be in a state of active metabolism. Biomolecules, probably proteins, exuded from phytoplankton appeared to be responsible for the loss of brevetoxins, either by irreversible complexation or by degradation. Selective removal of PbTx-2 and -1, but not PbTx-3, -9 or BTX-B5, by cultured phytoplankton revealed that brevetoxin removal is dependent upon the presence of an α,β-unsaturated aldehyde functionality. The mechanism of biotransformation appears to be common among phytoplankton, since members of various taxonomic groups including diatoms, dinoflagellates, and a cryptophyte each caused 75-90% decrease in PbTx-2 concentration, as did a generic protein (bovine serum albumin) added to seawater at high concentration. These findings support the concept of potentially using competitor phytoplankton species or compounds derived from phytoplankton as biocontrol agents for waterborne toxins associated with red tide.

The use of chemical compounds to suppress the growth of competitors is a competitive strategy known as allelopathy that can be readily observed with many phytoplankton species in lab- oratory studies. However, it is unclear how these allelopathic interactions are altered when the com- plexity of the system is increased to more closely mimic natural conditions. In the present study, we conducted laboratory experiments to decipher how the identity, abundance, and growth stage of competitors affect the outcome of allelopathic interactions with the red tide dinoflagellate Karenia brevis. Multiple chemical compounds produced by K. brevis were found to inhibit the growth of 4 phytoplankton competitors, although these competitors were susceptible to different combinations of compounds. We found that physiological state and cell concentration of competitors were important determinants of allelopathy, with early-stage (lag phase) cells more vulnerable to allelopathic effects than later growth stages for the diatom Skeletonema grethae. Despite being allelopathic to multiple competitors in the laboratory, in a microcosm experiment using plankton field assemblages, extracel- lular extracts of 2 strains of K. brevis had no effects on some taxa although they stimulated the growth of some diatoms. This suggests that in a species-rich ecological community under oligotrophic condi- tions, the relative importance of K. brevis allelopathy may not be as high as most laboratory studies predict.

Three antimalarial meroditerpenes have been isolated from two Fijian red macroalgae. The absolute stereochemistry of callophycolide A (1), a unique macrolide from Callophycus serratus, was determined using a combination of Mosher's ester analysis, circular dichroism analysis with a dimolybdenum tetraacetate complex, and conformational analysis using NOEs. In addition, two known tocopherols, β-tocopherylhydroquinone (4) and δ-tocopherylhydroquinone (5), were isolated from Amphiroa crassa. By oxidizing 5 to the corresponding δ-tocopherylquinone (6), antimalarial activity against the human malaria parasite Plasmodium falciparum was increased by more than 20-fold.

SummaryActin filament-disrupting marine macrolides are promising templates from which to design therapeutics against cancer and other diseases that co-opt the actin cytoskeleton. Typically, these macrolides form either a 1:1 or 2:1 actin-macrolide complex where their aliphatic side chain, or “tail,” has been reported to convey the major determinant of cytotoxicity. We now report the structure of the marine macrolide lobophorolide bound to actin with a unique 2:2 stoichiometry in which two lobophorolide molecules cooperate to form a dimerization interface that is composed entirely of the macrolide “ring” region, and each molecule of lobophorolide interacts with both actin subunits via their ring and tail regions to tether the subunits together. This binding mode imposes multiple barriers against microfilament stability and holds important implications for development of actin-targeting drugs and the evolution of macrolide biosynthetic enzymes.

Chemical signaling plays an important role in predator–prey interactions and feeding dynamics. Like other organisms that are sessile or slow moving, some marine sponges contain aversive compounds that defend these organisms from predation. We sought to identify and characterize a fish chemoreceptor that detects one of these compounds. Using expression cloning in Xenopus oocytes coexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, the beta-2 adrenergic receptor (β 2 AR), and fractions of a zebrafish cDNA library, we isolated a cDNA clone encoding receptor activity–modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), a novel coreceptor involved in signaling in response to triterpene glycosides. This coreceptor appears to be structurally and functionally related to RAMPs, a family of coreceptors that physically associate with and modify the activity of G protein–coupled receptors (GPCRs). In membranes from formoside-responsive oocytes, RL-TGR was immunoprecipitated in an apparent complex with β 2 AR. In HEK293 cells, coexpression of β 2 AR induced the trafficking of RL-TGR from the cytoplasm to the plasma membrane. These results suggest that RL-TGR in the predatory fish physically associates with the β 2 AR or another, more physiologically relevant GPCR and modifies its pharmacology to respond to triterpene glycosides found in sponges that serve as a potential food source for the fish. RL-TGR forms a coreceptor that responds to a chemical defense compound in the marine environment, and its discovery might lead the way to the identification of other receptors that mediate chemical defense signaling.

Four new bromophycolides, R–U (1–4), were isolated from the Fijian red alga Callophycus serratus and were identified by 1D and 2D NMR and mass spectroscopic analyses. These compounds expand the known structural variety of diterpene-benzoate macrolides and exhibited modest cytotoxicity toward selected human cancer cell lines. Bromophycolide S (2) also showed submicromolar activity against the human malaria parasite Plasmodium falciparum.

This article reviews recent reports on the structural revision of natural products. Through a critical assessment of the original and revised published structures, the article addresses why each structure was targeted for revision, discusses the techniques and key discrepancies that led to the proposal of the revised structure, and offers measures that may have been taken during the original structure determination to prevent error. With the revised structures in hand, weaknesses of original proposals are assessed, providing a better understanding on the logic behind structure determination.

Bromophycolides J-Q (1-8) were isolated from extracts of the Fijian red alga Callophycus serratus and identified with 1D and 2D NMR spectroscopy and mass spectral analyses. These diterpene-benzoate macrolides represent two novel carbon skeletons and add to the ten previously reported bromophycolides (9-18) from this alga. Among these 18 bromophycolides, several exhibited activities in the low micromolar range against the human malaria parasite Plasmodium falciparum.

Presented here is the optimization and development of a desorption electrospray ionization mass spectrometry (DESI-MS) method for detecting natural products on tissue surfaces. Bromophycolides are algal diterpene-benzoate macrolide natural products that have been shown to inhibit growth of the marine fungal pathogen Lindra thalassiae. As such, they have been implicated in antimicrobial chemical defense. However, the defense mechanisms are not yet completely understood. Precise detection of these compounds on algal tissue surfaces under ambient conditions without any disruptive sample processing could shed more light onto the processes involved in chemical defense of marine organisms. Conventional DESI-MS directly on algal tissue showed relatively low sensitivity for bromophycolide detection. Sensitivity was greatly improved by the addition of various anions including Cl(-), Br(-), and CF(3)COO(-) into the DESI spray solvent. Chloride adduction gave the highest sensitivity for all assayed anions. Density functional optimization of the bromophycolide anionic complexes produced during DESI supported this observation by showing that the chloride complex has the most favorable binding energy. Optimized DESI protocols allowed the direct and unambiguous detection of bromophycolides, including A, B, and E, from the surface of untreated algal tissue.

Two novel alpha-pyrone macrolides, neurymenolides A (1) and B (2), were isolated from the Fijian red alga Neurymenia fraxinifolia and characterized using a combination of NMR and mass spectral analyses. These molecules represent only the second example of alpha-pyrone macrolides, with 1 existing as interchanging atropisomers due to restricted rotation about the alpha-pyrone ring system. Neurymenolide A (1) displayed moderately potent activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).

Phytochemical analysis of Fijian populations of the green alga Tydemania expeditionis led to the isolation of two unsaturated fatty acids, 3(zeta)-hydroxy-octadeca-4(E),6(Z),15(Z)-trienoic acid (1) and 3(zeta)-hydroxy-hexadeca-4(E),6(Z)-dienoic acid (2), along with the known 3(zeta)-hydroxy-octadeca-4(E),6(Z)-dienoic acid (4). Investigations of the red alga Hydrolithon reinboldii led to identification of a glycolipid, lithonoside (3), and five known compounds, 15-tricosenoic acid, hexacosa-5,9-dienoic methyl ester, beta-sitosterol, 10(S)-hydroxypheophytin A, and 10(R)-hydroxypheophytin A. The structures of 1-3 were elucidated by spectroscopic methods (1D and 2D NMR spectroscopy and ESI-MS). Compounds 1, 2, and 4, containing conjugated double bonds, demonstrated moderate inhibitory activity against a panel of tumor cell lines (including breast, colon, lung, prostate and ovarian cells) with IC(50) values ranging from 1.3 to 14.4 microM. The similar cell selectivity patterns of these three compounds suggest that they might act by a common, but unknown, mechanism of action.

We tested whether interactions among phytoplankton competitors affect toxin dynamics involving the red tide dinoflagellate Karenia brevis, whose brevetoxins incapacitate and kill coastal wildlife. The addition of a live diatom, Skeletonema costatum, led to decreased concentrations of brevetoxin B (PbTx-2) associated with K. brevis cells in co-culturing experiments and with two of three natural bloom samples containing K. brevis. Similar decreases in PbTx-2 concentration, but not PbTx-3 concentration, occurred when a mixture of brevetoxins (without live K. brevis cells) was exposed to S. costatum, indicating that S. costatum metabolizes waterborne PbTx-2. Liquid chromatography–mass spectrometry (LC–MS) and ELISA analyses indicated that PbTx-2 is probably not transformed into other brevetoxins or into known brevetoxin metabolites, and instead is biotransformed by a previously unrecognized mechanism. Four different S. costatum strains from around the world caused similar loss of PbTx-2, suggesting that evolutionary experience with K. brevis is not a pre-requisite for the ability to metabolize PbTx-2. Additionally, phytoplankton-associated bacteria were found to play no role in the loss of PbTx-2, as bacteria-free S. costatum strains metabolized PbTx-2. Finally, loss of waterborne PbTx-2 caused by exposure to a dinoflagellate, a cryptophyte, and two additional diatom species indicates that this phenomenon is widespread among phytoplankton. Our results unexpectedly suggest that competing phytoplankton species present during K. brevis blooms, and possibly other red tides, could mediate bloom toxicity and therefore ecosystem-level consequences of red tides.

Cytotoxicity-guided fractionation of the green macroalga Tydemania expeditionis led to isolation of four sulfate-conjugated triterpenoids including one new lanostane-type triterpenoid disulfate, lanosta-8-en-3,29-diol-23-oxo-3,29-disodium sulfate (1), and three known cycloartane-type triterpenoid disulfates, cycloartan-3,29-diol-23-one 3,29-disodium sulfate (2), cycloart-24-en-3,29-diol-23-one 3,29-disodium sulfate (3), and cycloartan-3,23,29-triol 3,29-disodium sulfate (4). Extensive 1D and 2D NMR analyses in combination with X-ray crystallography established the structure and absolute configuration of 1 and allowed determination of the absolute configurations of 2–4 with a revision of previously assigned configuration at C-5. Each natural product was moderately cytotoxic in tumor cell and invertebrate toxicity assays. Of the natural products, only 4 exhibited significant antifungal activity at whole-tissue natural concentrations against the marine pathogen Lindra thalassiae. Comparison of the biological activities of natural products with their desulfated derivatives indicated that sulfation does not appear to confer cytotoxicity or antifungal activity.

Because competitive interactions may have led to adaptations enabling bloom-forming phytoplankton to dominate pelagic communities, we explored the allelopathic effects of one red tide dinoflagellate, Karenia brevis, on competing phytoplankton species. Exposure to waterborne compounds from natural K. brevis blooms resulted in growth inhibition or death for four of five co-occurring species tested, whereas compounds exuded by K. brevis cultures suppressed three of these same competitors (the diatoms Asterionellopsis glacialis and Skeletonema costatum and the dinoflagellate Prorocentrum minimum) plus one additional species (the dinoflagellate Akashiwo cf. sanguinea) that was unaffected by bloom exudates. K. brevis exudates lowered photosynthetic efficiency and damaged cell membranes of competing phytoplankton, but had no effect on competitor esterase activity, nor did they limit competitor access to iron. Overall, during blooms, K. brevis exudes potent allelopathic compounds, competitors vary in their susceptibility to K. brevis allelopathy, and K. brevis may achieve nearly monospecific blooms by lowering the photosynthetic efficiency of competitor species and increasing competitor membrane permeability, eventually resulting in competitor growth suppression or death.

Peptide toxins from animal venom have been used for many years for the identification and study of cation-permeable ion channels. However, no peptide toxins have been identified that interact with known anion-selective channels, including cystic fibrosis transmembrane conductance regulator (CFTR), the protein defective in cystic fibrosis and a member of the ABC transporter superfamily. Here, we describe the identification and initial characterization of a novel 3.7-kDa peptide toxin, GaTx1, which is a potent and reversible inhibitor of CFTR, acting from the cytoplasmic side of the membrane. Thus, GaTx1 is the first peptide toxin identified that inhibits a chloride channel of known molecular identity. GaTx1 exhibited high specificity, showing no effect on a panel of nine transport proteins, including Cl(-) and K(+) channels, and ABC transporters. GaTx1-mediated inhibition of CFTR channel activity is strongly state-dependent; both potency and efficacy are reduced under conditions of elevated [ATP], suggesting that GaTx1 may function as a non-competitive inhibitor of ATP-dependent channel gating. This tool will allow the application of new quantitative approaches to study CFTR structure and function, particularly with respect to the conformational changes that underlie transitions between open and closed states.

In order to test whether phytoplankton such as the red tide dinoflagellate Karenia brevis negatively affect rotifer grazers and what mechanisms are involved in these interactions, we conducted laboratory feeding experiments using K. brevis cultured from Gulf of Mexico coastal waters and two species of rotifers, one co-occurring and the other from an inland sea in Russia. The co-occurring rotifer Brachionus ibericus did not ingest K. brevis as a sole diet but tolerated K. brevis in a diet mixed with Rhodomonas lens, whereas the allopatric rotifer, Brachionus plicatilis, rejected K. brevis in either diet, although B. plicatilis started feeding on the mixed diet after a delay of 4 days. The deterrence of K. brevis was mediated by its cellular organic extracts and not by exudates, with compounds other than brevetoxins PbTx-2, PbTx-3, and PbTx-9 implicated in deterrence. The finding that closely related rotifers respond differently to K. brevis and to its chemical defense suggests that a shared evolutionary history may have led B. ibericus to tolerate K. brevis in a mixed diet.

Marine and terrestrial studies show that small, sedentary herbivores that utilize plants as both food and habitat can gain enemy-free space by living on hosts that are chemically defended from larger, generalist consumers. Although large herbivores are increasingly recognized as important consumers of macrophytes in freshwater communities, the potential indirect effects of herbivory on plant-associated macroinvertebrates have rarely been studied. Here, we show that the large, generalist consumers in a riverine system, Canada geese, Branta canadensis , and crayfish, Procambarus spiculifer , both selectively consumed riverweed, Podostemum ceratophyllum , over an aquatic moss, Fontinalis novae-angliae, even though moss comprised 89% of the total plant biomass on riverine rocky shoals. Moss supported twice as many plant-associated macroinvertebrates as riverweed, suggesting that it might provide a spatial refuge from consumption by these larger consumers. Bioassay-guided fractionation of moss extracts led to the isolation of a C18 acetylenic acid, octadeca-9,12-dien-6ynoic acid, that deterred crayfish feeding. In contrast to results with Canada geese and crayfish, both the amphipod Crangonyx gracilis and the isopod Asellus aquaticus consumed significant amounts of moss but rejected riverweed in laboratory feeding assays. Moreover, neither amphipod nor isopod feeding was deterred by the crude organic extract of Fontinalis , suggesting that these mesograzers tolerate or circumvent the chemical defenses that deterred larger consumers. Thus, herbivory by large, generalist herbivores may drive freshwater plant community structure towards chemically defended plants and favor the ecological specialization of smaller, less mobile herbivores on unpalatable hosts that represent enemy-free space.

Chemical cues regulate key ecological interactions in marine and terrestrial ecosystems. They are particularly important in terrestrial plant–herbivore interactions, where they mediate both herbivore foraging and plant defense. Although well described for terrestrial interactions, the identity and ecological importance of herbivore foraging cues in marine ecosystems remain unknown. Here we show that the specialist gastropod Elysia tuca hunts its seaweed prey, Halimeda incrassata, by tracking 4-hydroxybenzoic acid to find vegetative prey and the defensive metabolite halimedatetraacetate to find reproductive prey. Foraging cues were predicted to be polar compounds but instead were nonpolar secondary metabolites similar to those used by specialist terrestrial insects. Tracking halimedatetraacetate enables Elysia to increase in abundance by 12- to 18-fold on reproductive Halimeda, despite reproduction in Halimeda being rare and lasting for only ∼36 h. Elysia swarm to reproductive Halimeda where they consume the alga’s gametes, which are resource rich but are chemically defended from most consumers. Elysia sequester functional chloroplasts and halimedatetraacetate from Halimeda to become photosynthetic and chemically defended. Feeding by Elysia suppresses the growth of vegetative Halimeda by ∼50%. Halimeda responds by dropping branches occupied by Elysia, apparently to prevent fungal infection associated with Elysia feeding. Elysia is remarkably similar to some terrestrial insects, not only in its hunting strategy, but also its feeding method, defense tactics, and effects on prey behavior and performance. Such striking parallels indicate that specialist herbivores in marine and terrestrial systems can evolve convergent ecological strategies despite 400 million years of independent evolution in vastly different habitats.

Interactions among microscopic planktonic organisms underpin the functioning of open ocean ecosystems. With few exceptions, these organisms lack advanced eyes and thus rely largely on chemical sensing to perceive their surroundings. However, few of the signaling molecules involved in interactions among marine plankton have been identified. We report a group of eight small molecules released by copepods, the most abundant zooplankton in the sea, which play a central role in food webs and biogeochemical cycles. The compounds, named copepodamides, are polar lipids connecting taurine via an amide to isoprenoid fatty acid conjugate of varying composition. The bloom-forming dinoflagellate Alexandrium minutum responds to pico- to nanomolar concentrations of copepodamides with up to a 20-fold increase in production of paralytic shellfish toxins. Different copepod species exude distinct copepodamide blends that contribute to the species-specific defensive responses observed in phytoplankton. The signaling system described here has far reaching implications for marine ecosystems by redirecting grazing pressure and facilitating the formation of large scale harmful algal blooms.

TanDEM-X is a recent SAR mission, consisting of two almost identical spacecraft flying in close formation. The small distance between the two radar satellites allows two images to be acquired at the same time (bistatic images), strongly reducing the influence of temporal decorrelation, which is one of the major sources of error in repeat-pass interferometric analyses. For the first time, we successfully apply TanDEM-X data to observe topographic changes at active volcanoes by using the image pairs to generate high-resolution digital surface models (DSMs) for each transit of the satellites. Taking the difference between two bistatic DSMs allows us to assess substantial topographic changes and/or sudden ground displacements above the 1 m level. As the first test case, we used bistatic TanDEM-X data to assess topographic change due to the major Merapi 2010 eruption. The preliminary estimated volumetric loss of 19 × 106 m3 is reasonable; however, strong phase noise due to geometrical decorrelation and resulting unwrapping errors affect the result. To demonstrate that much smaller topographic changes are observable with TanDEM-X, we further analyzed data acquired before and after a small explosion at Volcan de Colima in June 2011. The estimated volume loss of 2 × 105 m3 fits well to ground truth data.

Bromophycolides C-I (1−7) were isolated from extracts of the Fijian red alga Callophycus serratus, and identified by NMR and mass spectral techniques. These novel natural products share a carbon skeleton and biosynthetic origin with previously identified bromophycolides A (8) and B (9), which form a rare group of diterpene-benzoate macrolides. Bromophycolides C-I (1−7) displayed modest antineoplastic activity against a range of human tumor cell lines.

The defining feature of the life cycle in monogonont rotifers such as Brachionus plicatilis (Muller) is alternation of asexual and sexual reproduction (mixis). Why sex is maintained in such life cycles is an important unsolved evolutionary question and one especially amenable to experimental analysis. Mixis is induced by a chemical signal produced by the rotifers which accumulates to threshold levels at high population densities. The chemical features of this signal were characterized using size exclusion, enzymatic degradation, protease protection assays, selective binding to anion ion exchange and C3 reversed phase HPLC columns, and the sequence of 17 N-terminal amino acids. These studies were carried out over two years beginning in 2003 using B. plicatilis Russian strain. When rotifer-conditioned medium was treated with proteinase K, its mixis-inducing ability was reduced by 70%. Proteinase K was added to medium auto-conditioned by 1 female ml−1 where typically 17% of daughters became mictic and mixis was reduced to 1%. A cocktail of protease inhibitors added to conditioned medium significantly reduced degradation of the mixis signal by natural proteases. Conditioned medium subjected to ultrafiltration retained mixis-inducing activity in the >10 kDa fraction, but the

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.

[structures: see text] Three diterpene-benzoate natural products, with novel carbon skeletons and an unusual proposed biosynthesis, were isolated from extracts of the Fijian red alga Callophycus serratus and identified by a combination of X-ray crystallographic, NMR, and mass spectral analyses. Bromophycolide A (1) displayed cytotoxicity against several human tumor cell lines via specific apoptotic cell death. This represents the first discovery of natural products incorporating a diterpene and benzoate skeleton into a macrolide system.

Monospecific blooms of phytoplankton can disrupt pelagic communities and negatively affect human health and economies. Interspecific competition may play an important role in promoting blooms, and so we tested (1) whether the outcome of competition between the red tide dinoflagellate Karenia brevis (ex Gymnodinium breve) and 12 cooccurring phytoplankters could be explained by allelopathic effects of compounds released by K. brevis and (2) whether waterborne, lipophilic molecules, including brevetoxins, are involved. Nine of 12 phytoplankton species were suppressed when grown with live K. brevis at bloom concentrations. K. brevis extracellular filtrates or lipophilic extracts of filtrates inhibited six of these nine species, indicating allelopathy. However, these inhibitory effects were weaker than those experienced by competitors exposed to live K. brevis. Brevetoxins at ecologically reasonable waterborne concentrations accounted for the modest inhibition by K. brevis of only one competitor, Skeletonema costatum. The addition of brevetoxins also caused significant autoinhibition, reducing the maximum concentration of K. brevis. Allelopathy is one mechanism by which K. brevis appears to exhibit competitive advantage over some sympatric phytoplankters, although unidentified compounds other than brevetoxins must be involved, in most cases. K. brevis was also susceptible to competitive exclusion by several species, including Odontella aurita and Prorocentrum minimum, known to thrive during K. brevis blooms. Although field experiments are required to assess whether allelopathy plays a fundamental role in bloom dynamics, our results indicate that allelopathy occurs widely but with species-specific consequences.

Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified that interact with known anion-selective channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR channels are expressed in epithelial cells and are associated with several genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the Cl−permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered by the lack of a high-affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion Leiurus quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing single-channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or adenosine 5′-(β,γ-imido)triphosphate, and block was strengthened on reduction of extracellular Cl−concentration, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore-block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function.

Worms and other marine invertebrates living in soft sediments commonly produce brominated natural products that have been hypothesized to function as defenses against consumers, but this hypothesis has not been tested directly. When 16 species of worms from a Georgia mud flat were fed to two sympatric fishes (Fundulus heteroclitus, Leiostomus xanthurus) and a crab (Callinectes similis), 15 species (94%) were palatable to all three predators. Only the hemichordate Saccoglossus kowalevskii was unpalatable to both fishes, but even it was readily consumed by the crab. Bioassay-guided chemical investigations demonstrated that Saccoglossus kowalevskiiwas rejected by fishes because it contained 2,3,4-tribromopyrrole at 0.2% of worm dry mass. This is the first direct test of brominated worm metabolites as defenses against sympatric consumers. The deterrence of 2,3,4-tribromopyrrole in S. kowalevskii may explain why densities of this worm increase 40-fold during seasons when predation is high and densities of palatable worms decline sharply. To more broadly examine the effects of brominated metabolites on worm palatability, we collected from North Carolina, Georgia, and Florida 14 species reported to produce brominated metabolites. These species were fed to sympatric consumers to assess palatability and also analyzed for brominated metabolites by gas chromatography mass spectrometry (GC/MS). Nine of the fourteen species contained brominated metabolites, but only two were unpalatable. In a final test, five additional brominated metabolites produced by marine worms were added to palatable foods at natural concentrations and at up to 20 3 natural concentrations. None deterred feeding at natural concentrations, one was deterrent at 5‐153 natural concentration, and four had no effect at even 203 natural concentration. Thus, while one worm was defended by a brominated compound, most worms containing brominated metabolites were palatable, and brominated natural products seldom functioned as chemical defenses against consumers.

Brown seaweeds (Fucales) produce phlorotannins that are often considered chemical defenses against herbivores. The many correlative and fewer direct tests conducted have shown effects of phlorotannins on herbivore feeding behavior to be variable. In an attempt to clarify the roles of phlorotannins versus other metabolites in defending brown algae, we conducted bioassay-guided fractionation of herbivore-deterrent extracts from the commonly studied brown alga Fucus vesiculo- sus. Feeding by the amphipods Ampithoe valida and A. longimana and the sea urchin Arbacia punc- tulata was suppressed by crude and water-soluble extracts of F. vesiculosus, but this deterrence was lost following storage or fractionation of the active, water-soluble extract. Phlorotannins in these extracts did not decompose in parallel with the loss of feeding deterrence. F. vesiculosus phloro- tannins were fed to herbivores at 3 to 12 × the isolated yield (or 4.2 to 16.8% of plant dry mass). No herbivore was deterred from feeding by concentrations of 3 or 6 ×, but A. valida (the only test herbi- vore that readily consumes F. vesiculosus in the field) was deterred at 12 × isolated yield. When juve- nile A. valida were raised on an artificial diet containing F. vesiculosus phlorotannins at 3 × isolated yield, the phlorotannin-rich diet significantly enhanced, rather than reduced, amphipod survivorship and growth relative to an equivalent diet without phlorotannins. Females ovulated only on the phlorotannin-rich diet. Compounds other than phlorotannins appear to defend the F. vesiculosus populations we investigated, but we were unable to identify these unstable compounds.

When offered a choice between brown seaweeds (Phaeophyta) from shallow inshore populations versus deeper offshore populations along the mid-Atlantic coast of the United States of America, the herbivorous amphipod Ampithoe longimana consistently preferred plants from the inshore populations. This was the case for three species (Dictyota menstrualis, Spatoglossum schroederi, and Sargassum filipendula) collected from each of a single inshore and offshore site, and for one species (D. menstrualis) collected from each of three inshore and three offshore sites. Bioassay-guided fractionation of chemical crude extracts from D. menstrualis suggested that the relative unpalatability of the offshore plants was due to the lipid-soluble secondary metabolites 4beta-hydroxydictyodial A and 18, O-dihydro-4beta-hydroxydictyodial A 18-acetate, along with minor compounds that were not fully identified. The inshore-offshore pattern did not appear to result from induction of defenses due to herbivory by mesograzers, as mesograzer densities were higher on the more palatable inshore plants. Herbivore feeding preferences for inshore versus offshore seaweeds matched the effects of those seaweeds on their fitness. When juvenile amphipods were raised on inshore versus offshore tissues of D. menstrualis, amphipod survivorship, growth, and ovulation were significantly suppressed on the offshore compared to the inshore tissues. Few previous investigations have studied intraspecific variance in seaweed palatability. We extend these by showing that between-population differences in palatability can persist for several years and by demonstrating that this variance is chemically based and has dramatic effects on herbivore fitness.

Numerous studies have investigated chemical defenses among sessile species growing on hard substrates, but few have addressed this for mobile species in soft-sediment communities. We investigated the palatability and potential chemical defenses of 11 worm species from soft-sediment systems in southern Florida, USA. Three species were unpalatable to the bluehead wrasse Thalassoma bifasciatum. The polychaete Cirriformia tentaculata and the hemichordate Ptychodera bahamensis were uniformly unpalatable. For the polychaete Eupolymnia crassicornis, the exposed tentacles were unpalatable, but the body, which remains protected in a deeply buried tube, was palatable. These unpalatable worms were chemically defended; extracts of C. tentaculata, P. bahamensis, and the tentacles of E. crassicornis deterred fish feeding. For C. tentaculata, bioassay-guided fractionation demonstrated that a mixture of 3 closely related alkylpyrrole sulfamates deterred fish at naturally occurring concentrations (2-n-hexylpyrrole sulfamate (1.6% of worm dry mass), 2-n-heptylpyrrole sulfamate (3.1% dry mass), and 2-n-octylpyrrole sulfamate (0.8% dry mass)). This appears to be the first documentation of characterized natural products defending a marine worm from consumers. For P. bahamensis and the tentacles of E. crassicornis, deterrent effects of crude extracts decomposed before specific compounds could be identified.

Ten fish mortality events, involving primarily Atlantic menhaden, occurred from early July through September 2000 in several bays and creeks in Delaware, USA. Two events involved large mortalities estimated at 1–2.5 million fish in Bald Eagle Creek, Rehoboth Bay. Samples from Indian Inlet (Bethany Beach), open to the Atlantic, as well as from an enclosed area of massive fish kills at nearby Bald Eagle Creek and Torque Canal were collected and sent to our laboratory for analysis. Microscopic examination of samples from the fish kill site revealed the presence of a single-cell Raphidophyte alga Chattonella cf. verruculosa at a maximum density of 1.04 × 107 cells/L. Naturally occurring brevetoxins were also detected in the bloom samples. Besides the Chattonella species, no other known brevetoxin-producing phytoplankton were present. Chromatographic, immunochemical, and spectroscopic analyses confirmed the presence of brevetoxin PbTx-2, and PbTx-3 and -9 were confirmed by chromatographic and immunochemical analyses. This is the first confirmed report in the United States of brevetoxins associated with an indigenous bloom in temperate Atlantic estuarine waters and of C. cf. verruculosa as a resident toxic organism implicated in fish kills in this area. The bloom of Chattonella continued throughout September and eventually declined in October. By the end of October C. cf. verruculosa was no longer seen, nor was toxin measurable in the surface waters. The results affirm that to avoid deleterious impacts on human and ecosystem health, increased monitoring is needed for brevetoxins and organism(s) producing them, even in areas previously thought to be unaffected. Key words: brevetoxins, Chattonella cf. verruculosa, Delaware, fish kills, harmful agal blooms. Environ Health Perspect 110:465–470 (2002). [Online 1 April 2002] http://ehpnet1.niehs.nih.gov/docs/2002/110p465-470bourdelais/abstract.html

We developed a competitive enzyme-linked immunosorbent assay (ELISA) to analyze brevetoxins, using goat anti-brevetoxin antibodies obtained after immunization with keyhole limpet hemocyanin-brevetoxin conjugates, in combination with a three-step signal amplification process. The procedure, which used secondary biotinylated antibodies, streptavidine-horseradish peroxidase conjugate, and chromogenic enzyme substrate, was useful in reducing nonspecific background signals commonly observed with complex matrices. This competitive ELISA detected brevetoxins in seawater, shellfish extract and homogenate, and mammalian body fluid such as urine and serum without pretreatment, dilution, or purification. We investigated the application of this technique for shellfish monitoring by spiking shellfish meat with brevetoxins and by analyzing oysters from two commercial shellfish beds in Florida that were exposed to a bloom of Karenia brevis (formerly Gymnodinium breve). We performed brevetoxin analysis of shellfish extracts and homogenates by ELISA and compared it with the mouse bioassay and receptor binding assay. The detection limit for brevetoxins in spiked oysters was 2.5 microg/100 g shellfish meat. This assay appears to be a useful tool for neurotoxic shellfish poisoning monitoring in shellfish and seawater, and for mammalian exposure diagnostics, and significantly reduces the time required for analyses.

Aquatic organisms produce compounds that deter consumers, alter prey behavior, suppress or kill target and nontarget species, and dramatically affect food-web structure, community composition, and the rates and pathways of biogeochemical cycles. Toxins from marine and freshwater phytoplankton create health hazards for both aquatic and terrestrial species and can significantly affect human activities and the economic vitality of local communities. A reasonable case can be made that phytoplankton metabolites such as dimethyl sulfide (DMS) link interaction webs that span hundreds to thousands of kilometers and connect production from oceanic phytoplankton to desert cacti and coyotes via zooplankton, fishes, and sea birds. The possible role of DMS in global heat budgets expands this effect even further. The ecosystem-wide and potentially global consequences of aquatic chemical cues is an underappreciated topic that warrants additional attention.