Polyunsaturated Aldehydes Research Articles

Assessment of sex-related variability of biomarkers in sea urchin (Paracentrotus lividus) from Bizerte lagoon, Tunisia

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants.

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

PUFAs and PUAs production in three benthic diatoms from the northern Adriatic Sea

The production of polyunsaturated aldehydes (PUAs) has been reported by many planktonic diatoms, where they have been implicated in deleterious effects on copepod reproduction and growth of closeby microbes or suggested as infochemicals in shaping plankton interactions. This study investigates the production of PUAs by diatoms commonly occurring in the microphytobenthic communities in temperate regions: Tabularia affinis, Proschkinia complanatoides and Navicula sp. Results highlight the production of PUAs by the three benthic diatoms during stationary and decline phases, with intracellular concentrations from 1.8 to 154.4 fmol cell−1, which are within the range observed for planktonic species. The existence of a large family of PUAs, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal, was observed. Since particulate and dissolved PUAs were positively correlated, together with cell lysis, equivalent concentrations may be released during late growth stages, which may affect benthic invertebrates grazing on them and other microalgae.

Defense related decadienal elicits membrane lipid remodeling in the diatom Phaeodactylum tricornutum.

Diatoms rapidly release extracellular oxylipins (oxygenated lipids) including polyunsaturated aldehydes in response to herbivory and other stresses. Oxylipins have several defense-related activities including inhibition of reproduction in herbivores and signaling to distant diatoms. Physiological changes in diatoms exposed to varying levels of oxylipins are only beginning to be understood. In this study, Phaeodactylum tricornutum cultures were treated with sublethal concentrations of the polyunsaturated aldehyde trans,trans-2,4-decadienal (DD) to assess effects on lipid composition and membrane permeability. In cells treated with DD for 3 hr, all measured saturated and unsaturated fatty acids significantly decreased (0.46–0.69 fold of levels in solvent control cells) except for 18:2 (decreased but not significantly). The decrease was greater in the polyunsaturated fatty acid pool than the saturated and monounsaturated fatty acid pool. Analysis of lipid classes revealed increased abundances of phosphatidylethanolamine and phosphatidylcholine at 3 and 6 hr. Concomitantly, these and other membrane lipids exhibited increased saturated and monounsaturated acyl chains content relative to polyunsaturated acyl chains compared to control cells. Evidence of decreased plasma membrane permeability in DD treated cells was obtained, based on reduced uptake of two of three dyes relative to control cells. Additionally, cells pre-conditioned with a sublethal DD dose for 3 hr then treated with a lethal DD dose for 2 hr exhibited greater membrane integrity than solvent pre-conditioned control cells that were similarly treated. Taken together, the data are supportive of the hypothesis that membrane remodeling induced by sublethal DD is a key element in the development of cellular resistance in diatoms to varying and potentially toxic levels of polyunsaturated aldehydes in environments impacted by herbivory or other stresses.

Effect of dissolved polyunsaturated aldehydes on the size distribution of transparent exopolymeric particles in an experimental diatom bloom

Diatoms constitute a widespread group of phytoplanktonic species that thrive in coastal waters and produce episodical blooms. During these blooms, these diatoms release several organic compounds into the environment. Two types of organic molecules released by diatoms are the polyunsaturated aldehydes (PUA) and exopolymeric substances (EPS). EPS can spontaneously form particles called transparent exopolymeric particles (TEP). Spatial and temporal distributions of both, PUA and TEP, are linked to phytoplankton abundance, and peaks of both have been reported after diatom bloom episodes in the photic zone. For the first time, we have inferred interactions between both kinds of compounds by analyzing the effects of a mix of dissolved PUA (heptadienal, octadienal and decadienal) on TEP production in experimental microcosms. Our results show that the presence of dissolved PUA significantly increased TEP size and generated larger aggregates (>200 μm) towards the end of the bloom period, probably by increasing TEP stickiness. Since TEP production in nature enhances particle aggregation and carbon export from euphotic to deeper zones in aquatic ecosystems, this could imply important effects on the vertical flow of materials in the pelagic system and changes in the size distribution of available food particles after bloom events of PUA-producer species.

New inter-correlated genes targeted by diatom-derived polyunsaturated aldehydes in the sea urchin Paracentrotus lividus

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants.

Toxicity of diatom polyunsaturated aldehydes to marine bacterial isolates reveals their mode of action

Diatoms produce and release polyunsaturated aldehydes (PUAs) during senescence in culture and at the end of blooms in nature and these compounds play different ecological roles, as infochemicals, allelochemicals and pheromones In order to elucidate the toxic effects of PUAs, we isolated six bacterial strains from the Mediterranean Sea during a diatom bloom and tested their tolerance to PUA in terms of growth and cell membrane properties. Based upon 16S rRNA sequencing, these bacteria were assigned to the genera Pseudomonas, Sufflavibacter, Halomonas, Vibrio, Idiomarina, and Labrenzia. Growth of these strains was reduced by 50% (EC50) at PUA concentrations ranging from 600 to 1700 μM of 2E,4E/Z-heptadienal (HEPTA), 400–800 μM of 2E, 4E/Z-octadienal (OCTA), and 70–400 μM of 2E, 4E/Z-decadienal (DECA). Two of these strains, Vibrio sp. and Halomonas, sp. were also investigated for membrane fatty acid composition in terms of adaptive modifications of their degree of saturation (ratio between saturated and unsaturated fatty acids) by GC-FID. A direct correlation between hydrophobicity and PUA toxicity was observed, and these bacteria were also found to react to PUAs by increasing the degree of saturation of their membranes fatty acids.Tested PUAs were 4-fold more toxic than the well-investigated n-alkanols, most probably due to their additional chemical aldehyde toxicity to disrupting proteins by the formation of Schiff's bases, and therefore, they act as very toxic and effective poison, probably accumulating in cytoplasmic membranes because of their high hydrophobicity.

Chytridiomycosis of Marine Diatoms-The Role of Stress Physiology and Resistance in Parasite-Host Recognition and Accumulation of Defense Molecules.

Little is known about the role of chemotaxis in the location and attachment of chytrid zoospores to potential diatom hosts. Hypothesizing that environmental stress parameters affect parasite-host recognition, four chytrid-diatom tandem cultures (Chytridium sp./Navicula sp., Rhizophydium type I/Nitzschia sp., Rhizophydium type IIa/Rhizosolenia sp., Rhizophydium type IIb/Chaetoceros sp.) were used to test the chemotaxis of chytrid zoospores and the presence of potential defense molecules in a non-contact-co-culturing approach. As potential triggers in the chemotaxis experiments, standards of eight carbohydrates, six amino acids, five fatty acids, and three compounds known as compatible solutes were used in individual and mixed solutions, respectively. In all tested cases, the whole-cell extracts of the light-stressed (continuous light exposure combined with 6 h UV radiation) hosts attracted the highest numbers of zoospores (86%), followed by the combined carbohydrate standard solution (76%), while all other compounds acted as weak triggers only. The results of the phytochemical screening, using biomass and supernatant extracts of susceptible and resistant host-diatom cultures, indicated in most of the tested extracts the presence of polyunsaturated fatty acids, phenols, and aldehydes, whereas the bioactivity screenings showed that the zoospores of the chytrid parasites were only significantly affected by the ethanolic supernatant extract of the resistant hosts.

A fast and direct liquid chromatography‐mass spectrometry method to detect and quantify polyunsaturated aldehydes and polar oxylipins in diatoms

AbstractPolyunsaturated aldehydes (PUAs) are a group of microalgal metabolites that have attracted a lot of attention due to their biological activity. Determination of PUAs has become an important routine procedure in plankton and biofilm investigations, especially those that deal with chemically mediated interactions. Here we introduce a fast and direct derivatization free method that allows quantifying PUAs in the nanomolar range, sufficient to undertake the analysis from cultures and field samples. The sample preparation requires one simple filtration step and the initiation of PUA formation by cell disruption. After centrifugation the samples are ready for measurement without any further handling. Within one chromatographic run this method additionally allows us to monitor the formation of the polar oxylipins arising from the cleavage of precursor fatty acids. The robust method is based on analyte separation and detection using ultra high performance liquid chromatography‐atmospheric pressure chemical ionization mass spectrometry (UHPLC‐APCI MS) and enables high throughput investigations by employing an analysis time of only 5 min. Our protocol thus provides an alternative and extension to existing PUA determinations based on gas chromatography‐mass spectrometry (GC‐MS) with shorter run times and without any chemical derivatization. It also enables researchers with widely available LC‐MS analytical platforms to monitor PUAs. Additionally, non‐volatile oxylipins such as ω‐oxo‐acids and related compounds can be elucidated and monitored.

Inhibitory effect of polyunsaturated aldehydes (PUAs) on the growth of the toxic benthic dinoflagellate Ostreopsis cf. ovata

Diatoms have been shown to produce and release a wide range of secondary metabolites that mediate interactions between individuals of different species. Among these compounds, different types of fatty acid derived long-chained polyunsaturated aldehydes (PUAs) have been related to multiple functions such as intra- or interspecific signals and adverse effect on the reproduction of marine organisms. Several studies have reported changes on growth, cell membrane permeability, flow cytometric properties and cell morphology in phytoplankton organisms exposed to PUAs, but little information is available on the effect of these compounds on benthic microalgae. Ostreopsis cf. ovata is a toxic benthic dinoflagellate which causes massive blooms along the Mediterranean coasts typically during the late summer period. In this study the effects of three toxic PUAs known to be produced by several algae (2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal) on the growth, cytological features and cell morphology of O. cf. ovata were investigated. Our results show a clear decrease of O. cf. ovata growth with longer-chain molecules than with shorter-chain ones, confirmed also by EC50 values calculated at 48h for 2E,4E-decadienal and 2E,4E-octadienal (6.6±1.5, 17.9±2.6μmolL−1 respectively) and at 72h for 2E,4E-heptadienal (18.4±0.7μmolL−1). Moreover, morphological analysis highlighted up to 79% of abnormal forms of O. cf. ovata at the highest concentrations of 2E,4E-decadienal tested (9, 18 and 36μmolL−1), a gradual DNA degradation and an increase of lipid droplets with all tested PUAs. Further studies are needed to better clarify the interactions between diatoms and O. cf. ovata, especially on bloom-forming dynamics.

Spatial distributions of polyunsaturated aldehydes and their biogeochemical implications in the Pearl River Estuary and the adjacent northern South China Sea

This study reports the first comprehensive exploration of the spatial patterns of dissolved and particulate polyunsaturated aldehydes (PUAs), their physical and biological controlling factors, and their potential biogeochemical influences in the Pearl River Estuary (PRE) of the northern South China Sea (NSCS). High levels of total particulate PUAs (0–41nM) and dissolved PUAs (0.10–0.37nM) were observed with substantial spatial variation during an intense summer phytoplankton bloom outside the PRE mouth. We found the particulate PUAs strongly correlated with temperature within the high chlorophyll bloom, while showing a generally positive correlation with chlorophyll-a for the entire region. Additionally, the Si/N ratio significantly correlated with the particulate PUAs along the estuary suggesting the important role of silica on PUA production in this region. The dissolved PUAs counterparts exhibited a positive correlation with chlorophyll-a within the high chlorophyll bloom, but a negatively one with temperature outside, reflecting the essential bio-physical coupling effects on the dissolved PUAs distributions in the ocean. Biogeochemical implications of PUAs on the coastal ecosystem include not only the deleterious restriction of high PUAs-producing diatom bloom on copepod population, but also the profound influence of particulate PUAs on the microbial cycling of organic carbon in the NSCS.

Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae).

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S.marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S.marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR-derived green fluorescence in S.marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA-exposed S.marinoi cells were treated with the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an opposite response was observed, with an increase in DHR-derived green fluorescence. A higher DHR-derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2-methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20min. These results indicate the involvement of O2 (•) generation and SOD activity in H2 O2 formation (with downstream ROS generation dependent from H2 O2 ) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA-exposed S.marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O2 (•-) and H2 O2 . This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.

Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7

Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments.

First Morphological and Molecular Evidence of the Negative Impact of Diatom-Derived Hydroxyacids on the Sea Urchin Paracentrotus lividus.

Oxylipins (including polyunsaturated aldehydes [PUAs], hydoxyacids, and epoxyalcohols) are the end-products of a lipoxygenase/hydroperoxide lyase metabolic pathway in diatoms. To date, very little information is available on oxylipins other than PUAs, even though they represent the most common oxylipins produced by diatoms. Here, we report, for the first time, on the effects of 2 hydroxyacids, 5- and 15-HEPE, which have never been tested before, using the sea urchin Paracentrotus lividus as a model organism. We show that HEPEs do induce developmental malformations but at concentrations higher when compared with PUAs. Interestingly, HEPEs also induced a marked developmental delay in sea urchin embryos, which has not hitherto been reported for PUAs. Recovery experiments revealed that embryos do not recover following treatment with HEPEs. Finally, we report the expression levels of 35 genes (involved in stress, development, differentiation, skeletogenesis, and detoxification processes) to identify the molecular targets affected by HEPEs. We show that the 2 HEPEs have very few common molecular targets, specifically affecting different classes of genes and at different times of development. In particular, 15-HEPE switched on fewer genes than 5-HEPE, upregulating mainly stress-related genes at a later pluteus stage of development. 5-HEPE was stronger than 15-HEPE, targeting 24 genes, mainly at the earliest stages of embryo development (at the blastula and swimming blastula stages). These findings highlight the differences between HEPEs and PUAs and also have important ecological implications because many diatom species do not produce PUAs, but rather these other chemicals are derived from the oxidation of fatty acids.

Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins.

Marine organisms possess a series of cellular strategies to counteract the negative effects of toxic compounds, including the massive reorganization of gene expression networks. Here we report the modulated dose-dependent response of activated genes by diatom polyunsaturated aldehydes (PUAs) in the sea urchin Paracentrotus lividus. PUAs are secondary metabolites deriving from the oxidation of fatty acids, inducing deleterious effects on the reproduction and development of planktonic and benthic organisms that feed on these unicellular algae and with anti-cancer activity. Our previous results showed that PUAs target several genes, implicated in different functional processes in this sea urchin. Using interactomic Ingenuity Pathway Analysis we now show that the genes targeted by PUAs are correlated with four HUB genes, NF-κB, p53, δ-2-catenin and HIF1A, which have not been previously reported for P. lividus. We propose a working model describing hypothetical pathways potentially involved in toxic aldehyde stress response in sea urchins. This represents the first report on gene networks affected by PUAs, opening new perspectives in understanding the cellular mechanisms underlying the response of benthic organisms to diatom exposure.

The deleterious effect of diatoms on the biomass and growth of early stages of their copepod grazers

Diatoms are key phytoplankton species in the world oceans and essential in the transfer of energy through marine food chains. Their beneficial role has been questioned after the discovery that some diatom species produce teratogenic compounds such as polyunsaturated aldehydes and other oxylipins that induce abortions, poor larval development and high offspring mortality in many invertebrates. To test whether maternal and post-embryonic diatom diets affected development in the copepod Paracartia latisetosa, we carried out experiments in which the PUA-producing diatom Skeletonema marinoi (SKE) was fed to this copepod species. Controls were run with the dinoflagellate, Prorocentrum minimum (PRO), which does not produce any of these compounds. Four treatments were tested: both mothers and progeny always fed on either SKE or PRO, and those in which mothers received either PRO or SKE, and neonates were switched to the other diet. Our results show that a SKE/SKE diet induced lower egg production and egg viability, as well as slower embryonic development than the control diet. Naupliar development was negatively affected with the diatom diet which blocked the metamorphosis of larvae at N3, 4days after hatching, compared to the control diet that allowed for normal naupliar development in 10days. Nauplii reared on PRO and spawned by females fed on SKE were able to complete development, but only after 11days, whereas larvae reared on SKE spawned by females fed on PRO were arrested at the N5 developmental stage, 12days after hatching. Apoptotic TUNEL-positive nauplii were detected with the SKE diet, indicating imminent death. Significant differences were also recorded by comparing biomass of equivalent naupliar stages obtained for each treatment, and the overall mean biomass female−1day−1 for each diet. Growth inhibition due to oxylipin-producing diatoms may have important consequences on prey–predator relationships and on the biomass flux through marine food chains.

Expression of death-related genes and reactive oxygen species production in Skeletonema tropicum upon exposure to the polyunsaturated aldehyde octadienal

<p>The effects of 4<em>E</em>/<em>Z</em>-octadienal (OCTA) on <em>ScDSP-1 </em>and <em>ScDSP-2 </em>gene expression and reactive oxygen species (ROS) production were investigated in the marine diatom <em>Skeletonema tropicum</em> (formerly <em>costatum</em>) using qRTPCR and flow cytometry. <em>ScDSP-1 </em>and <em>ScDSP-2 </em>genes have been previously shown to be involved in cell death in ageing cells and in response to photosynthetic stress. OCTA induced a differential, concentration-dependent <em>DSP</em> gene expression associated to ROS production, 821.6 and 97.7 folds higher for <em>ScDSP-1</em> and <em>ScDSP-2</em>, respectively. Among the concentrations tested, only 8 μM OCTA, which caused a reduction of 50% in cell concentrations at 24 h, was able to elicit an expression pattern consistent with a signalling role. Interestingly, only intermediate levels of reactive oxygen species (ROS) (<em>i.e</em>., 1.5±0.1 increase) were observed to be elicited by such concentration. These results suggest that ROS are key components of the molecular cascade triggered by polyunsaturated aldehydes (PUA) and leading to cell death. This could have implications for bloom final stages at sea, where PUA may act as effectors of diatom population dynamics through ROS acting as modulators.</p>

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum.

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.

Underestimation of microzooplankton grazing in dilution experiments due to inhibition of phytoplankton growth

AbstractMicrozooplankton dilution grazing experiments conducted in phytoplankton rich waters, particularly in polar and subpolar seas, often result in calculation of nonsignificant or negative grazing coefficients. We hypothesized that preparation of filtered seawater (FSW) from water containing high biomass of phytoplankton results in release of allelochemicals that inhibit phytoplankton growth, lowering the net growth of phytoplankton in the more diluted treatments. We tested this hypothesis during blooms of Skeletonema marinoi and Phaeocystis pouchetii in a nutrient‐enriched mesocosm in the Raunefjord, Norway. During the S. marinoi bloom, inhibition of phytoplankton growth occurred in the diluted treatments. Simultaneously the concentration of total as well as dissolved polyunsaturated aldehydes (PUAs) was elevated. Passage of the FSW through a carbon cellulose cartridge to remove dissolved organic material reduced, or eliminated, the inhibition. In the early phase of the P. pouchetii bloom that followed the diatom bloom in the mesocosm, PUA concentration was relatively low and the untreated FSW had a less drastic, but often significant, inhibitory effect on phytoplankton growth. Laboratory experiments with cultures of S. marinoi and P. pouchetii confirmed that material present in filtrate prepared from diluted cultures was self‐inhibitory. Many phytoplankters, particularly during late stages of a bloom, produce inhibitory metabolites that may be released during filtration of the relatively large volumes of seawater needed for dilution experiments. Under some conditions, dilution grazing experiments may underestimate phytoplankton growth coefficients and microzooplankton grazing coefficients.

The effect of dissolved polyunsaturated aldehydes on microzooplankton growth rates in the Chesapeake Bay and Atlantic coastal waters.

Allelopathy is wide spread among marine phytoplankton, including diatoms, which can produce cytotoxic secondary metabolites such as polyunsaturated aldehydes (PUA). Most studies on diatom-produced PUA have been dedicated to their inhibitory effects on reproduction and development of marine invertebrates. However, little information exists on their impact on key herbivores in the ocean, microzooplankton. This study examined the effects of dissolved 2E,4E-octadienal and 2E,4E-heptadienal on the growth rates of natural ciliate and dinoflagellate populations in the Chesapeake Bay and the coastal Atlantic waters. The overall effect of PUA on microzooplankton growth was negative, especially at the higher concentrations, but there were pronounced differences in response among common planktonic species. For example, the growth of Codonella sp., Leegaardiella sol, Prorodon sp., and Gyrodinium spirale was impaired at 2 nM, whereas Strombidium conicum, Cyclotrichium gigas, and Gymnodinium sp. were not affected even at 20 nM. These results indicate that PUA can induce changes in microzooplankton dynamics and species composition.