No Evidence for the Induction of Brown Algal Chemical Defense by the Phytohormones Jasmonic Acid and Methyl Jasmonate

Induced chemical defense reactions are widespread in marine brown algae. Despite the evidence that the biosynthesis of defense metabolites can be up-regulated upon herbivory, we do not know how this regulation of biosynthetic pathways to secondary metabolites is achieved in brown algae. In higher plants, the phytohormone jasmonic acid (JA) is crucial for the mediation of induced chemical defenses, and several findings of this metabolite from marine sources have been reported. We tested the hypothesis that JA or related metabolites play a role in induced brown algal defense. Quantification of oxylipins with a detection limit around 20ng g−1 algal tissue did not reveal the presence of JA in the seven examined brown algal species Dictyota dichotoma, Colpomenia peregrina, Ectocarpus fasciculatus, Fucus vesiculosus, Himanthalia elongata, Saccharina latissima (formerly Laminaria saccharina), and Sargassum muticum. Moreover, treatment with ecologically relevant concentrations of JA and methyl jasmonate did not lead to a significant change in the profile of medium- and non-polar metabolites of the tested algae. Only when high concentrations of ≥500μg ml−1 medium of the phytohormones were applied that a metabolic response which could be attributed to unspecific stress was observed. Bioassays with D. dichotoma that focused on medium- and non-polar compounds confirmed the lack of a biological role of JA and methyl jasmonate in the induction of algal induced chemical defenses. The phytohormone-treated samples did not exhibit any increased defense potential towards the amphipod Ampithoe longimana and the isopod Paracerceis caudata. JA and related phytohormones, known to be active in higher plants, thus appear to play no role in brown algae for induction of the defense chemicals studied her

The potential role of wound-activated volatile release in the chemical defence of the brown alga Dictyota dichotoma: Blend recognition by marine herbivores

Abstract.: The chemical defence potential against herbivores of certain Dictyotalean brown algae increases after tissue disruption. This wound activated defence has been explored in bioassays, but the metabolic pathways behind it are unknown. Here we describe a metabolic profiling approach to identify the activated defence metabolites. Before and after tissue damage of Dictyota dichotoma modified diterpenes, non-volatile medium polar metabolites as well as volatile compounds were profiled. While comparison of extracted intact and mechanically wounded algae revealed no significant differences in structure and distribution of semi-volatile and reversed phase LC/MS detectable metabolites, a strong release of gaseous volatiles was observed. Solid phase micro extraction (SPME) and GC/MS were used for identification and quantification of these biogenic gases. This showed that D. dichotoma released elevated amounts of trimethylamine (TMA) and dimethylsulphide (DMS) after mechanical tissue damage. To study the ecological significance of compounds released post injury and of the biosynthetically connected non-volatile acrylate, choice assays were performed with the amphipod Amphithoe longimana. Behavioural assays on artificial diets did not reveal any repellent role for the single isolated metabolites. In strong contrast, a mixture of TMA, DMS and acrylate significantly reduced the association of the herbivores with the treated food pellets.This shows that mixtures of these biogenic gases and acrylate are recognized by the herbivores and influence food selectio

Disruption-free Solid Phase Extraction of Surface Metabolites from Macroalgae

This is an Accepted Manuscript of a book chapter published by CRC Press in Protocols for Macroalgae Research on 28.04.2018, available online: https://www.crcpress.com/Protocols-for-Macroalgae-Research/Charrier-Wichard-Reddy/p/book/9781498796422 The surface chemistry of aquatic organisms is decisive for their biotic interactions. Metabolites in the spatially limited laminar boundary layer mediate processes, such as fouling, allelopathy and chemical defense against herbivores. However, very few methods are available for the investigation of such surface metabolites. Here we give a detailed protocol in which surfaces are extracted by means of C18 solid phase material, elution of the solid phase extraction material with solvent and analysis via liquid chromatography / mass spectrometry (LC/MS) and/or gas chromatography / mass spectrometry (GC/MS). The protocol introduced here is based on a previous publication (Cirri et al. 2016) where validation is described. The method is robust, picks up metabolites of a broad polarity range and is easy to handle. It was developed for the macroalgae Fucus vesiculosus, Caulerpa taxifolia and Gracilaria vermiculophylla, but can be easily transferred to other algae and to other aquatic organisms in general

Nitrogen utilization analysis reveals the synergetic effect of arginine and urea in promoting fucoxanthin biosynthesis in the mixotrophic marine diatom Phaeodactylum tricornutum

Fucoxanthin is a new dietary ingredient applied in healthy foods with specific benefits of body weight loss and liver fat reduction. The marine diatom Phaeodactylum tricornutum is a highly suitable species for fucoxanthin production. In the present study, aiming to promote fucoxanthin biosynthesis in mixotrophic P. tricornutum , NaNO 3 , tryptone, and urea were evaluated as nitrogen sources with 0.10 mol L −1 of glycerol as the organic carbon source for mixotrophic growth in shake flasks. Compared to NaNO 3 , the mixture of tryptone and urea (referred to as T+U, 1:1, mol N:mol N) as organic nitrogen sources could induce a higher biomass and fucoxanthin production. Through nitrogen utilization analysis, leucine, arginine, lysine, and phenylalanine in the T+U medium were identified as the amino acids that primarily support cell growth. Among those amino acids, arginine causes the highest rate of nitrogen utilization and cell growth promotion. After 12 days of cultivation, the highest biomass concentration (3.18 g L −1 ), fucoxanthin content (12.17 mg g −1 ), and productivity (2.68 mg L −1 day −1 ) were achieved using 25 mmol N L −1 of arginine and 5 mmol N L −1 of urea as nitrogen sources, indicating that arginine and urea performed synergistically on enhancing biomass and pigment production. This study provides new insights into the promotion of fucoxanthin biosynthesis by nitrogen utilization analysis and verifies the synergetic effect of arginine and urea on facilitating the development of a promising strategy for efficient enhancement of fucoxanthin production through mixotrophic cultivation of P. tricornutum

The oxylipin chemistry of attraction and defense in brown algae and diatoms

Max Planck Institute for Chemical Ecology, Jena, Germany. This review covers the research on brown algal pheromones from the first structural characterisation of an active principle in 1971 to the recent detailed insight into their biosynthesis. Development of analytical methods and bioassays that lead to the identification of a structural variety of different fatty acid-derived pheromones are reported. Special emphasis is focused on the inactivation of initially released pheromones through pericyclic reactions. The impact of pheromone-research on the defensive chemistry of brown algae and diatoms is discussed. 121 references are cited

Metabolomic Assessment of Induced and Activated Chemical Defence in the Invasive Red Alga Gracilaria vermiculophylla

In comparison with terrestrial plants the mechanistic knowledge of chemical defences is poor for marine macroalgae. This restricts our understanding in the chemically mediated interactions that take place between algae and other organisms. Technical advances such as metabolomics, however, enable new approaches towards the characterisation of the chemically mediated interactions of organisms with their environment. We address defence responses in the red alga Gracilaria vermiculophylla using mass spectrometry based metabolomics in combination with bioassays. Being invasive in the north Atlantic this alga is likely to possess chemical defences according to the prediction that well-defended exotics are most likely to become successful invaders in systems dominated by generalist grazers, such as marine macroalgal communities. We investigated the effect of intense herbivore feeding and simulated herbivory by mechanical wounding of the algae. Both processes led to similar changes in the metabolic profile. Feeding experiments with the generalist isopod grazer Idotea baltica showed that mechanical wounding caused a significant increase in grazer resistance. Structure elucidation of the metabolites of which some were up-regulated more than 100 times in the wounded tissue, revealed known and novel eicosanoids as major components. Among these were prostaglandins, hydroxylated fatty acids and arachidonic acid derived conjugated lactones. Bioassays with pure metabolites showed that these eicosanoids are part of the innate defence system of macroalgae, similarly to animal systems. In accordance with an induced defence mechanism application of extracts from wounded tissue caused a significant increase in grazer resistance and the up-regulation of other pathways than in the activated defence. Thus, this study suggests that G. vermiculophylla chemically deters herbivory by two lines of defence, a rapid wound-activated process followed by a slower inducible defence. By unravelling involved pathways using metabolomics this work contributes significantly to the understanding of activated and inducible defences for marine macroalgae

Selective silicate-directed motility in diatoms

Diatoms are highly abundant unicellular algae that often dominate pelagic as well as benthic primary production in the oceans and inland waters. Being strictly dependent on silica to build their biomineralized cell walls, marine diatoms precipitate 240 × 1012 mol Si per year, which makes them the major sink in the global Si cycle. Dissolved silicic acid (dSi) availability frequently limits diatom productivity and influences species composition of communities. We show that benthic diatoms selectively perceive and behaviourally react to gradients of dSi. Cell speed increases under dSi-limited conditions in a chemokinetic response and, if gradients of this resource are present, increased directionality of cell movement promotes chemotaxis. The ability to exploit local and short-lived dSi hotspots using a specific search behaviour likely contributes to micro-scale patch dynamics in biofilm communities. On a global scale this behaviour might affect sediment–water dSi fluxes and biogeochemical cycling

Quantification of Dissolved and Particulate Polyunsaturated Aldehydes in the Adriatic Sea

Polyunsaturated aldehydes (PUA) are supposed to play critical roles in chemically-mediated plankton interactions. Laboratory studies suggest that they act as mediators of chemical defense and chemical communication. PUA are oxylipins containing an α,β,γ,δ–unsaturated aldehyde structure element and are mainly found in diatoms. We present here a detailed surface mapping of PUA during a spring bloom of the diatom Skeletonema marinoi in the Adriatic Sea. We monitored dissolved PUA, as well as particulate PUA, which are produced by phytoplankton after cell disintegration. Our survey revealed a patchy distribution of PUA and shows that at most stations S. marinoi is the major contributor to the overall PUA. Our data also suggest that lysis of a diatom bloom can contribute significantly to the dissolved PUA concentrations and that other producers, which are smaller in cell size compared to diatoms, have to be taken into account as well if the total PUA content of marine samples is considered. The analyses of samples collected in deeper water suggests that diatom contribution to PUA decreases with depth, while smaller-sized unidentified organisms take place as dominant contributors to the PUA concentrations

Dynamics of Dissolved and Particulate Polyunsaturated Aldehydes in Mesocosms Inoculated with Different Densities of the Diatom Skeletonema marinoi

A survey of the production of polyunsaturated aldehydes (PUA) of manipulated plankton communities is presented here. PUA are phytoplankton-derived metabolites that are proposed to play an important role in chemically mediated plankton interactions. Blooms of different intensities of the diatom Skeletonema marinoi were generated in eight mesocosms filled with water from the surrounding fjord by adding different amounts of a starting culture and nutrients. This set-up allowed us to follow PUA production of the plankton community over the entire induced bloom development, and to compare it with the natural levels of PUA. We found that S. marinoi is a major source for the particulate PUA 2,4-heptadienal and 2,4-octadienal (defined as PUA released upon wounding of the diatom cells) during the entire bloom development. Just before, and during, the decline of the induced diatom blooms, these PUA were also detected in up to 1 nM concentrations dissolved in the water. In addition, we detected high levels of the PUA 2,4-decadienal that was not produced by the diatom S. marinoi. Particulate decadienal correlated well with the cell counts of the prymnesiophyte Phaeocystis sp. that also developed in the fertilized mesocosms. Particulate decadienal levels were often even higher than those of diatom-derived PUA, indicating that PUA sources other than diatoms should be considered when it comes to the evaluation of the impact of these metabolites

Associated bacteria affect sexual reproduction by altering gene expression and metabolic processes in a biofilm inhabiting diatom

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally coexisting bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations