Benthic cyanobacterial metabolites interact to reduce coral larval survival and settlement.

Benthic cyanobacterial mats (BCMs) are becoming increasingly abundant on coral reefs worldwide. High growth rates and prolific toxin production give them the potential to cause widespread coral recruitment failure through allelopathic effects, but few studies have made the link between their toxicity for coral larvae and in situ toxin concentrations. Here we investigated the allelopathic effects of the benthic cyanobacterium Anabaena sp.1 on larvae of the coral Pocillopora acuta. This cyanobacterium produces several non-ribosomal cyclic lipopeptides of the laxaphycin family with cytotoxic properties. Therefore, we measured the concentration of laxaphycins A and B in Anabaena mats and in the water column and tested their effects on coral larvae. We found that Anabaena crude extract reduces both larval survivorship and settlement and that laxaphycin B reduces settlement. When larvae were exposed to both laxaphycins, there was a reduction in both larval survival and settlement. In the natural reef environment, laxaphycin A and B concentrations increased with increasing proximity to Anabaena mats, with concentrations being consistently above LC50 and EC50 thresholds within a 1 cm distance of the mats. This study demonstrates that laxaphycins reduce the survival and inhibit the settlement of coral larvae at concentrations found near Anabaena mats in situ. It further shows a combined effect between two cyanobacterial metabolites. As BCMs become more common, more of their secondary metabolites might be released in the water column. Their occurrence will lead to a reduction in coral recruitment rates, contributing to the continuing decline of coral reefs and shift in community structure.

LC-HRMS-Driven Computational Toolbox to Assess Extraction Protocols Dedicated to Untargeted Analysis: How to Ease Analyzing Pesticide-Contaminated Soils?

Metabolomics is a powerful approach that allows for high throughput analysis and the acquisition of large biochemical data. Nonetheless, it still faces several challenging requirements, such as the development of optimal extraction and analytical methods able to respond to its high qualitative and quantitative requisites. Hence, the objective of the present article is to suggest a LC-HRMS-based untargeted profiling approach aiming to provide performant tools that help assess the performance and the quality of extraction methods. It is applied in a herbicide-contaminated soil metabolomics context. The trifactorial experimental design consists of 150 samples issued from five different extraction protocols, two types of soils, and three contamination conditions (contaminated soils with two different formulated herbicides against uncontaminated soils). Four performance and quality criteria are investigated using adapted LC-HRMS-driven computational tools. First, 861 metabolic features are annotated, and then the width of metabolome coverage and quantitative performance of the five different extraction protocols are assessed in all samples using various optimized configurations of heatmaps as well as van Krevelen diagrams. Then, the reproducibility of LC-HRMS profiles issued from the five extractions is studied by two different approaches: Euclidean distances and relative standard deviations. The two methods are examined and compared. Their advantages and limitations are thus discussed. After, the capacity of the different extractions to discriminate between contaminated and uncontaminated soils will be evaluated using orthogonal projections to latent structures-discriminant analysis. Different data scaling parameters are tested, and the results are explored and discussed. All of the suggested computational and visualization tools are performed using public-access platforms or open-source software. They can be readapted by metabolomics developers and users according to their study contexts and fields of application.

Characteristic response of formulation ingredients revealed by ultra high performance liquid chromatography-electrospray ionization-high resolution mass spectrometry-based untargeted screening of pesticides in soil.

Commercial solutions of pesticides consist of two main components: The active substance and the formulation ingredients. These ingredients, mainly composed of polymeric surfactants, are considered inert vis-à-vis the targeted organisms and nature. Nonetheless, a relatively low attention is given to their analysis and fate tracking in the environment. In this context, the current paper, embedded in a large study of fate and impact of formulated pesticides in soil, focuses on the analysis of these formulation ingredients. It mainly highlights and discusses the characteristic response of these ingredients in liquid chromatography-mass spectrometry-based untargeted screening of two commercial herbicides applied on soil. This characteristic response is based on different spectral and chromatographic aspects, as their amplified adducts and double-charged ions formation, or their "wavy" chromatographic profiles and the inversion of their elution order following the polymerization degree. These patterns are briefly discussed in order to explain them, and then thanks to their understanding, 12 different series (165 compounds) of formulation ingredients were outlined and discriminated from active substance and soil metabolites. After, high-resolution and tandem mass spectrometry data were investigated for rapid interseries and intraseries identification-by-chain. In addition, recommendations for methods development and hints on postanalytical data processing for identity determination of these ingredients are given in order to help in enhancing future studies. Limitations of the applied approach are also outlined, and some innovate suggestions are provided based on the described findings.

Synthesis and Biological Activity of Novel α-Conotoxins Derived from Endemic Polynesian Cone Snails.

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.

Antibacterial Activities and Life Cycle Stages of Asparagopsis armata: Implications of the Metabolome and Microbiome.

The red alga Asparagopsis armata is a species with a haplodiplophasic life cycle alternating between morphologically distinct stages. The species is known for its various biological activities linked to the production of halogenated compounds, which are described as having several roles for the algae such as the control of epiphytic bacterial communities. Several studies have reported differences in targeted halogenated compounds (using gas chromatography-mass spectrometry analysis (GC-MS)) and antibacterial activities between the tetrasporophyte and the gametophyte stages. To enlarge this picture, we analysed the metabolome (using liquid chromatography-mass spectrometry (LC-MS)), the antibacterial activity and the bacterial communities associated with several stages of the life cycle of A. armata: gametophytes, tetrasporophytes and female gametophytes with developed cystocarps. Our results revealed that the relative abundance of several halogenated molecules including dibromoacetic acid and some more halogenated molecules fluctuated depending on the different stages of the algae. The antibacterial activity of the tetrasporophyte extract was significantly higher than that of the extracts of the other two stages. Several highly halogenated compounds, which discriminate algal stages, were identified as candidate molecules responsible for the observed variation in antibacterial activity. The tetrasporophyte also harboured a significantly higher specific bacterial diversity, which is associated with a different bacterial community composition than the other two stages. This study provides elements that could help in understanding the processes that take place throughout the life cycle of A. armata with different potential energy investments between the development of reproductive elements, the production of halogenated molecules and the dynamics of bacterial communities.

Metabarcoding and Metabolomics Reveal the Effect of the Invasive Alien Tree Miconia calvescens DC. on Soil Diversity on the Tropical Island of Mo'orea (French Polynesia).

Miconia calvescens is a dominant invasive alien tree species that threatens several endemic plants in French Polynesia (South Pacific). While most analyses have been performed at the scale of plant communities, the effects on the rhizosphere have not been described so far. However, this compartment can be involved in plant fitness through inhibitory activities, nutritive exchanges, and communication with other organisms. In particular, it was not known whether M. calvescens forms specific associations with soil organisms or has a specific chemical composition of secondary metabolites. To tackle these issues, the rhizosphere of six plant species was sampled on the tropical island of Mo'orea in French Polynesia at both the seedling and tree stages. The diversity of soil organisms (bacteria, microeukaryotes, and metazoa) and of secondary metabolites was studied using high-throughput technologies (metabarcoding and metabolomics, respectively). We found that trees had higher effects on soil diversity than seedlings. Moreover, M. calvescens showed a specific association with microeukaryotes of the Cryptomycota family at the tree stage. This family was positively correlated with the terpenoids found in the soil. Many terpenoids were also found within the roots of M. calvescens, suggesting that these molecules were probably produced by the plant and favored the presence of Cryptomycota. Both terpenoids and Cryptomycota were thus specific chemicals and biomarkers of M. calvescens. Additional studies must be performed in the future to better understand if they contribute to the success of this invasive tree.

Untargeted metabolomics as a tool to monitor biocontrol product residues' fate on field-treated Prunus persica.

Evidence of chemical plant protection products' (PPPs) long-term impact has been found in all environmental compartments. Therefore, other types of PPPs are developed to complement chemical PPPs like PPPs from natural sources, namely biocontrol products (BPs). Little is known about those new BPs, and it is important to assess their potential long-term environmental impact. Recently, the Environmental Metabolic Footprinting (EMF) approach was developed. It permits studying sample's entire meta-metabolome (endometabolome and xenometabolome) through a kinetics tracking of metabolomes of treated and untreated samples. Those metabolomes are compared time-by-time to estimate the "resilience time" of the samples after treatment. The current study aims to investigate BP residues' dissipation on peach fruits (Prunus persica). For that, an untargeted Liquid Chromatography-Mass Spectrometry metabolomics approach based on the EMF was optimised to separate the xenometabolome of the PPP from the endometabolome of the fruits. This "new version" of the EMF approach is able to target the BP treatment residues' (xenometabolome) dissipation exclusively. Thus, it is able to determine the time needed to have no more residues in the studied matrix: the "dissipation interval". Field experiment was conducted on peach tree orchard against brown rot treated with (i) a plant extract BP (Akivi); (ii) a reference mineral extract BP (Armicarb®); and (iii) a Chemical reference treatment campaign. Formulated Akivi and its by-products' dissipation was monitored, a degradation kinetics appeared but the sampling did not last long enough to allow the determination of the "dissipation interval". Armicarb® and the Chemical reference's residues and by-products showed a persistence pattern along the sampling kinetics. These results indicate that the EMF approach, formerly developed on soil and sediment, is applicable for fruit matrices and can be used to investigate the fate of complex BP treatment on the matrix through the xenometabolome tracking on treated fruits.

Coral larval settlement preferences linked to crustose coralline algae with distinct chemical and microbial signatures.

The resilience of coral reefs is dependent on the ability of corals to settle after disturbances. While crustose coralline algae (CCA) are considered important substrates for coral settlement, it remains unclear whether coral larvae respond to CCA metabolites and microbial cues when selecting sites for attachment and metamorphosis. This study tested the settlement preferences of an abundant coral species (Acropora cytherea) against six different CCA species from three habitats (exposed, subcryptic and cryptic), and compared these preferences with the metabolome and microbiome characterizing the CCA. While all CCA species induced settlement, only one species (Titanoderma prototypum) significantly promoted settlement on the CCA surface, rather than on nearby dead coral or plastic surfaces. This species had a very distinct bacterial community and metabolomic fingerprint. Furthermore, coral settlement rates and the CCA microbiome and metabolome were specific to the CCA preferred habitat, suggesting that microbes and/or chemicals serve as environmental indicators for coral larvae. Several amplicon sequence variants and two lipid classes-glycoglycerolipids and betaine lipids-present in T. prototypum were identified as potential omic cues influencing coral settlement. These results support that the distinct microbiome and metabolome of T. prototypum may promote the settlement and attachment of coral larvae.

d-Peptidase Activity in a Marine Mollusk Detoxifies a Nonribosomal Cyclic Lipopeptide: An Ecological Model to Study Antibiotic Resistance.

In the marine environment, sessile cyanobacteria have developed chemical strategies for protection against grazers. In turn, herbivores have to circumvent these defenses and in certain cases even take advantage of them as shelter from their own predators. This is the case of Stylocheilus striatus, a sea hare that feeds on Anabaena torulosa, a cyanobacterium that produces toxic cyclic lipopeptides of the laxaphycin B family. S. striatus consumes the cyanobacterium without being affected by the toxicity of its compounds and also uses it as an invisibility cloak against predators. In this article, using different substrates analogous to laxaphycin B, we demonstrate the presence of an enzyme in the digestive gland of the mollusk that is able to biotransform laxaphycin B derivatives. The enzyme belongs to the poorly known family of d-peptidases that are suspected to be involved in antibiotic resistance.

Electrospray ionization and heterogeneous matrix effects in liquid chromatography/mass spectrometry based meta-metabolomics: A biomarker or a suppressed ion?

RATIONALE: Correct biomarker determination in metabolomics is crucial for unbiased conclusions and reliable applications. However, this determination is subject to several drifts, e.g. matrix effects and ion suppression in Liquid Chromatography/Mass Spectrometry (LC/MS)-based approaches. This phenomenon provokes critical issues for biomarker determination, particularly during comparative studies dealing with samples exhibiting heterogeneous complexities. METHODS: Occurrence of the issue was coincidentally noticed when studying the environmental impact of a complex bioinsecticide: Bacillus thuringiensis israelensis. The studied samples comprised insecticide-spiked sediments and untreated control sediments. QuEChERS extractions followed by LC/ESI-Q/ToF analyses were performed on sediments after 15 days of incubation. Meta-metabolomes containing pesticide xenometabolites and sediment endometabolites were analyzed in depth using XCMS-based computational data preprocessing. Multivariate statistical analyses (PCA, OPLS-DA) and raw data crosschecks were performed to search for environmental biomarkers. RESULTS: Multivariate analyses and raw data crosschecks led to the selection of nine metabolites as biomarker candidates. However, when exploring the mass spectra, co-elutions were noticed between seven of these metabolites and multi-charged macromolecules originating from the pesticide. Provoked false positives were thus suspected due to a potential ion suppression exclusively occurring in the spiked samples. A dilution-based approach was then applied. It confirmed five metabolites as suppressed ions. CONCLUSIONS: Ion suppression should be considered as a critical issue for biomarker determination when comparing heterogeneous metabolic profiles. Raw chromatograms and mass spectra crosschecks are mandatory to reveal potential ion suppressions in such cases. Dilution is a suitable approach to filter reliable biomarker candidates before their identification and absolute quantification.

Online Headspace-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry-based untargeted volatile metabolomics for studying emerging complex biopesticides: A proof of concept.

This work introduces a novel online Headspace-Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry-based untargeted metabolomics approach, suggested as an alternative tool to study the environmental fate of volatile xenometabolites in emerging complex biopesticides, e.g. the Myrica gale methanolic extract herbicide containing several unknown metabolites. A "living" microcosm sample was designed for non-destructive analysis by a 35-min HS-SPME automated extraction and a 36-min GC-MS run. A 38-day kinetics study was then applied on two groups of soil samples: control and spiked. Statistical tools were used for the comparative kinetics study. The Principal Component Analysis revealed and explained the evolution and the dissipation of the herbicide volatile xenometabolome over time. The time-series Heatmap and Multivariate Empirical Bayes Analysis of Variance allowed the prioritization of 101 relevant compounds including 22 degradation by-products. Out of them, 96 xenometabolites were putatively identified. They included 63 compounds that are identified as herbicide components for the first time. The Orthogonal Projections to Latent Structures Discriminant Analysis and its Cross-Validation test were used to assess the total dissipation of the herbicide volatile residues and method detection limit. The reproducibility of the method was also assessed. The highest inter-samples (n = 3) Peak Area RSD was 7.75 %. The highest inter-samples (n = 3) and inter-days (n = 8) Retention Time SD were 0.43 sec and 3.44 sec, respectively. The work presents a green, non-laborious and high-throughput approach. It required a small number of environmental samples (6 microcosms) that were analyzed 8 times and were not destroyed during the study.

Insights into the Natural Defenses of a Coral Reef Fish Against Gill Ectoparasites: Integrated Metabolome and Microbiome Approach.

Understanding natural defense mechanisms against parasites can be a valuable tool for the development of innovative therapies. We have previously identified a butterflyfish species (Chaetodon lunulatus) that avoids gill monogenean parasites while living amongst closely related parasitized species. The metabolome and microbiome of several sympatric butterflyfish species from the island of Moorea (French Polynesia) were previously described. In this study, we used the previously generated datasets in an attempt to identify metabolites and bacteria potentially involved in parasite defense mechanisms. We investigated the interplay between the gill mucus metabolome and microbiome of the non-susceptible C. lunulatus versus sympatric butterflyfish species that were always found parasitized in the Central and Eastern Indo-Pacific. After observing significant differences between the metabolome and bacteria of susceptible versus non-susceptible fish, we obtained the discriminant metabolites and operational taxonomic units (OTUs) using a supervised analysis. Some of the most important discriminant metabolites were identified as peptides, and three new peptides derived from ß-subunit hemoglobin from C. lunulatus (CLHbß-1, CLHbß-2, and CLHbß-3) were purified, characterized and synthesized to confirm their structures. We also identified specific bacterial families and OTUs typical from low-oxygen habitats in C. lunulatus gill mucus. By using a correlation network between the two datasets, we found a Fusobacteriaceae strain exclusively present in C. lunulatus and highly correlated to the peptides. Finally, we discuss the possible involvement of these peptides and Fusobacteriaceae in monogenean avoidance by this fish species.

Trichormamide C Structural Confirmation through Total Synthesis and Extension to Analogs.

The growing interest in marine natural substances as potential new drugs has made total synthesis a real asset for structure confirmation. Trichormamide C (1), a cyclic lipopeptide isolated from the cyanobacteria Oscillatoria sp., is characterized by the presence of nonproteinogenic amino acids in the sequence. Trichormamide C structural confirmation was carried out through the implementation of a flexible synthesis resulting in two new analogs (3 and 4).

Salen/salan metallic complexes as redox labels for electrochemical aptasensors.

This work presents the synthesis and characterization of salen/salan metal complexes for their future application as electrochemical labels in affinity sensors. Due to its stability and electrochemical properties, an oxovanadium salan complex was selected and coupled to an estradiol-specific aptamer. The response of the resulting aptasensor was shown to decrease with increasing estradiol concentration.

Environmental Metabolic Footprinting (EMF) vs. half-life: a new and integrative proxy for the discrimination between control and pesticides exposed sediments in order to further characterise pesticides' environmental impact.

Pesticides are regularly used for a variety of applications and are disseminated throughout the environment. These substances may have significant negative impacts. To date, the half-life, t1/2, was often used to study the fate of pesticides in environmental matrices (water, soil, sediment). However, this value gives limited information. First, it does not evaluate the formation of by-products, resulting in the need for additional experiments to be performed to evaluate biodegradation and biotransformation products. T1/2 also fails to consider the chemical's impact on biodiversity. Resilience time, a new and integrative proxy, was recently proposed as an alternative to t1/2, with the potential to evaluate all the post-application effects of the chemical on the environment. The 'Environmental Metabolic Footprinting' (EMF) approach, giving an idea of the resilience time, was used to evaluate the impact of botanicals on soil. The goal is to optimise the EMF to study the impact of a microbial insecticide, the Bacillus thuringiensis israelensis (Bti), on sediment. The difficulty of this work lies in the commercial solution of Bti that is really complex, and this complexity yields chromatograms that are extremely difficult to interpret; t1/2 cannot be used. No methodologies currently exist to monitor the impact of these compounds on the environment. We will test the EMF to determine if it is sensitive enough to tolerate such complex mixtures. A pure chemical insecticide, the α-cypermethrin, will be also studied. The article shows that the EMF is able to distinguish meta-metabolome differences between control and exposed (with Bti) sediments.

Evidence for photolytic and microbial degradation processes in the dissipation of leptospermone, a natural ß-triketone herbicide.

Bioherbicides appear as an ecofriendly alternative to synthetic herbicides, generally used for weed management, because they are supposed to have low side on human health and ecosystems. In this context, our work aims to study abiotic (i.e., photolysis) and biotic (i.e,. biodegradation) processes involved in the fate of leptospermone, a natural ß-triketone herbicide, by combining chemical and microbiological approaches. Under controlled conditions, the photolysis of leptospermone was sensitive to pH. Leptospermone has a half-life of 72 h under simulated solar light irradiations. Several transformation products, including hydroxy-leptospermone, were identified. For the first time, a bacterial strain able to degrade leptospermone was isolated from an arable soil. Based on its 16S ribosomal RNA (rRNA) gene sequence, it was affiliated to the Methylophilus group and was accordingly named as Methylophilus sp. LS1. Interestingly, we report that the abundance of OTUs, similar to the 16S rRNA gene sequence of Methylophilus sp. LS1, was strongly increased in soil treated with leptospermone. The leptospermone was completely dissipated by this bacteria, with a half-life time of 6 days, allowing concomitantly its growth. Hydroxy-leptospermone was identified in the bacterial culture as a major transformation product, allowing us to propose a pathway of transformation of leptospermone including both abiotic and biotic processes.