Influence of ion source geometry on the repeatability of topographically guided LAESI-MSI

[Image: see text] Spatially resolving the relative distribution of analyte molecules in biological matter holds great promise in the life sciences. Mass spectrometry imaging (MSI) is a technique that can provide such spatial resolution but remains underused in fields such as chemical ecology, as traditional MSI sample preparation is often chemically or morphologically invasive. Laser ablation electrospray ionization (LAESI)-MSI is a variation of MSI particularly well-suited for situations where chemical sample preparation is too invasive but provides new challenges related to the repeatability of measurement outcomes. We assess the repeatability of LAESI-MSI by sampling a droplet of [ring-(13)C(6)]l-phenylalanine with known concentration and expressing the resulting variability as a coefficient of variation, c(v). In doing so, we entirely eliminate variability caused by surface morphology or underlying true sample gradients. We determine the limit of detection (LOD) for(13)C(6)-Phe by sampling from droplets with successively decreasing but known concentration. We assess the influence of source geometry on the LOD and repeatability by performing these experiments using four distinct variations of sources: one commercial and three custom-built ones. Finally, we extend our study to leaf and stem samples Arabidopsis thaliana and Gossypium hirsutum. We overcome the challenges of LAESI associated with three-dimensional surface morphology by relying on work previously published. Our measurements on both controlled standard and realistic samples give strong evidence that LAESI-MSI’s repeatability in current implementations is insufficient for MSI in chemical ecology

Spatially resolved in vivo plant metabolomics by laser ablation-based mass spectrometry imaging (MSI) techniques: LDI-MSI and LAESI

This short review aims to summarize the current developments and applications of mass spectrometry-based methods for in situ profiling and imaging of plants with minimal or no sample pre-treatment or manipulation. Infrared-laser ablation electrospray ionization and UV-laser desorption/ionization methods are reviewed. The underlying mechanisms of the ionization techniques–namely, laser ablation of biological samples and electrospray ionization–as well as variations of the LAESI ion source for specific targets of interest are described

Linking metabolite production to taxonomic identity in environmental samples by (MA)LDI-FISH

One of the greatest challenges in microbial ecology remains to link the metabolic activity of individual cells to their taxonomic identity and localization within environmental samples. Here we combined mass-spectrometric imaging (MSI) through (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) with fluorescence in situ hybridization (FISH) to monitor antibiotic production in the defensive symbiosis between beewolf wasps and ‘Streptomyces philanthi' bacteria. Our results reveal similar distributions of the different symbiont-produced antibiotics across the surface of beewolf cocoons, which colocalize with the producing cell populations. Whereas FISH achieves single-cell resolution, MSI is currently limited to a step size of 20–50 μm in the combined approach because of the destructive effects of high laser intensities that are associated with tighter laser beam focus at higher lateral resolution. However, on the basis of the applicability of (MA)LDI-MSI to a broad range of small molecules, its combination with FISH provides a powerful tool for studying microbial interactions in situ, and further modifications of this technique could allow for linking metabolic profiling to gene expression

Metabolome and transcriptome related dataset for pheromone biosynthesis in an aggressive forest pest Ips typographus

Eurasian spruce bark beetle, Ips typographus, is an aggressive pest among spruce vegetation. I. typographus host trees colonization is mediated by aggregation pheromone, consisting of 2-methyl-3-buten-2-ol and cis-verbenol produced in the beetle gut. Other biologically active compounds such as ipsdienol and verbenone have also been detected. 2-Methyl-3-buten-2-ol and ipsdienol are produced de-novo in the mevalonate pathway and cis-verbenol is oxidized from α-pinene sequestrated from the host. The pheromone production is presumably connected with further changes in the primary and secondary metabolisms in the beetle. To evaluate such possibilities, we obtained qualitative metabolomic data from the analysis of beetle guts in different life stages. We used Ultra-high-performance liquid chromatography-electrospray ionization-high resolution tandem mass spectrometry (UHPLC-ESI-HRMS/MS). The data were dereplicated using metabolomic software (XCMS, Camera, and Bio-Conductor) and approximately 3000 features were extracted. The metabolite was identified using GNPS databases and de-novo annotation in Sirius program followed by manual curation. Further, we obtained differential gene expression (DGE) of RNA sequencing data for mevalonate pathway genes and CytochromeP450 (CyP450) genes from the gut tissue of the beetle to delineate their role on life stage-specific pheromone biosynthesis. CyP450 gene families were classified according to subclasses and given individual expression patterns as heat maps. Three mevalonate pathway genes and five CyP450 gene relative expressions were analyzed using quantitative real-time (qRT) PCR, from the gut tissue of different life stage male/female beetles, as extended knowledge of related research article (Ramakrishnan et al., 2022). This data provides essential information on pheromone biosynthesis at the molecular level and supports further research on pheromone biosynthesis and detoxification in conifer bark beetles

Metabolic profiling of rhizobacteria Serratia plymuthica and Bacillus subtilis revealed intra- and interspecific differences and elicitation of plipastatins and short peptides due to co-cultivation

Rhizobacteria live in diverse and dynamic communities having a high impact on plant growth and development. Due to the complexity of the microbial communities and the difficult accessibility of the rhizosphere, investigations of interactive processes within this bacterial network are challenging. In order to better understand causal relationships between individual members of the microbial community of plants, we started to investigate the inter- and intraspecific interaction potential of three rhizobacteria, the S. plymuthica isolates 4Rx13 and AS9 and B. subtilis B2g, using high resolution mass spectrometry based metabolic profiling of structured, low-diversity model communities. We found that by metabolic profiling we are able to detect metabolite changes during cultivation of all three isolates. The metabolic profile of S. plymuthica 4Rx13 differs interspecifically to B. subtilis B2g and surprisingly intraspecifically to S. plymuthica AS9. Thereby, the release of different secondary metabolites represents one contributing factor of inter- and intraspecific variations in metabolite profiles. Interspecific co-cultivation of S. plymuthica 4Rx13 and B. subtilis B2g showed consistently distinct metabolic profiles compared to mono-cultivated species. Thereby, putative known and new variants of the plipastatin family are increased in the co-cultivation of S. plymuthica 4Rx13 and B. subtilis B2g. Interestingly, intraspecific co-cultivation of S. plymuthica 4Rx13 and S. plymuthica AS9 revealed a distinct interaction zone and showed distinct metabolic profiles compared to mono-cultures. Thereby, several putative short proline-containing peptides are increased in co-cultivation of S. plymuthica 4Rx13 with S. plymuthica AS9 compared to mono-cultivated strains. Our results demonstrate that the release of metabolites by rhizobacteria alters due to growth and induced by social interactions between single members of the microbial community. These results form a basis to elucidate the functional role of such interaction-triggered compounds in establishment and maintenance of microbial communities and can be applied under natural and more realistic conditions, since rhizobacteria also interact with the plant itself and many other members of plant and soil microbiota

Initial studies of mating disruption of the tomato moth, Tuta absoluta (Lepidoptera : Gelechiidae) using synthetic sex pheromone

The potential of the synthetic major component of T. absoluta (Meyrick) sex pheromone for mating disruption was studied in small plots (0.01 hectares) with fresh-market tomato crop. The effects of the application of the sex pheromone 3E,8Z,11Z-14: Ac (from 0 to 80 g a. i./ha) were assessed on male orientation to pheromone baited traps, mating in cages and plant damage. The highest levels of interruption in male orientation (60-90%) were found in plots treated with 35 to 50 g/ha of sex pheromone. However, no treatment with pheromone was capable of significantly reducing the percentage of mined leaflets or bored fruits or the frequency of mating in cages compared to the control plots. The failure in mating disruption technique may be attributed to the composition of the synthetic pheromone, doses used, high pest population density, and mated female migration to the area treated