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

Dopamine drives Drosophila sechellia adaptation to its toxic host

Many insect species are host-obligate specialists. The evolutionary mechanism driving the adaptation of a species to a toxic host is, however, intriguing. We analyzed the tight association of Drosophila sechellia to its sole host, the fruit of Morinda citrifolia, which is toxic to other members of the melanogaster species group. Molecular polymorphisms in the dopamine regulatory protein Catsup cause infertility in D. sechellia due to maternal arrest of oogenesis. In its natural host, the fruit compensates for the impaired maternal dopamine metabolism with the precursor l-DOPA, resuming oogenesis and stimulating egg production. l-DOPA present in morinda additionally increases the size of D. sechellia eggs, what in turn enhances early fitness. We argue that the need of l-DOPA for successful reproduction has driven D. sechellia to become an M. citrifolia obligate specialist. This study illustrates how an insect's dopaminergic system can sustain ecological adaptations by modulating ontogenesis and development. DOI: http://dx.doi.org/10.7554/eLife.03785.00

Plant-based natural product chemistry for integrated pest management of Drosophila suzukii

Since the first reports of damage by Drosophila suzukii, the spotted-wing Drosophila (SWD), over a decade ago in Europe, widespread efforts have been made to understand both the ecology and the evolution of this insect pest, especially due to its phylogenetic proximity to one of the original model organisms, D. melanogaster. In addition, researchers have sought to find economically viable solutions for the monitoring and management of this agricultural pest, which has now swept across much of Europe, North America and Asia. In a new direction of study, we present an investigation of plant-based chemistry, where we search for natural compounds that are structurally similar to known olfactory cues from parasitoid wasps that in turn are welldescribed ovipositional avoidance cues for many Drosophila species. Here we test 11 plant species across two plant genera, Nepeta and Actinidia, and while we find iridoid compounds in both, only those odorants from Actinidia are noted to be detected by the insect antenna, and in addition, found to be behaviorally active. Moreover, the Actinidia extracts resulted in oviposition avoidance when they were added to fruit samples in the laboratory. Thus we propose the possible efficacy of these plants or their extracted chemistry as a novel means for establishing a cost-effective integrated pest management strategy towards the control of this pest fly

A new glance at the chemosphere of macroalgal–bacterial interactions: In situ profiling of metabolites in symbiosis by mass spectrometry

Symbiosis is a dominant form of life that has been observed numerous times in marine ecosystems. For example, macroalgae coexist with bacteria that produce factors that promote algal growth and morphogenesis. The green macroalga Ulva mutabilis (Chlorophyta) develops into a callus-like phenotype in the absence of its essential bacterial symbionts Roseovarius sp. MS2 and Maribacter sp. MS6. Spatially resolved studies are required to understand symbiont interactions at the microscale level. Therefore, we used mass spectrometry profiling and imaging techniques with high spatial resolution and sensitivity to gain a new perspective on the mutualistic interactions between bacteria and macroalgae. Using atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionisation high-resolution mass spectrometry (AP-SMALDI-HRMS), low-molecular-weight polar compounds were identified by comparative metabolomics in the chemosphere of Ulva. Choline (2-hydroxy-N,N,N-trimethylethan-1-aminium) was only determined in the alga grown under axenic conditions, whereas ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was found in bacterial presence. Ectoine was used as a metabolic marker for localisation studies of Roseovarius sp. within the tripartite community because it was produced exclusively by these bacteria. By combining confocal laser scanning microscopy (cLSM) and AP-SMALDI-HRMS, we proved that Roseovarius sp. MS2 settled mainly in the rhizoidal zone (holdfast) of U. mutabilis. Our findings provide the fundament to decipher bacterial symbioses with multicellular hosts in aquatic ecosystems in an ecologically relevant context. As a versatile tool for microbiome research, the combined AP-SMALDI and cLSM imaging analysis with a resolution to level of a single bacterial cell can be easily applied to other microbial consortia and their hosts. The novelty of this contribution is the use of an in situ setup designed to avoid all types of external contamination and interferences while resolving spatial distributions of metabolites and identifying specific symbiotic bacteria