Bacterial ectosymbionts in cuticular organs chemically protect a beetle during molting stages.

In invertebrates, the cuticle is the first and major protective barrier against predators and pathogen infections. While immune responses and behavioral defenses are also known to be important for insect protection, the potential of cuticle-associated microbial symbionts to aid in preventing pathogen entry during molting and throughout larval development remains unexplored. Here, we show that bacterial symbionts of the beetle Lagria villosa inhabit unusual dorsal invaginations of the insect cuticle, which remain open to the outer surface and persist throughout larval development. This specialized location enables the release of several symbiont cells and the associated protective compounds during molting. This facilitates ectosymbiont maintenance and extended defense during larval development against antagonistic fungi. One Burkholderia strain, which produces the antifungal compound lagriamide, dominates the community across all life stages, and removal of the community significantly impairs the survival probability of young larvae when exposed to different pathogenic fungi. We localize both the dominant bacterial strain and lagriamide on the surface of eggs, larvae, pupae, and on the inner surface of the molted cuticle (exuvia), supporting extended protection. These results highlight adaptations for effective defense of immature insects by cuticle-associated ectosymbionts, a potentially key advantage for a ground-dwelling insect when confronting pathogenic microbes.

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.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

Author Correction: The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification to species level of live single microalgal cells from plankton samples with matrix-free laser/desorption ionization mass spectrometry.

INTRODUCTION: Marine planktonic communities are complex microbial consortia often dominated by microscopic algae. The taxonomic identification of individual phytoplankton cells usually relies on their morphology and demands expert knowledge. Recently, a live single-cell mass spectrometry (LSC-MS) pipeline was developed to generate metabolic profiles of microalgae. OBJECTIVE: Taxonomic identification of diverse microalgal single cells from collection strains and plankton samples based on the metabolic fingerprints analyzed with matrix-free laser desorption/ionization high-resolution mass spectrometry. METHODS: Matrix-free atmospheric pressure laser-desorption ionization mass spectrometry was performed to acquire single-cell mass spectra from collection strains and prior identified environmental isolates. The computational identification of microalgal species was performed by spectral pattern matching (SPM). Three similarity scores and a bootstrap-derived confidence score were evaluated in terms of their classification performance. The effects of high and low-mass resolutions on the classification success were evaluated. RESULTS: Several hundred single-cell mass spectra from nine genera and nine species of marine microalgae were obtained. SPM enabled the identification of single cells at the genus and species level with high accuracies. The receiver operating characteristic (ROC) curves indicated a good performance of the similarity measures but were outperformed by the bootstrap-derived confidence scores. CONCLUSION: This is the first study to solve taxonomic identification of microalgae based on the metabolic fingerprints of the individual cell using an SPM approach.

The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom.

Flagellated oomycetes frequently infect unicellular algae, thus limiting their proliferation. Here we show that the marine oomycete Lagenisma coscinodisci rewires the metabolome of the bloom-forming diatom Coscinodiscus granii, thereby promoting infection success. The algal alkaloids ß-carboline and 4-carboxy-2,3,4,9-tetrahydro-1H-ß-carboline are induced during infection. Single-cell profiling with AP-MALDI-MS and confocal laser scanning microscopy reveals that algal carbolines accumulate in the reproductive form of the parasite. The compounds arrest the algal cell division, increase the infection rate and induce plasmolysis in the host. Our results indicate that the oomycete manipulates the host metabolome to support its own multiplication.

Live Single-Cell Metabolomics With Matrix-Free Laser/Desorption Ionization Mass Spectrometry to Address Microalgal Physiology.

Unicellular phototrophic algae can form massive blooms with up to millions of individual cells per milliliter in freshwater and marine ecosystems. Despite the temporal dominance of bloom formers many algal species can co-exist and compete for nutrients and space, creating a complex and diverse community. While microscopy and single cell genomics can address the taxonomic inventory, the cellular metabolome has yet to be thoroughly explored to determine the physiological status of microalgae. This might, however, provide a key to understand the observed species diversity in the homogeneous environment. Here, we introduce an effective, rapid and versatile method to analyze living single cells from aqueous substrata with laser-desorption/ionization mass spectrometry (LDI-MS) using a simple and inexpensive matrix-free support. The cells deposited on a cultivation-medium wetted support are analyzed with minimal disturbance as they remain in their natural viable state until their disruption during LDI-MS. Metabolites desorbed from single cells are analyzed on High-Resolution Mass Spectrometry (HR-MS) using the Orbitrap FT-MS technology to fingerprint cellular chemistry. This live single-cell mass spectrometry (LSC-MS) allows assessing the physiological status and strain-specifics of different microalgae, including marine diatoms and freshwater chlorophytes, at the single-cell level. We further report a reliable and robust data treatment pipeline to perform multivariate statistics on the replicated LSC-MS data. Comparing single cell MS spectra from natural phytoplankton samples and from laboratory strains allows the identification and discrimination of inter and intra-specific metabolic variability and thereby has promising applications in addressing highly complex phytoplankton communities. Notably, the herein described matrix-free live-single-cell LDI-HR-MS approach enables monitoring dynamics of the plankton and might explain why key-players survive, thrive, avoid selective feeding or pathogenic virus and bacteria, while others are overcome and die.

Exodermis and endodermis are the sites of xanthone biosynthesis in Hypericum perforatum roots.

Xanthones are specialized metabolites with antimicrobial properties, which accumulate in roots of Hypericum perforatum. This medicinal plant provides widely taken remedies for depressive episodes and skin disorders. Owing to the array of pharmacological activities, xanthone derivatives attract attention for drug design. Little is known about the sites of biosynthesis and accumulation of xanthones in roots. Xanthone biosynthesis is localized at the transcript, protein, and product levels using in situ mRNA hybridization, indirect immunofluorescence detection, and high lateral and mass resolution mass spectrometry imaging (AP-SMALDI-FT-Orbitrap MSI), respectively. The carbon skeleton of xanthones is formed by benzophenone synthase (BPS), for which a cDNA was cloned from root cultures of H. perforatum var. angustifolium. Both the BPS protein and the BPS transcripts are localized to the exodermis and the endodermis of roots. The xanthone compounds as the BPS products are detected in the same tissues. The exodermis and the endodermis, which are the outermost and innermost cell layers of the root cortex, respectively, are not only highly specialized barriers for controlling the passage of water and solutes but also preformed lines of defence against soilborne pathogens and predators.

Sample Preparation for Mass Spectrometry Imaging of Plant Tissues: A Review.

Mass spectrometry imaging (MSI) is a mass spectrometry based molecular ion imaging technique. It provides the means for ascertaining the spatial distribution of a large variety of analytes directly on tissue sample surfaces without any labeling or staining agents. These advantages make it an attractive molecular histology tool in medical, pharmaceutical, and biological research. Likewise, MSI has started gaining popularity in plant sciences; yet, information regarding sample preparation methods for plant tissues is still limited. Sample preparation is a crucial step that is directly associated with the quality and authenticity of the imaging results, it therefore demands in-depth studies based on the characteristics of plant samples. In this review, a sample preparation pipeline is discussed in detail and illustrated through selected practical examples. In particular, special concerns regarding sample preparation for plant imaging are critically evaluated. Finally, the applications of MSI techniques in plants are reviewed according to different classes of plant metabolites.

Privatization of cooperative benefits stabilizes mutualistic cross-feeding interactions in spatially structured environments.

Metabolic cross-feeding interactions are ubiquitous in natural microbial communities. However, it remains generally unclear whether the production and exchange of metabolites incurs fitness costs to the producing cells and if so, which ecological mechanisms can facilitate a cooperative exchange of metabolites among unrelated individuals. We hypothesized that positive assortment within structured environments can maintain mutualistic cross-feeding. To test this, we engineered Acinetobacter baylyi and Escherichia coli to reciprocally exchange essential amino acids. Interspecific coculture experiments confirmed that non-cooperating types were selectively favoured in spatially unstructured (liquid culture), yet disfavoured in spatially structured environments (agar plates). Both an individual-based model and experiments with engineered genotypes indicated that a segregation of cross-feeders and non-cooperating auxotrophs stabilized cooperative cross-feeding in spatially structured environments. Chemical imaging confirmed that auxotrophs were spatially excluded from cooperative benefits. Together, these results demonstrate that cooperative cross-feeding between different bacterial species is favoured in structured environments such as bacterial biofilms, suggesting this type of interactions might be common in natural bacterial communities.

Correcting mass shifts: A lock mass-free recalibration procedure for mass spectrometry imaging data.

Mass spectrometry imaging (MSI) has become widely popular because of its potential to map the spatial distribution of thousands of compounds in a single measurement directly from tissue surfaces. With every MSI experiment, it is important to maintain high mass accuracy for correct identification of the observed ions. Many times this can be compromised due to different experimental factors, leading to erroneous assignment of peaks. This makes recalibration a crucial preprocessing step. We describe a lock mass-free mass spectra recalibration method, which enables to significantly reduce these mass shift effects. The recalibration method is applied in three steps: First, we decide on an order to process all the spectra. Herein, we describe three different methods for ordering the spectra-minimum spanning tree (MST), topological greedy (TG), and crystal growth (CG). Second, we construct a reference (consensus) spectrum, from the ordered spectra, and third, all spectra are individually corrected against this consensus spectrum. The performance of the recalibration method is demonstrated on three imaging datasets acquired from matrix-assisted laser desorptionionization (MALDI) and laser desorption/ionization (LDI) mass spectrometry imaging of whole-body Drosophila melanogaster fly. The applied recalibration method is shown to strongly reduce the observed mass shifts in the imaging datasets. Among the three ordering methods, CG and MST perform comparatively better than TG and highly decrease the overall standard deviation of the mass error distribution. Lock mass correction of MSI data is practically difficult, as not all spectra contain the selected lock mass peak. Our method eliminates this need.

Mass spectrometry imaging of surface lipids on intact Drosophila melanogaster flies.

The spatial distribution of neutral lipids and semiochemicals on the surface of six-day-old separately reared naive Drosophila melanogaster flies has been visualized and studied using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and laser-assisted desorption/ionization (LDI)-TOF imaging (MSI). Metal targets were designed for two-dimensional MSI of the surface of 3-D biological objects. Targets with either simple grooves or profiled holes designed to accurately accommodate the male and female bodies were fabricated. These grooves and especially holes ensured correct height fixation and spatial orientation of the flies on the targets after matrix application and sample drying. For LDI-TOF to be used, the flies were arranged into holes and fixed to a plane of the target using fast-setting glue. In MALDI-TOF mode, the flies were fixed as above and sprayed with a lithium 2,5-dihydroxybenzoate matrix using up to 100 airbrush spray cycles. The scanning electron microscopy images revealed that the deposits of matrix were homogenous and the matrix formed mostly into the clusters of crystals (40-80 µm) that were separated from each other by an uncovered cuticle surface (30-40 µm). The MSI using target with profiled holes provided superior results to the targets with simple grooves, eliminating the ion suppression/mass deviation due to the 3-D shape of the flies. Attention was paid to neutral lipids and other compounds including the male anti-attractant 11-cis-vaccenyl acetate for which the expected distribution with high concentration on the tip of the male abdomen was confirmed. The red and blue mass shift (PlusMinus1 colour scale) was observed associated with mass deviation predominantly between ±0.2 and 0.3 Da. We use in-house developed software for mass recalibration, to eliminate the mass deviation effects and help with the detection of low-intensity mass signals.

Poly[N-(2-hydroxypropyl)methacrylamide]-based tissue-embedding medium compatible with MALDI mass spectrometry imaging experiments.

Traditional tissue-sectioning techniques for histological samples utilize various embedding media to stabilize the tissue on a sectioning target and to provide a smooth cutting surface. Due to the ion suppression effect in MALDI ionization and number of background peaks in the low-mass region, these media are not suitable for mass spectrometry imaging (MSI) experiments. To overcome this, droplets of water are often used to mount the tissue on a sectioning target, but the ice block formed around the tissue does not provide a good support for sectioning of fragile samples. In this work, we propose a novel embedding media, compatible with MALDI ionization and MSI experiments, based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). Using a reversible addition-fragmentation chain transfer polymerization technique, well-defined pHPMA polymer with narrow mass distribution was prepared. Benefits of the resulted pHPMA-based embedding media were tested on different tissue samples.

Scanning electron microscopic imaging of surface effects in desorption and nano-desorption electrospray ionization.

Scanning electron microscopy was used to investigate rivulets that are formed on the analyzed surface during desorption electrospray ionization (DESI) experiment. Ferromagnetic nanoparticles added to the spray solvent in a form of colloid solution functioned as an additional surface probe. The existence of the rivulets was confirmed on glass and newly demonstrated on two different types of porous polytetrafluoroethylene (PTFE). The results show that in standard DESI set-up the rivulets are arranged into very regular shapes. Same rivulets were obtained in DESI experiments without high voltage on the sprayer. However, no such rivulets or any other regular patterns were found on a surface in nano-DESI (nanospray DESI without the carrier nebulizing gas) experiments. This indicates that symmetrical rivulets are created by the hydrodynamical rather than electrostatic forces. It was also demonstrated that blocking the rivulets by a simple physical barrier did not influence known surface charging effects.