Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core).

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+ /K+ -ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.

Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging.

Spatial metabolomics describes the spatially resolved analysis of interconnected pathways, biochemical reactions, and transport processes of small molecules in the spatial context of tissues and cells. However, a broad range of metabolite classes (e.g., steroids) show low intrinsic ionization efficiencies in mass spectrometry imaging (MSI) experiments, thus restricting the spatial characterization of metabolic networks. Additionally, decomposing complex metabolite networks into chemical compound classes and molecular annotations remains a major bottleneck due to the absence of repository-scaled databases. Here, we describe a multimodal mass-spectrometry-based method combining computational metabolome mining tools and high-resolution on-tissue chemical derivatization (OTCD) MSI for the spatially resolved analysis of metabolic networks at the low micrometer scale. Applied to plant toxin sequestration in Danaus plexippus as a model system, we first utilized liquid chromatography (LC)-MS-based molecular networking in combination with artificial intelligence (AI)-driven chemical characterization to facilitate the structural elucidation and molecular identification of 32 different steroidal glycosides for the host-plant Asclepias curassavica. These comprehensive metabolite annotations guided the subsequent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) analysis of cardiac-glycoside sequestration in D. plexippus. We developed a spatial-context-preserving OTCD protocol, which improved cardiac glycoside ion yields by at least 1 order of magnitude compared to results with untreated samples. To illustrate the potential of this method, we visualized previously inaccessible (sub)cellular distributions (2 and 5 µm pixel size) of steroidal glycosides in D. plexippus, thereby providing a novel insight into the sequestration of toxic metabolites and guiding future metabolomics research of other complex sample systems.

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches.

Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.

Potentially Poisonous Plastic Particles: Microplastics as a Vector for Cyanobacterial Toxins Microcystin-LR and Microcystin-LF.

The potential of microplastics to act as a vector for micropollutants of natural or anthropogenic origin is of rising concern. Cyanobacterial toxins, including microcystins, are harmful to humans and wildlife. In this study, we demonstrate for the first time the potential of microplastics to act as vectors for two different microcystin analogues. A concentration of up to 28 times from water to plastic was observed for the combination of polystyrene and microcystin-LF achieving toxin concentrations on the plastic of 142 ± 7 µg g-1. Based on the experimental results, and assuming a worst-case scenario, potential toxin doses for daphnids are calculated based on published microplastic ingestion data. Progressing up through trophic levels, theoretically, the concentration of microcystins in organisms is discussed. The experimental results indicate that adsorption of microcystins onto microplastics is a multifactorial process, depending on the particle size, the variable amino acid composition of the microcystins, the type of plastic, and pH. Furthermore, the results of the current study stressed the limitations of exclusively investigating microcystin-LR (the most commonly studied microcystin congener) as a model compound representing a group of around 250 reported microcystin congeners.

3D-surface MALDI mass spectrometry imaging for visualising plant defensive cardiac glycosides in Asclepias curassavica.

Mass spectrometry-based imaging (MSI) has emerged as a promising method for spatial metabolomics in plant science. Several ionisation techniques have shown great potential for the spatially resolved analysis of metabolites in plant tissue. However, limitations in technology and methodology limited the molecular information for irregular 3D surfaces with resolutions on the micrometre scale. Here, we used atmospheric-pressure 3D-surface matrix-assisted laser desorption/ionisation mass spectrometry imaging (3D-surface MALDI MSI) to investigate plant chemical defence at the topographic molecular level for the model system Asclepias curassavica. Upon mechanical damage (simulating herbivore attacks) of native A. curassavica leaves, the surface of the leaves varies up to 700 µm, and cardiac glycosides (cardenolides) and other defence metabolites were exclusively detected in damaged leaf tissue but not in different regions of the same leaf. Our results indicated an increased latex flow rate towards the point of damage leading to an accumulation of defence substances in the affected area. While the concentration of cardiac glycosides showed no differences between 10 and 300 min after wounding, cardiac glycosides decreased after 24 h. The employed autofocusing AP-SMALDI MSI system provides a significant technological advancement for the visualisation of individual molecule species on irregular 3D surfaces such as native plant leaves. Our study demonstrates the enormous potential of this method in the field of plant science including primary metabolism and molecular mechanisms of plant responses to abiotic and biotic stress and symbiotic relationships.

Targeting Kinases in Fasciola hepatica: Anthelminthic Effects and Tissue Distribution of Selected Kinase Inhibitors.

Protein kinases have been discussed as promising druggable targets in various parasitic helminths. New drugs are also needed for control of fascioliasis, a food-borne trematode infection and worldwide spread zoonosis, caused by the liver fluke Fasciola hepatica and related species. In this study, we intended to move protein kinases more into the spotlight of Fasciola drug research and characterized the fasciolicidal activity of two small-molecule inhibitors from human cancer research: the Abelson tyrosine kinase (ABL-TK) inhibitor imatinib and the polo-like 1 (PLK1) inhibitor BI2536. BI2536 reduced viability of 4-week-old immature flukes in vitro, while adult worms showed a blockade of egg production. Together with a significantly higher transcriptional expression of PLK1 in adult compared to immature worms, this argues for a role of PLK1 in fluke reproduction. Both fluke stages expressed ABL1-TK transcripts at similar high levels and were affected by imatinib. To study the uptake kinetic and tissue distribution of imatinib in F. hepatica, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for the first time in this parasite. Drug imaging revealed the accumulation of imatinib in different fluke tissues from 20 min to 12 h of exposure. Furthermore, we show that imatinib is metabolized to N-desmethyl imatinib by F. hepatica, a bioactive metabolite also found in humans. Besides the vitellarium, gastrodermal tissue showed strong signal intensities. In situ hybridization demonstrated the gastrodermal presence of abl1 transcripts. Finally, we assessed transcriptional changes of physiologically important genes in imatinib-treated flukes. Moderately increased transcript levels of a gene encoding a multidrug resistance protein were detected, which may reflect an attempt to defend against imatinib. Increased expression levels of the cell cycle dependently expressed histone h2b and of two genes encoding superoxide dismutases (SODs) were also observed. In summary, our pilot study demonstrated cross-stage activity of imatinib but not BI2536 against immature and adult F. hepatica in vitro; a fast incorporation of imatinib within minutes, probably via the oral route; and imatinib-induced expression changes of physiologically relevant genes. We conclude that kinases are worth analyzing in more detail to evaluate the potential as therapeutic targets in F. hepatica.