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International audience; Nosema ceranae is an emerging and invasive gut pathogen in Apis mellifera and is considered as a factor contributing to the decline of honeybee populations. Here, we used a combined LC-MS and NMR approach to reveal the metabolomics changes in the hemolymph of honeybees infected by this obligate intracellular parasite. For metabolic profiling, hemolymph samples were collected from both uninfected and N. ceranae-infected bees at two time points, 2 days and 10 days after the experimental infection of emergent bees. Hemolymph samples were individually analyzed by LC-MS, whereas each NMR spectrum was obtained from a pool of three hemolymphs. Multivariate statistical PLS-DA models clearly showed that the age of bees was the parameter with the strongest effect on the metabolite profiles. Interestingly, a total of 15 biomarkers were accurately identified and were assigned as candidate biomarkers representative of infection alone or combined effect of age and infection. These biomarkers included carbohydrates (α/β glucose, α/β fructose and hexosamine), amino acids (histidine and proline), dipeptides (Glu-Thr, Cys-Cys and γ-Glu-Leu/Ile), metabolites involved in lipid metabolism (choline, glycerophosphocholine and O-phosphorylethanolamine) and a polyamine compound (spermidine). Our study demonstrated that this untargeted metabolomics-based approach may be useful for a better understanding of pathophysiological mechanisms of the honeybee infection by N. ceranae.

International audience; Oxidized unsaturated fatty acids - i.e. eicosanoids and other oxylipins - are lipid mediators involved in the regulation of numerous physiological functions such as inflammation, blood coagulation, vascular tone and endothelial permeability. They have raised strong interest in clinical lipidomics in order to understand their role in health and diseases and their use as biomarkers. However, before the clinical translation, it is crucial to validate the analytical reliability of oxylipins. This notably requires to assess the putative artificial formation or degradation of oxylipins by (unsuitable) blood handling during plasma generation, storage and sample preparation. Using a liquid chromatography-mass spectrometry method covering 133 oxylipins we comprehensively analyzed the total (free + esterified) oxylipin profile in plasma and investigated the influence of i) addition of additives during sample preparation, ii) different storage times and temperatures during the transitory stage of plasma generation and iii) long-term storage of plasma samples at -80 degrees C. Addition of radical scavenger butylated hydroxytoluene reduced the apparent concentrations of hydroxy-PUFA and thus should be added to the samples at the beginning of sample preparation. The concentrations of all oxylipin classes remained stable (within analytical variance of 20%) during the transitory stage of plasma generation up to 24 h at 4 degrees C or 4 h at 20 degrees C before centrifugation of EDTA-whole blood and up to 5 days at - 20 degrees C after plasma separation. The variations in oxylipin concentrations did not correlate with storage time, storage temperature or stage of plasma generation. A significant increase of potentially lipoxygenase derived hydroxy-PUFA compared to immediate processing was only detected when samples were stored for longer times before centrifugation, plasma separation as well as freezing of plasma revealing residual enzymatic activity. Autoxidative rather than enzymatic processes led to a slightly increased concentration of 9-HETE when plasma samples were stored at - 80 degrees C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 degrees C supporting the application of oxylipin profiling in clinical research.

This research has been supported by the ANR French program MetaboHUB. We would like to thank Mélanie Pétéra for her valuable technical assistance; AbstractIntroduction: Active microorganisms have been recently discovered in clouds, thus demonstrating the capacity of microorganisms to exist in harsh environments, including exposure to UV and oxidants, osmotic and cold shocks, etc. It is important to understand how microorganisms respond to and survive such stresses at the metabolic level.Objectives: The objective of this work is to assess metabolome modulation in a strain of Pseudomonas syringae isolated from cloud water and facing temperature downshift from 17 to 5 °C by identifying key molecules and pathways of the response/adaptation to cold shock.Methods: Bacterial extracts from suspensions of cells grown at 17 °C and further incubated in microcosms at 5 and 17 °C to mimic cloud conditions were analysed by combining LC-MS and NMR; the results were evaluated in comparison to similar suspensions kept at constant temperature. The differences in the metabolome profiles were deciphered using multivariate statistics (PLS-DA).Results: Key cold shock biomarkers were observed, including cryoprotectants (trehalose, glucose, glycerol, carnitine, glutamate), antioxidants (glutathione and carnitine) and their precursors, alkaloids (bellendine and slaframine) and metabolites involved in energy metabolism (ATP, carbohydrates). Furthermore, new short peptides (nine dipeptides and a tetrapeptide) were found that have no known function.Conclusions: This study shows that in response to cold temperatures, Pseudomonas syringae PDD-32b-74 demonstrates numerous metabolism modifications to counteract the impacts of low temperatures.