A combined LC-MS and NMR approach to reveal metabolic changes in the hemolymph of honeybees infected by the gut parasite Nosema ceranae.

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/ß glucose, α/ß 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.

Co-occurrence of microcystin and anatoxin-a in the freshwater lake Aydat (France): Analytical and molecular approaches during a three-year survey.

Cyanobacterial mass occurrence is becoming a growing concern worldwide. They notably pose a threat to water users when cyanotoxins are produced. The aim of this study was to evaluate the occurrence and the dynamics of two cyanotoxins: microcystin (MC) and anatoxin-a (ANTX-a), and of two of the genes responsible for their production (respectively mcyA and anaC) during three consecutive bloom periods (2011, 2012 and 2013) in Lake Aydat (Auvergne, France). MC was detected at all sampling dates, but its concentration showed strong inter- and intra-annual variations. MC content did not correlate with cyanobacterial abundance, nor with any genera taken individually, but it significantly correlated with mcyA gene abundance (R2=0.51; p=0.042). MC content and mcyA gene abundance were maximal when cyanobacterial abundance was low, either at the onset of the bloom or during a trough of biomass. The LC-MS/MS analysis showed the presence of ANTX-a in the 2011 samples. To our knowledge, this is the first report of the presence of this neurotoxin in a French lake. The presence of ANTX-a corresponded to the only year for which Anabaena did not dominate the cyanobacterial community alone, and several cyanobacterial genera were present, including notably Aphanizomenon. anaC gene detection by PCR was not coherent with ANTX-a presence, both gene and toxin were never found for a same sample. This implies that molecular tools to study genes responsible for the production of anatoxin-a are still imperfect and the development of new primers is needed. This study also highlights the need for better monitoring practices that would not necessarily focus only on the peak of cyanobacterial abundance and that would take cyanotoxins other than MC into account.