Honeybees play an important role in pollinating native plants and agricultural crops and produce valuable hive products. Within the last decade, honeybee colonies have been reported to be in decline, due to both biotic and abiotic stress factors including pathogens and pesticides. This study evaluated the impact of different isolates of Nosema spp.[Nosema apis spores (NA), Nosema ceranae from Apis mellifera from France (NF), N. ceranae from Apis cerana from Thailand (NC1), and N. ceranae from A. mellifera from Thailand (NC2)] on the different gut sections of newly emerged adult A. mellifera bees. With an attempt to decipher the early impact of Nosema spp. on the first barrier against Nosema infection, we used off-gel bottom-up proteomics on the different anatomical sections of the gut four days post inoculation. A total of 2185 identified proteins in the esophagus, 2095 in the crop, 1571 in the midgut, 2552 in the ileum, and 3173 in the rectum were obtained. Using label-free quantification, we observed that the response of the host varies according to the Nosema spp. (N. apis versus N. ceranae) and the geographical origin of Nosema. The proteins in the midgut of A. mellifera, orally inoculated with spores of N. ceranae isolated from France, were the most altered, when compared with controls, exhibiting 50 proteins down-regulated and 16 up-regulated. We thereby established the first massspectrometry-based proteomics of different anatomical sections of the gut tissue of Nosema-infected A. mellifera four days post inoculation, following infection by different isolates of Nosema spp. that provoked differential host responses. We reported an alteration of proteins involved in the metabolic pathways and specifically eight proteins of the oxidative phosphorylation pathway. More importantly, we propose that the collagen IV NC1 domain-containing protein may represent an early prognostic marker of the impact of Nosema spores on the A. mellifera health status. Data are available via ProteomeXchange with the identifier PXD021848.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue.More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.