The honey bee Apis mellifera is exposed to a variety of biotic and abiotic stressors, such as the highly prevalent microsporidian parasite Nosema (Vairimorpha) ceranae and neonicotinoid insecticides. Both can affect honey bee physiology and microbial gut communities, eventually reducing its lifespan. They can also have a combined effect on the insect’s survival. The use of bacterial probiotics has been proposed to improve honey bee health, but their beneficial effect remains an open question. In the present study, western honey bees were experimentally infected with N. ceranae spores, chronically exposed to the neonicotinoid thiamethoxam, and/or supplied daily with the homofermentative bacterium Pediococcus acidilactici MA18/5M thought to improve the honey bees’ tolerance to the parasite. Deep shotgun metagenomic sequencing allowed the response of the gut microbiota to be investigated with a taxonomic resolution at the species level. All treatments induced significant changes in honey bee gut bacterial communities. Nosema ceranae infection increased the abundance of Proteus mirabilis, Frischella perrara, and Gilliamella apicola and reduced the abundance of Bifidobacterium asteroides, Fructobacillus fructosus, and Lactobacillus spp. Supplementation with P. acidilactici overturned some of these alterations, bringing back the abundance of some altered species close to the relative abundance found in the controls. Surprisingly, the exposure to thiamethoxam also restored the relative abundance of some species modulated by N. ceranae. This study shows that stressors and probiotics may have an antagonistic impact on honey bee gut bacterial communities and that P. acidilactici may have a protective effect against the dysbiosis induced by an infection with N. ceranae.

Varroa destructor, a major ectoparasite of the Western honey bee Apis mellifera, is a widespread pest that damages colonies in the Northern Hemisphere. Throughout their lifecycle, V. destructor females feed on almost every developmental stage of their host, from the last larval instar to the adult. The parasite is thought to feed on hemolymph and fat body, although its exact diet and nutritional requirements are poorly known. Using artificial Parafilm™ dummies, we explored the nutrition of V. destructor females and assessed their survival when fed on hemolymph from bee larvae, pupae, or adults. We compared the results with mites fed on synthetic solutions or filtered larval hemolymph. The results showed that the parasites could survive for several days or weeks on different diets. Bee larval hemolymph yielded the highest survival rates, and filtered larval plasma was sufficient to maintain the mites for 14 days or more. This cell-free solution therefore theoretically contains all the necessary nutrients for mite survival. Because some bee proteins are known to be hijacked without being digested by the parasite, we decided to run a proteomic analysis of larval honey bee plasma to highlight the most common proteins in our samples. A list of 54 proteins was compiled, including several energy metabolism proteins such as Vitellogenin, Hexamerin, or Transferrins. These molecules represent key nutrient candidates that could be crucial for V. destructor survival.

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.

Pollinators, including Bombus terrestris, are crucial for maintaining biodiversity in ecosystems and for agriculture. Deciphering their immune response under stress conditions is a key issue for protecting these populations. To assess this metric, we analyzed the B. terrestris hemolymph as an indicator of their immune status. Hemolymph analysis was carried out using mass spectrometry, MALDI molecular mass fingerprinting was used for its effectiveness in assessing the immune status, and high-resolution mass spectrometry was used to measure the impact of experimental bacterial infections on the “hemoproteome”. By infecting with three different types of bacteria, we observed that B. terrestris reacts in a specific way to bacterial attacks. Indeed, bacteria impact survival and stimulate an immune response in infected individuals, visible through changes in the molecular composition of their hemolymph. The characterization and label-free quantification of proteins involved in specific signaling pathways in bumble bees by bottom-up proteomics revealed differences in protein expression between the non-experimentally infected and the infected bees. Our results highlight the alteration of pathways involved in immune and defense reactions, stress, and energetic metabolism. Lastly, we developed molecular signatures reflecting the health status of B. terrestris to pave the way for diagnosis/prognosis tools in response to environmental stress.