The honeybee (Apis mellifera) has to cope with multiple environmental stressors, especially pesticides. Among those, the herbicide glyphosate and its main metabolite, the aminomethylphosphonic acid (AMPA), are among the most abundant and ubiquitous contaminant in the environment. Through the foraging and storing of contaminated resources, honeybees are exposed to these xenobiotics. As ingested glyphosate and AMPA are directly in contact with the honeybee gut microbiota, we used quantitative PCR to test whether they could induce significant changes in the relative abundance of the major gut bacterial taxa. Glyphosate induced a strong decrease in Snodgrassella alvi, a partial decrease of a Gilliamella apicola and an increase in Lactobacillus spp. abundances. In vitro, glyphosate reduced the growth of S. alvi and G. apicola but not Lactobacillus kunkeei. Although being no bee killer, we confirmed that glyphosate can have sublethal effects on the honeybee microbiota. To test whether such imbalanced microbiota could favor pathogen development, honeybees were exposed to glyphosate and to spores of the intestinal parasite Nosema ceranae. Glyphosate did not significantly enhance the effect of the parasite infection. Concerning AMPA, while it could reduce the growth of G. apicola in vitro, it did not induce any significant change in the honeybee microbiota, suggesting that glyphosate is the active component modifying the gut communities.

The gut of the European honeybee Apis mellifera is the site of exposure to multiple stressors, such as pathogens and ingested chemicals. Therefore, the gut microbiota, which contributes to host homeostasis, may be altered by these stressors. The abundance of major bacterial taxa in the gut was evaluated in response to infection with the intestinal parasite Nosema ceranae or chronic exposure to low doses of the neurotoxic insecticides coumaphos, fipronil, thiamethoxam, and imidacloprid. Experiments were performed under laboratory conditions on adult workers collected from hives in February (winter bees) and July (summer bees) and revealed season-dependent changes in the bacterial community composition. N. ceranae and a lethal fipronil treatment increased the relative abundance of both Gilliamella apicola and Snodgrassella alvi in surviving winter honeybees. The parasite and a sublethal exposure to all insecticides decreased the abundance of Bifidobacterium spp. and Lactobacillus spp. regardless of the season. The similar effects induced by insecticides belonging to distinct molecular families suggested a shared and indirect mode of action on the gut microbiota, possibly through aspecific alterations in gut homeostasis. These results demonstrate that infection and chronic exposure to low concentrations of insecticides may affect the honeybee holobiont.

To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.

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.

The microsporidian Vairimorpha (Nosema) ceranae is one of the most common parasites of the honeybee. A single honeybee carries many parasites and therefore multiple alleles of V. ceranae genes that seem to be ubiquitous. As a consequence, nucleotide diversity analyses have not allowed discriminating genetic structure of parasite populations. We performed deep loci-targeted sequencing to monitor the haplotype frequencies of genome markers in isolates from discontinuous territories, namely the tropical islands of the South West Indian Ocean. The haplotype frequency distribution corroborated the suspected tetraploidy of the parasite. Most major haplotypes were ubiquitous in the area but with variable frequency. While oceanic isolates differed from European and Asian outgroups, parasite populations from distinct archipelagoes also differed in their haplotype distribution. Interestingly an original and very divergent Malagasy isolate was detected. The observed population structure allowed formulating hypotheses upon the natural history of V. ceranae in this oceanic area. We also discussed the usefulness of allelic distribution assessment, using multiple informative loci or genome-wide analyses, when parasite population is not clonal within a single host.

In La Réunion, the established honeybee subspecies Apis mellifera unicolor, an endemic subspecies of African lineage, is facing considerable challenges. Since the introduction of the Varroa destructor mite in 2017 high colony losses have been recorded. We investigated the dynamics of V. destructor and two viruses, the Deformed Wing Virus (DWV), known to be transmitted by the mite, and the Chronic Bee Paralysis Virus (CBPV), in A. m. unicolor. Colonies from two apiaries located at 300 and 900 m a.s.l were monitored twice for one year without any acaricide treatment. The brood area, V. destructor infestation rates, DWV and CBPV prevalence and load were recorded monthly. A. m. unicolor maintained brood rearing throughout the year. Varroa destructor infestation resulted in high colony mortality (up to 85 %) and high phoretic mite rates (up to 52 mites per hundred bees). The establishment of DWV in colonies occurred after that of V. destructor and the mite infestation rate had a significant effect on the virus prevalence and load. CBPV appeared only transiently throughout the surveys. The data showed that, in tropical colonies with permanent brood rearing, V. destructor and DWV can reach high levels, but are still subject to seasonal variations that appear to be influenced by environmental conditions. This suggests that beekeeping practices could be adapted by favouring sites and periods for transhumance or acaricide treatment.