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The honeybee, Apis mellifera, is undergoing a worldwide decline whose origin is still in debate. Studies performed for twenty years suggest that this decline may involve both infectious diseases and exposure to pesticides. Joint action of pathogens and chemicals are known to threaten several organisms but the combined effects of these stressors were poorly investigated in honeybees. Our study was designed to explore the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid. Five days after their emergence, honeybees were divided in 6 experimental groups: (i) uninfected controls, (ii) infected with N. ceranae, (iii) uninfected and exposed to fipronil, (iv) uninfected and exposed to thiacloprid, (v) infected with N. ceranae and exposed 10 days post-infection (p.i.) to fipronil, and (vi) infected with N. ceranae and exposed 10 days p.i. to thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. Surprisingly, exposures to fipronil and thiacloprid had opposite effects on microsporidian spore production. Analysis of the honeybee detoxification system 10 days p.i. showed that N. ceranae infection induced an increase in glutathione-S-transferase activity in midgut and fat body but not in 7-ethoxycoumarin-O-deethylase activity. After exposure to sublethal doses of fipronil or thiacloprid a higher mortality was observed in N. ceranae-infected honeybees than in uninfected ones. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

Summary The causes underlying the increased mortality of honeybee colonies remain unclear and may involve multiple stressors acting together, including both pathogens and pesticides. Previous studies suggested that infection by the gut parasite Nosema ceranae combined with chronic exposure to sublethal doses of the insecticide fipronil generated an increase in oxidative stress in the midgut of honeybees. To explore the impact of these two stressors on oxidative balance, we experimentally infected bees with N. ceranae and/or chronically exposed to fipronil at low doses for 22 days, and we measured soluble reactive oxygen species (ROS) and ROS damage by quantifying both protein and lipid oxidation in the midgut. Our results revealed a disruption of the oxidative balance, with a decrease in both the amount of ROS and ROS damage in the presence of the parasite alone. However, protein oxidation was significantly increased in the N. ceranae/fipronil combination, revealing an increase in oxidative damage and suggesting higher fipronil toxicity in infected bees. Furthermore, our results highlighted a temporal order in the appearance of oxidation events in the intestinal cells and revealed that all samples tended to undergo protein oxidation during ageing, regardless of treatment. We developed protocols for A. mellifera in order to quantify soluble peroxides, lipid peroxidation and protein oxidation during infection with N. ceranae and intoxication with fipronil or exposed to the N‐acetylcysteine antioxidant. The parasite alone appeared to induce a decrease in oxidative state of intestinal cells, but associated with fipronil, it might increase its toxicity by generating, on the contrary, a rise in intestinal protein oxidation of the bee.

Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae . While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae .

In ecosystems, a variety of biological, chemical and physical stressors may act in combination to induce illness in populations of living organisms. While recent surveys reported that parasite-insecticide interactions can synergistically and negatively affect honeybee survival, the importance of sequence in exposure to stressors has hardly received any attention. In this work, Western honeybees ( Apis mellifera ) were sequentially or simultaneously infected by the microsporidian parasite Nosema ceranae and chronically exposed to a sublethal dose of the insecticide fipronil, respectively chosen as biological and chemical stressors. Interestingly, every combination tested led to a synergistic effect on honeybee survival, with the most significant impacts when stressors were applied at the emergence of honeybees. Our study presents significant outcomes on beekeeping management but also points out the potential risks incurred by any living organism frequently exposed to both pathogens and insecticides in their habitat.

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.

Despite their ubiquity, the diversity and dynamics of Microsporidia in aquatic ecosystems have long been overlooked. Here, we applied 18S rRNA gene metabarcoding targeting the V1‐V3 region for Microsporidia and V4 region for other eukaryotes to sedimentary ancient DNA to reconstruct the temporal dynamics of Microsporidia and their eukaryotic hosts over the past 138 years in Lake Aydat (France). We detected remarkably high microsporidian diversity, with 1976 Operational Taxonomic Units (OTUs). However, 54% of these OTUs could not be assigned at the genus level, indicating substantial, as yet undescribed diversity. The remaining OTUs were affiliated predominantly with three genera: Crispospora, Pseudoberwaldia, and Vittaforma. Despite only one formally described species in each of these genera, we show a high species‐level diversity forming putative freshwater lake‐specific clades, which are likely associated with new hosts. Using co‐occurrence network analysis combined with published host records, we identify rotifers as candidate reservoirs for lacustrine Microsporidia. The microsporidian community underwent substantial temporal changes, marked by a major turnover in the early 2000s. This shift coincides with decades of strong anthropogenic pressure on Lake Aydat, including untreated sewage inputs, nutrient enrichment from agro‐pastoral activities, and wetland destruction. As obligate intracellular parasites, Microsporidia are tightly linked to the ecology and population dynamics of their hosts, suggesting that the observed community shifts primarily reflect changes in host communities. Sedimentary ancient DNA study revealed 138 years of hidden microsporidian (unicellular eukaryotic parasite) diversity and host–parasite dynamics in a freshwater lake. A major community shift in the 2000s, linked to anthropogenic pressures, shows how paleogenomics reveals the diversity and long‐term ecological trajectories of these previously overlooked aquatic parasites.

Among the various “omics” approaches that can be used in toxicology, volatolomics is in full development. A volatolomic study was carried out on soil bacteria to validate the proof of concept, and this approach was implemented in a new model organism: the honeybee Apis mellifera. Emerging bees raised in the laboratory in pain-type cages were used. Volatolomics analysis was performed on cuticles, fat bodies, and adhering tissues (abdomens without the digestive tract), after 14 and 21 days of chronic exposure to 0.5 and 1 µg/L of fipronil, corresponding to sublethal doses. The VOCs analysis was processed using an HS-SPME/GC-MS method. A total of 281 features were extracted and tentatively identified. No significant effect of fipronil on the volatolome could be observed after 14 days of chronic exposure. Mainly after 21 days of exposure, a volatolome deviation appeared. The study of this deviation highlighted 11 VOCs whose signal abundances evolved during the experiment. Interestingly, the volatolomics approach revealed a VOC (2,6-dimethylcyclohexanol) that could act on GABA receptor activity (the fipronil target) and VOCs associated with semiochemical activities (pheromones, repellent agents, and compounds related to the Nasonov gland) leading to a potential impact on bee behavior.

Among the various “omics” approaches that can be used in toxicology, volatolomics is in full development. A volatolomic study was carried out on soil bacteria to validate the proof of concept, and this approach was implemented in a new model organism: the honeybee Apis mellifera. Emerging bees raised in the laboratory in pain-type cages were used. Volatolomics analysis was performed on cuticles, fat bodies, and adhering tissues (abdomens without the digestive tract), after 14 and 21 days of chronic exposure to 0.5 and 1 µg/L of fipronil, corresponding to sublethal doses. The VOCs analysis was processed using an HS-SPME/GC-MS method. A total of 281 features were extracted and tentatively identified. No significant effect of fipronil on the volatolome could be observed after 14 days of chronic exposure. Mainly after 21 days of exposure, a volatolome deviation appeared. The study of this deviation highlighted 11 VOCs whose signal abundances evolved during the experiment. Interestingly, the volatolomics approach revealed a VOC (2,6-dimethylcyclohexanol) that could act on GABA receptor activity (the fipronil target) and VOCs associated with semiochemical activities (pheromones, repellent agents, and compounds related to the Nasonov gland) leading to a potential impact on bee behavior.

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.