To Treat or Not to Treat Bees? Handy VarLoad: A Predictive Model for Varroa destructor Load.

The parasitic Varroa destructor is considered a major pathogenic threat to honey bees and to beekeeping. Without regular treatment against this mite, honey bee colonies can collapse within a 2-3-year period in temperate climates. Beyond this dramatic scenario, Varroa induces reductions in colony performance, which can have significant economic impacts for beekeepers. Unfortunately, until now, it has not been possible to predict the summer Varroa population size from its initial load in early spring. Here, we present models that use the Varroa load observed in the spring to predict the Varroa load one or three months later by using easily and quickly measurable data: phoretic Varroa load and capped brood cell numbers. Built on 1030 commercial colonies located in three regions in the south of France and sampled over a three-year period, these predictive models are tools designed to help professional beekeepers' decision making regarding treatments against Varroa. Using these models, beekeepers will either be able to evaluate the risks and benefits of treating against Varroa or to anticipate the reduction in colony performance due to the mite during the beekeeping season.

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.

Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees.

Due to the widespread use of pesticides and their persistence in the environment, non-target organisms are chronically exposed to mixtures of toxic residues. Fungicides, herbicides and insecticides are all found at low doses in the diet of pollinators such as honey bees, but due to the lack of data on the toxicological effects of these mixtures, determining their risk is difficult to assess. We therefore developed a study combining the identification of common pollen-bound pesticide mixtures associated with poor colony development and tested their effects on bee behavior and physiology. We exposed bees to the identified pesticide mixtures during the first days of their adult life, a crucial period for physiological development. Using optic bee counters we recorded the behavior of bees throughout their lives and identified two pesticide mixtures that delay the onset of foraging and slow-down foraging activity. Furthermore, one of these mixtures hampers pollen foraging. As bee longevity is strongly influenced by the time spent foraging, bees exposed to these pesticide mixtures outlived control bees. Physiological analysis revealed that perturbations of the energetic metabolism preceded the altered behavior. In conclusion, we found that early-life exposure to low doses of pesticide mixtures can have long-term effects that translate into longer-lived but slower and less efficient bees. These surprising findings contrast with the commonly reported increase in bee mortality upon pesticide exposure, and demonstrate that exposure that may seem harmless (e.g., very low doses, pesticides not intended to kill insects) can have undesirable effects on non-target organisms.

Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

Lethal and sub-lethal effects of thymol on honeybee (Apis mellifera) larvae reared in vitro.

BACKGROUND: Thymol offers an attractive alternative to synthetic chemicals to keep Varroa under control. However, thymol accumulates in bee products and is suspected of having adverse effects on colonies and especially on larvae. In this study, we investigated the effects of acute and chronic exposure to thymol on larvae reared in vitro with contaminated food and compared results to the theoretical larval exposure based on the amount of pollen and honey consumed by larvae during their development. RESULTS: The laboratory assays reveal that, first, the 48 h-LD50 of thymol introduced into larval food is 0.044 mg larva(-1) . Second, the 6 day-LC50 is 700 mg kg(-1) food. A significant decrease of larval survival and mass occurred from 500 mg thymol kg(-1) food (P < 0.0001). Finally, vitellogenin expression, which reached a maximum at the fifth instar larvae, is delayed for individuals exposed to 50 mg thymol kg(-1) food (P < 0.0006). That is 10 times higher than the theoretical level of exposure. CONCLUSION: Based on the level of thymol residue found in honey and pollen, these results suggest that the contamination of food by thymol represents no notable risk for the early-developing larvae.

Comparative susceptibility of three Western honeybee taxa to the microsporidian parasite Nosema ceranae.

Genetic diversity of a host species is a key factor to counter infection by parasites. Since two separation events and the beginning of beekeeping, the Western honeybee, Apis mellifera, has diverged in many phylogenetically-related taxa that share common traits but also show specific physiological, behavioural and morphological traits. In this study, we tested the hypothesis that A. mellifera taxa living in a same habitat should respond differently to parasites like Nosema ceranae, a microsporidia living in host's midgut. We used the Poulin and Combes' concept of virulence to compare the susceptibility of three A. mellifera taxa to N. ceranae infection. Three criteria were measured 10 days post-infection (dpi): the host mortality, the host sugar consumption and the development success of the parasite (i.e. number of spores produced). Interestingly, we showed that the observed variation in susceptibility to infection by N. ceranae is not linked to honeybee taxa but results from the variability between colonies, and that those differences are probably linked to genetic variations. The use of these three criteria allows us to conclude that the differences in susceptibility are mediated by a genetic variability in honeybee workers from resistance to tolerance. Finally, we discuss the consequences of our findings for beekeeping management.

Parasite-insecticide interactions: a case study of Nosema ceranae and fipronil synergy on honeybee.

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.

Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae.

BACKGROUND: 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. METHODOLOGY/FINDING: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y.

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.

Microsporidia: a model for minimal parasite-host interactions.

Microsporidia are emerging fungi-like intracellular parasites of economic, veterinary and medical importance. The strategy they use to invade their host is related to the rapid extrusion of a unique and highly specialized organelle, the polar tube, which allows the injection of the infectious spore content within a target cell. This original process seems to be dependent on initial interactions between parasite and host cell components. The extreme reduction and compaction of most microsporidian genomes resulted in the loss of many metabolic pathways, which makes these parasites highly dependent on their host. Recent significant advances have been made in the understanding of mammal and insect immune responses against microsporidian infections with the involvement of both adaptive and innate immunity.

Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2.

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.