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Neonicotinoids as thiamethoxam and thiacloprid are suspected to be implicated in the decline of honey bee populations. As nicotinic acetylcholine receptor agonists, they disturb acetylcholine receptor signaling in insects, leading to neurotoxicity and are therefore globally used as insecticides. Several behavioral studies have shown links between neonicotinoid exposure of bees and adverse effects on foraging activity, homing flight performance and reproduction, but the molecular aspects underlying these effects are not well-understood. In the last years, several studies through us and others showed the effects of exposure to neonicotinoids on gene expression in the brain of honey bees. Transcripts of acetylcholine receptors, hormonal regulation, stress markers, detoxification enzymes, immune system related genes and transcripts of the energy metabolism were altered after neonicotinoid exposure. To elucidate the link between homing flight performance and shifts in gene expression in the brain of honey bees after neonicotinoid exposure, we combined homing flight activity experiments applying RFID technology and gene expression analysis. We analyzed the expression of endocrine factors, stress genes, detoxification enzymes and genes linked to energy metabolism in forager bees after homing flight experiments. Three different experiments (experiment I: pilot study; experiment II: “worst-case” study and experiment III: laboratory study) were performed. In a pilot study, we wanted to investigate if we could see differences in gene expression between controls and exposed bees (experiment I). This first study was followed by a so-called “worst-case” study (experiment II), where we investigated mainly differences in the expression of transcripts linked to energy metabolism between fast and slow returning foragers. We found a correlation between homing flight duration and the expression of cytochrome c oxidase subunit 5A, one transcript linked to oxidative phosphorylation. In the third experiment (experiment III), foragers were exposed in the laboratory to 1 ng/bee thiamethoxam and 8 ng/bee thiacloprid followed by gene expression analysis without a subsequent flight experiment. We could partially confirm the induction of cytochrome c oxidase subunit 5A, which we detected in experiment II. In addition, we analyzed the effect of the feeding mode (group feeding vs. single bee feeding) on data scattering and demonstrated that single bee feeding is superior to group feeding as it significantly reduces variability in gene expression. Based on the data, we thus hypothesize that the disruption of energy metabolism may be one reason for a prolongation of homing flight duration in neonicotinoid treated bees.

Summarizing all our results, we can draw the following picture (Fig.6). The expression of HCV proteins induces an ER stress response. The consequence of prolonged ER stress is the release of Ca2+ from the ER into the cytosol. Cytosolic Ca2+-transients activate Ca2+ dependent kinases, which phosphorylate the transcription factor CREB. Phosphorylated CREB binds to the CRE-element in the PP2Ac promoter and induces transcription. Elevated PP2Ac level inhibits the enzymatic activity of PRMT1. Two consequences of reduced PRMT1 activity are the hypomethylation of STAT 1 and the hypomethylation of NS3 helicase. Hypomethylated STAT1 shows increased association with PIAS1 and decreased binding to IFNa-target genes upon IFNa treatment. The consequence of the inhibited IFNa-signaling is a reduced antiviral response, which contributes to the development of chronic liver infection, which later on can progress to liver cirrhosis and HCC. On the other hand, the unwinding activity of hypomethylated NS3 is increased compared to methylated NS3. The higher unwinding activty of NS3 causes an increase in viral replication. Therefore the upregulation of PP2Ac has two advantages for the virus: reduced cellular antiviral response and increased viral replication. Perspectives: The promising results of the improved IFNa signaling in UHCV 57.3 cells (express HCV proteins in the absence of tetracycline) after AdoMet/Betaine treatment and the fact that the expression of HBV proteins in Huh7.93 cells induces obviously the same IFNa inhibiting mechanism (increased PP2Ac levels, hypomethylated STAT1 and decreased induction of IFNa target genes), it opens the interesting perspective of correcting the HBV induced defect in IFNa signaling also by treating cells with AdoMet and betaine. We would expect, that the AdoMet/betaine treatment would at least partially restore the IFNa signaling in Huh7.93 cells and would lead to an increased induction of antiviral genes compared to Huh7.93 cells not treated with AdoMet/Betaine. AdoMet and Betaine are two compounds, which are already on the market as over the counter drugs, and which are used to treat liver diseases. Now that we could show improved IFNa signaling in UHCV57.3 cell pretreated with AdoMet and betaine, we plan to apply these drugs to HCV patients in a clinical study. The plan is to retreat nonresponders with a combination of AdoMet, Betaine, IFNa and rabavirin. To increase the activity of PRMT1 by manipulating the methionine/AdoMet cycle is one possibility to correct the inhibition of the IFNa signaling by HCV. Another possibility would be to target the first event, induction of ER stress by HCV. Without the induction of ER stress during expression of HCV proteins, Ca2+ would not leak out of the ER and the phosphorylation of CREB would not occur, so the level of PP2A would not rise and therefore there would be no inhibition of PRMT1. One possibility to avoid ER stress is the treatment of cells with chemical chaperones. Chemical chaperones are a group of low-molecular-weight componds, which can reverse the mislocalization and/or aggregation of proteins(138). Polyos such as glycerol, trimethylamines such as trimethlyamine N-oxide (TMAO), amino acid derivatives and other compounds like 4- phenylbutyric acid (PBA) and membrane-permeable forms of enzyme antagonists belong to the chemical chaperones. The mechanisms by which chemical chaperones function are not fully understood but are thought to include stabilization of improperly folded proteins, reduction of aggregation, prevention of nonproductive interactions with other resident proteins and alteration of the activity of endogenous chaperones in such a way that the affected proteins are more efficiently transported to the appropriate intracellular or extracellular destination. An interesting experiment would be the expression of viral proteins in UHCV57.3 cells for 24h in the presence of chemical chaperones, and check afterwards for the level of PP2Ac and for the induction of ER stress markers like BiP or spliced XBP1. We would expect, that the increased level of chaperones in the ER prevent the induction of ERstress and the Ca2+ -transients in the cytosol. Therefore the transcription factor CREB does not get phosphorylated, and the PP2Ac level stays constant even in the presence of viral proteins.