Schlüsselwort: gelelectrophoresis

This work investigated the influence of the varroacide formic acid on the honeybee Apis mellifera as well as its parasite Varroa destructor. Varroosis, in combination with other factors, leads to partially massive honeybee colony losses. These are of enormous ecological and economic importance, making an efficient drug treatment of the bee colonies obligatory. Formic acid is currently considered the most reliable and relatively simple treatment method due to its numerous advantages over other active substances, although damage to the honeybee may occur during this treatment. In order to increase the understanding of the mechanism of action of formic acid and to reduce the negative effects on the honeybee in the future by an adapted application, the reaction to 60% formic acid in honeybees and varroa mites was investigated using comparative molecular biological and biochemical methods. In the present study, molecular endogenous effects of formic acid treatment on gene expression in honeybee and varroa mite were investigated. For this purpose, RNA-Seq was applied and the results were subsequently validated by RT-qPCR analyses. Subsequent proteome analyses were performed to detect the correlating proteins in order to determine whether these detected transcriptional changes are also reflected in the protein pattern. In this work corresponding experiments were carried out for the varroa mite, the least studied model. Since even the concentration of a protein in a system does not provide valid details about its activity and function, the formic acid-specific activity of selected enzymes was examined in functional assays in the last part of the work. For this purpose, an activity assay of Cytochrom C oxidase was established in a microtiter plate system. Further investigations of the enzymatic activity of some important candidate proteins will follow by their recombinant expression with subsequent functional assays. The results of transcriptome analysis in honeybees indicate that the known higher formic acid sensitivity of the younger larval stages compared to the newly hatched worker bees is due to a lower detoxification capacity as a result of a reduced equipment with appropriate detoxification enzymes compared to adult bees. However, a detection of formic acid-induced candidate genes in honey bees could not be achieved in varroa mites. This could indicate different formic acid metabolism strategies between these two organisms. On this basis, the persistent problem of unwanted damage to bees during formic acid treatment could be solved by developing new formulations and/or applications that target specific target structures in the mites. The proteome analysis of formic acid-treated varroa mites indicated an imbalance in proteostasis due to restricted protein synthesis combined with increased protein degradation. This indicates a significant loss of mass and could explain a damaging effect of formic acid on the varroa mites. The formic acid-exposed varroa mites showed an induction of heat shock proteins, an indicator of oxidative stress. This is presumably caused by a specific inhibition of the respiratory chain and the citrate cycle, which is proven in our data. As a result, damage to cellular macromolecules leads to the ageing of the organism and ultimately to cell death. The proteome analysis further revealed an increased concentration of several candidate proteins associated with detoxification, which however did not correlate with the transcriptome analysis. Overall, the investigation of the proteins with the components of the respiratory chain provided the presumed primary target site of formic acid action as well as the defence mechanisms responsible for formic acid-generated toxicity, which according to our data mainly include heat shock proteins and detoxification enzymes. The initial results of the activity studies of Cytochrom C oxidase indicate an inhibition by formic acid. These results confirm previous findings from the literature and further data sets of this work. Thus, formic acid damage appears to be a consequence of oxidative stress produced by inhibition of cellular respiration with lactic acidosis. In summary, our data show for the first time molecular effects of formic acid treatment on honeybees and simultaneously on varroa mites. Based on the findings of this study, future treatment strategies can be specifically adapted to the varroa mite, thereby significantly increasing the success of treatment in varroa control; the negative effects on honeybees, which have frequently occurred up to now, can be reduced by a better understanding of the molecular processes.