Figure 1.
A. Structures of the dialkoxybenzenes; their codenames are explained in ref [17]. B. Structures of the 5(2′-methoxyethyl) cyclopent-2-en-1-alkoxy diethers (cy{R1,1} compounds). C. Synthesis of the cy{R1,1} compounds. Abbreviations: rt = room temperature; TBDMSCl = tert-butyl dimethylsilyl chloride; THF = tetrahydrofuran.
Figure 2.
Electrophysiology with the Varroa foreleg.
A. Electrophysiology setup of an isolated Varroa foreleg that was stimulated with the headspace volatiles of freshly caught honey bees in a jar. B. Typical traces of Varroa foreleg responses to air (left) and honey bee volatiles (right). C. Varroa foreleg electrophysiological response amplitude. Comparison between the responses to the headspaces of different numbers of bees: no bee (empty jar), 1, 5 and 10 bees. ANOVA repeated measures: bars marked by different letters are significantly different, p<0.05, n = 6.
Figure 3.
Electrophysiological screening of the compounds.
A. Order of the Varroa foreleg stimulations and terminology used for the corresponding responses. The time interval between each stimulus was 30 s, unless otherwise stated. The stimuli were: Air, Headspace of five nurse bees (bee stimulus), Bee stimulus together with the compound (Bee stimulus + comp) or of the hexane control (Bee stimulus + hexane). In italics, below the stimuli, are the names of the values presented in the results. B. Initial screen of the Varroa foreleg electrophysiological response to different stimuli, all loaded at 10 µg in the stimulus cartridge (normalized values against the response to air %, average+SE). For the bee stimuli, the headspace from 5 nurse bees was used. Bars marked by different letters are significantly different, ANOVA repeated measures, p<0.05, n = 10.C. Testing of the individual components of the blend HCO-2169 at 10 µg doses (n = 10). D. Experiment with the three isomers of diethyoxybenzene at 10 µg doses (n = 6).
Figure 4.
Dose responses of long-term inhibitory compounds cy{4,1}, 3b{2,2} and cy{2,2}.
The responses of the Varroa forelegs to stimulation with different amounts of each compound and with the headspace from 5 nurse bees (normalized values against the response to air %, average+SE). Bars within each dose, marked by different letters, are significantly different, ANOVA repeated measures, p<0.05, n = 7.
Figure 5.
Detailed evaluation of the long-term inhibitory effect of the most active compounds.
The effect of 0.1 µg cy{4,1} (A) or 3b{2,2} (B), with and without a simultaneous stimulus of the headspace volatiles from 5 nurse bees, on the electrophysiological response of the Varroa foreleg. The data are normalized values (%, average+SE): bars marked by different letters are significantly different, ANOVA repeated measures, p<0.05, p<0.05, n = 6. The longevity of the inhibitory effect of 0.1 µg cy{4,1} (C) or 0.1 µg 3b{2,2} (D) on Varroa foreleg electrophysiological responses. The time interval between the mixed stimulus (Bee + compound) and the pure bee stimulus was varied. Values are normalized against the response to air (%, average+SE): bars marked by different letters are significantly different, ANOVA repeated measures, p<0.05; n = 6.
Figure 6.
The effect of selected compounds on Varroa host choice between a nurse and a forager bee.
A. Experimental setup. The test compound did not contact the mite, and the mite could move around and choose between a freshly killed nurse or forager. B. Effect of cy{4,1}: data are the percentage of Varroa that selected a particular host in the presence of hexane (control) or disrupting compound cy{4,1} at different doses (0.01 µg, 0.1 µg, 10 µg). Numbers within the bars show the number of Varroa choosing each of the hosts. C. Effect of 3b{2,2}. D. Effect of 3c{2,2}.
Figure 7.
The effect of selected compounds on Varroa ability to reach any host.
Effect of 3 selected compounds on the percentage of mites reaching any of the hosts in the choice bioassay, 180 min from the beginning of the experiment. The data are percentage of viable mites in the presence of hexane (control) or disrupting compound at each of three tested doses (0.01 µg, 0.1 µg, 10 µg) Chi-square test, ns.
Figure 8.
Active space and structure-activity of host choice alteration activity.
A. Overlay of energy minimized conformers of cy{4,1} and 3b{2,2}. The Connolly molecular surface of the overlaid molecules is shown in light blue. Hydrogen atoms and lone pairs have been omitted on the structures, but are included in the surfaces. Distances: a∼8.6 Å, b∼10 Å, c∼8.5 Å, d∼6.9 Å, e∼5.9 Å. B and C. Examples of the two structure-activity correlations found. B. Correlation between the highest occupied molecular orbital (HOMO) energy and the difference in short-term inhibition (%) between the “Bee before” and “Bee + compound” treatments (Δ STI (%)). Only the aromatic compounds (3c series, 3a{2,2}, 3b{2,2} and DEET) are included. C. Correlation between the polar accessible surface area (ASA_P) and the difference in long-term inhibition (%) between the “Bee before” and “Bee after” treatments (Δ LTI (%)).