Behavioral Assays

The behavioral response of M. viciae to the components of the alarm pheromone was investigated under the conditions reported in Sun Y.F. et al. (2012) for A. pisum, using a Y-tube. Briefly, an airflow of 0.5 L/min was introduced into each arm of the glass Y-tube olfactometer through a glass stimulus chamber (odor source adapter) attached to each of the two arms. In each test, 1 μl of hexane solution of each chemical compound, concentration 0.5%, was placed in the glass stimulus chamber of the “treatment” arm. As a control, 1 μl of hexane was placed in the glass stimulus chamber of the “control” arm of the olfactometer. Groups of twenty wingless adult aphids were introduced at the bottom of the Y-shaped copper wire and allowed to walk to either arm at the Y-junction. After 15 min, the number of aphids in the treatment and control sides of the olfactometer were counted. Six replications with each compound were performed. Tested compounds were (E)-β-farnesene (Bedoukian Research, Danbury, CT, United States), (±)-α-pinene, β-pinene, (-)-α-pinene, (+)-limonene, hexane (Sigma-Aldrich-Fluka) and a mixture comprising (E)-β-farnesene 14.2%, (-)-α-pinene 11.8% and β-pinene 74% (Francis et al., 2005). The behavioral responses to all the analyzed compounds and mixture were compared by one-way analysis of variance (ANOVA) and Tukey’s HSD multiple comparisons test using GraphPad Prism 6.00 software for Windows (GraphPad Software) (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗ p < 0.001).