Team:KU Leuven/Journal/Ecology

After reading a lot of articles about MeS attracting of ladybugs, we wanted to test it ourselves. At Biobest, we could perform preference experiments with two-spotted ladybugs, therefore we made a special platform. We made different dilutions of MeS in ethanol and used pure ethanol as a control in our experiments. All our experiments were performed in a chemical safety cabinet to avoid distribution of the volatile MeS and ethanol.

After the disappointing results of our insect experiments at BioBest, we wanted to find out the cause of the random movement of the ladybugs. We thought about different concentrations and solvents, a different set-up and so on. After talking to Tim Belien of pcfruit, he offered us their Y-tube olfactometer and their lab, to perform some more experiments in different conditions. After reading some articles, we contacted the authors for some help. With some great information over the solvent and concentrations, we begun our experiments at pcfruit.
We started the first day by making different dilutions of methyl salicylate in hexane. We compared the behaviour of adult ladybugs on these concentrations with a control, pure hexane, in a Y-tube olfactometer. The next day, we tried to dilute the methyl salicylate in paraffin oil. This was not easy, because of the viscosity. We compared again all the MeS concentrations with the control in a Y-tube olfactometer.

The effects of (E)-β-farnesene (EBF) are described thoroughly in different articles, in contrary to their responses to methyl salicylate. Methyl salicylate (MeS) is a critical signal molecule in induced plant defenses. It activates the plant’s defense mechanisms and therefore it should have a negative effect on the aphids. When the plant defense mechanisms are activated, the plant will protect its most valuable parts, their reproductive parts and terminal leaves. Therefore, we hope to see a redistribution of the aphids to the lower leaves after induction with MeS.
We started our experiment with the induction of the plants with different concentrations of MeS: 10^-9, 8*10^-10, 4*10^-9, 10^-10 and 10*10^-11. We made the dilutions in water and ethanol (EtOH). We made a mistake in the water dilutions, the highest concentration MeS is not 10^-9, but 10^-8. Since the plants have two different defence pathways depending on the site of infection, the roots or the leaves, we used two different induction methods. We induced the plants through the roots with water and via spraying with EtOH on the leaves. We performed each induction method five times with every MeS concentration and used water induction as a control. After an induction of 48h, we placed fifteen aphids on the top of each plant.

We examined the amount of aphids and the distribution of these aphids 3 and 7 days after putting them on the plants. After this last aphid count, we used the plants for a cafeteria experiment with predators. We wanted to examine the effect of the different MeS inductions on the predator preference. In this experiment we used Macrolophus, a recently identified natural enemy of the aphids. A plant of every MeS concentration and a control were put in a circle and the predators were released in the middle of the circle. The amount of predators on every plant was counted after 45 min. Afterwards, the predators were shaken off the plant. This was repeated twice. To determine the effect on a longer time span, we counted the aphids also after 24h.

After the cafeteria experiment, we also wanted to determine the effect of MeS on the root growth. Therefore, we washed all the roots and measured the most important parameters: the primary root, the width of the top of the primary root and the longest secondary root.