Team:KU Leuven/Project/Ecological/wetlab

In order to validate the BanAphids' model, we decided to investigate the effect of the two substances, E-beta-farnesene (EBF) and Methyl Salicylate (MeS) on aphids. Once we started our project, we noticed that we were not the only ones approaching this agricultural problem from this angle. Research groups like Rothamsted are currently investigating the use of EBF to repel aphids as well but in the form of GM crops. We also found that other areas of the industry were also interested in our project such as Biobest, worldwide leader in sustainable crop protection and pcfruit whose mission is to research prospects for biological fruit growing, new crop protection methods, etc. We were invited to perform our insect experiments at Biobest and pcfruit to validate the BanAphids model. We attempted to determine the working concentrations of our two substances in pure form and to examine the preference of aphids and their predators under different conditions and environments. With the data we have collected so far, we can observe that these two substances indeed do have an effect on aphids.

What we wanted the insects to do!

Through many years of research, it has been found that E-beta-farnesene (EBF) is the essential compound of an aphid's alarm pheromone and that the general response to this signal is a change in gene expression that will agitate the aphids causing them to leave the plant. We tried to prove this using the synthetic compound EBF (Sigma-Aldrich, farnesene, mixture of isomers). EBF also works as an insect attractant. We also tried to prove the kairomone effect of Methyl Salicylate (Sigma-Aldrich, Methyl Salicylate ≥99%) through the use of the synthetic compound. We experimented with three different set-ups to try and determine a range working concentrations of MeS and EBF on the insects. A description of the natural enemies, ladybugs (adult and larvae), green lacewings (larvae) and parasitic wasps (adult), and aphids we received from biobest and employed in our experiments is given here.

Natural Enemies

1. The predatory insects were subjected to a choice experiment. Starting from a determined point on our set-up (see video), the insects were released and given a choice between one of the five MeS concentrations and a control solution which was the solvent used (in this case ethanol). The insects were given 1-3 mins to make a choice. After our first day at Biobest we had very disappointing results, there was no clear reaction to be seen to either the MeS or the control solution. This set-up was performed under a fume hood and this might have resulted in the rapid elimination of MeS.

2. With advice from Biobest we tried some troubleshooting, we diluted MeS and EBF in other solvents, in particular, hexane and paraffin oil. We chose to use paraffin oil because MeS is a volatile and paraffin oil would allow the slow release of this volatile, giving the insects more time to respond to MeS. Under the advice from Prof. Felix Wäckers and dr. Veerle Mommaerts, we also tried to prime the insects first. Practically, priming the insects means that we let a few predator/parasitoid insects loose on a washed aphid-infested leaf. The insects that showed interest in the leaf, this means that the insect was walking around in search of aphids, were further used in the behavioural experiments. We also tried a different set-up, with a fan, mimicking a wind tunnel, that blows in the direction of the insects so that the MeS fumes are blown toward the insect. In these set of experiments we used an aphid infested leaf as a control compared to response to this leaf after introduction of one of the five MeS or EBF concentration. This however did not demonstrate any clear behaviour form the insects other than random movement. We contemplated yet again which parameters we could change, we thought that the insects were maybe more interested in their new 'strange' environment, the set-up, than the substances but other than changing the set-up, the concentrations we used and the solvent, we didn't see many other variables that could have affected our results.

3. After talking to Tim Belien of pcfruit, he offered us their Y-tube olfactometer and their lab. We compared again all the five MeS concentrations with the control in a Y-tube olfactometer. The lady bug adults were first released into the Y-tube to become habituated with the set-up, before being exposed to MeS or EBF. We could see an obvious attraction of three ladybugs in a row at a MeS concentration of 10^-10. But after changing the side of the Y-tube in which MeS was released, we could not see the attraction anymore. We think that the Y-tube olfactometer was not clean enough and that there was still some MeS on the other tube as well. We saw repulsion of all ladybugs at a MeS concentration of 10^-7, demonstrating the upper limit of MeS concentrations. These experiments gave us an indication of the working MeS concentrations.

Repellent effect of EBF on Aphids

Several different EBF concentrations were released in proximity of aphids. We first used pure EBF because we were afraid that there was no response

What we expected the plants to do!

Through many years of research, it has been found that E-beta-farnesene (EBF) is the essential compound of an aphid's alarm pheromone and that the general response to this signal is a change in gene expression that will agitate the aphids causing them to leave the plant. We tried to prove this using the synthetic compound EBF (Sigma-Aldrich, farnesene, mixture of isomers). EBF also works as an insect attractant. We also tried to prove the kairomone effect of Methyl Salicylate (Sigma-Aldrich, Methyl Salicylate ≥99%) through the use of the synthetic compound. We experimented with three different set-ups to try and determine a range working concentrations of MeS and EBF on the insects. A description of the natural enemies, ladybugs (adult and larvae), green lacewings (larvae) and parasitic wasps (adult), and aphids we received from biobest and employed in our experiments is given here.

Natural Enemies

1. The predatory insects were subjected to a choice experiment. Starting from a determined point on our set-up (see video), the insects were released and given a choice between one of the five MeS concentrations and a control solution which was the solvent used (in this case ethanol). The insects were given 1-3 mins to make a choice. After our first day at Biobest we had very disappointing results, there was no clear reaction to be seen to either the MeS or the control solution. This set-up was performed under a fume hood and this might have resulted in the rapid elimination of MeS.

2. With advice from Biobest we tried some troubleshooting, we diluted MeS and EBF in other solvents, in particular, hexane and paraffin oil. We chose to use paraffin oil because MeS is a volatile and paraffin oil would allow the slow release of this volatile, giving the insects more time to respond to MeS. Under the advice from Prof. Felix Wäckers and dr. Veerle Mommaerts, we also tried to prime the insects first. Practically, priming the insects means that we let a few predator/parasitoid insects loose on a washed aphid-infested leaf. The insects that showed interest in the leaf, this means that the insect was walking around in search of aphids, were further used in the behavioural experiments. We also tried a different set-up, with a fan, mimicking a wind tunnel, that blows in the direction of the insects so that the MeS fumes are blown toward the insect. In these set of experiments we used an aphid infested leaf as a control compared to response to this leaf after introduction of one of the five MeS or EBF concentration. This however did not demonstrate any clear behaviour form the insects other than random movement. We contemplated yet again which parameters we could change, we thought that the insects were maybe more interested in their new 'strange' environment, the set-up, than the substances but other than changing the set-up, the concentrations we used and the solvent, we didn't see many other variables that could have affected our results.

3. After talking to Tim Belien of pcfruit, he offered us their Y-tube olfactometer and their lab. We compared again all the five MeS concentrations with the control in a Y-tube olfactometer. The lady bug adults were first released into the Y-tube to become habituated with the set-up, before being exposed to MeS or EBF. We could see an obvious attraction of three ladybugs in a row at a MeS concentration of 10^-10. But after changing the side of the Y-tube in which MeS was released, we could not see the attraction anymore. We think that the Y-tube olfactometer was not clean enough and that there was still some MeS on the other tube as well. We saw repulsion of all ladybugs at a MeS concentration of 10^-7, demonstrating the upper limit of MeS concentrations. These experiments gave us an indication of the working MeS concentrations.

Repellent effect of EBF on Aphids

Several different EBF concentrations were released in proximity of aphids. We first used pure EBF because we were afraid that there was no response