Team:KU Leuven/Project/Ecological/wetlab

To validate our BanAphids model, we needed to investigate the effect of E-β-farnesene (EBF) and Methyl Salicylate (MeS) on aphids and plants. We need to know the working concentrations of our two substances in pure form and to examine the behaviour of aphids and their predators under different conditions and environments. Moreover, we studied the impact of pure MeS and/or EBF on plant growth.

We noticed quickly that this agricultural problem is looked at by various companies, although from a different angle. Rothamsted, a UK based research station, is currently investigating the use of EBF to repel aphids as well, but in the form of GM crops. Locally, several companies were interested in our project. Biobest, worldwide leader in sustainable crop protection and pcfruit, a research company focussing on biological fruit growing and new crop protection methods both invited us to discuss our project.
After fruitful visits, Biobest and pcfruit opened their research departments for us. We could perform our insect experiments there to validate our BanAphids model. With the data we have collected so far, we can observe that these two substances indeed do have an effect on aphids.

Defining the working range

E-β-farnesene (EBF) is the most common compound of the aphid's alarm pheromones. The general response to this stress-signal is a change in gene expression and subsequent protein expression, causing the aphids to become restless, form wings and leave the plant. Here, we used synthetic EBF (Sigma-Aldrich, farnesene, mixture of isomers) to study these effects. Check out our E-beta-farnesene construction page for the characterization of the EBF producing biobrick. The result from a BanAphid exposure on aphids can also be seen here below.

Methyl salicylate (MeS) on the other hand attracts the natural predators of the aphids. We tried to prove this kairomone effect of methyl salicylate with a synthetic variant of MeS (Sigma-Aldrich, methyl salicylate ≥99% pure).

We experimented with three different set-ups to determine a working range of MeS and EBF. Our BanAphids are built to produce an alternative source of EBF and MeS but with the same effects as the synthetic counterparts. We also needed the aphids' natural enemies (ladybugs, green lacewings and parasitic wasps), which we received from biobest. Their description is given here.

Attracting Natural Enemies with synthetic MeS

1. The aphid's natural enemies were subjected to a choice experiment. Starting from a determined point on our set-up, 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 to 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 could be the result of performing the experiments under a fume hood, this might have resulted in the rapid elimination of MeS.

2. With advice from Biobest we did 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. dr. 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 were walking around in search of aphids, were further used in the behavioural experiments. We also tried the experiment 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 a washed aphid-infested leaf as a control compared to responses to the infested leaf introduced with one of the five MeS or EBF concentrations . This however did not demonstrate any clear behaviour form the insects other than random movement. We contemplated 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 0,1 ng/ml. 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 100 ng/ml, demonstrating the upper limit of MeS concentrations. These experiments gave us an indication of the working MeS concentrations.

Aphid repellent effect of BanAphid EBF

In our first setup we tried releasing several EBF concentrations below a sweet pepper plant, infested with aphids. The behaviour of the aphids was observed for an hour after the release of the alarm pheromone EBF. We first used pure EBF because we were afraid that there would be no response. In the beginning of the experiment, we could see some movement of the aphids, but not for all of them. After 20 min, they were back on the plant sucking sap. The extremely high concentration of EBF most likely made them insensitive to an EBF effect.
We then decided to test EBF concentrations that are more realistic to what individual aphids could produce. Luckily, our bacterial EBF construct was ready to go! So we decided to perform a second aphid experiment with our BanAphids, to read more about these setup, click here. We saw a positive result from one of these setups, as can be seen on the video. Aphids are sitting calmly on the leaf (sedentary insects) and from the moment the EBF producing BanAphids are released, we see the aphids becoming mobile.

What we expected the plants to do

The effects of EBF on aphids are described thoroughly in different articles, contrary to their responses to MeS. MeS is a critical signal molecule in the induction of plant defence mechanisms and therefore it should have a negative effect on the aphids. According to the optimal defence theory, a plant will protect its most valuable parts when under attack, for instance, their reproductive parts and terminal leaves. We therefore expected to see a redistribution of the aphids to the lower leaves after induction with MeS. A plant has different defence pathways depending on the site of infection (i.e. roots or leaves). We therefore used two different induction methods. We induced the plants with five different concentrations of MeS through the roots with water and via spraying with ethanol on the leaves. If you would like to find out how to practically induce a plant, please look at our protocol. 48h (day 2 of the experiment) post-MeS induction, 15 of the smallest aphids were placed on the head of the plant so that the following generation would be the F1 generation and the plants were placed individually in nets. The distribution and total number of aphids were examined on day 7 and day 10.

Total aphid population - root induced

On day 7 and 10, we examined the total amount of aphids on the plants. We expected to see no change in the total amount of aphids on the plants (15 aphids), since the aphids should not be able to reproduce yet. We indeed see that the amount of aphids is around 15 aphids per plant, demonstrating that replication has not occurred yet neither amongst the control plants nor at the MeS induced plant. We do see a small trend at day 7: aphids on the plants induced with the lowest MeS concentrations (0,01 and 0,1 ng/ml) seemed to show no difference when compared to the control. Amongst the plants induced with the highest MeS concentrations (0,4; 0,8 and 1 ng/ml), there seem to be a slightly higher amount of aphids. Their origin is unclear, possibly we did not put the youngest aphids possible on the leaf so some may have reproduced in the days past after all. This needs to be compared to data from day 10 before we can draw any conclusions. On some plants there is much more than 15 aphids but this is probably the result of contamination while placing the aphids on the plant. On day 10 we see that now the lower concentrations of MeS contain more aphids than the higher MeS concentrations and the control.

Total aphid population - leaf induced

We see here approximately the same results as the total aphid population via root induction. On day 7 we see again a slight increase in aphid population amongst the plants induced with the highest MeS concentrations (0,4; 0,8 and 1 ng/ml), but on day 10 we see that the aphid population seems to be the highest amongst plants induced with the lowest MeS concentrations (0,01 and 0,1 ng/ml).

Is the aphid population affected?

On day 7, there is a small variation in aphid population but not so different from the control. This is expected since reproduction should not be possible yet. On day 10, we see that the plants induced with highest MeS concentrations (0,4; 0,8 and 1 ng/ml) are less populated than the control plant. This could be a demonstration of the plant's defence against aphids, reducing the reproductive capacity of the aphids. We also noticed an increased amount of winged aphids on these highest MeS concentration induced plants compared to the lowest concentrations and the control. This indicates that the aphids were motivated to leave the plant. The plants induced with the lowest MeS concentrations (0,01 and 0,1 ng/ml) did not show much change in aphid population compared to the control. It is possible that these concentrations of MeS were too low to induce the plant's defence mechanisms; the plant remained neutral and results don't differ much from the control. Finally, our observations showed similar results, irrespective of the chosen induction method (roots or leaves).

Aphid distribution on the plant - root induced

After counting all the aphids on the plant, we calculated the number of aphids on the head of the plant. Normally, aphids prefer young leaves since they have the least "woody" elements. these young leaves are most prominent at the the head of the plant. According to the optimal defence theory, we expect the MeS induced plant to protect it's most valuable parts, this includes the head of the plant, so that these parts become unattractive for aphids and they redistribute themselves. We can see that the control has the highest percentage of aphids in the top of the plant, compared to the MeS induced plants. The difference is most clear with plants induced through the roots.

Aphid distribution on the plant - leaf induced

Induction of the plants with MeS results in a redistribution of the aphids to the lower leaves, just like the optimal defence theory predicts. The younger leaves are more valuable for the plants and are therefore better provided with defence mechanisms. The redistribution of the aphids is more clear in the plants induced through the roots, in contrary to the plants induced via spraying on the leaves. The percentage of aphids in the head of the plant in leaf-induced control plants is significantly less than in root-induced control plants. It is difficult to explain why this is, but it does also mean that aphid distribution doesn't differ much from the control amongst the leaf-induced plants. In the root-induced plants, the percentage of aphids on the head of the plant is , as expected, less than in the control plant. Possibly, induction through the roots feeds immediately into the vertical axis (root to head) which is a better route for stimulating the plant defence mechanism than a horizontal route (via the leaves).

Fitness impact of the MeS induction

During the experiment we noticed a clear difference in plant growth rates between the two methods of induction. The plants induced via their leaves were much bigger than the plants induced through their roots, even though the plants induced via the leaf suffered actual ethanol damage.

Predator preference to MeS induced plants

After the last aphid count on day 10, 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. The predators were subject to a choice experiment. A plant of every MeS concentration and a control was placed in a circle and approximately 15 Macrolophus were released in the middle of the circle. To know exactly how we performed this experiment, click here. The number of predators on every plant was counted after 45 min. Next, the predators were shaken off the plant. This was repeated twice. To determine the effect on a longer time span, we also counted the aphids after 24h.

After 45 mins, root-induced plants with the highest MeS concentrations (0,8 and 1 ng/ml) contained more predators than the control plant, conversely the leaf-induced plants had less predators with higher concentrations. After 24 hours, the levels of predators present on induced vs. control plants equalised for both conditions. The results shown in the graph are the mean percentage of the three counts.

It is difficult to draw a conclusion. It is possible that the different methods of induction stimulate a different activation of defence mechanism, therefore leading to changes in the interactions with aphids and predators. Possibly the MeS induction effect has petered out by this time (day 13-14). We could test this by a secondary MeS induction at day 7. Once again, more work for the future.