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Team:KU Leuven/Project/Ecological/wetlab
From 2013.igem.org
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
Insect & Plant Experiments
In order to validate the BanAphids model, we decided to investigate the effect of the two substances, E-β-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. 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 our 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-β-farnesene (EBF) is the most common compound of the aphid's alarm pheromones. 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 determine a range working concentrations of MeS and EBF on the insects. A description of the natural enemies (ladybugs, green lacewings and parasitic wasps) and aphids, we received from biobest and employed in our experiments, is given here.
One of the set-ups we used.
Second set-up, with wind tunnel
Y-tube olfactometer set-up
Ladybug experiment in Y-tube.
Natural Enemies
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 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 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 an 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 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 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.
Repellent effect of EBF on Aphids
Several different EBF concentrations were released below an sweet pepper infested plant. The behaviour of the aphids was observed for an hour after the release of the alarmpheromone. We first used pure EBF because we were afraid that there was no response. In the beginning of the experiment, we could se some movement of the aphids, but not for them all. After 20 min, they were again on the plant sucking sap. Probably, the high concentration of EBF made them all insensitive for other lower concentrations.
What we expected the plants to do
The effects of EBF on aphids are described thoroughly in different articles, in 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, the roots or the 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 and found that all the plants still had around 15 aphids. 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 see that the amount of aphids is around 15 aphids demonstrating that replication has not occurred yet amongst the control plants and the MeS induced plant. We see that amongst the plants induced with the highest MeS concentrations (0,4; 0,8 and 1 ng/ml) that there seems to be a trend of slightly higher amount of aphids. The 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. 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.
MeS induction via the roots on day 7 - total aphid population
MeS induction via the roots on day 10 - total aphid population
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 a slight increase in aphid population, again 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). This is similar to what can be seen when the plants are induced via the roots.
MeS induction via the leaf on day 7 - total aphid population
MeS induction via the leaf on day 10 - total aphid population
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, in reducing their reproductive capacity. 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 so that these results don't differ much from the control. These conclusions don't differ much between the two different induction methods.
Aphid distribution on the plant - root induced
After counting all the aphids on the plant, we calculated the number of aphids in the head of the plant. Normally, aphids prefer the head of the plant, on the young leaves. 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, but the difference is the most clear with the plants induced through the roots.
MeS induction via the roots on day 7 - aphid distribution
MeS induction via the roots on day 10 - aphid distribution
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, this also means that amongst the leaf-induced plants, the distribution doesn't differ much from the control. We see in the root-induced plants that the percentage of aphids in the head of the plant is less than in the control plant. This acts as a good control in this case.
MeS induction via the leaf on day 7 - aphid distribution
MeS induction via the leaf on day 10 - aphid distribution
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.
Leaf vs. root induced plant
Root induced plant vs. root induced control plant
We were therefore curious about the effect of MeS on the root growth. All the roots of the induced plants were washed, dried and weighed. The most important parameters were measured: the primary root, the width of the top of the primary root and the longest secondary root. To learn more about the procedure, click here. Just as there were differences in plant growth, the same was observed in the growth of the roots. The plants induced through their roots had much shorter and thinner roots in contrast to the plants induced via the leaves. The decrease in growth of the plants induced via the roots can be the result of the method of induction. The other possibility is that root induced plants activate aboveground and belowground mechanisms and therefore more resources are used for the plant defences instead of the plant growth.
By comparing the leaf induced control plant and the root induced control plant, we can see that the root induction results in smaller plants due to the thinner and shorter roots. This suggests that root induction does cause a certain amount of damage to the plant and this can account for a certain percentage of the reduction in growth amongst the root induced plants. However after comparing the roots of the root induced control plant and a MeS root induced plant, we can see that the MeS induced plant has smaller and thinner roots than the control. This suggests that the decreased growth is probably due to the MeS induction accounting for the remaining percentage. Induction of the plant defences requires a lot of energy, so that the plant suffers a fitness impact, having less energy to spend on their growth. There are no clear differences observable in root length or thickness between the different MeS leaf induced plants compared to the control. This yet again demonstrates the difference in effect between the two induction methods, and that it could account for a difference in behaviour.
Comparison of root induced plant with its control
Comparison between leaf control and root control
Comparison between the two induction methods
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, after which, 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.
Predator preference after 45 min
Predator preference after 24 hours
After 45 mins, it can be observed that the plants induced with the highest MeS concentrations (0,8 and 1 ng/ml) contain more predators than the control plant. After 24 hours, the effects of MeS induction are more visible. The most predators can be found on the plant induced with 0,8 ng/ml, the other plants don't differ much from the control. The results shown in the graph are the mean percentage of the three counts.
Predator preference after 45 min
Predator preference after 24 hours
The results from the plants induced via the leaf are less clear, and it is difficult to draw any conclusions. It is possible that due to the different method of induction that there is a different activation of defence mechanism therefore leading to changes in its interaction with aphids and predators. After 45 min, all the MeS induced plants contain less predators than the control. After 24 hours, the predators are almost equally divided between all the plants and the effect of MeS is not visible.
