Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Larvae of the small white cabbage butterfly pie scrape are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, and Brussels sprouts, and many others. Interestingly, plants can evolve resistance to herbi force, such as bedo tappe, and in this video we will test the resistance of the model plant species rabbit opposite Ana to this tissue, Jin insect using two methods.

Hi, I'm Martin Navos, a postdoctoral fellow in George Jander's lab at the Bo Thompson Institute for Planned Research. Today I'd like to show you how to set up a choice and a no choice. Experiment with Arab Opsis Ana as a model plan, researching resistance against pest feeders.

Rearing of the caterpillars takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Peter. Scrape one for the larvae, and the other contains the adults the butterflies, in order to demonstrate the involvement of plant hormones and toxic plant chemicals.

In resistance to this insect test, I'd like to demonstrate two experiments. The first will involve the de determination of the role of jasmonic acid. A plant hormone often indicated in resistance to insects.

In a no choice experiment, caterpillar growth can be compared on wild type and mutant plants impaired in production of just mon acid. The hypothesis would be that larvae gain more weight on the mutant plant. So let's get started with the first experiment.

So we're gonna do a no choice experiment with the plants, a Revit opsis elliana to measure the weight gain of purest rapha larvae. So what we first have to do is we have to carefully enclose these plants. In one of these cups, we cut off the bottom of this plastic cup.

So in order to enclose, I use the back end of this fine paint brush. So without damaging leaves, we're gonna enclose these plants. So we have a no choice experiment here between the rabbit opss, the wild type, and the mutant plant.

The mutant is jasmonic acid impaired mutant, and probably more susceptible to feeding. This is a no choice experiment because one larvae can only choose from one genotype of plants. So we're going to add one larvae to each cup.

It's important to start off with larvae that just have hatched. So these are called L one larvae. So here's one that has just hatched.

Alright, so I'm gonna carefully place this larvae on the test band. So next step is to enclose these plants with their one larva, and I use a fine mesh and an oleic band to close em up so they won't crawl out. So then we wait a few days, usually seven days before we measure the larva weight gain.

So after seven days of feeding, we take out the larvae and weigh em on this scale and use the flexible forceps carefully pick up the larvae and gonna record their weight. So this guy almost weighs 250 milligrams. I'm gonna compare the weight of this caterpillar that was on the mutant plant with the weight of the caterpillar that was on the well type plant.

As you can immediately see, there's a lot more plant tissue left of the well type plant. So we assume that the larvae is, has gained less weight. The larvae on the wild type plant only weighs about a hundred milligrams.

So there's a huge difference both in the amount of tissue that has been eaten and the weight gain of the larvae. So that's it for the no choice experiment. Let's move on to the choice experiment, shall we?

Specialist pests identify and are attracted by host cues that are toxic to generalist orbi force. In the case of pi, rapid glucosinolates are used as an over position lene signal. Here I will use wild type and mutant rabbit opsis impaired in glucosinolate production.

To demonstrate a choice experiment, before you start this experiment, one needs to be able to distinguish male and female butterflies. Here's an example, female bureaus rapid butterflies have two dots on each wing while males carry only one, sometimes males can be mistaken for a female if they have a second, often faint marking. So let's set up the choice experiment.

In this case, we have both wild type and a mutant plant. The mutant plant is impaired in a production of glucosinolates. Again, gluconates are toxic to generalist early reports, but specialists like this pra that we're working with is attractive to Lucas simulate.

So these are the five larva stages of penis ape, shining with the smallest L one first larva stage, gaining weight until the fifth larva they'll find. Then it transforms into a pupa and the complete transformation takes place and these larvae will result in adult butterflies. For this choice experiment, we're using this cage I put in both the wild tide and the mutant plant, and now I'm gonna add one female butterfly to it.

To assess the acai, I usually catch females that are fertile. So to make sure that they're fertile, I catch females that are actively lea eggs, I carefully place my hand around it and transfer it into the test cage. So we leave the butterfly in for 24 hours and then count the number of eggs laid on each of these genotypes.

So some useful tips when you're doing these type of experiments is, first of all, when you select a female butterfly, don't use it for subsequent experiments cause there might be an effect of learning. Secondly, one can only compare plants that have similar growth phenotypes. So if you have plants that don't have a similar growth phenotype, you can take similar size leaves and mount those on top of an LMI flask.

So after, after 24 hours have passed, we can take out the plants first. The wall type plant, as you can see, it has a lot of eggs on there, and the mutant plant, the mutant that which lacks glucosinolates. So it lacks an over position or acai stimulus.

As you can see, there's hardly any eggs or very few, at least on the mutant. While there's a lot of eggs on the wild tap plant. So I've just shown you how to set up experiments to determine resistance against insects.

The first experiment was a no choice. Experiments where we compared larval weight gain on mutant versus well type plants. The second experiment, a choice experiment.

We compared acla by a female butterfly also on a mutant versus a wall type plant. Thanks a lot. That's it.

I hope it's useful for your experiments.