Plants may upregulate the production of many different secondary metabolites in response to insect feeding. One response of tobacco plants to herbivory are being not upon by insects is to increase the production of the neurotoxic alkaloid nicotine, using nicotine. As an example, I will demonstrate how to set up an experiment to assess the toxicity of plant secondary metabolites against aphids.
Certain lineages of MiSiS persky, the green peach aphid have adapted to specialize on cultivated tobacco. Here we hypothesize that these tobacco adapted lineages have increased resistance to nicotine over non-adaptive lineages. Hi, my name is John Ramsey.
I'm a grad student in the lab of George Jander at the Bo Thompson Institute for Plant Research. Today I'm gonna show you a procedure to measure the toxicity of plant metabolites against aphids. This procedure involves preparing an artificial diet to which the plant metabolite of interest.
In this case, nicotine is added at varying concentrations, caging aphids, such that they feed on this diet and counting aphids after a number of days to measure the survivorship and fecundity on different concentrations of nicotine. So let's get started. With the experiment.
We can rear aphids on artificial diets, taking advantage of their feeding style in which they insert a specialized S style into the plant vasculature and consume a diet of flow sap. We prepare a liquid diet similar to that which the A encounters were feeding on a plant consisting of sucrose and amino acids to this solution. We then add varying concentrations of the plant metabolite.
We are interested in assay the toxicity of, in this case nicotine. In this experiment, four adult aphids are enclosed in a small plastic cup. They feed from liquid diet pipetted between two layers of biofilms stretched over the top of the cup after three days.
Feeding toxicity of diets containing different concentrations of nicotine is compared to control diets by counting adult and larval aphids in each cup. Now it's time to prepare the experimental diet. I have here a solution of sucrose and 20 amino acids.
Aphids can survive and reproduce normally on this diet for at least a week. Now I've aliquot this diet in one milliliter, Alec Watts containing variant concentrations of nicotine from zero to 500 micromolar in these fendor tubes, then four aphids are selected either red or green and put into these experimental cups. A paintbrush is used to delicately lift aphids off the leaf and put them into the experimental cup.
When selecting aphids, it's important to choose insects of the same general size and color darker aphid, such as this one may be older and not appropriate for this experiment. Then I'll stretch the first layer of phim over the top of the cup to contain the aphids. Next, i'll aliquot 100 microliters of the liquid diet between the two paraform layers and then stretch the second paraform layer securing it around the edges of the cup.
I've poked these air holes to make sure that there's good airflow up through the experiment, and I'll put this cup in this tube holder and place it on a rack in the insect room under a long day photo period under lights. So here we can look at the results from an experiment, which I set up three days ago in the same way as the experiment that I've just demonstrated. I have one cup, each of green and red aphids on zero micromolar, 10 micromolar, 100 micromolar, and 1000 micromolar or one millimolar nicotine diet.
And you can see for the control diet zero micromolar nicotine, both the red and the green aphids are surviving and reproducing. Likewise, on the 10 micromolar diet, we have a large number of red aphid progeny. We still have green adults, but they don't have very many progeny.
So they may be feeling the effects of nicotine, even at as low a concentration as 10 micromolar. Here there are no surviving adult green aphids on the hundred micromolar nicotine diet, but the red aphids are surviving and reproducing at the same level as the control. You can see on the diet containing 1000 micromolar nicotine.
Neither the green nor the red aphids are able to survive. So based on this, we can consider the possibility that one of the adaptations that has allowed the red MiSiS lineage to infest tobacco in the field is an enhanced ability to detoxify nicotine. We've just shown you how to set up an experiment to use artificial diets to measure the toxicity of plant secondary metabolites against aphis.
When setting up these experiments important to remember to choose insects which not only are the same size and color, but also come from plants at similar developmental stages. So that's it. Thanks for watching and good luck with your experiments.
