Overview
To understand the insect test in an easy way, the testee selection, and experiment design would be briefly described. The results consist of three parts-feeding assay pre-test, the fusion protein improvement test, and preference test.
Testee selection
In the test of Pantide toxicity, we chose tobacco cutworm as the testee for the insect appetite tests because all the Pantide targets lepidopterans. We hope that we can observe the difference of the three toxins in vivo. Tobacco cutworms, which are one of the major pests in vegetable farms, impact on Crucifers in farmland around the world. In the serious pest damage, the density of tobacco cutworms is approximately up to two hundred million per ha[1]. Therefore, we used five tobacco cutworms to mimic the much more severe situation in our insect experiment.
Experiment Design
All the experiments were to check the functions of Pantide for leaf protection, so the observation of the results would focus on the remained area of the leaf disks.
In the following experiments, we used E. coli BL21 Rosetta-Gami strain to produce Pantide. We aimed to evaluate the remained leaf area applying Pantide. In this insect test, Pantide was diluted into three concentrations to observe the trends of dose response and to confirm the quality of PANTIDE. All the following experiments have three dilution ratios including 1/125 x, 1/25x and 1/5x. Three repeated tests were done in each experiment. See the procedure of insect experiment on Protocols.
Hypothesis
According to the mechanism of Pantide, Pantide is supposed to perform its toxicity in the nervous system of the larvae. We might see larvae paralyze and finally die. So that we may define Pantide as a biological pesticide.
Insect Test Result
Feeding Assay Pre-test
To know the qualitative dose response of Pantide, we prepared the samples with the sonicated LB solution containing Pantide-expressed E. coli Rosetta gami strain and diluted it with the three concentration. This experiment is the pre-test that shows us whether the amount of Pantide is sufficient enough to perform the toxicity against the larvae. We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply Bacillus thuringiensis, which is the most widely-used bioinsecticide. We preserved all the result of the remained leaves sealing with the glass paper and calculated the percentage of the remained area on the leaves. Here are the feeding assay results.
Figure 1. Remained leaf disks in the pre-test with the Hv1a and Hv1a-lectin.
