Introduction

To determine the efficacy of a plant-protective insecticidal agent it must be tested on its targeted pest as well as in combination with the plant it is expected to protect. To achieve this, experiments are performed in vitro to screen through thousands of potentially active compounds or microbial strains. This is done by exposing the animal, in this case caterpillars, to an environment pre-treated with the insecticidal agent(s). Two common assays are used to assess the efficacy: the Diet Assay and the Leaf Disk assay. Both of these assays are performed in multi well plates so as to test multiple agents at different concentrations and with repetition to gain statistical confidence in the results.

MCAM and Agriscience

The MCAM™ gives the researcher the ability to image the full well plate in a single acquisition while maintaining the resolution needed to see the contents of the individual wells in high detail. With this, the acquisition is not only a record created for archival purposes, but it can be analyzed using Ramona Optics custom software to automatically gather important metrics from the well plate. Results can be aggregated through the well plate giving the researcher unbiased information needed to determine the effectiveness of the treatment. This decreases the time a researcher must spend analyzing a single well plate from minutes to seconds, allowing them to test more compounds and find effective insecticidal agents more quickly. In the examples described below, the Ramona team collaborated with scientists at AgBiome, Inc. to co-develop a proprietary algorithm suitable for the particular insect bioassay needs. Such capabilities can be uniquely developed for other interested researchers, using the Ramona tools described.

Diet Assay

In the Diet Assay the caterpillar is placed on a food mixture including the agents to be tested. At the end of the exposure period (a few days), the caterpillar is inspected to measure its growth and/or activity level. An effective insecticidal agent would cause the caterpillar to not grow and to be inactive or even to die. An ineffective agent would allow the caterpillar to grow and remain active. Depending on plating conditions (single vs multi animals per well), researchers run this type of bioassay experiment in well plates of either 96 or 24 individual wells. The MCAM™ can be used to image the full well plate in a single acquisition while also having the resolution to measure the size of the caterpillars in each well, as shown in Figure 1.

In the Diet Assay, caterpillars can be differentiated from the background to find the area of the caterpillar in each well. Building off user-defined thresholds, the caterpillars can be divided into growth stages to better distinguish the effect of the tested agent on the growth of the caterpillars. The results of each column are then grouped to show the average effect of common treatments on the caterpillars.

The experiment shown in Figure 2 displays the natural variation in caterpillar growth without any pre-treatments to the diet. With treatment of an effective agent it is expected that the variation in the caterpillar’s areas would be increased. Final areas of the caterpillars would be expected to range from Day 0 sizes to Day 5 sizes, for caterpillars exposed to effective agents or those exposed to ineffective agents (or negative controls), respectively. The MCAM™’s analysis software can quickly report these variations to give researchers the data they need on the effectiveness of their tested insecticidal agents.

Leaf Disk Assay

The Leaf Disk Assay is an in vitro test to ensure an insecticidal agent works to protect a host plant from the targeted pest, in this case caterpillars. In this assay a small section of a leaf (about a centimeter in diameter) is removed from the plant containing (or treated with) the compound studied. These leaf disks are then placed in a multi well plate (24, 48 or 96) with the caterpillars and left for 3 days. After this period, the caterpillars are removed and the leaves are inspected to measure the amount of leaf that was eaten, as shown in Figure 3.

In the Leaf Disk Assay, leaves can be differentiated from the background to find the area of the leaf present in each well plate. Comparing the area of the leaf present on day 0, Figure 4.a, to that of the area remaining on day 3, Figure 4.b, of the assay provides the amount of leaf eaten by the caterpillar.

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The experiment shown in Figure 4 displays the variation of leaf areas at the end of the assay. A successful insecticidal agent would result in wells similar to those in row G (second from bottom), with little leaf area removed, while an unsuccessful one would allow the caterpillar to eat a majority of the leaf as seen in row F (third from bottom). The MCAM™’s analysis software can quickly report these variations to give researchers the data they need on the effectiveness of their tested agents.

Conclusion

The MCAM™ has the ability to image a full well plate in a single snapshot. Even with this large field of view, resolution is maintained so that the contents of the well can be viewed with high detail. These images are great for archival purposes, but the true benefit comes when the imaging is combined with our custom analysis software. Here we presented the results of two commonly used assays. Our integrated solution can greatly speed up the measurement of well plates for these assays, allowing researchers to work through more plates per day, and derive data pointing to the most effective insecticidal agents more quickly.

Click here to learn more about AgBiome, the North-Carolina-based Agriscience company collaborating with Ramona to perfect this assay on our Multi-Camera Array Microscope (MCAM™).

AgBiome will be presenting a poster titled “Image analysis for high-throughput screening of bacterially-derived insecticidal toxins,” during this year’s Entomology Society of America Conference, Tuesday November 7, 2023, from 5:30 PM — 6:30 PM.

See more about Ramona at ramonaoptics.com