The overall goal of this demonstration is to describe how to mark insects with protein for dispersal or migration research. Included in this demonstration are methods on how to detect the marks by a sandwich or an indirect enzyme-linked immunosorbent assay. This method can help answer key questions in the field of insect dispersal research.
It can be used to study insect migration patterns, insect host plant preferences, and spread of invasive species. The advantages of this technique are that the detection method is simple and inexpensive. Suitable for high throughput, and the animals can be easily marked directly in their natural habitat.
Generally individuals new to this method will struggle, because it seems more difficult than it is, because most field researchers do not have the experience with this simple technique. I first had the idea for this method in 1989. We were rearing big-eyed bug predators on an artificial diet.
We were concerned that these bugs would not be effective predators on a Lygus bug pest after being reared in captivity for a long time on an unnatural diet. In other words, they would be domesticated. We needed a safe, effective, and inexpensive method to distinguish them after their release into the field from their native counterparts.
None of the current marking methods fit these criteria. A previous experience I had using the ELISA procedure to conduct predator gut content analyses for Lygus specific protein remains, gave me the idea of applying a foreign protein mark on the lab-reared predator. In turn, we could analyze the field collected big-eyed bugs for Lygus remains and for the foreign protein remains.
To begin, collect about 100 insects of interest from a laboratory colony or from the field, and divide them into two clean rearing containers. Load one container with a regular 20 milliliter diet packet, to serve as a negative control. For the other, lace a diet packet with a milligram of chicken IgG-IgY.
Add one milliliter of solution and mix thoroughly. If needed, use whatever the insects are normally fed in place of the packet. After 24 hours of being reared on these diet packets, supply both groups of insects with normal unmarked food.
At the desired time intervals, collect 20 individual insects from each cohort. Freeze them at minus 20 degrees Celsius, until all the required cohorts are collected for the experiment. To prepare a 96 well plate for the ELISA, to each well add 50 microliters of rabbit anti-chicken IgY primary antibody diluted one in 500 in Tris-Buffered Saline, or TBS.
Let the loaded plate incubate for at least an hour at room temperature or overnight at four degrees Celsius. Later, empty the wells, and add 300 microliters of block solution to each well. This is a one percent non-fat dry milk solution.
Let the plate incubate for 30 minutes at room temperature. During the block incubation, transfer individual frozen insects to micro centrifuge tubes loaded with one milliliter of TBS. Then, thoroughly homogenize each insect with a clean pestle.
When the plate is ready, load each well with 100 microliters of one homogenized insect sample. Each 96 well plate accommodates 80 specimens. Leave the first and last column of the plate empty for controls.
In the first column, load seven 100 microliter aliquots of TBS alone, and load one well with 100 microliters of TBS with egg white positive control. In the last column, load 100 microliters of eight individual unmarked insect preparations. Allow the samples to incubate at room temperature for at least an hour.
After the incubation, carefully wash the wells three times with Phosphate Buffered Saline Tween, or PBS-Tween. Through our experience we have learned that most errors occur during the wash steps. Splashing can lead to a high occurrence of false positive reactions, so be careful.
After the washes, add 50 microliters of rabbit anti-chicken IgG-IgY conjugated to horseradish peroxidase, and diluted one to 10, 000 in one percent milk solution. Allow the solution to incubate in the wells for at least one hour at room temperature. Later, wash out the plate three times with PBS-Tween as before.
Then, load the wells with 50 microliters of TMB substrate. You may start to see a color change right away. Let the plate incubate for 10 minutes before reading it with a microplate spectrophotometer.
Read the plate at 650 nanometers and record the values. To externally mark insects, prepare one liter plastic bottles with access holes, and divide 100 insects into two bottles. Then, spray the insects in one container with one milliliter of distilled water, which serves as negative control.
Next, attach a medical nebulizer to an air outlet, and adjust the air flow so that the nebulizer produces vapor. Spray the other insects with one milliliter of five percent chicken egg white solution. The spraying process should take between two and five minutes to complete.
Then, cork the holes and let the solution dry completely at room temperature. Once dried, transfer the insects back to their normal rearing containers, with their standard diet and maintain them as usual. Later, collect the insects at predetermined time intervals for testing.
To check the external mark retention, use a procedure identical to the ELISA assay for testing the internal mark retention, with minor modifications. After the insects are transferred to micro centrifuge tubes with TBS, let them soak on an orbital shaker at 120 RPM for an hour, instead of homogenizing them. After soaking the insects, pipette 100 microliters of the solution into the plate wells, and let the plate incubate for at least an hour.
After washing out the insect samples, add 300 microliters of PBS with one percent BSA to each well. After 30 minutes at room temperature, wash the wells twice with PBS-Tween. Then, to each well, add 50 microliters of rabbit anti-egg albumin primary antibody, diluted one in eight thousand in PBS-BSA silhouette.
Incubate the plate for at least an hour, and wash out the wells as before. Then, to each well, add 50 microliters of goat anti-rabbit IgG conjugated to horseradish peroxidase, and diluted one in two thousand, in PBS-BSA silhouette. Let the plate incubate for at least an hour, at room temperature and then proceed with the protocol as previously described.
Retention of the internal mark was tested at four time points. Overall, the insects treated without protein yielded consistently low ELISA values. Whereas, all of the insects fed the protein enriched diet yielded consistently strong ELISA values.
External marks were tested at four time points as well. The insects that were topically marked with egg whites yielded consistently strong values. Predictably, the water spritzed negative controls registered very low ELISA values.
After watching this video, you should have a good understanding of how to mark insects, and analyze them for the presence of the mark. Once mastered, this technique can be done in four hours and 30 minutes if it is performed properly. Over the course of a day, 16 assay plates with well over 1000 specimens can be processed.
While attempting this procedure, it's important to remember that the assay is extremely sensitive. As such, it is imperative that all surfaces, containers, and equipment are clear of protein contamination. After its development, this technique paved the way for researchers to explore local and area wide dispersal patterns of insects in agricultural, urban, forest, and other natural habitats.
We have demonstrated on small scale example here, however, the technique has been used on a much larger scale to tag the insects directly in their natural habitat using commercial spray equipment. We have been amazed on the creative applications of our collaborators.
