A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

The overall goal of this experiment is to assess the effectiveness of insecticides in killing bed bug eggs and first instars. This method can help answer key questions in the entomology field, such as the effects of toxicological assays on bed bug eggs and small nymphs. The main advantage of this technique is that it is designed for treating a difficult insect life stage with insecticides, particularly the egg stage.

To begin the experiment, pull five to 10 mated well-fed adult female bed bugs from a colony and place the females into a Petri dish with filter paper. Prepare 10 replicates to ensure there will be enough eggs for the insecticide assay. Check the dishes every day to see if the females have laid eggs.

Once the females have laid eggs, take the females out of the Petri dish and put them into new Petri dishes with a new filter paper daily. Record the data on the old and new Petri dishes. Allow the eggs to age four to five days before starting the assay.

Once the eggs have aged appropriately, remove the bed bug eggs gently from the filter papers using soft-tip forceps. Gently scrape the bottom of the egg attached to the filter paper using the forceps and then gently grab the egg without applying too much pressure to avoid tossing the bed bug eggs. Place the removed bed bug eggs into their respective groups inside the plastic Petri dishes.

Rub the bottom of each Petri dish on the outside with a fabric softener sheet to limit static electricity from flinging bed bug eggs. Next, prepare serial dilutions of insecticides to get a range of five different concentrations and a control solution with tap water only each to a volume of 20 mL. Take 50 mL centrifuge tubes precut at the mL line and then cut fine mesh into squares large enough to fit into the cap.

Screw the mesh tightly eliminating any bumps into the centrifuge tubes using the centrifuge tube cap. Next, using a paintbrush, place a group of bed bug eggs onto the mesh inside of a centrifuge tube. Dip the group of bed bug eggs inside of the cut centrifuge tube into an insecticide concentration for five seconds.

Remove the centrifuge tube out of the insecticide solution. Place the centrifuge tube directly onto the laboritory tissue to dry the mesh with the eggs inside. After drying, remove the group of eggs out of the centrifuge tube using a small fine-bristled paint brush and place them into a clean Petri dish with a clean filter paper.

Record the egg mortality for 14 days to ensure all eggs have had sufficient time to hatch. Correct for mortality in the control treatment using Abbott's formula. Insert recorded mortality with the corresponding concentrations into the analysis software to calculate LC50 values.

Separate a group of five adult male and five adult female bed bugs fed the previous day and place them into a Petri dish with filter paper. Allow the adult bed bugs to mate and lay eggs for eight days. Next remove the adults from the Petri dishes and allow the eggs to hatch into first instars within the Petri dishes.

Collect the unfed first instars of the same age using a paintbrush, then place them into new clean Petri dishes until the insecticide assay. Pipet 150 microliter aliquots of each of five previous prepared dilutions of insecticide and water for the control group onto individual filter papers. Evenly distribute the insecticide by applying droplets around the entire edge and center of the filter paper.

Place the treated filter papers individually on top of individual hardboard panels. Release groups of first instars onto the individual filter papers while still wet with insecticide and cover the first instars with the bottom of a Petri dish inverted and place a weight on top of the Petri dish to prevent the first instar bed bugs from escaping. Record the mortality of first instar bed bugs after 24 hours of exposure to all insecticide concentrations.

Correct for mortality in the control treatment using Abbott's formula. Insert the recorded mortality and concentrations into the analysis software to calculate LC50 values. The percent response or mortality of Harlin strain bed bug eggs to five different concentrations of metocloprid beta-cyfluthrin increased as the dose increased.

Mortality reached 100%at the highest concentration tested. After watching this video, you should have a better understanding of how to evaluate insecticide efficacy against bed bug eggs and first instars.