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

Summary

Insecticide evaluations are often targeted against adult insects, rather than immature stages. Here, we present a protocol for evaluating insecticides against bed bug eggs with a comparison to the first nymphal bed bug stage. These protocols could be adjusted for other insects to evaluate insecticide efficacy in non-adult life stages.

Abstract

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or concentration-response curve, where mortality increases as the dose or concentration of an insecticide is increased. Standard lethal concentration (LC₅₀) and lethal dose (LD₅₀) tests that result in 50% mortality of a test population can be challenging for evaluating toxicity of insecticides against non-adult insect life stages, such as eggs and early instar or nymphal stages. However, this information is essential for understanding insecticide efficacy in all bed bug life stages, which affects control and treatment efforts. This protocol uses a standard dipping bioassay modified for bed bug eggs and a contact insecticidal assay for treating nymphal first instars. These assays produce a concentration-response curve to further quantify LC₅₀ values for insecticide evaluations.

Introduction

Bed bugs are a significant urban pest that can cause blood loss, skin irritations, sleeplessness, depression, and anxiety in their human hosts ^1,2,3,4,5,6. The costs of eliminating and controlling bed bugs are high and often times require multiple visits to a home by a pest control company⁷. Multiple visits are usually required because of the cryptic behavior of bed bugs and the difficulties associated with killing them. In particular, bed bug eggs ....

Protocol

NOTE: This protocol includes two insecticide assays for separately treating bed bug eggs and first instars. Both protocols were conducted using the same insecticides, however, the protocols had to be adapted to ensure insecticide exposure and to easily manipulate the specimens.

1. Egg Dipping Insecticide Assay

1. Pull mated, well-fed adult female bed bugs from a colony and place the females (5-10) into a Petri dish with filter paper. Have at least ten replicates to ensure that there will be enough eggs for the insecticide assay. Check to see if the females have laid eggs every day.
   1. Once they lay eggs, take the females o....

Results

The eggs were dipped into 5 different concentrations of imidacloprid/ β-cyfluthrin (µL/mL). The first instars were placed onto treated surfaces of five different concentrations of imidacloprid/β-cyfluthrin (Figure 2).

We used three different populations of C. lectularius: Harlan, Richmond, and Epic Center (Table 1). The Harlan strain is susceptible to pyrethroid insecticide.......

Discussion

A critical step in this assay is to ensure that no eggs that are removed from a surface are damaged prior to the assay. Many insects cement their eggs to a substrate, therefore, a preliminary test may be needed to ensure that removal does not cause mortality. This test can be conducted with several replications of a treated group (bed bugs removed from filter paper) compared to a control group (bed bugs not removed). Similar low rates of mortality (or no mortality) between the treated and control group will ensure that .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petri dishes	Fisher Scientific Inc.	08-757-105	Plastic
Filter paper	Whatman	1001-042
Featherweight forceps	Fisher Scientific Inc.	4750
Temprid SC (imidacloprid/bifenthrin)	Bayer CropScience
Transport GHP (acetamiprid/bifenthrin)	FMC Corp.
Centrifuge tube		06-443-18	50 mL; flat-top threaded
Fine mesh		7250C
KimWipe		06-666A	11 cm x 21 cm
Small paint brush			Any small paint brush
Hardboard panels			Composite wood or tile would be sufficient from a home improvement store
Metal weights			Any type of metal screw that will hold the weight of the Petri dish down onto a surface