A critical review of current laboratory methods used to evaluate mosquito repellents

Abstract

Pathogens transmitted by mosquitoes threaten human health around the globe. The use of effective mosquito repellents can protect individuals from contracting mosquito-borne diseases. Collecting evidence to confirm and quantify the effectiveness of a mosquito repellent is crucial and requires thorough standardized testing. There are multitudes of methods to test repellents that each have their own strengths and weaknesses. Determining which type of test to conduct can be challenging and the collection of currently used and standardized methods has changed over time. Some of these methods can be powerful to rapidly screen numerous putative repellent treatments. Other methods can test mosquito responses to specific treatments and measure either spatial or contact repellency. A subset of these methods uses live animals or human volunteers to test the repellency of treatments. Assays can greatly vary in their affordability and accessibility for researchers and/or may require additional methods to confirm results. Here I present a critical review that covers some of the most frequently used laboratory assays from the last two decades. I discuss the experimental designs and highlight some of the strengths and weaknesses of each type of method covered.

Keywords: contact repellency; laboratory assays; methods review; mosquito attractants; mosquito repellents; repellent efficacy; spatial repellency; standardized methods.

Figure 1

Laboratory repellency assays without an attractant source. (A) Diagram of the general format of a Tube assay. Shown is a transparent tube containing female mosquitoes. Both ends of the tube are capped to prevent mosquitoes from escaping the assay. The interior side of each cap contains a filter paper. The beige cap represents the untreated filter paper, and the pink cap represents the treated filter paper. (B) Diagram of the Close Proximity Response assay. Shown is a mesh-sided cage containing one female mosquito. A modified pipette tip containing a filter paper is shown being held up against the mesh region of the cage the mosquito is resting at. The pink box in the pipette tip represents a treated filter paper. (C) Diagram of the Excito-Repellency Test Chamber assay. Shown are two connected cages referred to as chambers. The left chamber is the main chamber and contains female mosquitoes. The main chamber has two treated papers shown in pink. The right chamber is the receiving chamber where repelled mosquitoes can relocate.

Figure 2

Laboratory repellency assays with an attractant source. (A) Diagram of Y-tube Olfactometer assay. Shown is a transparent Y-tube that is laid down horizontally with a small fan placed at the base of the “Y”. Each chamber has a small door that can be rotated open or close. The holding chamber contains acclimating mosquitoes. The pink circle on the hand represents a treatment. The location of the hand alternates between chambers for each replicate. (B) Diagram of a Taxis Cage. Shown are three cages connected by doors that can be opened or closed by the remote-controlled motor. Each cage is labeled either toward, middle, or away in relation to the volunteer’s location. (C) Diagram of a Taxis Cage located in a Wind Tunnel. Shown is a volunteer sitting near a Taxis Cage. The volunteer’s pink shirt represents a treatment. (D) Diagram of a Surface Landing assay. Shown is a mosquito-infested cage with a heated plate located underneath it. The pink area of the heated plate represents a treatment. (E) Diagram of a Feeding assay. Shown is a cage filled with mosquitoes. A plexiglass window is used to film mosquito feeding and behavior. A heated feeder filled with blood is located at the top of the cage. (F) Diagram of a blood feeder used in the Feeding assay. The red region represents the blood within the feeder and the parafilm is treated, indicated by the pink font. (G) Diagram of the Arm-In-Cage assay. Shown is a volunteer with their arm inserted in a mosquito-infested cage. (H) Diagram of the volunteer’s arm that is used in the Arm-In-Cage assay. Shown is a sleaved arm. The white border represents the cutout region where the volunteer’s skin is exposed to mosquitoes. The exposed skin is treated which is shown in pink.