The World Health Organization protocol for the arm-in-cage (AIC) test for measuring mosquito landing rates calls for 200–250 host-seeking female mosquitoes placed in a 0.064-cubic-meter cage. A new study published in the Journal of Medical Entomology, however, finds that typical landing rates in the AIC test differ significantly from landing rates found in large-room or field tests, which may more accurately simulate real-world conditions. (Photo originally published in Moreno-Gómez et al 2021, Journal of Medical Entomology)

By John P. Roche, Ph.D.

Mosquitoes spread many human diseases, and the Asian tiger mosquito (Aedes albopictus) spreads 26 arboviruses, including dengue, Zika, and chikungunya. Being active in the morning and early evening, Asian tiger mosquitoes are more likely to spread disease than mosquitoes that are out only at night when fewer people are outdoors.

Repellents are important public health tools for reducing mosquito-borne diseases, and they routinely undergo testing to ensure their efficacy. Field testing of repellents is dangerous because it risks infection by pathogens, so lab testing using pathogen-free mosquitoes is preferred. For lab tests to be valuable, however, they need to accurately approximate mosquito exposure in the field.

Mara Moreno-Gómez

In a new study, Mara Moreno-Gómez of Henkel Ibérica in Barcelona, Spain, and colleagues measured mosquito landing rates in the lab and field to learn how to recreate a realistic landing rate in the lab. “This work is essential to develop safer yet reliable alternatives to field testing,” Moreno-Gómez says, “given that the latter exposes study participants to health risks.” The study results were published in January in the Journal of Medical Entomology.

The human landing catch (HLC) method is commonly used to estimate mosquito landing frequency. An alternative procedure, the landing rate count (LRC) method, is less often used but offers the advantage of not changing the density of mosquitoes in the vicinity by capturing them. Because it offers a more accurate way to compare landing rates between the lab and the field, Moreno-Gómez and her colleagues chose the LRC method for their study.

Studies that sample mosquito landing rates have been done in the field, in rooms in a lab, or by placing an arm in a cage containing mosquitoes—known as an arm-in-cage (AIC) test. The goal of these methods is to determine what is called the repellent’s complete protection time (CPT)—the duration of time from the start of when someone is exposed to mosquitoes until a mosquito lands on their skin. The longer the CPT, the more effective the repellent.

A subject stands in an experimental room used to assess the rate at which mosquitoes land on the skin of subjects’ lower legs. A new study published in the Journal of Medical Entomology sought to determine the rate at which Asian tiger mosquitoes (Aedes albopictus) landed on people in field tests and lab tests, with a goal of making tests of mosquito repellents in the lab more accurately aligned with realistic conditions in the field. (Photo originally published in Moreno-Gómez et al 2021, Journal of Medical Entomology)

Lab AIC tests need to accurately approximate mosquito exposure in the field. Previous research comparing tests in the field with AIC tests in the lab found that the lab tests may be using an unrealistically high density of mosquitoes, leading to an underestimate of CPT, which in turn would lead to an underestimate of the effectiveness of a repellent.

In their current study, Moreno-Gómez and colleagues performed a field test, a room test, and a lab AIC test. In the field test, 16 subjects stood in outdoor locations in the suburbs of Padua in northeastern Italy and measured how many Ae. albopictus mosquitoes landed on their lower legs within five minutes. The investigators conducted 221 five-minute sample periods between the hours of 9 a.m. and 6 p.m. Aedes albopictus is the only dangerous mosquito that is active in the daytime in the Padua region.

In the room test, the investigators measured how many times a mosquito landed on a person’s lower legs in three- or five-minute intervals in a 30-square-meter room. They used three different densities of individual Ae. albopictus mosquitoes: 15–20, 25–30, and 45–50.

The AIC test used 10 subjects utilizing the World Health Organization protocol. Two hundred colony-bred mosquitoes were placed in a cage measuring 0.064 cubic meters. Subjects placed their exposed forearm in a cage and counted the number of times that a mosquito landed on their skin during a one-minute interval.

In the field test, the mean landing rate over a period of five minutes was 9.5 landings per minute. The maximum landing rate was 26.8 landings per minute.

The investigators found that, in the room test, the lowest landing rate occurred when using 15–20 mosquitoes, as would be expected. With 15–20 mosquitoes and three minutes of exposure, the landing rate was 30.4 landings per minute. With 15–20 minutes and five minutes of exposure, the landing rate was 32.3 landings per minute.

In the AIC test, on the other hand, the investigators discovered that the average landing rate was 229 landings per minute.

Moreno-Gómez and her colleagues used the maximum rate of 26.8 landings per minute from the field as the guideline to determine the most accurate testing regimen to use in the lab. A comparable landing rate in the room test was found using 15–20 mosquitoes in a 30-square-meter room over a period of three minutes. The average landing rate of 229 landings per minute in the AIC test, however, was dramatically higher than that seen in the field, indicating that the AIC test would considerably underestimate CPT. The implication is that the AIC test should be refined to reduce the number of mosquitoes used in the cage to more closely approximate mosquito density in the field.

A new study published in the Journal of Medical Entomology sought to determine the rate at which Asian tiger mosquitoes (Aedes albopictus) landed on people in field tests and lab tests, with a goal of making tests of mosquito repellents in the lab more accurately aligned with realistic conditions in the field. The study found that typical landing rates in the arm-in-cage test differ significantly from landing rates found in large-room or field tests. (Image originally published in Moreno-Gómez et al 2021, Journal of Medical Entomology)

“To our knowledge, this is the first study of its kind to compare the landing rates of a mosquito species, Aedes albopictus, in both the field and the laboratory,” Moreno-Gómez says. “By better simulating outdoor conditions and more accurately estimating CPT, the results of this study can help improve and refine the evaluation standards used for testing repellents in the laboratory.”

In regard to future research, Moreno-Gómez says, “The next step should be to conduct additional studies to adjust the densities used in the AIC test so that the resulting landing rates better represent what is observed in the field. Furthermore, this study focused exclusively on Aedes albopictus. Future work should include other mosquito species with a view to more broadly improving current guidelines for mosquito repellent testing.”

This study highlights that it is important for experimental studies to examine their assumptions and accurately approximate conditions found in the real world. In addition to future work on how to adjust the AIC test to make it more accurate, and testing the efficacy of repellents on other mosquitoes, mosquito investigators could also coordinate with studies of the efficacy of repellents against other arthropod vectors of disease, such as ticks.

John P. Roche, Ph.D., is an author, biologist, and educator dedicated to making rigorous science clear and accessible. Director of Science View Productions and Adjunct Professor at the College of the Holy Cross, Dr. Roche has published over 200 articles and has written and taught extensively about science. For more information, visit https://authorjohnproche.com/.