Mosquitoes in the genus Anopheles commonly spread Plasmodium, the parasite that causes malaria. (Anopheles gambiae shown here.) Researchers at Kasetsart University in Bangkok, Thailand, tested a technique called the forced oviposition method to produce enough mosquito offspring from wild-caught female Anopheles mosquitos to test for insecticide resistance. (Photo by James Gathany, CDC Public Health Image Library)

By John P. Roche

Mortality from malaria in Thailand has been reduced by almost 90 percent in the past 20 years, a tremendous success. This reduction has been in large part due to the use of long-lasting insecticide nets and indoor residual spraying with insecticides targeting the Anopheles mosquitoes that spread the disease. But, with continued exposure over time, mosquitoes can evolve resistance to specific classes of insecticides, and 32 million people in the Thailand are still at risk of contracting malaria.

Assessing resistance requires exposing field mosquitoes to the insecticide in question, but it is often difficult to obtain enough mosquitoes from the field for this purpose. Amonrat Panthawong, Ph.D., and Theeraphap Chareonviriyaphap, Ph.D., of Kasetsart University in Bangkok and colleagues tested a way to solve this problem—a technique for producing sufficient numbers of mosquitoes from wild-caught female mosquitoes to test for insecticide resistance. They report their findings in a study published in June in the Journal of Medical Entomology.

Classes of insecticides used against Anopheles include organochlorines, organophosphates, carbamates, and pyrethroids. Pyrethroids are the most commonly used. They are safe to humans, and they have been approved by the World Health Organization (WHO) for use in nets. But resistance to pyrethroid insecticides is developing in many mosquito populations, and resistance has been detected in most countries in southeast Asia. There are alternative insecticides that can be used where resistance to pyrethroids has evolved, including neonicotinoids, but information on the degree of resistance is needed to inform such decisions, and inducing egg laying in field-caught mosquitoes has proven difficult.

The technique Panthawong and colleagues used is known as the forced oviposition [laying eggs] method, which was first developed by John Morgan and colleagues in 2010 and was modified by the investigators for this study. The method involves confining individual blood-fed female mosquitoes in small vials with a moistened piece of filter paper; Morgan and colleagues speculate the stress of confinement induces the mosquitoes to lay their eggs.

Panthawong and colleagues’ goal was to test if it was possible to use the forced oviposition method to produce a new generation of Anopheles mosquito offspring from mosquitoes caught in the wild—what biologists call a first filial (F1) generation—to test for insecticide resistance. They used five field strains and four lab strains of Anopheles to test their forced oviposition method. The field strains were An. dirus, An. epiroticus, An. harrisoni, An. maculatus, and An. barbirostris. The lab strains were An. dirus, An. epiroticus, An. minimus, and An. cracens.

The investigators gathered female mosquitoes that had already taken a blood meal, placed them in paper cups or cages covered with nets, and provided cotton wool soaked with 10 percent sucrose solution. The females were maintained there for five days. Then females were moved into 35-milliliter plastic vials with a cotton ball covered with a moistened piece of filter paper at the bottom of the vial on which to lay eggs. Vials were checked each day to see if eggs had been laid. When eggs were laid, the resulting larvae were then reared. The variables measured were the number of females that laid eggs, the total number of eggs, the number of hatching eggs, the number of larvae, the number of pupae, and the number of adults that emerged.

Plastic vials used to test the forced oviposition method to produce mosquitoes for testing insecticide resistance. Pieces of moistened filter paper were placed on the bottom of the vials for the female mosquitoes to use for egg laying. (Image originally published in Panthawong et al 2021, Journal of Medical Entomology)

Panthawong and colleagues found that the mean number of eggs laid per female mosquito for field populations ranged from 36.3 for An. harrisoni to 147.6 for An. barbirostris, and that the mean number of eggs laid per female mosquito for lab populations ranged from 39.3 for An. cracens to 93.6 for An. dirus.

The mean rate of egg laying was close between field and lab populations, but there was a lot of variability in the rate of egg laying among individual species, ranging from 12 percent for An. cracens to 66 percent for An. dirus. The pupation rate ranged from 43.9 percent for An. harrisoni to 98.7 percent for An. maculatus. The rate of adult emergence was high, with 85 to 100 percent of pupae emerging into adults. The number of adults generated by the method was sufficient for insecticide resistance testing; for example, more than 4,400 F1 adults were generated by An. maculatus.

This study showed clear support for the efficacy of the in-tube forced oviposition method. Panthawong and colleagues concluded that their technique “is a successful strategy to generate sufficient numbers of F1 progeny for further laboratory studies and analysis.”

The investigators found that the proportion of females that laid eggs was only 8–18 percent in field-collected An. dirus and lab-reared An. epiroticus and An. cracens. Previous work by Morgan et al. in 2010 and Nepomichene et al. in 2017 found that An. arabiensis and An. mascarensis laid more eggs in 1.5 milliliter tubes than in 30x30x30 centimeter cages. This study used 35 milliliter vials. In this study, Panthawong and colleagues suggested that smaller containers may induce more environmental stress on the female mosquitoes, causing them to lay more eggs. Thus, further research and testing of the forced ovulation method could consider using smaller containers.

The forced oviposition technique is promising for testing for the presence of insecticide resistance in mosquito populations in the field. It can also be used to generate mosquitoes to test the efficacy of proposed control measures and to study the mechanisms of resistance, such as the upregulation of enzymes that detoxify insecticides, which develop in mosquitoes.

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/.