Insecticide Resistance in Mosquitoes

culex-1-final copy.jpgThis series is about mosquitoes (Culex pipiens complex) and insecticide resistance. It ran live on the site over two weeks in 2015 as Linda Kothera, a mosquito biologist at the Center for Disease Control (CDC) in Fort Collins, Colorado answered reader questions.
culex-2-small copyMosquitoes are vectors of human and other animal diseases. For example, Culex pipiens mosquitoes can carry the West Nile virus. Humans and horses are what are called dead-end hosts, meaning the virus doesn’t amplify in them. Birds, especially corvids (crows, blue jays, magpies, etc.) are considered amplifying hosts and the disease is spread by mosquitoes taking blood meals from infected birds, then biting humans. Insecticides are used to reduce the number of mosquitoes in the environment, and thus control transmission of the disease. Larvicides, which are designed to affect the aquatic juvenile stage of the life cycle, are added to areas of standing water. Adulticides are sprayed to kill adults. Adulticides are applied at night, when most beneficial insects are not actively flying, and are often used in doses of as little as an ounce per acre (about the size of a football field). Mosquito control districts vary in how much insecticide application they engage in. Where Linda lives, for example, adulticiding doesn’t take place until there are West Nile virus-positive mosquitoes detected during surveillance (the regular trapping and testing of mosquitoes). Repellants (i.e. “bug spray”) are used to keep mosquitoes from landing on humans and attempting to feed.

culex-3 copyThere’s naturally occurring genetic diversity in any population, resistance happens when an individual, or group has a small variation that helps them survive better. So, over tens to hundreds of generations, the result is a population better adapted to their environment. If the mosquitoes’ environment includes an insecticide, some part of that population will become resistant over time. There are many ways that these changes could occur, so there are multiple genes that affect insecticide resistance. Some genes and mechanisms are well-characterized, but because Culex pipiens complex mosquitoes prefer what they euphemistically call “organically-enriched” larval habitats (i.e. they love water with lots of animal waste or rotting vegetation) it is thought that genes associated with helping larvae cope with such conditions could help them adapt to the pressures of insecticides. Here are some examples of insecticide resistance in Culex pipiens: Something called kdr (“knock down resistance”) is an example of one mechanism of resistance called Target Site Mutations, and results from a single base pair change in the genetic code for one of the sodium channel genes. The mutation changes the shape of the receptor targeted by the toxin in pyrethroid insecticides, and they are rendered ineffective. The other major mechanism that causes resistance is known as metabolic resistance. Here, genes are either up-regulated or they exist in multiple copies. Either way, more gene product is made, which detoxifies or sequesters the toxin in the insecticide. Genes in the esterase family sometimes exist in multiple copies and are associated with metabolic resistance.

culex-4 copyDifferent colors of mosquitoes in this picture represent a single base pair change, which is one of the mechanisms that can cause variability in a population, that can cause the different genes to insecticide resistance that the individual might have. Linda’s lab at the CDC is studying this genetic diversity. For example, adults that have been found to be resistant (they test them by exposing them to standard amounts of insecticides) could have the kdr mutation, as well as multiple copies of two esterase genes. They extract that individual’s DNA and are interested in the differences in the DNA sequences of both known resistance genes such as kdr and esterases, as well as several other genes. They compare the genetic variation of resistant individuals to individuals that are susceptible to the insecticide. This co-variation in a set of genes in resistant vs. susceptible mosquitoes can give them a better picture of what genes are involved in resistance.

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They are using a kind of next generation sequencing technology called targeted sequencing to sequence up to 100 genes in several individuals simultaneously. This project is ongoing, and they’re still figuring out the limits of how many individuals they can run at once, but it’s probably around 25-30. They plan to use the data to determine the genetic differences that exist between susceptible and resistant individuals and aren’t due to other factors. They expect to find differences in some genes that have not been previously well-characterized, and plan to develop diagnostic assays for these genes so that the CDC or other agencies tasked with mosquito control can test their mosquitoes for resistance. These are useful tools for local vector control programs studying the Culex pipiens mosquitoes.

culex-6-small copyMosquito control districts in the U.S. work with state public health departments to ensure that if West Nile virus is detected, the public is notified and efforts are made to control the mosquito population. Nevertheless, it’s still important to use some common sense practices for keeping mosquitoes out of your home and reduce the number of bites that you get. No one likes mosquito bites, right? Clear standing water, such as that found in old tires and outdoor potted plant saucers, use screens on windows and use mosquito repellant, especially at dawn and dusk, even if there is no current outbreak. Here are the CDC’s recommendations.

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