Can you tell which of these mosquitoes is Aedes aegypti and which is Aedes albopictus? (Click to zoom.) Even for professional entomologists, visually identifying wild-caught mosquitoes to species requires rearing them to either fourth instar larvae or adults. A new set of improvements to a DNA-testing method developed by the CDC, however, will enable faster identification of either (or both) species’ presence in a collected sample of first instar larvae. And, to answer the question, that’s Ae. albopictus on the left and Ae. aegypti on the right. (Photo credits: James Gathany, CDC Public Health Image Library)

A crucial step in management of mosquito-borne diseases is knowing exactly what kind of mosquitoes are present in any given locale. Are they garden-variety species that aren’t carriers of human pathogens, or are they potentially dangerous vectors such as Aedes aegypti or Aedes albopictus?

Identifying and discerning between those two species is important as well, since their capacity for transmitting particular disease pathogens varies. Existing methods for identifying species requires setting ovitraps (which attract adult female mosquitoes to lay eggs) and rearing collected eggs into fourth instar larvae or adults for visual identification—which takes at least four days. DNA testing methods also exist, but they come with some complications. However, efforts by researchers at the U.S. Centers for Disease Control and Prevention (CDC) and Western Carolina University have resulted in significant improvements in the DNA testing methods that will make them faster and more accessible for mosquito labs around the country.

Reported in July in the Journal of Medical Entomology, the new process is a “duplex real-time PCR assay.” PCR (polymerase chain reaction) allows scientists to amplify DNA samples for various purposes, including species identification, and has been in use for more than 20 years. But the new protocol developed by a team led by Linda Kothera, Ph.D., a microbiologist at the CDC in the lab of Harry M. Savage, Ph.D., speeds up the process for discerning Aedes vectors in a few important ways, by allowing the use of:

• two fluorescent dyes, one each for Ae. aegypti and Ae. albopictus, to allow identification of either or both species in a single test, rather than doing so in separate tests
• “real-time” PCR, which removes a separate visualization step necessary in traditional PCR tests
• combined DNA samples, meaning DNA from multiple mosquito specimens can be pooled together, rather than tested individually
• first-instar larvae, which can be reared from eggs in a few hours, rather than a few days
• DNA samples from sonicated larvae, which can be done with a common jewelry sonicator, a less-expensive option compared to DNA extraction kits (though any DNA-extraction method will work).

These improvements “streamline the process and cut down on the amount of time needed to identify species composition,” says Kothera. “The new assay is more sensitive to small amounts of DNA than previous assays that used regular PCR.”

PCR assays used in this way test for specific DNA segments that are unique to a species, to serve as the signal for the presence of the species in the sample. For distinguishing between Ae. aegypti and Ae. albopictus, Kothera’s team targeted genes driving the mosquitoes’ sense of smell.

“I hypothesized that Aedes aegypti, because of its strong preference for human hosts, could have different DNA sequences in its odor receptor genes compared to Ae. albopictus, which will bite humans but takes bloodmeals from other hosts as well. In other words, I thought these genes would be more or less species specific,” Kothera says.

That hypothesis proved successful, and the odorant receptor gene fragments sought in the test will signal the presence of either species in the sample, and Kothera and colleagues also checked the test against seven other species of Aedes mosquitoes to ensure their presence wouldn’t lead to false positives.

By sharing the new method in the Journal of Medical Entomology, Kothera, Savage, and their co-author, Brian Byrd, Ph.D., at Western Carolina University’s Environmental Health Program, hope to enable mosquito labs far and wide to improve their capacity for detecting mosquito vectors.

“An accurate ascertainment of species composition is important for effective vector control. Although morphology can be used to distinguish species at certain life stages, rearing mosquitoes in controlled conditions requires time, space, and laboratory resources. PCR-based identification methods reduce the amount of time needed to identify species, with real-time assays eliminating the need for a separate visualization step. Our assay identifies Ae. aegypti and Ae. albopictus from sonicated first-instar larvae, and can serve as a tool to inform vector control agencies while they conduct surveillance and work to limit the spread of mosquito-borne pathogens,” they write.