In regions where bed nets are common, some mosquitoes have shifted their feeding times to avoid the nets. A new study examines two strains of mosquitoes in the Culex pipiens species complex to dig into the genetic basis for this behavioral shift. (Photo by Ary Farajollahi, Bugwood.org)

By Andrew Porterfield

Andrew Porterfield

Mosquitoes and other blood-feeding insects can cause a host of dangerous human diseases, including malaria, dengue, Zika, West Nile, chikungunya, and yellow fever. Since these diseases have so far evaded effective vaccines, insecticides and insecticide-treated bed nets have been among the most effective ways to reduce their spread.

However, scientists have noted that these insecticide strategies create strong selection pressures in mosquitoes, driving the development of insecticide resistance and behavioral changes among mosquitoes. Behavior changes include altering blood feeding times, as mosquitoes have changed to bite humans after they exit mosquito nets in the morning hours and before they enter them in the evening.

Molecular circadian clocks that control these behaviors are genetically controlled feedback loops that respond to light-dark cycles and temperature changes. Also, flight and feeding behavior are closely related, with flying time an indicator of increased feeding among mosquitoes. This behavior has been seen to change, seemingly in response to insecticide use, but its genetic basis has not been studied closely.

Paul Hickner, Ph.D.

A team of researchers from the University of Notre Dame and the U.S. Department of Agriculture’s Agricultural Research Service looked for the genetic basis that could underlie variation in timing of mosquito blood feeding. By studying the behavior and genetics of two strains of mosquitoes in the Culex pipiens species complex, the researchers discovered a cluster of previously unknown genes that could detail the genetic basis behind mosquitoes’ day-night calibration for blood feeding. Their results were published in August in the Journal of Medical Entomology.

Previous studies had shown that C. pipiens generally feed at twilight and overnight, but female flight activity was seen to vary considerably among strains of mosquito. The researchers compared the behavior and genetics of two Culex strains: the Shasta strain (from Shasta County, California), an older isolate that feeds any time of day or night, and the Trinidad strain, a newer strain from the West Indies that feeds only at night.

For the genetic part of the study, the researchers first mated a Trinidad male with a Shasta female to create an intercross generation (F2). Crosses create individuals or strains with different fractions of the genome inherited from the parental lines. Both the phenotypes (in this case, feeding times and flight activity) and genotypes (genes that may be associated with these behaviors) were examined. The researchers then conducted quantitative trait locus (QTL) mapping, a statistical analysis that attempts to determine an area on a chromosome that is connected to the phenotype (feeding times and flight activity). QTL analysis does not necessarily identify specific genes—only the region of a chromosome that seems to affect the trait being observed. In this experiment, 95 F2 progeny mosquitoes were used for QTL analysis.

Genetic linkage maps were used to identify the genomic location of clock genes and other genes shown in previous work to play some role in feeding or locomotion. Then, flight activity of 32 females from both strains were observed over seven days, recording the frequency and time a mosquito broke an infrared beam during flight.

The researchers found a single QTL on chromosome 2 that was behind the differences in blood feeding time between the two strains. The QTL region was 67.4 Mb (mega bases), and contained 2,062 genes. Of those, 21 were involved with behavior regulation, feeding behavior, circadian behavior, locomotory behavior, or sleep. Flight activity was also significantly different between two strains, with the Trinidad strain active at night, dusk, and dawn, and Shasta also active during daytime.

Surprisingly, known clock genes, such as period, timeless, clock, cycle, PAR-domain protein 1, vrille, discs overgrown, and cryptochromes 1 and 2, were not in the QTL. “It is possible they have a small effect on biting time that could not be detected in our study,” says lead author Paul Hickner, Ph.D., research entomologist at USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, Texas, and previously a postdoctoral researcher at Notre Dame. “Identification of small-effect QTLs is challenging and often requires the analysis of a very large number of F2 progeny.”

How—and how quickly—this difference between strains evolved remains unknown, Hickner says, “because we don’t know exactly which genes are involved. Culex pipiens are generally twilight and/or nocturnal feeders, but there was standing genetic variation in the underlying genes within the Shasta population that could produce, in the right combination, blood feeding at any time of day.”

Could Culex develop these behavior changes without genetic change? Unlikely, Hickner says. “Blood feeding requires many physiological processes such as flight, sensory perception, metabolism, and detoxification. These are generally correlated in part by the circadian clock. Blood feeding at any time of day could represent a “disengagement” of these processes with the clock.”

The adaptation of daytime feeding in nocturnal mosquitoes is not limited to C. pipiens, the researchers write. “The Pimperena strain of Anopheles gambiae is almost exclusively a nocturnal blood feeder, while other A. gambiae laboratory strains feed during the day.”

Hickner and colleagues add, “Further studies are needed to determine if the genetic mechanisms for the shift in blood-feeding times in laboratory colonies are related to those underlying the observed shifts in field populations. A better understanding of the genetic basis of these behaviors could help identify measures to address these challenges in the prevention of vector-borne diseases.”

Andrew Porterfield is a writer, editor, and communications consultant for academic institutions, companies, and nonprofits in the life sciences. He is based in Camarillo, California. Follow him on Twitter at @AMPorterfield or visit his Facebook page.