**2. Materials and method**

#### **2.1 Study areas**

We chose the survey sites based on the theoretical range of the primary vector of Zika virus, *Ae. aegypti,* as described by the CDC [8]*.* The surveyed area including

### *Survey for a Vector of Zika Virus and Two Other Mosquito Species in Four Ecoregions… DOI: http://dx.doi.org/10.5772/intechopen.94499*

most of Missouri south of the Missouri River as well as a few places north of the river on the western side of the state near and inside Kansas City, MO. This large region included four ecoregions: the Central Dissected Till Plains, the Osage Plains, the Ozark Highlands and the Mississippi River Alluvial Basin [6]. Only two surveyed counties were in the Central Dissected Till Plains, both near Kansas City. Most of the surveyed region lies within the Ozark Highlands, a region south of the Missouri River and covered with heavily forested hills. The western part of the surveyed region includes part of the Osage Plains region, a fertile prairie land with several streams and rivers. The southeastern portion of the surveyed region (the "Bootheel" of Missouri) includes parts of the Mississippi Alluvial Plain and is bordered by the Mississippi river. It supports large tracts of agriculture, including rice, soybean and cotton crops. The counties included in the survey (as well as the ecoregion for each) are depicted in **Figure 1**. Due to interest in potential vectors of human disease, the survey focused on locations near human habitations with many artificial containers, especially automobile salvage yards, used tire shops, and cemeteries. We also collected larvae from these sites and those data will be reported elsewhere. We resurveyed some sites as many as six times during three summers. Trapping occurred between June 3 and September 23 in 2016, between July 17 and October 29 in 2017, and between June 19 and August 19 in 2018.

We used three types of traps: the Fay-Prince Omnidirectional trap, the BG Sentinel trap and the Centers for Disease Control miniature light trap. All traps were baited with approximately five pounds of dry ice in a plastic cooler with a hole in the bottom to let the gas disperse, but the BG Sentinel trap also used a

#### **Figure 1.**

*Missouri counties where mosquito survey was performed are shaded gray (summers 2016–2018) (counties noted with a 1 are in the dissected till plains ecoregion. Those noted with a 2 are in the Osage Plains Ecoregion. Counties noted with a 3 are in the Ozarks Highlands ecoregion and those noted with a 4 are in the Mississippi River Alluvial Plain ecoregion).*

commercial lure (BG Lure) as it was designed to do. We placed the traps with baits on the sites in early afternoon and retrieved them in late morning. All traps were at least 50 paces apart from other traps. Trap contents were placed in a large cooler with a small amount of ice, then transported to the laboratory in Springfield, Missouri, where all mosquitoes were killed by freezing. Laboratory workers then pinned the female specimens and identified them microscopically using dichotomous keys in Darsie and Ward [9] and Burkett-Cadena [10].
