Rapid Differentiation of Vector Species: Development of Microsatellite Markers for Population Genetics of Biting Midges and a Potential Tool for Species Identification of Culicoides Sonorensis

Abstract

Background: Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, a SNP analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance. Methods: The aim of the current study was to develop a reliable and affordable way of differentiating the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers which were also tested for species differentiation.Results: A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recover the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately differentiating the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested. Conclusions: These microsatellites provide an affordable way in which to differentiate the species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America.

Immigration and seasonal bottlenecks: High inbreeding despite high genetic diversity in an oscillating population of the biting midge,Culicoides sonorensis(Diptera: Ceratopogonidae)

“…A total of 13 microsatellite markers ( C43, C45, C47, C65, C226, C230, C244, C508, C728, C838, C927, C1241 , and C2085 ) from Shults et al . (2022) (Shults et al 2021a) were used in this study. The forward primer of each pair was 5′-fluorescently labeled with 6-NED, VIC, PET, or FAM.…”

“…(2022) (Shults et al 2021a) were used in this study. The forward primer of each pair was 5′-fluorescently labeled with 6-NED, VIC, PET, or FAM.…”