Development of microsatellite markers for population genetics of biting midges and a potential tool for species identification of Culicoides sonorensis Wirth & Jones

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 Latreille 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 Wirth & Jones 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, single-nucleotide polymorphism (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 distinguishing between 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 recovered the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately distinguishing between 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 to distinguish between the sibling 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. Graphical Abstract

A Review of the Vector Status of North American Culicoides (Diptera: Ceratopogonidae) for Bluetongue Virus, Epizootic Hemorrhagic Disease Virus, and Other Arboviruses of Concern

“…This work also discovered that C. albertensis, C. sonorensis, and C. variipennis share a single mitochondrial haplogroup, preventing the molecular identification of these species using common mt barcoding genes. The same delimitation patterns found in Shults et al [30••] were also recovered using a set of newly developed microsatellite markers [74]. One of these markers appears to have species-specific amplification in C. sonorensis, and could be used to develop a single-tube assay to detect this vector species in pools of Culicoides samples.…”

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

“…Genomic DNA was extracted from these individuals using a modified Gentra Puregene extraction protocol (Gentra Systems, Inc. Minneapolis, MN, USA). 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 2022) were used in this study. The forward primer of each pair was 5′-fluorescently labeled with 6-NED, VIC, PET, or FAM.…”

“…Regardless, the cyclical nature of these populations results in pronounced seasonal fluctuations in adult abundance and rates of migration. Most biting midge species studied so far exhibit long-distance dispersal, often wind-mediated (Ducheyne et al 2007, Sanders and Carpenter 2014, Jacquet et al 2016), leading to high gene flow among populations and an absence of population differentiation even at continental scales (Jacquet et al 2015, Onyango et al 2015a, Shults et al 2021). Despite the absence of genetic differentiation among most populations, high levels of inbreeding coefficient ( F IS ) and heterozygote deficiencies are commonly found in Culicoides species (Onyango et al 2015b, Mignotte et al 2021, Shults et al 2021).…”

Cutaneous filarioid nematodes of dogs in the United States: Are they emerging, neglected, or underdiagnosed parasites?

“…Biting midges (Ceratopogonidae) should also be further investigated as possible vectors since they are known to transmit multiple Onchocerca species in the US including the equine O. cervicalis, and the bovine O. gutturosa (88)(89)(90)(91) as well as others worldwide (1,92). Although a wide range of Culicoides species are present in O. lupi endemic areas of the southwestern US, many of which are documented to feed primarily on humans (93), two subspecies within the Culicoides variipennis complex, Culicoides v. sonorensis and Culidoides v. occidentalis (94), are known to feed on both dogs and humans (93), and Culidoides v. variipennis has been shown to naturally and experimentally transmit O. cervicalis to horses in the US (95,96). Additionally, the apparent distribution of Culidoides v. occidentalis (94) encompasses the known endemic areas in the southwestern US coinciding with human, companion animal, and coyote cases (70,81).…”