Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera: Ceratopogonidae), using multi-locus DNA microsatellites

Onyango et al. Veterinary Research (2015) 46:108 DOI 10.1186/s13567-015-0250-8 RESEARCH ARTICLE VETERINARY RESEARCH Open Access Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera: Ceratopogonidae), using multi-locus DNA microsatellites Maria G Onyango1,2, Nigel W Beebe3,4, David Gopurenko5,6, Glenn Bellis7, Adrian Nicholas6, Moses Ogugo8, Appolinaire Djikeng8,9, Steve Kemp8, Peter J Walker1 and Jean-Bernard Duchemin1* Abstract Bluetongue virus (BTV) is a major pathogen of ruminants that is transmitted by biting midges (Culicoides spp.). Australian BTV serotypes have origins in Asia and are distributed across the continent into two distinct episystems, one in the north and another in the east. Culicoides brevitarsis is the major vector of BTV in Australia and is distributed across the entire geographic range of the virus. Here, we describe the isolation and use of DNA microsatellites and gauge their ability to determine population genetic connectivity of C. brevitarsis within Australia and with countries to the north. Eleven DNA microsatellite markers were isolated using a novel genomic enrichment method and identified as useful for genetic analyses of sampled populations in Australia, northern Papua New Guinea (PNG) and Timor-Leste. Significant (P < 0.05) population genetic subdivision was observed between all paired regions, though the highest levels of genetic sub-division involved pair-wise tests with PNG (PNG vs. Australia (FST = 0.120) and PNG vs. Timor-Leste (FST = 0.095)). Analysis of multi-locus allelic distributions using STRUCTURE identified a most probable two-cluster population model, which separated PNG specimens from a cluster containing specimens from Timor-Leste and Australia. The source of incursions of this species in Australia is more likely to be Timor-Leste than PNG. Future incursions of BTV positive C. brevitarsis into Australia may be genetically identified to their source populations using these microsatellite loci. The vector’s panmictic genetic structure within Australia cannot explain the differential geographic distribution of BTV serotypes. Introduction Bluetongue (BT) is an economically important viral disease throughout tropical and temperate regions of the world, posing a threat to the livestock industries, through production losses and negative impacts on trade [1]. The disease affects primarily sheep and goats. Cattle can also be infected but rarely show signs of disease [2]. Biting midges (Culicoides spp.) are vectors of bluetongue virus (BTV). In Australia, C. actoni Smith, C. brevitarsis Kieffer, C. fulvus Sen and Das Gupta are proven vectors of BTV and several others including C. brevipalpis Delfinado, C. dumdumi Sen and Das Gupta C. oxystoma Kieffer, C. * Correspondence: 1 CSIRO Health & Biosecurity Australian Animal Health Laboratory, 5 Portalington Road, Geelong, Victoria 3220, Australia Full list of author information is available at the end of the article peregrinus Kieffer and C. wadai Kitaoka are regarded as potential vectors [3,4]. Of these species, C. brevitarsis is the most widely distributed throughout northern and eastern parts of the continent [5,6], and is considered to be the major vector, employing cattle and buffalo dung as breeding sites [4,7]. BTV appears to have been introduced to Australia from Southeast Asia on multiple occasions by infected wind-borne vectors [8,9]. Indeed, 10 of the 26 known BTV serotypes have been detected in Australia through intensive surveillance during the past 30 years and there is evidence that at least four of these serotypes were introduced since the surveillance programme commenced [10]. The absence of clinical bluetongue disease in Australia, despite evidence of widespread infection in cattle, has been attributed to the limited distribution of © 2015 Onyango et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Onyango et al. Veterinary Research (2015) 46:108 C. brevitarsis to non-sheep breeding regions, primarily in the south of the continent and the relatively low pathogenicity of Australian BTV serotypes. Surveillance has indicated that the distribution of BTV serotypes in Australia is asymmetric with all 10 serotypes detected in the far northern region and only two serotypes (BTV-1 and BTV-21) enzootic in the southern portions of the eastern states. The factors influencing the distribution of serotypes are unknown and there is concern that introductions of exotic BTV strains from Southeast Asia via windborne Culicoides could destabilize the current situation [9]. A recent study of long-distance dispersal of Culicoides midges using an aerial migration model, indicated that migration of Culicoides into northern Australia from Timor-Leste (TL) and Papua New Guinea (PNG) is possible with Timor considered the most likely source of incursions [11]. Recent phylogeographic analyses [12,13] generally support those contentions and further indicate C. brevitarsis likely entered Australia and PNG separately from independent southeast Asian sources, in recent historical times [13]. Results of those prior genetic studies were based on analyses of a single maternally inherited gene and are potentially biased by a variety of evolutionary, demographic and sampling processes [14,15]. Additional population genetic analyses using multiple independent loci are needed to test hypotheses concerning the origins of recent arrivals of midge species in Australia. The first aim of this study was to develop a technical workflow for identifying DNA microsatellite markers de novo from small organisms such as Culicoides from which limited quantities of genomic DNA can be extracted. The second aim was to identify and compare allelic diversity of microsatellite loci among C. brevitarsis in Australia and neighbouring countries (PNG and TL) that are suspected sources of Culicoides spp. entering Australia. In this latter aim, we also sought to determine the levels of population genetic connectivity among the regions and infer the likely source(s) of midges in Australia during historical and current times. Materials and methods Insect sampling and DNA preparation A total of 141 samples were collected using light trap or sweep net, preserved in 70% ethanol and identified from sites in the Northern Territory (NT), Queensland (QLD), New South Wales (NSW), PNG and TL (Figure 1A) as described by Gopurenko et al. [13]. Sp (...truncated)