Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): results from an international ring trial targeting four species of the subgenus Avaritia

Abstract

BackgroundBiting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly.MethodsIn this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe.ResultsA total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction.ConclusionsThe results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Primera cita de <em>Culicoides paradoxalis</em> Ramilo & Delécolle, 2013 (Diptera, Ceratopogonidae) en España

“…Culicoides species of subgenus Avaritia (Culicoides obsoletus, Culicoides scoticus, Culicoides imicola, Culicoides chiopterus and Culicoides dewulfi) are thought to be the primary vectors of BTV and SBV in the Mediterranean region, based on abundance and host preference, vector competence studies, and isolation or detection of virus in field-collected midges (see Garros et al, 2014). Similarly, species of subgenus Culicoides are implicated as vectors of BTV (Caracappa et al, 2003;Vanbinst et al, 2009;Romón et al, 2012) and SBV (De Regee et al, 2012;Elbers et al, 2013;Balenghien et al, 2014).…”

Mechanistic model for predicting the seasonal abundance of Culicoides biting midges and the impacts of insecticide control

“…The few species-level adult surveillance datasets that exist across Europe tend to be available at lower monthly or weekly temporal resolutions, are not publicly available and still do not distinguish between C. obsoletus and C. scoticus [32, 75]. While male members of the subgenus Avaritia in western Europe can be identified reliably based upon marked differences in their genitalia, the routine morphological identification of the females that are important in transmission is less straightforward (see [76] and references therein). This has led to the development of PCR-based identification assays for the species of the subgenus Avaritia , particularly required for distinguishing between two widespread and abundant species, C. obsoletus ( s.s. ) and C. scoticus .…”

Bluetongue: control, surveillance and safe movement of animals

“…The species C. obsoletus and C. scoticus have a large Palaearctic distribution and females are highly morphologically close. Microscope mounting is needed and therefore differentiation based on morphology is difficult, requires crossing multiple criteria to be reliable, and then is time consuming (Garros et al., ). Due to this, all European national entomological surveillance programs include data on the both species grouped together.…”

“…Reversely in Corsica, C. obsoletus/C. scoticus are secondary to C. imicola, and on the Mediterranean coast, Culicoides newsteadi is dominant (Balenghien et al, 2012).…”