Morphometric discrimination of two sympatric sibling species in the Palaearctic region, Culicoides obsoletus Meigen and C. scoticus Downes & Kettle (Diptera: Ceratopogonidae), vectors of bluetongue and Schmallenberg viruses

Abstract

Background: Some Palaearctic biting midge species (subgenus Avaritia) have been implicated as vectors of bluetongue virus in northern Europe. Separation of two species (C. obsoletus and C. scoticus) is considered difficult morphologically and, often, these female specimens are grouped in entomological studies. However, species-specific identification is desirable to understand their life history characteristics, assess their roles in disease transmission or measure their abundance during arboviral outbreaks. This study aims to investigate whether morphometric identification techniques can be applied to female C. obsoletus and C. scoticus individuals trapped at different geographical regions and time periods during the vector season.

Methods: C. obsoletus and C. scoticus were collected using light-suction traps from the UK, France and Spain, with two geographical locations sampled per country. A total of 759 C. obsoletus/C. scoticus individuals were identified using a molecular assay based on the cytochrome c oxidase subunit I gene. Fifteen morphometric measurements were taken from the head, wings and abdomen of slide-mounted specimens, and ratios calculated between these measurements. Multivariate analyses explored whether a combination of morphometric variables could lead to accurate species identification. Finally, Culicoides spp. collected in France at the start, middle and end of the adult vector season were compared, to determine whether seasonal variation exists in any of the morphometric measurements.

Results: The principal component analyses revealed that abdominal characteristics: length and width of the smaller and larger spermathecae, and the length of the chitinous plates and width between them, are the most reliable morphometric characteristics to differentiate between the species. Seasonal variation in the size of each species was observed for head and wing measurements, but not abdominal measurements. Geographical variation in the size of Culicoides spp. was also observed and is likely to be related to temperature at the trapping sites, with smaller individuals trapped at more southern latitudes.

Conclusions: Our results suggest that female C. obsoletus and C. scoticus individuals can be separated under a stereomicroscope using abdominal measurements. Although we show the length and width of the spermathecae can be used to differentiate between the species, this can be time-consuming, so we recommend undertaking this using standardized subsampling of catches.

Keywords: Arbovirus; BTV; Bluetongue virus; Culicoides obsoletus; Culicoides scoticus; Morphology; Morphometrics; Obsoletus Group; Obsoletus complex; Sibling species.

Fig. 1

Location of field-trapping sites for Culicoides samples used for morphometric and molecular identification, in France (Calvados sampled 27^th April, 12^th July and 3^rd November 2010; Landes 26^th April 2010), Spain (Caldes de Malavella sampled 16^th June 2011; Avià 21^st May 2009) and the UK (Bala sampled 14^th July 2011; Devon 4^th November 2011)

Fig. 2

Morphometric measurements of Culicoides. a & b the head: where a) is length of eight basal flagellar segments of the antenna; b) is the length of the five apical flagellar segments of the antenna; c) is the length of flagellomere 10; d) is the length of flagellomere 11; e) is the length of the third palpal segment; and f) is the width of the third palpal segment. c the wing: where a) is the costa length; b) is the width of the wing; and c) is the length of the wing (arculus to tip). d & e the abdomen: where a) is the length of spermatheca 1; b) is the width of spermatheca 1; c) is the length of spermatheca 2; d) is the width of spermatheca 2; e) is the length between the chitinous plates; and f) is the width of the chitinous plates

Fig. 3

A scree plot highlighting the relationship between the eigenvalues and the number of axis in a principal component analysis of a 15 morphometric measurements of C. obsoletus and C. scoticus; and b eight ratios derived from morphometric measurements of those individuals

Fig. 4

Results of principal component analysis on a morphological measurements of C. obsoletus and C. scoticus; and b morphometric ratios of C. obsoletus and C. scoticus