Infection, Genetics and Evolution 21 (2014) 110–117

Contents lists available at ScienceDirect

Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid

Wing geometry as a tool for discrimination of Obsoletus group (Diptera: Ceratopogonidae: Culicoides) in France L. Hajd Henni a, F. Sauvage b,c, C. Ninio a, J. Depaquit a, D. Augot a,⇑ a

Usc-VECPAR, ANSES-LSA, EA 4688, SFR Cap Santé, Université de Reims Champagne-Ardenne, F-51100 Reims, France Université de Lyon, F-69000 Lyon, France c Université Lyon 1, CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622 Villeurbanne, France b

a r t i c l e

i n f o

Article history: Received 25 May 2013 Received in revised form 8 October 2013 Accepted 10 October 2013 Available online 26 October 2013 Keywords: Culicoides chiopterus Culicoides dewulfi Culicoides obsoletus Culicoides scoticus Wing geometry DNA

a b s t r a c t In Europe, Culicoides chiopterus, Culicoides dewulfi, Culicoides obsoletus and Culicoides scoticus, which belongs to the subgenus Avaritia and Obsoletus group are the most proficient Bluetongue and Schmallenberg vectors. Within this group, correct identification based on morphological traits is difficult but essential to assess disease transmission risk. The development of new tools has revolutionized taxonomy (i.e. geometric morphometrics and molecular biology). Wing morphology is of primary importance to entomologists interested in systematics. Here, we report phenotypic differentiation patterns among the species above mentioned using a landmark-based geometric morphometric approach that efficiently identified C. chiopterus and C. dewulfi. Wing shape of the C. scoticus sample exhibited large specific variability. Based on landmarks and phylogenetic analyses (Maximum Parsimony), we suggest that Obsoletus group in Europe includes only C. obsoletus and C. scoticus. C. dewulfi and C. chiopterus are clearly excluded. Their shape seems closer to C. obsoletus that is why we suggest that only these two species should be grouped in the Obsoletus group. In addition, the concordance between phenetic clusters and phylogenies inferred from molecular data based on a fragment of the mtDNA COI gene and rDNA 28S suggests the existence of a strong signal in wing shape. These findings encourage us to use this powerful tool in taxonomic studies. Ó 2013 Published by Elsevier B.V.

1. Introduction Bluetongue (BTV) and Schmallenberg (SBV) virus are emerging viral diseases in Northern Europe (Thiry et al., 2006; EFSA, 2012). These diseases affecting domestic and wild ruminants were reported for the first time in Northern Europe in August 2006 and winter 2011, respectively. The biting midges responsible for the transmission of BTV and SBV belong to the genus Culicoides. According to the lack of economically important disease they transmit, the biting midges of Europe have long been neglected, and have only become a focus of research since 2006 with the unexpected outbreaks of BTV and SBV. Several entomological monitoring programs were developed to identify possible BTV and SBV vectors. Recent studies suggested the following species as putative BTV vectors: Culicoides obsoletus, Culicoides scoticus, Culicoides chiopterus, Culicoides dewulfi, Culicoides pulicaris, Culicoides achrayi (Hoffmann et al., 2009) and SBV vectors: C. Obsoletus group (=C. obsoletus, C. scoticus, C. chiopterus, C. dewulfi) (Rasmussen et al., 2012), C. obsoletus complex, C. obsoletus s.s., C. dewulfi, C. chiopterus and C. pulicaris (De Regge et al., 2012). Systematics of the genus Culicoides has relied mainly ⇑ Corresponding author. Tel.: +33 26 91 37 06. E-mail address: [email protected] (D. Augot). 1567-1348/$ - see front matter Ó 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.meegid.2013.10.008

on morphological characters. Identification of Culicoides to species level is difficult even for specialist taxonomists and sibling species within species complexes can be indistinguishable with classical morphological methods (Meiswinkel et al., 2004; Pagès et al., 2005). Molecular (Dallas et al., 2003; Perrin et al., 2006) and morphometric tools (Pagès et al., 2005, 2009; Augot et al., 2010; Muñoz-Muñoz et al., 2011) have been employed to identify precisely different species of subgenera Avaritia and Culicoides. Wing morphology is of primary importance to entomologists interested in systematics. The wing has been used in morphological identification of Culicoides species with an important discrimination level (Wirth et al., 1985, 1988). A key has been developed on 58 Culicoides species of the Iberian Peninsula using only the characteristic pattern of the wing (Rawlings, 1996). The level of differentiation was epidemiologically valuable. Geometric morphometrics is a powerful and cheap characterizing tool for many organisms including sand flies (Dujardin et al., 2002); triatomines (Villegas et al., 2002; Feliciangeli et al., 2007; Dujardin et al., 2007); tsetse flies (Camara et al., 2006) and recently biting midges (Muñoz-Muñoz et al., 2011). The main goal of the present study was the identification of four species (C. chiopterus, C. dewulfi, C. obsoletus and C. scoticus) using landmark-based geometric morphometrics on wing traits. To identify with certainty the four species, our study was supported by

111

L.H. Henni et al. / Infection, Genetics and Evolution 21 (2014) 110–117

molecular analyses based on mitochondrial DNA. These four species belong to Obsoletus group (Nielsen and Kristensen, 2011). Moreover, inside the Obsoletus group, two sympatric sibling species exist (C. scoticus and C. obsoletus) generally noted Obsoletus complex (Venail et al., 2012). The results are particularly important to assess risk of transmission of diseases (i.e. BTV and SBV). To our knowledge, no phylogenetic study has been carried out to investigate the evolutionary history in Obsoletus group with a marker that presents a low mutation rhythm. So, a second objective of this study was to evaluate the molecular phylogeny of Obsoletus group using the D1D2 domains of the 28S ribosomal DNA (Augot et al., In preparation). By introducing morphometric techniques, we also reevaluated the taxonomic status of C. dewulfi, C. chiopterus C. obsoletus and C. scoticus. 2. Materials and methods 2.1. Sample collection Culicoides were collected during 2008–2011 from three French departments (Table 1): Aisne (code beginning with the letter P), Ardennes (BF, N) and Gard (D). Midges were caught outdoor by UV CDC miniature light traps (John W. Hock Company). Traps were set approximately 1 h before sunset until 1 h after sunrise under favorable climatic conditions (absence of heavy rain and/or wind). Midges were stored in 96% ethanol before morphological and molecular analyses. The sampling also included specimens previously characterized (n = 79) (Augot et al., 2010) to which new specimens have been added (n = 36). 2.2. Specimen identification Initial identification of Culicoides was based on wing spot pattern and other morphological keys (Institute for Animal Health hosted website, 2013). Wings, head and abdomen of individual midges were mounted in Chloral gum after clearing in boiling Marc–André solution on microscope sides for morphological identification (Delécolle, 1985; Kremer and Rebholtz, 1977; Augot et al., 2010). 115 Females individuals Culicoides from the subgenus Avaritia were dissected and identified as: 21 C. dewulfi (BF, n = 16; P, n = 5); 21 C. chiopterus (N, n = 20); 58 C. obsoletus (D, n = 10; P, n = 48); 16 C. scoticus (P, n = 16).

Sequencing and phylogenetic analyses were based on a fragment of the mtDNA COI gene and rDNA 28S. PCR were performed in a 50 ml volume using 5 ml of extracted DNA solution and 50 pmol of the primers: (i) LepF (50 -ATT CAA CCA ATC ATA AAG ATA TTG G-30 ) and LepR (50 -TAA ACT TCT GGA TGT CCA AAA AAT CA-30 ) for the mtDNA COI gene analysis (Augot et al., 2010). Amplification conditions (Costa et al., 2007) were as follows: after an initial denaturation step at 94 °C for 3 min, 5 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 90 s, and extension at 68 °C for 60 s were followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 51 °C for 90 s, and extension at 68 °C for 60 s and a final extension at 68 °C for 10 min by using Taq polymerase (50 , Germany). (ii) C0 1 (50 -ACCCGCTGAATTTAAGCAT-30 ) and D2 (50 -TCCGTGT TTCAAGACGGG-30 ) for the rDNA 28S analysis (Depaquit et al., 1998). Amplification conditions were as proposed by the above mentioned authors: an initial denaturation step at 94 °C for 3 min, followed by 35 cycles of (denaturation at 94 °C for 30 s, annealing at 58 °C for 90 s, and extension at 68 °C for 60 s) and a final extension at 68 °C for 10 min by using Taq polymerase (5, Germany). Amplicons were analysed by electrophoresis in 1.5% agarose gel containing ethidium bromide. Following amplification, the PCR products were sequenced bidirectionally, using the primers by Beckman Coulter Genomics (Takeley, United Kingdom) or Eurofins (Germany). Phylogenetic analysis was performed using the neighbour-joining (NJ) method (Kimura 2-parameter) and maximum parsimony (MP) method (using a Branch and Bound search option) with MEGA software version 3.1 (Kumar et al., 2004). The bootstrap probabilities of each node were calculated using 1000 and 500 replicates to assess the robustness of the NJ and MP methods, respectively. A NJ tree of K2P distances was created to provide a graphic visualization of clustering group among different species (Saitou and Nei, 1987). For comparison with NJ method, we included COI sequences of Genbank: C. chiopterus (HQ824412) C. dewulfi (HQ824416); C. obsoletus (HQ824371); C. scoticus (HQ824385) all from Switzerland. A specimen of Culicoides nubeculosus (JQ620128) also was processed as out-group (Ander et al., 2012). For 28S sequences with MP method, only three specimens per species of this study were analyzed. A specimen of C. nubeculosus was processed as out group (Augot et al., In preparation).

2.3. DNA extraction, PCR amplification and sequencing 2.4. Morphometrics analysis Total DNA was extracted for each dissected midges from thorax, legs and the anterior part of the abdomen using the QIAmp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany), following manufacturer’s instructions with a final elution volume of 100 ll.

2.4.1. Samples preparation and data collection Culicoides wings were detached from the thorax, placed on a microscopic slide within a Chloral gum under the cover slip. A

Table 1 Samples of Culicoides collected in three departments (Aisne, Ardenne, Gard) in France. Department

Locality

Latitude

Code of specimen

Number of wings per species

Aisne

Montigny-laCour Signy-le Petit

49°590 69°00 N, 4°010 4500 E 49°530 46.7800 N, 4°190 20.1900 E 49°470 33.910 N, 4°160 50.7800 E 49°480 50.9400 N, 4°140 05.2100 E 49°430 24.3500 N, 4°220 22.2500 E 44°000 31.900 N, 3°420 58.600 E

P

19 C. scoticus, 48 C. obsoletus, 6 C. dewulfi 21 C. chiopterus

Ardenne

Rumigny Hannapes 0

Signy-l Abbaye Gard

Sumène

N BF44, BF52, BF57, BF62, BF64, BF65, BF66, BF76, BF77, BF108, BF136, BF141 BF97, BF98

12 C. dewulfi

BF123, BF124

2 C. dewulfi

D

10 C. obsoletus

2 C. dewulfi

112

L.H. Henni et al. / Infection, Genetics and Evolution 21 (2014) 110–117

digital camera was used to capture the wing images. Scaling was performed using micrometric calibration slide. In order to find the most discriminating landmarks, two sets of nine and ten landmarks (Fig. 1) covering most of the wing surface were selected and digitized using a twofold magnification lens and the free software CLIC (http://bioinfo-prod.mpl.ird.fr/morphometrics/clic/index. html.). The set of ten landmarks keep the first nine and add one more. Measures were repeated for the first nine landmarks. All landmarks were recorded on one wing per individual (left or right indistinctly) (Muñoz-Muñoz et al., 2011) by the same person for more consistency (Bookstein, 1991).

2.4.2. Repeatability Random error measurement is common in morphometric analysis, and it can cause serious statistical problems (Arnqvist and Martensson, 1998). To detect this kind of error, we used the repeated records for the first nine landmarks of all individuals, and we quantified their repeatability by species and coordinate as the ratio between the individual variability (computed as the sum over all specimens of squared differences between individual coordinates and their individual mean) and the total variability (computed as the sum of squared differences between individual coordinates and the overall mean for the given species at the given coordinate) (Corner et al., 1992). To perform the size and shape analysis described in the next paragraph, the position of a landmark for a given specimen was replaced by the average of both raw repeat measures as the specimen positions during recording remain the same (Von Cramon-Taubadel et al., 2007).

2.4.3. Statistical analysis of size and shape We explored wing size and shape variations from the 115 specimens using the landmark-based geometric morphometric method (Bookstein, 1991). The Generalized Procrustes Analysis or GPA (Rohlf and Slice, 1990) optimally translates, rotates and uniformly scales the sets of landmarks from sampled midges. GPA and thin-plate spline analysis (Bookstein, 1991) produced size and shape variables, allowing us to separate both components of the morphometric variations. Individual wing size is estimated by the ‘‘centroid size’’ (CS), i.e. the square root of the sum of squared distances of a set of landmarks from their centroid (Rohlf and Slice, 1990). We tested homoscedasticity and normality of CS values by species using Levene and Shapiro–Wilks tests, respectively. The means of CS values were compared between species through one-way ANOVA and Tukey HSD post hoc tests. The Tukey’s honestly significant difference (HSD) procedure for unequal sample size was applied to test for

differences in species pairs comparisons (Spjotvoll and Stoline, 1973). The GPA also gives data, which describes the individual deviations from the consensus configuration, once corrected for size. The shape components constitutes the W’ matrix (i.e. the non-uniform components called ‘partial warps’ and the uniform components of shape) computed by tpsRelw software (Rohlf, 2006). Sometimes, shape is also studied using the ‘relative warps’, which are the principal components from a principal component analysis on partial warps. To test the accuracy of the approximation of the fitted coordinate configurations from Kendall’s shape space by the projection onto tangent space, we computed the correlation between Procrustes and Euclidian distances using tpsSmall (Rohlf, 2003). In order to explore the relationships between shape and size, we proceeded to multivariate regressions of shape by centroid sizes and in order to see this relation according to species, we proceeded to multivariate analyses of covariance (MANCOVA) between shape and size or shape and canonical scores by species. Actually, the MANCOVA between shape and size, using CS values here, by species assessed interspecific allometry to test for a common pattern (Villegas et al., 2002). Statistical significance was obtained by the Wilks statistics and permutation test procedure (Good, 2000). To evaluate the possible environmental effect of trapping site on shape changes, another MANCOVA with ‘‘population’’ as factor was computed within the C. obsoletus species, which was the unique one captured in more than one site. We used the canonical variate analysis (CVA) on partial warps to explore the shape variation in pairwise form within the same species and among the species using MorphoJ (Klingenberg, 2011). We used the percentage of accurate predicted species from the CVA to evaluate the discriminatory power of wing shape from our set of landmarks Computational statistics were performed using the free software R (http://cran.r-project.org/), the tps soft series (Rohlf, 2003, 2007) and MorphoJ (Klingenberg, 2011). Data about the same ten landmarks from seventeen specimens of the species C. nubeculosus were added and the GPA over the entire set of data was performed again. The results were used to build a phenetic tree of the relationships between species according to shape. The C. nubeculosus sample represented the outgroup for the tree construction. Procrustes distances (the square root of the sum of squared differences between corresponding points) between each pair of the five species were computed and the tree was constructed using the neighbor-joining algorithm (MOG module, version 104.01 from the CLIC package by Dujardin, available at http://mome-clic.com/the-clic-package/ calling PHYLIP, the software from Felsenstein, and NJPLOT by Perriere and Gouy for tree editing).

3. Results 3.1. Molecular analysis

Fig. 1. Location of 10 landmarks on a wing (drawing of Delécolle, 1985) used in the morphometrical analysis of the Obsoletus group (C. chiopetrus, C. dewulfi, C. obsoletus and C. scoticus).

Samples from 115 specimens were sequenced for COI mtDNA and 12 specimens for 28S rDNA as follow: C. chiopterus (N380, N383, N387); C. dewulfi (Bf123, Bf141, P3C8), C. obsoletus (D212, P3C20, P5C5), C. scoticus (P6C12, P6C25, P7C8) belonging to the series. Sequences obtained are available in GenBank under Accession Nos. C. chiopterus (KF802219–KF802237), C. dewulfi (HM022876– M022881; KF802203-KF802218), C. obsoletus (HM022792– HM022856) and C. scoticus (HM022857–HM022875) for COI and C. chiopterus (KF826474–KF826476), C. dewulfi

L.H. Henni et al. / Infection, Genetics and Evolution 21 (2014) 110–117

(KF826471–KF826473), C. obsoletus (KF826477–KF826479), C. scoticus (KF826480–KF826482) and C. nubeculosus (KF286367) for 28S. Molecular comparisons after alignment were based on 385 bp for the COI and 656 bp for 28S (including gaps). 3.2. Analysis of COI sequences The membership of each sample in the various branches was strongly supported by bootstrap values (Fig. 2a). The analysis also yielded moderate bootstrap support (44%) for the clustering of C. obsoletus, C. scoticus and C. chiopterus together. The low intraspecific divergences as follow: C. dewulfi (0,000), C. chiopterus (0,006), C. obsoletus (0,007) and C. scoticus (0,002). The interspecific K2P values for different species are: 0; 270 (between C. dewulfi and C. chiopterus), 0,226 (between C. dewulfi and C. obsoletus), 0,211 (between C. dewulfi and C. scoticus), 0,278 (between C. dewulfi and C. nubeculosus), 0,165 (between C. chiopterus and C. obsoletus), 0,159 (between C. chiopterus and C. scoticus), 0,218 (between C. chiopterus and C. nubeculosus), 0,133 (between C. obsoletus and C. scoticus), 0,210 (between C. obsoletus and C. nubeculosus) and 0,215 (between C. scoticus and C. nubeculosus). 3.3. Analysis of 28S sequences The branch and bound search provided three trees equiparcimonious, 648 steps long. Their strict consensus is given in Fig. 2b. Consistency index = 0,78; Retention index = 0,89

a

113

The intraspecific divergences are as follow: C. dewulfi (0,001), C. chiopterus (0,002), C. obsoletus (0,002) and C. scoticus (0,001). The intraspecific K2P values for different species are: 0; 030 (between C. dewulfi and C. chiopterus), 0,030 (between C. dewulfi and C. obsoletus), 0,030 (between C. dewulfi and C. scoticus), 0,071 (between C. dewulfi and C. nubeculosus), 0,013 (between C. chiopterus and C. obsoletus), 0,014 (between C. chiopterus and C. scoticus), 0,056 (between C. chiopterus and C. nubeculosus), 0,010 (between C. obsoletus and C. scoticus) and 0,054 (between C. obsoletus and C. nubeculosus). 3.4. Morphometrical analysis The quantification of the random measurement error by landmark coordinate and species in our data revealed a high level of precision and repeatability in the collection of landmarks, which guaranteed the reliability of the wing size and shape analyses (Table 2). 3.4.1. Size analyses The null hypothesis of homoscedasticity of CS values among species appeared acceptable from the Levene test (F = 1.398; df = 3, 111; P = 0.247). Shapiro–Wilks tests conducted did not reject the normal distributions of CS values within the four studied species (P-values ranged from 0.305 for C. obsoletus to 0.688 for C. chiopterus). Hence, variations in wing size were analyzed through parametric statistics. ANOVA comparing mean CS between species and the Tukey HSD post hoc test revealed that all pairwise differences between C. scoticus and the three other species were

100

44 100 21 100

100

JQ620128-C. nubeculosus 0.02

b

57 82

P3C20 P5C5 D212

41

P7C8 52

P6C12

87 69

P6C25 N383 N380

92 56

N387 Bf 123 Bf 141

99 33

P3C8

Fig. 2. Trees obtained from nucleotide analysis of: (a) COI (with NJ method); (b) 28S rDNA (with MP metod) sequences of the 4 species of Obsoletus group: boostrap values are shown in nodes (1000 replicates for COI and 500 replicates for 28S).

114

L.H. Henni et al. / Infection, Genetics and Evolution 21 (2014) 110–117

Table 2 Percentage of the total variability in landmark coordinates tracing to random measurement error. Results are given by species and landmark coordinate (two coordinates per landmark). Species

X1

Y1

X2

Y2

X3

Y3

X4

Y4

X5

Y5

X6

Y6

X7

Y7

X8

Y8

X9

Y9

C. C. C. C.

0.12 0.23 0.07 0.14

0.34 0.17 0.16 0.13

0.38 0.74 0.22 0.24

0.26 0.51 0.19 0.19

0.24 0.38 0.11 0.24

0.12 0.13 0.16 0.09

0.28 1.32 0.19 0.12

0.22 0.27 0.15 0.21

0.12 0.13 0.03 0.04

0.1 0.19 0.1 0.12

0.16 0.17 0.07 0.08

0.21 0.17 0.09 0.09

0.11 0.17 0.09 0.09

0.18 0.1 0.1 0.13

0.1 0.14 0.09 0.07

0.16 0.17 0.09 0.06

0.06 0.09 0.04 0.09

0.22 0.24 0.17 0.21

chiopterus dewulfi obsoletus scoticus

significant (P-values 6 0.021). Mean CS in the studied species ranged from 328 lm in C. obsoletus to 374 lm in C. scoticus. C. chiopterus and C. dewulfi presented intermediate CS averages with 335 lm and 350 lm, respectively. CS values of C. scoticus being larger than the others (Fig. 3). All other pairwise differences were not significant (P > 0.057), meaning than size is comparable within C. obsoletus, C. chiopterus and C. dewulfi or that statistical power was too low in our data to detect the differences. We tested for a difference in size between both populations of C. obsoletus (ten individuals from the Gard department in Southern France vs. 48 individuals from the Aisne department in Northern France). The comparison of CS means was not significant (t = 1.035; df = 56; P = 0.305).

350 320

330

340

CS mean

360

370

380

3.4.2. Shape analyses The correlation between Procrustes and Euclidian distances appeared almost perfect (r = 1; slope = 0.9998; MS error = 0.000001) and validated the analysis of shape variables using multivariate statistical techniques. The MANCOVAs considering CS values and species revealed that the pattern of allometry can be considered similar between species as the model with interactions between size and species did not seem really better than the model with a common slope for all species (kwilks = 0.54; F = 1.30; df = 48, 275; P = 0.10). However, this analysis suggested a trend toward divergent slopes of wing shape on CS values. Solid arguments established that size and species are significant factors. The comparison of the multivariate regression of shape variation on size and the MANCOVA between shape and independent variables

size and species testified to the strong effect of species (kwilks = 0.10; F = 6.78; df = 48, 283; P . Jirakanjanakit, N., Leemingsawat, S., Thongrungkiat, S., Apiwathnasorn, C., Singhaniyom, S., Bellec, C., Dujardin, J.P., 2007. Influence of larval density or food variation on the geometry of the wing of Aedes (Stegomyia) aegypti. Trop. Med. Int. Health 12 (11), 1354–1360. Kiehl, E., Walldorf, V., Klimpel, S., Quraishy, S.A., Mehlhorn, H., 2009. The European vectors of Bluetongue virus: are there species complexes, single species or races in Culicoides obsoletus and C. pulicaris detectable by sequencing ITS-1, ITS-2 and 18S-rDNA? Parasitol. Res. 105, 331–336. Klingenberg, C.P., 2011. MorphoJ: an integrated software package for geometric morphometrics. Mol. Ecol. Res. 11, 353–357. Kremer, M., Rebholtz, C., 1977. Systematics of the obsoletus group of Culicoides (subgenus Avaritia) in the Palaearctic Region, with remarks on some types. Mosq. News 37, 278. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163. Lane, R.P., 1981a. Seasonal variation in size of some British Culicoides (Diptera: Ceratopogonidae). Entomol. Mon. Mag. 117, 235–240. Lane, R.P., 1981b. Allometry of size in three species of biting midges (Diptera: Ceratopogonidae, Culicoides). J. Nat. Hist. 15, 775–788. Mathieu, B., Cêtre-Sossah, C., Garros, C., Chavernac, D., Balenghien, T., Carpenter, S., Setier-Rio, Vignes-Lebbe, R., Ung, V., Candolfi, E., Delécolle, J.C., 2012. Development and validation of IIKC: an interactive identification key for Culicoides (Diptera: Ceratopogonidae) females from the Western Palaearctic region. Parasite Vect. 5, 137. http://dx.doi.org/10.1186/1756-3305-5-137. Meiswinkel, R., Gomulski, L.M., Delécolle, J.C., Goffredo, M., Gasperi, G., 2004. The taxonomy of Culicoides vector complexes - unfinished business. Vet. Ital. 40, 151–159. Muñoz-Muñoz, F., Talavera, S., Pagès, N., 2011. Geometric morphometrics of the wing in the subgenus Culicoides (Diptera: Ceratopogonidae): from practical implications to evolutionary interpretations. J. Med. Entomol. 48 (2), 129–139. Nielsen, S.A., Kristensen, M., 2011. Morphological and molecular identification of species of the Obsoletus group (Diptera: Ceratopogonidae) in Scandinavia. Parasitol. Res. 109 (4), 1133–1141. Nolan, D.V., Carpenter, S., Barber, J., Mellor, P.S., Dallas, J.F., Mordue Luntz, A.J., Piertney, S.B., 2007. Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe. Vet. Microbiol. 124, 82–94. Pagès, N., Sarto i Monteys, V., 2005. Differentiation of Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae) based on mitochondrial cytochrome oxidase subunit I. J. Med. Entomol. 42, 1026–1034. Pagès, N., Muñoz-Muñoz, F., Talavera, S., Sarto, V., Lorca, C., Núñez, J.I., 2009. Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions. Vet. Parasitol. 165, 298–310. Perrin, A., Cetre-Sossah, C., Mathieu, B., Baldet, T., Delecolle, J.C., Albina, E., 2006. Phylogenetic analysis of Culicoides species from France based on nuclear ITS1rDNA sequences. Med. Vet. Entomol. 20, 219–228. Rasmussen, L.D., Kristensen, B., Kirkeby, C., Rasmussen, T.B., Belsham, G.J., Bødker, R., Bøtner, A., 2012. Culicoids as vectors of Schmallenberg virus. Emerg. Infect. Dis. 18 (7), 1204–1206. http://dx.doi.org/10.3201/eid1807.120385. Rawlings, P., 1996. A key, based on wing patterns of biting midges (Genus Culicoides Latreille Diptera: Ceratopogonidae) in the Iberian Peninsula, for use epidemiological studies. Graellsia 52, 57–71. Rohlf, F. J. 2003. tpsSmall, version 1.20. State University, New York. Rohlf, F.J., 2006. A comment on phylogenetic correction. Evolution 60, 1509–1515. Rohlf, F. J. 2007. tpsRegr, version 1.35. State University, New York. Rohlf, F.J., Slice, D., 1990. Extensions of the Procrustes method for the optimal superimposition of landmarks. Syst. Zool. 39, 40–59. Saitou, N., Nei, M., 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Schwenkenbecher, J.M., Mordue (Luntz), A.J., Piertney, S.B., 2009. Phylogenetic analysis indicates that Culicoides dewulfi should not be considered part of the Culicoides obsoletus complex. Bull. Entomol. Res. 99, 371–375. Scientific Report of EFSA, 2012. ‘‘Schmallenberg’’ virus: analysis of the epidemiological data and assessment of impact. EFSA J. 10 (6), 2768. Smith, H., Mullens, R.A., 2003. Seasonal activity, size, and parity of Culicoides occidentalis (Diptera: Ceratopogonidae) in a coastal Southern California salt marsh. J. Med. Entomol. 40, 352–355. Spjotvoll, E., Stoline, M.R., 1973. Extension of t-method of multiple comparison to include cases with unequal sample sizes. J. Am. Stat. Ass. 68, 975–978. Thiry, E., Saegerman, C., Guyot, H., Kirten, P., Losson, B., Rollin, F., Bodmer, M., Czaplicki, G., Toussaint, J.F., De Clercq, K., Dochy, J.M., Dufey, J., Gilleman, J.L., Messeman, K., 2006. Bluetongue in Northern Europe. Vet. Rec. 159 (10), 327. Venail, R., Balenghien, T., Guis, H., Tran, A., Baldet, T., Setier-Rio, M.L., Delécolle, J.C., Mathieu, B., Martinez, D., Languille, J., Garros, C., 2012. Assessing diversity and abundance of vector populations at a national scale: example of Culicoides surveillance in France after bluetongue virus emergence. In Parasitol. Res. Monograph. Volume 3. Edited by Mehlhorn H: Springer-Verlag Berlin Heidelberg 77–102 [Mehlhorn H (Series Editor): Arthropods as Vectors of Emerging Diseases].

L.H. Henni et al. / Infection, Genetics and Evolution 21 (2014) 110–117 Villegas, J., Feliciangeli, M.D., Dujardin, J.P., 2002. Wing shape divergence between Rhodnius prolixus from Cojedes (Venezuela) and R. robustus from Me´rida (Venezuela). Infect. Genet. Evol. 2, 121–128. Von Cramon-Taubadel, N., Frazier, B.C., Lahr, M.M., 2007. The problem of assessing landmark error in geometric morphometrics: theory, methods, and modifications. Am. J. Phys. Anthropol. 134 (1), 24–35.

117

Wirth, W.W., Dyce, A.L., Peterson, B.V., 1985. An atlas of wing photographs, with a summary of the numerical characters of the neartic species of Culicoides (Diptera: Ceratopogonidae). Contrib. Amer. Ent. Inst. 22, 1–46. Wirth, W.W., Dyce, A.L., Spinelli, G.R., 1988. An atlas of wing photographs, with a summary of the numerical characters of the neotropical species of Culicoides (Diptera: Ceratopogonidae). Contrib. Amer. Ent. Inst. 25, 2–72.