• Biochemical markers;
  • biting midges;
  • bluetongue;
  • Obsoletus complex;
  • population genetics;
  • seasonal abundance;
  • temporal dynamics


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Culicoides species belonging to the Obsoletus complex (Diptera: Ceratopogonidae) have been indicated as primary bluetongue (BT) vectors in many European countries and their possible involvement in the maintenance and overwintering of BT viruses has been suggested, even in regions where Culicoides imicola Keiffer is the main vector. The Obsoletus complex includes two predominant taxa, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle. However, the role played by each species in the epidemiology of BT is still unknown. Taxonomic identification is mainly based on the morphology of male genitalia and the lack of other reliable diagnostic features makes the screening of trap-collected vector populations, mainly females, particularly difficult. Although molecular markers have facilitated species identification, little information is yet available on the biology, abundance and population dynamics of the two taxa. The aim of this work was to investigate the genetic profile and temporal distribution of C. obsoletus and C. scoticus by using isozyme electrophoresis applied to adult midges, collected weekly at two selected farms in southern Sardinia. A total of nine enzyme loci were analysed and five of them provided diagnostic allozyme markers (Hk, Mdh, Pgi, Idh-1 and Idh-2). Nei's genetic distance between the two taxa was in the range of other well-separated taxa (D = 1.792), supporting their status as true species. Culicoides scoticus represented almost 61% of the 562 specimens analysed; its genetic structure was characterized by a very low level of intra-population variation (mean heterozygosity He = 0.019) and higher genetic divergence between populations (FST = 0.0016) than in C. obsoletus. The latter species had significantly more heterozygotes (He = 0.123), a higher percentage of polymorphic loci, and no inter-population differentiation (FST0). We suggest that different biological and ecological constraints, such as breeding habitat requirements, may contribute to shaping the genetic profiles of C. scoticus and C. obsoletus. However, enough gene flow was maintained between populations of each species as no spatial and temporal structuring was sustained by Fisher's exact probability test (P > 0.5). The seasonal distributions of C. scoticus and C. obsoletus only partially overlapped: both species were mainly found early in the year, when the main vector, C. imicola, was present in low numbers, and peaked in abundance in April and May. Culicoides scoticus was predominant until May, decreased rapidly in the following months and increased again in winter, whereas C. obsoletus decreased more slowly and was still present in early summer. Consequently, C. scoticus may be a good candidate for playing a role in the transmission and maintenance of BT virus in Sardinia, as well as in other Mediterranean countries, during the months of late winter and early spring when the seroconversion of sentinel animals is still occurring in the absence of the main vector.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In the last decade European countries have experienced recurrent outbreaks of bluetongue (BT), an arboviral disease which affects ruminants and can cause severe epidemics, particularly in some breeds of sheep (Mellor & Wittmann, 2002; Saegerman et al., 2008). Because of its economic impact, which also results from limitations to the international trade in animals, BT was included in the former List A of epizootic diseases by the Office International des Épizooties (OIE).

The main vector of BT in the Mediterranean basin is Culicoides imicola Keiffer (Diptera: Ceratopogonidae), but other Culicoides species, mainly belonging to the Obsoletus and Pulicaris complexes, have been indicated as effective and important vectors in regions and countries where C. imicola is present at low frequencies or even absent, such as Cyprus, Bulgaria, Greece and central and southern Italy (Mellor & Pitzolis, 1979; Mellor & Wittmann, 2002; Caracappa et al., 2003; Torina et al., 2004; De Liberato et al., 2005; Savini et al., 2005). More recently, BT has spread to central and northern Europe, outside the present geographical range of C. imicola, confirming the importance of these indigenous Culicoides species as main BT vectors (Saegerman et al., 2008; Wilson & Mellor, 2009). During the 2006 outbreak in Germany, the Obsoletus complex, which accounted for 90% of the midges analysed, was, in fact, the only one found positive for BT virus (BTV) (serotype 8) (Mehlhorn et al., 2007).

The Obsoletus complex includes at least three species, Culicoides obsoletus (Meigen), Culicoides scoticus Downes & Kettle and Culicoides montanus Shakirjanova. An unidentified species A, present in mainland Italy, has also been identified based on DNA sequence analysis (Gomulski et al., 2005). However, the taxonomic and phylogenetic relationships within the complex are still debated (Meiswinkel et al., 2004; Gomulski et al., 2005; Mathieu et al., 2007; Nolan et al., 2007; Kiehl et al., 2009; Schwenkenbecher et al., 2009). Morphological identification is, indeed, limited by the availability of clearly distinctive features. Attempts have been made to find reliable diagnostic characters in females (Pagès & Sarto I Monteys, 2005), but, so far, identification is still accomplished mainly through the analysis of male genitalia, thus making field studies on trap-collected Culicoides (mainly females) particularly difficult.

Species-specific molecular probes of the mitochondrial cytochrome oxidase subunit I (COI) and the rRNA genes internal transcribed spacers (ITS) have been developed for unequivocal identification of each species in the complex (Pagès & Sarto I Monteys, 2005; Gomulski et al., 2005; Mathieu et al., 2007; Nolan et al., 2007) and were recently applied to study species abundance in Germany (Balczun et al., 2009).

However, despite the increasing importance of members of the Obsoletus complex as primary BT vectors in several European countries, information on the biology and population dynamics of each species within the complex is still scanty and their roles in the epidemiology of BT are yet to be clarified. In fact, most of the papers published so far have dealt with the complex as a whole because of the difficulty in the morphological identification of individual specimens.

In Sardinia, BT was first reported in August 2000, since then various BTV serotypes (1, 2, 4, 16 and 8) have circulated, causing recurrent outbreaks (Office International des Épizooties, 2009). Culicoides imicola is the main BT vector on the island (Goffredo et al., 2003), whereas species of the Obsoletus complex are widespread, but less abundant (Goffredo et al., 2004; Pili et al., 2006). Two species of the complex, C. obsoletus and C. scoticus, were identified based on the analysis of male genitalia (Pili et al., 2006), but their possible involvement in the transmission and maintenance of BTV when C. imicola is less abundant or even absent is still unknown.

The aim of this work was to investigate the genetic profiles of C. obsoletus and C. scoticus by using isozyme electrophoresis applied to adult midges collected in two farms in southern Sardinia. A number of wild-caught Culicoides males were identified to species level using morphological and molecular markers, and were used to search for diagnostic allozyme markers at eight loci. Large samples of females collected during 2004 were then electrophoretically tested to gain insight into the population structure of each species, its relative abundance and temporal and spatial distributions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Sample collection and species identification

Insect collections were made using Pirbright type light traps in two selected farms in southern Sardinia: Muravera (MV) (39°20′10.8′′ N, 09°33′53.6′′ E) on the east coast, and Villaperuccio (VP) (39°06′56.8′′ N, 08°40′52.1′′ E), situated 12 km inland from the west coast. Traps were located 1.5 m above the ground, close to sheep shelters, and operated from 1 h before sunset to 1 h after sunrise. Insects were collected in plastic cups containing water with a few drops of surfactant or in collecting nets. Weekly captures were made from January 2003 to December 2004. The maximum trap catch per month was calculated as a measure of species abundance (Baylis et al., 1997).

Culicoides specimens were separated from other insects and midges belonging to the Obsoletus complex were identified based on the wing pattern (Boorman, 1989; Rowlings, 1996). Male genitalia were dissected and mounted on slides, as were some females according to Boorman & Rowland (1988). After identification, specimens were preserved in 70% alcohol or frozen at −80 °C, depending on the collecting method.

The number of monthly samples used for the allozyme electrophoresis ranged from one to 72, depending on the total number of trap-collected adults.

Allozyme electrophoresis

Seven enzyme systems were tested by polyacrylamide gel electrophoresis. About 600 frozen specimens were individually homogenized in 15 µL of 10% sucrose, 0.1% Triton X-100®, 0.2 M Tris citrate buffer. When both allozyme and DNA analyses were performed, individual midges were cut in two parts and half of the body was used for each analysis. The following enzyme systems were analysed: adenylate kinase (AK); glucosephosphate isomerase (GPI); glycerophosphate isomerase (GPD); hexokinase (HK); isocitrate dehydrogenase (IDH-1 and IDH-2); malate dehydrogenase (MDH), and phosphoglucomutase (PGM).

Staining protocols were carried out according to Pasteur et al. (1988). Allozymes were ranked in order of increasing mobility from the origin. Relative fronts (RF) were obtained using the most common C. imicola allelomorphs as reference. Zimograms were interpreted according to the known quaternary structure of proteins. Genetic and statistical analyses were performed using biosys-1 (Swofford & Selander, 1981) and genepop 1.2 (Raymond & Rousset, 1995) software. Hardy–Weinberg equilibrium was tested for each locus in all populations using Levene's correction for small samples (Levene, 1949). Overall significance was obtained by Fisher's combined probabilities test (Fisher, 1970). The genetic differentiation of populations was analysed using F statistics (Weir & Cocherham, 1984) and an unbiased estimate of Fisher's exact test on R × C contingency tables was performed employing the Markov chain method of Guo & Thompson (1992). Unbiased genetic similarity (I) and distance (D) measures were obtained using Nei's algorithms (Nei, 1972, 1978).

Diagnostic DNA assays

For species cross-identification, DNA was extracted from the head and thorax of single individuals, whereas the abdomen was used for allozyme analysis. Tissues were rinsed in STE (50 mm NaCl, 10 mm Tris pH 8.0, 1 mm EDTA) (Sebastiani et al., 2001), and then homogenized in 30 µL of fresh STE for DNA extraction. Polymerase chain reaction (PCR) was performed according to Gomulski et al. (2005), using species-specific nuclear ITS2 primers.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In the course of the 2-year survey, a total of 6223 specimens were classified as belonging to the Obsoletus complex, corresponding to 5.1% of all Culicoides midges (121 990) collected in 2003 and 2004; this is a low percentage compared with the >46.5% of the catch accounted for by the main BT vector, C. imicola. However, if we consider only collections from January to May, this percentage increases to 28.3%, whereas that for C. imicola decreases to only 8.3% of the total catch. In both years, major peaks in abundance of the Obsoletus complex, measured as maximum catch per month, were recorded in April and May; the frequency of these midges decreased consistently over the summer and increased again during the last months of the year.

Differences in monthly catches were observed between sites and years: collections were more abundant in the eastern coastal locality of Muravera (MV), with peaks in the range of thousands (Fig. 1), than in the western inland site (VP), where the number of adult midges never exceeded a few hundred (Fig. 2). Higher numbers of midges were trapped during 2004 than in the previous year, coinciding with the end of a long period of drought (Servizio Agrometereologico della Sardegna, 2004).


Figure 1. Maximum monthly catches of Culicoides obsoletus s.l. (inline image) and Culicoides imicola (inline image) in Muravera (logarithmic scale).

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Figure 2. Maximum monthly catches of Culicoides obsoletus s.l. (inline image) and Culicoides imicola (inline image) in Villaperuccio (logarithmic scale).

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Culicoides obsoletus and C. scoticus were the only species of the Obsoletus complex identified by characteristic features of the genitalia in 47 trap-collected males. Species status was confirmed by PCR amplification of ITS2 species-specific sequences. Morphological and molecular cross-identification was the basis for searching diagnostic allozyme markers by gel electrophoresis using the same specimens.

Eight loci were identified by seven enzyme systems and diagnostic alleles were found at four of them: Hk, Idh-1, Idh-2 and Mdh. The Hk locus was monomorphic for alternative alleles in the two species, whereas Idh-1, Idh-2 and Mdh were polymorphic and the frequency of the most common allele was >0.78 (Table 1). More than 98% of individuals within each species were also homozygous for alternative alleles at locus Pgi (Table 1). Pgm was polymorphic but less informative than the other loci because allele frequencies did not differ significantly between populations of C. scoticus from VP and C. obsoletus from VP and MV (Fisher's test, P > 0.07). Nevertheless, overall genotype frequencies, analysed using Fisher's exact probability test, were significantly different between taxa at all loci, including Pgm (P = 0.0019). The other two loci, Ak and Gpd, were monomorphic for the same allele in both species.

Table 1.  Allelic frequencies in Culicoides scoticus and Culicoides obsoletus populations in Muravera and Villaperuccio.
LocusAllele C. scoticus C. obsoletus
  1. Sample sizes are shown in parentheses.

  2. MV, Muravera; VP, Villaperuccio.

Hk  (200)(127)(120)(113)
  100 0.0000.0001.0001.000
  95 1.0001.0000.0000.000
Idh-1  (220)(117)(89)(87)
  138 0.0000.0000.0060.006
  111 0.0000.0000.8990.856
  106 0.0050.0040.0000.000
  97 0.0000.0000.0960.138
  94 0.9860.9910.0000.000
  80 0.0090.0040.0000.000
Idh-2  (232)(119)(123)(109)
  176 0.0000.0000.0000.005
  137 0.0000.0000.7820.794
  125 0.0000.0080.0000.000
  118 0.0000.0000.2100.188
  109 0.0000.0000.0080.014
  107 0.0040.0000.0000.000
  103 0.9850.9880.0000.000
  70 0.0110.0040.0000.000
Mdh  (174)(108)(112)(98)
  119 0.0000.0000.0050.000
  88 0.0000.0000.0320.005
  78 0.0030.0000.0000.000
  64 0.0000.0000.9630.995
  57 0.9971.0000.0000.000
Pgi  (239)(128)(123)(111)
  183 0.0020.0000.0000.000
  146 0.9920.9960.0080.005
  100 0.0060.0040.9920.991
  56 0.0000.0000.0000.005
Pgm  (177)(93)(120)(96)
  97 0.0110.0330.0540.073
  84 0.9860.9620.9460.927
  72 0.0030.0040.0000.000

Single females, collected in 2004 and morphologically identified as belonging to the Obsoletus complex, were tested at the eight loci. Between 87 and 239 individuals were analysed per locus, and a total of 562 individuals were included in the final analysis. Based on the electrophoretic pattern, 342 specimens were identified as C. scoticus and 220 as C. obsoletus, indicating the prevalence of the first species in the study area. Ak and Gpd were non-informative and were excluded from the following analysis.

The genetic structure of C. scoticus was characterized by a very low level of variation, with mean expected heterozygosity (He) ranging from 0.018 to 0.020 in the two populations of MV and VP (Table 2); most individuals were homozygous at all loci. More variability was observed in C. obsoletus populations, expressed by a higher frequency of heterozygotes (He0.12) and percentage of polymorphic loci (50%).

Table 2.  Genetic structure of Culicoides scoticus and Culicoides obsoletus populations in Muravera and Villaperuccio.
SpeciesPop.Specimens/locus, mean (SE)Alleles/locus, mean (SE)Polymorphic loci, %*Observed heterozygosity, mean (SE)Expected heterozygosity, mean (SE)
  1. *Both 0.95 and 0.99 criterions for polymorphic loci are indicated (a locus is considered polymorphic if the frequency of the most common allele does not exceed 0.95 or 0.99).

  2. SE, standard error; MV, Muravera; VP, Villaperuccio.

C. scoticus MV203 (10.8)2.5 (0.3)0/500.016 (0.005)0.018 (0.005)
 VP120.3 (2.2)2.2 (0.4)0/330.018 (0.009)0.020 (0.011)
C. obsoletus MV109.3 (6.2)2.3 (0.3)50/670.086 (0.036)0.121 (0.053)
 VP102.3 (4.2)2.5 (0.4)50/500.111 (0.050)0.125 (0.058)

Deviation from Hardy–Weinberg equilibrium was assessed in the MV populations of both species because of an excess of homozygotes mainly at the Idh loci (P < 0.02). However, when all loci where considered, Hardy–Weinberg equilibrium was rejected only for C. obsoletus from MV (P = 0.0004), whereas VP populations of both species were in equilibrium at all loci (P > 0.6). Heterozygote deficit contributed to positive FIS values obtained at most loci in both species. Overall FIS across all loci was 0.12 for C. scoticus and 0.20 for C. obsoletus, indicating deviation from panmixia and the presence of inbreeding. Genetic differentiation within each species was not significant (P > 0.5) and corresponded to FST values of 0.0016 in C. scoticus and −0.0015 in C. obsoletus. The negative FST obtained in the latter species reflects only the computation method employed and should be interpreted as a zero value (i.e. a complete lack of differentiation between populations). In both species, genotypic frequencies did not change significantly (P > 0.06) during the year (January–December 2004, excluding months in which not enough data were available).

Divergence between the two taxa, estimated using Fisher's exact probability test, was significant for all loci in all pairwise comparisons (overall P across loci <0.000001), including Pgm (for this locus P≤ 0.01). Average Nei's genetic similarity (I) and distance (D) between the taxa were 0.167 and 1.792, respectively. If the two monomorphic loci Ak and Gpd were included in the analysis, the similarity index increased to 0.387.

Using the electrophoretic identification of C. scoticus and C. obsoletus in monthly collections of adult midges, seasonal differences in the relative abundance of each species were analysed (Table 3). Although both species were mainly collected in the winter–spring period, C. scoticus was prevalent in the samples during March–May and dropped to a few specimens in the following months, whereas C. obsoletus was always less frequent at both sites, except in June and July (Table 3).

Table 3.  Number of Culicoides scoticus and Culicoides obsoletus identified by diagnostic allozyme markers and total monthly catches of Obsoletus complex.
C. scoticus C. obsoletus Total catch C. scoticus C. obsoletus Total catch

If species ratios obtained from the analysed samples were related to the total number of Obsoletus complex midges collected during 2004, both species appeared to peak in abundance in the same period (April in MV and May in VP). Culicoides scoticus remained prevalent until May, rapidly disappeared in the following months and increased again in winter, whereas C. obsoletus decreased more slowly and was still present in June and July (Fig. 3).


Figure 3. Distribution of Culicoides scoticus (inline image) and Culicoides obsoletus (inline image) in total monthly catches in (A) Muravera and (B) Villaperuccio.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Species identification

Electrophoretic analysis of eight enzyme loci confirmed the presence in Sardinia of two species of the Obsoletus complex, C. scoticus and C. obsoletus, previously identified according to the morphology of male genitalia (Pili et al., 2006).

Diagnostic alleles at loci Hk, Mdh, Idh-1 and Idh-2 unambiguously identified the two species. More than 99% of the analysed midges were also homozygous for two alternative alleles at locus Pgi; the remaining midges were heterozygous for the same alleles. Genotype frequencies were significantly different at all loci (P = 0.0019), including the Pgm locus, at which alleles were shared by both taxa.

Species divergence

Overall genetic differentiation between C. scoticus and C. obsoletus was highly significant (Fisher's exact test, P < 0.000001). Divergence between the two species, expressed as Nei's coefficient of genetic distance, was high even if the monomorphic Ak and Gpd loci were included in the analysis (D = 0.950). Nei's genetic identity for all loci (I = 0.387) was close to the estimates obtained from the analysis of 21 isozyme loci in other well-separated species of Culicoides from North America [I = 0.379 between Culicoides gigas Root & Hoffman and Culicoides variipennis (Coquillet)] and much lower than among members of the Culicoides variipennis complex (I in the range of 0.739–0.814) (Tabachnick, 1992). This evidence strongly supports the claim that these two taxa warrant true species status, which was recently questioned on the basis of rDNA sequences analysis (Kiehl et al., 2009).

Genetic profile

The genetic structure of C. scoticus, estimated by excluding the monomorphic Ak and Gpd loci, was characterized by a very low level of variation (He = 0.019 ± 0.007), with most individuals being homozygous at all loci. Culicoides obsoletus showed higher variability with significantly more heterozygotes (He = 0.123 ± 0.054) and polymorphic loci, as found in other Culicoides species (Tabachnick, 1992).

Genetic divergence between populations of C. scoticus (FST = 0.0016) was higher than in C. obsoletus (FST = −0.0015). However, differentiation was not significant in either species, indicating that enough gene flow is maintained, probably through the passive transport of midges via wind currents, which is considered an important route of BT spread in Europe (Wittmann & Baylis, 2000; Alba et al., 2004).

Why does C. scoticus display less genetic intra-population variation and higher heterogeneity between populations than C. obsoletus? As the genetic structure of a species is an expression of its biology and ecology, differences in life history and breeding habitat requirements may explain the distinct genetic profiles of the two species. Data from this study indicate that C. scoticus is most abundant in late winter to early spring, whereas C. obsoletus is prevalent in early summer. During the spring, ephemeral breeding habitats are more frequent in Sardinia than permanent ones, coinciding with the autumn–winter rainy season. In late spring to early summer, precipitation is usually scanty and permanent habitats become predominant. Species breeding in temporary habitats are more affected by genetic drift that results from founder effects, as well as extinction and colonization events, thus favouring loss of heterozygosity and increasing population differentiation. By contrast, permanent breeding sites allow for the maintenance of gene flow and heterozygosity while reducing divergence between populations. Differences in the habitat preferences of the two species are yet to be established. Preliminary data from this laboratory indicate that breeding sites at least partially overlap because larvae of the two species have been identified in the same breeding sites using molecular markers (E. Pili, unpublished data, 2009). Alternatively, the prevalence of these species in ephemeral or permanent breeding sites may not reflect a matter of choice, but, rather, one of the availability of suitable grounds in those months in which each species is most abundant. In other words, climatic constraints may contribute to shaping the genetic structure of these species, as has been shown in species of the Variipennis complex (Tabachnick et al., 1996). A thorough study of the composition of larval populations in different breeding habitats will be necessary to clarify such issues.

Distribution and abundance

Culicoides species of the Obsoletus complex have gained considerable attention as efficient vectors: firstly, because they were implicated in the spread of BT in northern and central Europe (Mehlhorn et al., 2007; Saegerman et al., 2008; Wilson & Mellor, 2009), and, secondly, for their possible role in the maintenance and overwintering of BT viruses (Calvete et al., 2008; Wilson et al., 2008).

In Sardinia, as well as in most Mediterranean regions, C. imicola is the main vector of BT (Goffredo et al., 2003). Mainly present from late summer to early winter, this species was found at very low frequencies, or was even absent, from January until March in the 2003–2004 survey. During these months, C. imicola was always less abundant than the Obsoletus complex in both study areas, particularly in the first quarter of 2003. An increasing number of adults were captured from September 2003 onward, coinciding with rainfall frequency 20% above the mean and the end of a decade characterized by strong drought (Servizio Agrometereologico della Sardegna, 2004). Thus, in the rainy spring of 2004, because of the availability of more breeding sites, C. imicola was still present at the beginning of the year, but numbers of C. obsoletus s.l. were as much as 10 times higher than numbers of the main vector. Prevalences of the Obsoletus complex during this part of the year have been reported in other Mediterranean regions, such as Spain and the Balearic Islands (Ortega et al., 1999; Miranda et al., 2003), and a possible involvement in the maintenance of BTV has been proposed (Sarto I Monteys & Saiz-Ardanaz, 2003). Cases of sentinel animal seroconversion or BTV infections were reported in Sardinia between January and April (Calistri et al., 2004; Sulis et al., 2004; Istituto Zooprofilattico Sperimetale dell’Abruzzo e del Molise, 2007), when the main vector was practically absent and the Obsoletus complex represented the second most abundant vector species. If these data suggest a possible involvement of the Obsoletus complex in the maintenance of BTV, the genetic characterization of trap-collected Culicoides points to a major role for C. scoticus, which was more abundant than C. obsoletus in both MV and VP collections, as it was in Corsica (Baldet et al., 2004) and mainland Italy (Gomulski et al., 2005), particularly in the first 3–4 months of the year. For this reason, C. scoticus could be a good candidate for the transmission and maintenance of BTV in Sardinia in late winter to early spring when C. imicola is present at a lower frequency or even absent.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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