Parasitol Res (2014) 113:2435–2443 DOI 10.1007/s00436-014-3873-1
ORIGINAL PAPER
Biting midges monitoring (Diptera: Ceratopogonidae: Culicoides Latreille) in the governate of Monastir (Tunisia): species composition and molecular investigations D. Slama & E. Chaker & B. Mathieu & H. Babba & J. Depaquit & D. Augot
Received: 26 February 2014 / Accepted: 24 March 2014 / Published online: 14 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The results of entomological studies carried out in the governate of Monastir (Tunisia) in 2009–2010 (captures and emergences from muds) focusing on Culicoides species are presented in the present study. Identification of Culicoides at the species level is based on morphological characters, and a molecular study has been carried out based on mitochondrial DNA cytochrome C oxidase I gene (COI) and D1 and D2 domains of the 28S rDNA. The DNA sequences reported here are related to 10 species (on 25 known) of Culicoides described in Tunisia: Culicoides cataneii-gejgelensis, Culicoides circumscriptus, Culicoides imicola, Culicoides jumineri, Culicoides kingi, Culicoides langeroni, Culicoides newsteadi, Culicoides paolae, Culicoides puncticollis and Culicoides sahariensis. DNA sequencing of the COI gene and D1D2 domains discriminated all morphologically determined species. The choice of D1D2 domains considered as a conserved region is informative for Culicoides species identification. The molecular analyses of COI has grouped both C. circumscriptus, D. Slama : E. Chaker : H. Babba Faculty of Pharmacy, Clinical Biology Department, Laboratory of Medical and Molecular Parasitology-Mycology code LR12ES08, Monastir, Tunisia B. Mathieu Institute of Parasitology and Tropical Pathology, University of Strasbourg, Strasbourg, France D. Slama : J. Depaquit : D. Augot (*) ANSES, Université de Reims Champagne-Ardenne, SFR Cap santé, EA4688-USC «Transmission vectorielle et épidémiosurveillance de maladies parasitaires» (VECPAR), 51100 Reims, France e-mail:
[email protected] E. Chaker Laboratory of Parasitology and Mycology, TunisLa rabta, Tunisia D. Slama : H. Babba Laboratory of Maternity and Neonatology Center of Monastir, Monastir, Tunisia
C. puncticollis within two clusters and C. newsteadi within five subclusters. However, C. newsteadi shows relatively deep intraspecific divergence using COI sequences. Keywords Culicoides cataneii-gejgelensis . C. circumscriptus . C. imicola . C. jumineri . C. kingi . C. langeroni . C. newsteadi . C. paolae . C. puncticollis . C. sahariensis . mtDNA . rDNA . Monastir . Tunisia
Introduction Blue-tongue disease (BT) is an infectious arthropod-borne viral disease, which affects domestic ruminants. Several Culicoides species serve as Blue-tongue virus (BTV) vectors in different areas of the world (Mellor et al. 2000). Among these species, it is generally accepted that Culicoides imicola is the major BTV vector in southern Europe and the Mediterranean Basin. At least six BTV strains belonging to five serotypes (BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16) were continuously recorded in the Mediterranean Basin from 1998 to 2005. The route of introduction of different serotypes in Europe was divided in two ways: (i) in the East by Israel and Turkey and (ii) in the West by Maghreb’s region (Saegerman et al. 2008). In Tunisia, entomological studies report an increasing number of species during the last decades: 17 species including 10 new for Tunisia (Chaker and Kremer 1982); 22 species, with three new for this country: Culicoides paolae, C. imicola and Culicoides newsteadi (Chaker et al. 2005); and finally, 25 species of which 7 were identified for the first time: Culicoides punctatus, Culicoides fascipennis, Culicoides subfasciipennis, Culicoides santonicus, Culicoides submaritimus, Culicoides univittatus and Culicoides indistinctus (Sghaier et al. 2009). The same pattern is observed in Algeria (Djerbal and Délecolle 2009). In this context, Maghreb’s region seems to be a key region to anticipate
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new BTV phenomenon and a crossroads for Culicoides species movements. This paper describes the use of morphological and molecular techniques to identify Culicoides species in the governate of Monastir (Tunisia). Actually, three molecular markers were available for molecular identification: the cytochrome C oxidase I gene (Dallas et al. 2003) of mitochondrial DNA (mtDNA) and the internal transcribed spacer I (ITS1) (Perrin et al. 2006) and II (ITS2) (Gomulski et al. 2006). The sequences obtained with ITS1 are generally of low quality (Nielsen and Kristensen 2011). Polymerase chain reaction (PCR) products, with ITS2, seem to include many different sequences even from one individual sample (Matsumoto et al. 2009). Consequently, ITS1 and ITS2 are little employed for Culicoides identification. COI has emerged and served as “gold standard” for DNA barcode used in routine for Culicoides studies. But, a high intra or interspecific divergence was commonly observed with COI in European Culicoides populations (Pagès et al. 2009; Muñoz-Muñoz et al. 2011; Ander et al. 2012; Lassen et al. 2012). This paper proposes to evaluate the informativity of the D1 and D2 regions of the 28S ribosomal DNA (rDNA). These coding regions have never been employed for the taxonomy of Culicoides, but they appear to contain major phylogenetic information among Strepsiptera (Hwang et al. 1998), Anopheles (Raghavendra et al. 2009) or Phlebotomine sandflies (Depaquit et al. 1998).
Materials and methods Study sites and selection of specimens Biting midges were collected between 2009 and 2010 using light traps (home-made CDC miniature and OVI traps) and emergence in the laboratory from mud collected in different sites in the Center of Tunisia (Table 1). The head, wings and genitalia of individual biting midges were cut off within a drop of ethanol, cleared in boiling Marc-André solution, and mounted between slide and cover slide. The corresponding thorax for each specimen was stored in a vial at −20 °C before DNA extraction. Culicoides species were morphologically identified and separated using their wing patterns according to the key of Campbell and Pelham-Clinton (1960), Chaker and Kremer (1982) and Delécolle (1985).
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AAAGATATTGG-3′) and LepR (5′-TAAACTTCTGGATG TCCAAAAAATCA-3′) (Hajibabaei et al. 2006). The primers LepF and LepR were used under the following thermal profile (Costa et al. 2007): an initial denaturation step at 94 °C for 3 min; followed by 5 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 90 s and extension at 86 °C for 60 s; then 35 cycles of denaturation at 94 °C for 30 s, annealing at 51 °C for 90 s and extension at 86 °C for 60 s; and a final extension at 68 °C for 10 min by using Taq polymerase (5′, QIAGEN GmbH). Polymerase chain reactions (PCR) of D1/D2 domains were performed in a 50-μl volume using 5 μl of extracted DNA solution and 50 pmol of the primers C′1 (5′-ACCCGCTGAA TTTAAGCAT-3′) and D2 (5′-TCCGTGTTTCAAGACGGG3′) in the conditions indicated by Depaquit et al. (1998). Amplification conditions were as proposed by the latter 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). The goal of the present study is not to provide a phylogenetic study but to measure the intra and interspecific variability within our sampling for the two considered molecular markers. Direct sequencing of both DNA strands was performed using the primers used for PCR. The sequences were analysed using the Staden Package (Bonfield et al. 1995; Bonfield and Staden 1996). Their alignment was performed using the Bioedit software (Hall 1999). Distance analysis was performed using the neighbour-joining (NJ) method (Kimura 2-parameter=K2P) with MEGA software version 3.1 (Kumar et al. 2004). The bootstrap values of each node were calculated using 1,000 replicates to assess the robustness of the NJ method. Forcipomyia sp. was selected as an outgroup according to its phylogenetic position (Miller et al. 1997; Beckenbach and Borkent 2003). A total of 172 sequences of Culicoides COI are available in Genbank (Table 2), and the sequences from 42 individual midges including the outgroup obtained in this study, were analysed. Specimens sharing similar sequences are indicated in Table 2.
Results Morphological identification of Culicoides species
DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from Culicoides specimens using the QIAamp DNA Mini Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s instructions. A fragment of cytochrome C oxidase (COI) gene was amplified using the primers LepF (5′-ATTCAACCAATCAT
Ten species of the genus Culicoides were identified morphologically from a total of 8.920 captured and emerged (Table 3) Culicoides specimens: Culicoides cataneii-gejgelensis, Culicoides circumscriptus, C. imicola, Culicoides jumineri, Culicoides kingi, Culicoides langeroni, C. newsteadi, C. paolae, Culicoides puncticollis and Culicoides sahariensis.
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Table 1 Collection of Culicoides species used in this study Culicoides species
Collection locality
Geographic coordinate
Type of collect
Code of specimen
Sequences COI
C. cataneii-gejgelensis
Oued Khniss-kniss
35° 42′ 44″/10° 49′ 0
E
B2CM132
Yes
Yes
Oued Khniss-kniss
E
B2Cf111
Yes
Yesa
Oued Khniss-kniss
E
B2Cf34
No
Yesa
E
C. circumscriptus
a
Yesa
E
B2Cf11
Yes
Yesa
E
B2Cf43
No
Yesa
E
B28Cf3
Yes
Yesa
Oued Hamdoune
35° 46′ 48″/10° 49′ 59″
E
B28Cf2
Yes
Yesa
Elhelia
35° 42′ 44″/10° 49′ 0
E
B8Cf5
Yes
Yesa
E
B7Cf58
Yes
No
E
B7Cf58
No
Yesa
LT
S6CM9
Yesa
Yesa
LT
S3Cf5
Yesa
Yesa
S4Cf3
a
Yesa
Châaba-khniss
35° 46′ 48″/10° 49′ 59″
Khniss
LT
Khniss
Yes
a
LT
S3Cf8
Yes
Yes
LT
S6Cf111
No
Yesa Yes
Bir Zira Khniss
35° 42′ 44″/10° 49′ 0
LT
S3CM33
Yesa
Skanes
35° 45′ 02″/10° 33′ 99″
E
B2CM18
Yesa
Yesa
Châaba
35° 46′ 48″/10° 49′ 59″
LT
S3CM71
Yes
Yesa
LT
S3Cf124
Yesa
Yesa
a
Yesa
LT
C. kingi
Yes
35° 46′ 23″/10° 39′ 64″
Khniss
C. jumineri
B2Cf90
a
Oued Hamdoune
Bir Zira-kheniss C. imicola
D1D2
S3CM1
Yes
a
LT
S3Cf20
Yes
Yes
LT
S1Cf2
Yes
No
Bir Zira Khniss
35° 42′ 44″/10° 49′ 0
E
B2Cf12
Yes
Yes
Oued Frina
35° 42′ 44″/10° 49′ 0
LT
S3Cf84
Yes
No
LT
S3Cf167
Yes
Yes
C. langeroni
Bir Zira Khniss
35° 42′ 44″/10° 49′ 0
LT
S6Cf114
Yes
Yesa
LT
S6Cf115
Yes
Yesa
C. newsteadi
Khniss
35° 46′ 48″/10° 49′ 59″
LT
S3CM42
Yes
Yes
LT
S6Cf51
Yes
Yes
LT
S1Cf1
No
Yes
LT
S3Cf116
Yesa
Yesa
a
Yesa
a
Châaba-khniss C. paolae
Khniss Skanes
35° 46′ 48″/10° 49′ 59″ 35° 45′ 02″/10° 33′ 99″
LT
35° 46′ 48″/10° 49′ 59″
LT
S3Cf66
Yes
Yesa
Khniss
35° 46′ 48″/10° 49′ 59″
LT
S3Cf68
Yesa
Yesa
LT
S3Cf67
Yesa
Yesa
a
Yesa
a
Yesa
a
Yesa
a
LT Bir Zira-khniss
35° 46′ 48″/10° 49′ 59″
E
S3CM59 S7CM1 B7Cf60
Yes Yes Yes
E
B7Cf64
Yes
Yesa
E
B7CM49
Yesa
Yesa
E
B7Cf62
Yesa
Yesa
E
B7Cf61
a
Yes
Yesa
35° 46′ 48″/10° 49′ 59″
E
B2Cf37
Yes
Yesa
Oued Hamdoune
35° 46′ 23″/10° 39′ 64″
E
B28Cf6
Yes
Yesa
Oued khniss-khniss
35° 46′ 48″/10° 49′ 59″
E
B2Cf36
Yes
Yesa
Oued Frina-kheniss
35° 46′ 48″/10° 49′ 59″
E
B1Cf1
No
Yesa
E
B1CM9
Yes
Yesa
Bir Zira-kheniss
C. sahariensis
Yes
Khniss
LT C. puncticollis
S5CM1
Oued khniss-khniss
35° 46′ 48″/10° 49′ 59″
E emerging, LT light traps a
Specimens having similar sequences; for COI: C. puncticollis (B7Cf61=B7Cf62=B7Cf64); (B7Cf60=B7CM49); for D1D2: C. cataneii-gejgelensis (B2Cf43=B2Cf111); (B2Cf90=B2Cf34=B2Cf11); C. imicola (S6Cf111=S6CM9=S3Cf5=S4Cf3); C. jumineri (S3CM1=S3Cf124); (B2CM18= S3CM71)
2438 Table 2 Accession numbers of the COI sequences from Genbank used in the molecular analyses
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Species
Country
Access numbers use in the first analysis C. circumscriptus Spain Switzerland Sweden C. imicola Algeria Greece Israel Italy Portugal South Africa
C. newsteadi
C. puncticollis
Number
HM241850-HM241855 HQ824460-HQ824465 JQ620049-JQ620054 EU189055-EU189057 AJ549388-AJ549392 AF078097-AF078100; AJ867223; AJ549393-AJ549414; AF080531-AF080535 AJ867224; AJ867225; AJ867232-AJ867234 AF079975-AF079979; AJ49415-AJ49426; AJ867229-AJ867231 AF069249; AF069231-AF069233
Spain
DQ868882-DQ868894; AF080536-AF080540; AJ867227; AJ867228; AF080527-AF080530; AF083044; DQ871030
Denmark Italy Spain Sweden Denmark
JF766320; JF766324 AM236738- AM236746 GQ338915-GQ338922 JQ620112-JQ620126; JF766328-JF766332 JQ683314-JQ683333
Access numbers use in the final analysis C. circumscriptus Spain Switzerland Sweden C. imicola Algeria Greece Israel Italy Portugal South Africa
HM241850-HM241853 HQ824460; HQ824461; HQ824463 JQ620049; JQ620050; JQ620051; JQ620053 EU189055-EU189057 AJ549388 AF078097; AF078098; AF078100; AJ867223; AJ549413; AF080533 AJ867224; AJ867232-AJ867234 AF079976; AF079977; AF079978; AF079979; AJ549415; AJ549417; AJ867230; AJ867231 AF069231
Spain
DQ868882; DQ868883; DQ868884; DQ868886; DQ868888; DQ868889; DQ868891; DQ868892; AF080540; AF080527; AF080528; AF080529; AF083044; AF080536; AF080537; AF080538; AF080539
C. newsteadi
Denmark Italy Spain Sweden
C. punticollis
Denmark
JF766320; JF766324 AM236738; AM236746 GQ338916-GQ338921 JQ620112; JQ620117; JQ620119; JQ620120; JQ620125; JQ620126 JQ683314
In our collection seven subgenera are represented: Avaritia, Beltranmyia, Culicoides, Monoculicoides, Oecacta, Remmia, Synhelea, and others one are incertae sedis (C. langeroni and C. paolae).
NJ analysis Sequences obtained are available in Genbank under the following accession numbers: C. cataneii-gejgelensis (KJ729965,
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Table 3 Total number of the Culicoides species trapped in Tunisia Subgenera
Avaritia Beltranmyia Culicoides Monoculicoides Oecacta Synhelea Remmia Miscellaneous Total
Culicoides species
Number
C. imicola C. circumscriptus C. newsteadi C. puncticollis C. jumineri C. cataneii/C. gejgelensis C. sahariensis C. kingi C. paolae C. langeroni 10
Light trap
Emergence
3670 27 151 1 441 6 – 22 785 3 5106
– 2802 – 211 54 235 492 17 – 3 3814
KJ729966, KJ729967, KJ729968), C. circumscriptus (KJ729969, KJ729970, KJ729971, KJ729972), C. imicola (KJ729973, KJ729974, KJ729975, KJ729976), C. jumineri (KJ729977, KJ729978, KJ729979, KJ729980, KJ729981, KJ729988), C. kingi (KJ729983, KJ729984, KJ29985), C.
langeroni (KJ729986, KJ729987), C. newsteadi (KJ729989, KJ729990), C. paolae (KJ729991, KJ729992, KJ729993, KJ729994, KJ729995, KJ729996, KJ729997), C. puncticollis (KJ729998, KJ729999, KJ730000, KJ730001, KJ730002), C. sahariensis (KJ730003, KJ730004, KJ730005, KJ730006) for COI and C. cataneii-gejgelensis (KF286388, KF286389, K J 7 3 0 0 0 8 , K J 7 3 0 0 0 9 , K J 7 3 0 0 1 0 , K J 7 3 0 0 11 ) , C. circumscriptus (KF286353, KJ30013), C. imicola (KF286345, KJ730014, KJ730015, KJ730016, KJ730017, KJ730018), C. jumineri (KF286383, KF286384, KJ730012, KJ730019, KJ730020), C. kingi (KF286338, KJ730021), C. langeroni (KJ730022), C. newsteadi (KF286356, KF286357, KJ730023), C. paolae (KF286385, KF286386), C. puncticollis (KF286369, KJ730024), C. sahariensis (KF286387) for 28S. Molecular comparisons after alignment were based on 407 bp for COI and 630 bp for 28S (including gaps). COI polymerase chain reaction amplification and sequences of Culicoides The interspecific K2P values for different species ranged from 0.155 (between Culicoides jumineri and Culicoides kingi) to 0.266 (between Culicoides newsteadi and C. paolae).
Table 4 Estimation of pairwise distance of the Culicoides species trapped in Tunisia for the COI domain of the mtDNA and D1D2 region of the rDNA and respectively (sequences including Forcipomyia sp. as outgroup)
COI sequences 1-C. cataneii-gejgelensis 2-C. circumscriptus 3-C. imicola 4-C. jumineri 5-C. kingi 6-C. langeroni 7-C. newsteadi 8-C. paolae 9-C. sahariensis 10-Forcipomyia sp. 11-C. puncticollis D1-D2 sequences 1-C. cataneii-gejgelensis 2-C. circumscriptus 3-C. imicola 4-C. jumineri 5-C. kingi 6-C. langeroni 7-C. newsteadi 8-C. paolae 9-C. puncticollis 10-C. sahariensis 11-Forcipomyia sp.
1
2
3
4
5
6
7
8
9
10
0.194 0.224 0.161 0.190 0.212 0.210 0.263 0.186 0.215 0.193
0.198 0.194 0.176 0.210 0.229 0.234 0.231 0.223 0.173
0.186 0.163 0.210 0.234 0.209 0.238 0.200 0.198
0.162 0.210 0.199 0.262 0.166 0.211 0.194
0.205 0.205 0.246 0.200 0.205 0.192
0.216 0.281 0.219 0.231 0.201
0.275 0.202 0.217 0.243
0.249 0.233 0.233
0.213 0.203
0.223
0.019 0.027 0.020
0.024 0.013
0.018
0.062 0.032 0.032 0.033 0.055 0.040 0.159
0.060 0.022 0.032 0.027 0.051 0.029 0.155
0.059 0.024 0.039 0.036 0.056 0.036 0.148
0.055 0.020 0.037 0.028 0.050 0.032 0.158
0.062 0.079 0.062 0.056 0.051 0.0154
0.037 0.029 0.049 0.035 0.155
0.038 0.057 0.037 0.149
0.045 0.031 0.142
0.042 0.148
0.144
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Fig. 1 Neighbour-joining tree based on Kimura two-parameter genetic distances on the COI mtDNA gene sequences of Culicoides
Molecular comparisons after alignment were based on 407 bp (including gaps). The pairwise distances of COI sequences are ranging between 71.9 and 83.9 % (Table 4). The NJ analysis showed strong bootstrap support (96– 99 %) for 14 lineages corresponded to different species or populations of species (Fig. 1).
Specimens morphologically identified as C. imicola are dispatched in two separate clusters: the main one includes specimens from South Africa, Algeria, Greece, Italy and Portugal and the other one(s) include specimens from Israel and Spain. C. puncticollis and C. circumscriptus each form two clusters. C. puncticollis are grouped with the sequences published from Denmark and Tunisia; whereas C. circumscriptus
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2441 S3Cf 20
68 61 83 19
S3CM1
C. jumineri
B2CM18
S3CM33
S3Cf124
B2Cf 43 39
C. cataneii-C. gejgelensis
B2Cf 90
100 81
B2CM132
60
C. circumscriptus
B7CF58
42 29
S3Cf 8 98
B18Cf20
C. langeroni
S6Cf 114
C. imicola
S6Cf 111 S3CM42
71
S1Cf 1
100
C. newsteadi
67 S6Cf 51
81
S3Cf 66
48
B1Cf 1
C. paolae
C. sahariensis
B7Cf 60
C. puncticollis
B2CF12 100 S3Cf 167
B7Cf64
C. kingi S6CM14
Forcipomyia sp. S3Cf167
0.02
Fig. 2 Neighbour-joining tree based based on Kimura two-parameter genetic distances on the D1D2 domain of rDNA gene sequences of Culicoides
with the sequences from Switzerland, Spain, Sweden and Tunisia. Specimens morphologically identified as C. newsteadi are included in five different branches with the sequences published from Denmark, Tunisia and Sweden. D1D2 polymerase chain reaction amplification and sequences of Culicoides A limited intraspecific nucleotide diversity was observed among the species morphologically identified as C. cataneiigegjelensis (0.006), C. imicola (0.001), C. jumineri (0.002), C. kingi (0.003) and C. newsteadi (0.005). The NJ analysis showed strong bootstrap support (83–100 %) for 10 lineages, each of which corresponded to a single species (Fig. 2). Although, some nodes are supported by low bootstrap values indicating that these clusters are not strong. The pairwise distances of D1D2 sequences are presented in Table 4.
Discussion A total of 10 species have been reported in the governate of Monastir (Tunisia). In the present study, specimens of all species were found to form distinct clusters, and the degree of molecular divergence of the two selected markers was sufficient to support the specific status of each cluster.
Among several genes of interest tested for species characterization, mtDNA and rDNA have commonly been selected to study molecular evolution of biting midges. Mitochondrial DNA has been widely used in studies of population genetics, phylogeography and phylogeny because it provides easy access to a homologous gene and it has easy amplification and sequencing, set with rapid evolution and with little or no recombination (Hurst and Jiggins 2005). The rDNA molecule is divided in domains rapidly evolving (called divergent or variable domains), interspersed among extraordinarily conserved segments usable to hybridize primers. Each rDNA domain has a different rate of base substitution, with conserved and variable regions included in each of the long 18S and 28S sequences, and similarly, each one of the spacers also evolves at a different speed (Jorgensen and Cluster 1988). The usefulness of D1 and D2 domains of 28S varies depending on the organisms analysed, from being able to distinguish between species up to only furnishing significant information at intermediate and high taxa (subfamilies, families, orders or classes). In our study, DNA sequences of the D1 and D2 rDNA domains discriminated perfectly all the morphologically determined species. Even if the D1D2 sequences of most of the species recorded in Africa remain unknown, the use of primers fitting in highly conserved region amongst sequenced species, they should serve for the identification of the majority of Culicoides species. The interspecific comparisons ranging from 0.013 to 0.079 reinforce the potential interest of these domains for evolutionary systematic concerning Culicoides.
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In our study, the COI sequences discriminated all the morphologically determined species. A low intraspecific sequence divergence (0.024) in specimens morphologically identified was observed for C. imicola. These species clustered in two groups, with a major group including specimens from Africa, the Mediterranean Basin and Europe. The genetic structure of C. imicola seems to be relatively stable and could explain the low intraspecific divergence observed. The molecular analysis of COI has grouped both C. puncticollis (specimens from Denmark and from Tunisia) and C. circumscriptus (from Switzerland, Spain, Sweden and Tunisia) within two clusters. Culicoides circumscriptus appears to have different populations according to morphological observations (Kremer and Delécolle 1974; Chaker et al. 1980). Future analyses on an important sampling are needed to well define the borders of the intraspecific variations. Finally, C. newsteadi specimens were split into five clusters. We detected a high intraspecific sequence divergence (0.149) in specimens morphologically identified as C. newsteadi. The same pattern was observed by Pagès et al. (2009), Muñoz-Muñoz et al. (2011), Lassen et al. (2012) and Ander et al. (2012). In our study, we have observed the same haplotypes (C. newsteadi N3; C. newsteadi N4; C. newsteadi N5) than those reported by Ander et al. (2012). C. newsteadi from Tunisia are grouped with C. newsteadi N3. Incontestably, the status of C. newsteadi specimens is not solved. A high diverging was reported in the sequences of Culicoides of the mt COI: Culicoides dk1 diverging by 14.3 to 17.2 % from other subgenus Culicoides species and Culicoides Kalix and Culicoides dk3, which diverged by 5.9 % from each other and showed 12.5 to 17.6 % divergence to subgenus Culicoides specimens (Lassen et al. 2012); cryptic species of Culicoides obsoletus were reported differing by 10–11 % at nucleotide level (Wenk et al. 2012). How can these divergences be explained? One explanation could be that databases such as Genbank may contain errors as C. newsteadi or C. segnis and C. reconditus (Ander et al. 2012). It is likely that several haplotypes or cryptic species will be detected using molecular tools. But at present, no new species of C. obsoletus has been described. Culicoides paradoxalis, a new species belonging to the subgenus Culicoides, has been described in France and Portugal (Ramilo et al. 2013). A genetic distance was reported between C. paradoxalis and Culicoides lupicaris (16.6 %), and a high distance was found between C. paradoxalis and C. newsteadi (21 %). Moreover, cryptic diversity has also been demonstrated within Culicoides pulicaris, Culicoides fagineus and C. newsteadi using the COI marker which remains to be resolved (Pagès et al. 2009). Then, which value gives to this marker? One explanation of this observed genetic variability could be the existence of mtDNA sequences that integrated in the nuclear genome (nuclear mtDNA, ‘NUMT’) and evolved as pseudogenes, but this phenomenon is scare
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(Wenk et al. 2012). Or mtDNA rearrangement occurs with non-coding sequences (Matsumoto et al. 2009). Moreover, the effects of inherited symbiont populations and phylogeographic and phylogenetic studies were reviewed by Hurst and Jiggins (2005). They concluded that direct and indirect selection is sufficiently common to make inferences from mtDNA data unreliable. They noted that (i) a single population may be infected with more than one strain or species of parasitic inherited microorganism, (ii) different populations may show different infection status, and (iii) inherited microorganisms could influence the mtDNA sequences of their hosts. The use of mtDNA markers alone to explore phylogenetic relationships between closely related taxa is unsafe and dangerous (Hurst and Jiggins 2005). Consequently, relationships between taxa inferred from COI can be considered as doubtful, as well as their grouping into clusters. We cannot exclude that it may concern C. newsteadi and C. obsoletus. Mitochondrial sequences alone are insufficient for elucidating the overall taxonomy in Culicoides species. Combining with D1D2 domains of rDNA to a mitochondrial marker like COI or cytochrome b (Augot et al. 2013) should be an interesting alternative. Acknowledgments We thank Sylvette Gobert for proofreading this manuscript. The authors would like to thank Jean Claude Delécolle for helping with Culicoides determination.
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