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Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria
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Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria Berrayah Hakima , Hwal-Su Hwang , Kyeong-Yeoll Lee PII: DOI: Reference:
S0001-706X(18)31267-1 https://doi.org/10.1016/j.actatropica.2019.105261 ACTROP 105261
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
6 October 2018 7 May 2019 5 November 2019
Please cite this article as: Berrayah Hakima , Hwal-Su Hwang , Kyeong-Yeoll Lee , Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105261
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Highlights
Bluetongue virus is transmitted by Culicoides species.
Molecular diagnosis was conducted in Culicoides midges collected in Algeria.
The presence of 14 species were identified in Algeria
Geographic distribution and potential role of bluetongue virus transmission was discussed in identified species.
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Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria
Berrayah Hakimaa,b, Hwal-Su Hwang b, Kyeong-Yeoll Leeb,c,d
a
Head of General Pathology Department, Internal Audit at the Regional
Veterinary Laboratory of Tlemcen, Algeria b
Division of Applied Biosciences, College of Agriculture and Life Sciences,
Kyungpook National University, Daegu, Republic of Korea c
Institute of Plant Medicine, Kyungpook National University, Daegu, Republic of
Korea d
Agricultural Science and Technology Institute, Kyungpook National University,
Daegu, Republic of Korea
Correspondence: Kyeong-Yeoll Lee E-mail: [email protected] Tel.: 82-53-950-5759 Fax: 82-53-950-6758
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ABSTRACT Bluetongue is a serious vector-borne viral disease that infects wild and domestic ruminants. The causative virus is transmitted by midges of the genus Culicoides, which consists of at least 1,350 species worldwide. Since 1998, bluetongue disease has spread to Europe and northern Africa, including Algeria. To better understand the distribution of Culicoides species in Algeria, adult midges were collected from 17 different regions in Algeria from 2009 to 2015. At first, 492 specimens were grouped into 52 batches by wing patterns and geographic area of Algeria. Analysis of 60 nucleotide sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene showed that the presence of 14 species including five unknown species, which were belonged to seven distinct subgenera. At least five species (C. imicola, C. obsoletus, C. puncticollis, C. kingi, and C. newsteadi) were discussed as potential vectors of bluetongue virus (BTV). The present study provides important insights into the genetic diversity of Culicoides and the potential spread of BTV in Algeria.
Keywords: Bluetongue virus, Culicoides, Genetic diversity, Genotype, Vector
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1. Introduction Bluetongue is a vector-borne viral disease of ruminants, transmitted by certain species of biting midges of the genera Culicoides (Maan et al., 2012; Foxi et al., 2016). The etiological agent is a double-stranded RNA virus belonging to the genus Orbivirus of the family Reoviridae (Borden et al., 1971). Bluetongue disease was first reported in South Africa 120 years ago (Henning, 1956). Since 1998, it has spread to North Africa and Europe (Caporale, 2008). It is a notifiable disease to the World Organization for Animal Health (2014). At least 26 bluetongue virus (BTV) serotypes have been recognized worldwide (Zientara et al., 2014). BTV infects wild and domestic ruminants, mainly sheep, and causes an acute to chronic disease with variable rates of mortality (van der Sluijs et al., 2013). The economic impacts are not only due to direct losses of animals from death but also serious sequelae in surviving animals, which severely restricts trade, affecting farmers’ livelihoods. Culicoides is a highly diversified genus that contains at least 1,350 species within 34 subgenera (Borkent, 2017). However, the vector’s competence to transmit the virus can be influenced by environmental conditions, such as climate change (Paweska et al., 2002; Mellor, 2004; Wilson and Mellor, 2008), and requires that the species feed wholly or largely upon ruminants and occurs in abundance. As a result, only several species are known to be potential or proven vectors of BTV in different regions. Examples include C. imicola and C. bolitinos in Africa, C. imicola and C. fulvus in Asia, C. brevitarsis and C. fulvus in -4-
Australia, C. sonorensis in North America, and C. insignis and C. pusillus in Central and South America (Baldet et al., 2005). The C. obsoletus complex and the C. pulicaris complex transmit BTV in Northern Europe (Bessell et al., 2016). The hematophagous females can transmit a variety of filarial worms, protozoans, and viruses to humans and wild or domestic animals (Borkent, 2004; Meiswinkel et al., 2004b). The expanding distribution of BTV is also facilitated by transplacental transmission and by oral transmission (Wilson and Mellor, 2009; van der Sluijs et al., 2013). In North African countries, BTV was reported in 1999 in Tunisia, and a year later, in Algeria, with 28 outbreaks between July and September 2000 (Hamida, 2000). After the first outbreak in Algeria, the disease clinically affected 2,661 of 21,175 susceptible sheep, in 24 localities in Jijel, which is located in the northeast of the country. The disease continued to spread and reached six districts in the eastern and central parts of the country by the end of the epidemic (Hamida, 2000). Of the several bluetongue outbreaks reported in Algeria, various BTV serotypes have been identified, including serotype 2 (BTV-2) in 2000, and BTV-1 in 2006 and 2008, with new outbreaks in 2009 and 2010 (Madani et al., 2011). In 2014, BTV-1 and BTV-4 were circulating simultaneously in the same temporospatial point (Kardjadj et al., 2016). The increasing prevalence of the disease in Algeria highlights the vector’s competence and persistence and suggests the importance of the identification and surveillance of the vector.
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Identification of Culicoides species in Algeria has been based mostly on the observation of morphological characteristics (EpiReg-Maghreb, 2008; Mathieu, 2011; Borkent, 2014, 2017). However, modern techniques for DNA sequencing are powerful tools for species identification and revealing the genetic variation of small-sized species, including midges (Cêtre-Sossah et al., 2004; Cuellar et al., 2016; Augot et al., 2017; Bakhoum et al., 2018). The present study determined the species identity and geographic distribution of Culicoides midges in Algeria at the morphological and molecular levels and discussed potential vector species of BTV and further transmission of bluetongue in Algeria.
2. Materials and methods
2.1. Insect collection Adult midges were collected from 17 different geographic localities in Algeria from 2009 to 2015 (Table 1; Fig. 1). Midges were collected from areas of either livestock sheds or riversides using 12-V blacklight traps. We did not obtain any records of BTV outbreak in collecting sites in this study. Collected midges were preserved in plastic tubes containing 70% ethanol for further analysis.
2.2. Morphological identification Morphological identification was conducted using the interactive identification keys for female Culicoides (IIKC; Mathieu et al., 2012) available -6-
online at http://www.iikculicoides.net (Fig. 2). The main characteristics, such as veins and colors of the wings of each species, were observed under a light microscope (Olympus, Tokyo, Japan).
2.3. Polymerase chain reaction (PCR) analysis Genomic DNA was extracted from each individual using a PureLink™ Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s recommendations. DNA concentrations were determined using a nanophotometer (Implen, München, Germany). PCR was performed using specific primers for the amplification of COI sequence fragments of Culicoides species
(Mathieu
et
al.,
2012):
forward
primer
(C1J1718)
5′-
GGAGGATTTGGAAATTGATTAGT-3′ and reverse primer (C1N2191) 5′CAGGTAAAATTAAAATATAAACTTCTGG-3′. The amplification reaction mixture) contained 15 µL dNTP premix, 2 µL of each primer, 11 µL of template DNA lysate (30 ng), and a complementary volume of nuclease-free water. A 30µL aliquot of the mixture was amplified in a SimpliAmp® and Veriti® 96-well thermal cycler, under touch-up program for COI amplification: 95 °C for 5 s, 5 cycles (94 °C, 40 s; 45 °C, 40 s; 72 °C, 60 s), followed by 45 cycles (94 °C, 40 s; 50 °C, 40 s; 72 °C, 1 s) and final elongation at 72 °C for 7 min (Mathieu et al., 2012). The PCR products (5 μL) were run on 1% agarose gel and then stained with ethidium bromide for visualization under UV light.
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2.4. DNA sequence analysis PCR products were separated by gel electrophoresis using 1% lowmelting-point agarose, then excised from the gel and purified using a Wizard PCR Preps DNA purification system (Promega, Madison, WI, USA). The purified PCR products were cloned using a T-Blunt vector (Solgent, Daejeon, Korea) and sequenced analyzed at the Solgent Sequencing Facility (Solgent). The GenBank database at the National Center for Biotechnology Information (NCBI) was searched using the BLASTN algorithm (Schaffer et al., 2001), and nucleotide sequences were aligned using ClustalW (Thompson et al., 1994). At least three individuals were analyzed from each group, classified by morphological identification of species. For some species, only 1 or 2 specimens were examined due to the absence of available specimens.
2.5. Clade analysis The determined sequences were manually edited with Chromas® (version 2.31) to produce a consensus sequence of ~400 bp. The sequences were aligned using the multiple sequence comparison by log-expectation (MUSCLE) program in BioEdit© (version 7.2.5). The sequences were compared with publicly available sequences in the NCBI database to establish their relationship. In addition, List of Culicoides species previously reported in Algeria were summarized (Table 3).
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3. Results
3.1. Morphological identification Based on the wing pigmentation patterns of 492 specimens, samples were grouped into 52 batches by wing patterns and geographic area of Algeria.
3.2. Molecular identification Sixty representative specimens from the 52 batches partitioned by species and regions of Algeria were used to amplify 523 bp of partial COI sequences. The 60 sequences identified were submitted to the GenBank database. BLAST analyses showed the highest identity of each sequence (Table 2). The COI sequence variation among all the samples ranged from 0.00% to 22.32% (Table S1). At least 14 species were determined, including nine identified species and five unidentified species. The nine identified species were C. imicola, C. obsoletus, C. kingi, C. puncticollis, C. circumscriptus, C. cataneii, C. sahariensis, C. langeroni, and C. newsteadi. Most of these species were at least 98% identical to specimens reported from other countries in North Africa and Europe. However, C. cataneii was 97.01–97.44% identical to KJ29968 from Tunisia, C. sahariensis was 97.22– 97.44% identical to KJ730003 from Tunisia, and C. langeroni was 96.15% identical to KJ729987 from Tunisia.
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The five unidentified specimens were allocated as Culicoides sp. A, B, C, D, and E (Table 2). A GenBank database analysis showed these species shared the highest identities of 92.37% for Culicoides sp. A to C. imicola (AJ867227) of Morocco; 87.66% for Culicoides sp. B to C. enderleini (KJ833698) of Senegal; 88.46–89.53% for Culicoides sp. C to C. jumineri (KJ729978, KJ729988) of Tunisia; 93.80% for Culicoides sp. D to C. longipennis (MF105764) of Turkey, and 91.86–92.27% for Culicoides sp. E to C. shaklawensis (KT624129) of Slovakia. Intraspecific variation ranges were determined in Algerian populations of some species. For example, the intraspecific variation range of C. imicola was 0.0–1.93% (n = 12), 0.21–1.93% for C. kingi (n = 5), 0.64–2.36% for C. puncticollis (n = 10), 0.21–1.72% for C. circumscriptus (n = 7), 0.0–3.0% for C. sahariensis (n = 5), 0.86–2.36% for C. newsteadi (n = 5), and 0.0–1.72% for Culicoides sp. C (n = 5), as shown in Table S1. While species of C. imicola, C. kingi, C. puncticollis, C. circumscriptus, C. sahariensis, and C. newsteadi were recognized as occurring in various regions of Algeria, C. obsoletus, C. cataneii, C. langeroni, and Culicoides sp. A, B, D, and E were present in only one or two regions of Algeria. Culicoides sp. A and B were even more restricted, occurring only in the Adrar (Q) region, which is a desert area of southern Algeria.
3.3. Phylogenetic relationship of Culicoides species - 10 -
To determine the relationship of the identified species within the genus Culicoides, neighbor-joining analysis of the 409-bp COI sequences (after removing the 5′- and 3′-end regions) was conducted using the 60 sequences identified in this study and the most identical sequences of corresponding Culicoides species obtained from the GenBank database (Table 2; Fig. 2). According to the subgeneric classification of Borkent (2016), 14 species identified in this study were classified into seven subgenera; Avaritia, Remmia, Beltranmyia, Monoculicoides, Sensiculicoides, Oecacta, and Culicoides (Fig. 2). Three species (C. imicola, C. obsoletus, Culicoides sp. A) were grouped into the subgenus Avaritia cluster (Fig. 2). Culicoides imicola exhibited variabilities of 7.94–8.80%, at least 15%, and 16.09–16.31% with Culicoides sp. A (KX853276), C. obsoletus (KX853233), and Culicoides sp. B, respectively (Table S1). Two species (C. kingi, Culicoides sp. B) grouped in the subgenus Remmia and were closely related to the subgenus Avaritia. Two sequences (KX853277, KX853278) of Culicoides sp. B were identical and showed variabilities of 12.66% with C. enderleini (KJ833698), 14–15% with C. kingi, but just 8.37% with Culicoides sp. A. Both C. circumscriptus and C. puncticollis were closely related to each other, with only 16–17% COI sequence variation between them, and belonged to the subgenera Beltranmyia and Monoculicoides, respectively. Five species (C. langeroni, C. cataneii, Culicoides sp. C, Culicoides sp. D, Culicoides sp. E) were - 11 -
closely related to each other and belonged to the subgenus Sensiculicoides. Both C. sahariensis and C. newsteadi were closely related to each other and grouped in the subgenera Oecacta and Culicoides, respectively.
4. Discussion The molecular analysis of Culicoides species in Algeria determined the presence of at least 14 species, including five unidentified species. Both C. imicola and C. obsoletus belong to the subgenus Avaritia and are considered as vectors of BTV. Previous studies have indicated that C. imicola is the major vector of BTV in Africa (Baldet et al., 2005) and the Mediterranean basin (Carpenter et al., 2015), while C. obsoletus is considered to be a putative vector of BTV in northern Europe (Ninio et al., 2011). In the present study, C. imicola was collected from nine different locations (A, B, E, F, H, I, L, N, and P in Table 1) of Algeria. Although they were widely distributed around the country, intraspecific divergence was at least 1.93%. This result suggests that the wide distribution of vector species poses a potentially dangerous situation for the spread of BTV throughout the country. While C. obsoletus has been previously identified in Algeria in 1952 (EpiReg-Maghreb, 2008), the present study revealed that its presence was molecularly identified from Ain Defla (B), and morphologically identified from two additional locations, Tizi Ouzou (E) and Souk Ahras (K). Culicoides kingi was grouped in the subgenus Remmia and closely related to the subgenus Avaritia (Borkent, 2014). Culicoides kingi is one - 12 -
of the C. schultzei species complex (Cornet and Brunhes, 1994). However, this species complex is known to play a minor role in BTV transmission than C. imicola (Meiswinkel et al., 2004a). This species was distributed in northern interior regions (E and L) and the southern region (P) of Algeria. Both C. puncticollis and C. circumscriptus are widely distributed in Algeria but might not be genetically diverse because they had 98–99% similarity to individuals from Tunisia and European countries. Both species have been reported in Algeria previously (EpiReg-Maghreb, 2008). The transmission of BTV by C. puncticollis, but not C. circumscriptus, has been documented (Meiswinkel et al., 2004a; Wilson and Mellor, 2009). One sample collected from Constantine (F) in northeastern Algeria aligned with C. langeroni from Tunisia, sharing 95% identity and belonging to the subgenus Sensiculicoides. It was first reported in Algeria in 1921 (Mathieu, 2011). Both C. sahariensis and C. newsteadi were clustered together but belonged to the subgenera Oecacta and Culicoides, respectively (Borkent, 2014). This study showed that C. sahariensis was distributed in coastal, internal, and southern regions of Algeria, and genetically close to a Tunisian strain. Culicoides newsteadi shares high identity with C. halophilus from northern Europe, including Sweden, Slovakia, and Denmark. Recent molecular evidence has demonstrated that C. halophilus is identical to C. newsteadi N3 within the C. newsteadi species complex (Soren and Kristensen, 2015; Foxi et al., 2016). Culicoides newsteadi was reported in Algeria in 1921 (EpiReg-Maghreb, 2008), and the present study - 13 -
revealed its presence at two coastal locations Mostaganem (A) and Tizi Ouzou (E), and the western highland Bayadh-Rogassa (M). This species is identified as a BTV-competent species in Italy (Foxi et al., 2016). This study determined the COI sequences of five unidentified specimens in Algeria, allocated as Culicoides sp. A, B, C, D, and E. These species identities are uncertain because their COI sequence variations were more than 9% when compared with the most similar sequences in the GenBank database. For example, Culicoides sp. C shared 88.46–89.53% similarity to C. jumineri (KJ729988) of Tunisia. Five specimens were collected from Tizi Ouzou (E) and Constantine (F), which are located in the northern region of Algeria. The similarity of seven COI sequences (KJ729977–KJ729982 and KJ729988) of C. jumineri in the GenBank database was 91.82% (data not shown). Our results showed that the intraspecific variation of five Algerian individuals was 1.72% (Table S1). These results suggest that Culicoides sp. C is outranged to the genetic variation of C. jumineri. Further study is required to determine the identity of this species. Culicoides sp. E shared 91.86–92.27% sequence similarity to C. shaklawensis of Slovakia, which is the only sequence of this species listed in the GenBank database. It has not been reported previously in Algeria, which is in agreement with the distribution map in IIKC. This species was collected from Tizi Ouzou (E) and Constantine (F) in Algeria. Its potential role in the transmission of BTV is not known.
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The phylogenetic analysis of 14 species determined from the present study showed clustering of seven subgenera, namely Avaritia, Remmia, Beltranmyia, Monoculicoides, Sensiculicoides, Oecacta, and Culicoides. Five species (C. langeroni, C. cataneii, Culicoides sp. C, Culicoides sp. D, Culicoides sp. E) were closely related to each other and belonged to the subgenus Sensiculicoides. However, this subgenus showed great genetic diversity among species that are grouped into various subclades. All the subgenera were monophyletic, as mentioned previously (Borkent, 2016; Augot et al., 2017). This clustering is consistent with information from the world species catalog of biting midges (Borkent, 2014, 2016). Due to its large geographical area, Algeria might experience a high transboundary and trans-Mediterranean exchange of infected vectors with neighboring countries, such as Morocco and Tunisia. There is 1,622 km of coastline along the Mediterranean basin that is exposed to Europe, which allows spread and invasion between Africa and Europe, especially northern Africa. Simultaneously, there is movement of people and goods between North African and Asian countries. Among the 1,350 known Culicoides species, approximately 50 species are recognized as being involved in BTV transmission (Wilson and Mellor, 2009). Previously, 45 Culicoides species have been identified in Algeria, and 50 and 23 different Culicoides species in Morocco and Tunisia, respectively (EpiRegMaghreb, 2008). The present study confirmed the existence of at least five viruscompetent species in Algeria (C. imicola, C. obsoletus, C. puncticollis, C. kingi, - 15 -
and C. newsteadi). However, the geographical distribution of these five species appears to cover numerous regions in the context of our collected samples. Previous studies have demonstrated the biological sensitivity of Culicoides vectors and BTV to climate is due to the dynamic and climate-mediated vector ability of Culicoides species (Purse et al., 2008). Algeria is affected by temperature increases and an increased frequency of flood events owing to climate change (Sahnoune et al., 2013), creating a favorable environment for midge populations and increasing the chance for BTV to spread to new regions of the country. In conclusion, the present study reaffirmed the identification of at least 14 Culicoides species and their geographic distribution in Algeria, with the existence of at least five BTV-competent species spread over a relatively large distribution area. Thus, the potential impact of BTV dispersal is real. In the midst of climate change in Algeria, further studies are recommended to increase the knowledge regarding the distribution and competence of Culicoides species in the transmission of BTV, considering that bluetongue disease is highly climatesensitive.
Funding This work was supported by the Research Program for Exportation Support of Agricultural Products, Animal and Plant Quarantine Agency, Republic of Korea [grant number Z-1543086-2017-21-01]. - 16 -
Acknowledgments We thank Dr. Jae-Kyoung Shim at Kyungpook National University, Republic of Korea, for her help with DNA sequencing and data analysis.
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Mathieu, B., Cêtre-Sossah, C., Garros, C., Chavernac, D., Balenghien, T., et al., 2012. Development and validation of IIKC: an interactive identification key for Culicoides (Diptera: Ceratopogonidae) females from the Western Palaearctic region. Parasites Vectors 5, 137. doi:10.1186/1756-3305-5-137. Meiswinkel, R., Gomulski, L.M.,Delécolle, J.C., Goffredo, M., Gasperi, G., 2004a. The taxonomy of Culicoides vector complexes-unfinished business. Veterinaria Italiana 40,151-159. Meiswinkel, R., Venter, G.J., Nevill, E.M., 2004b. Vectors: Culicoides spp. In: Coetzer, J.A.W., Tustin, R., (Eds.), Infectious Diseases of Livestock (second ed.), Oxford University Press, Cape Town, pp. 93-136. Mellor. P.S., 2004. Environmental influences on arbovirus infections and vectors. In: Gillespie, S.H., Smith, G.L., Osbourn, A., (Eds.), SGM Symposium, 63. Microbe–Vector Interactions in Vector-Borne Diseases, Cambridge University Press, Cambridge, pp. 181-197. Ninio, C., Augot, D., Delecolle, J.C., Dufour, B., Depaquit, J., 2011. Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France. Parasit. Res.108, 657–663. Paweska, J.T., Venter, G.J., Mellor, P.S., 2002. Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos. Med. Vet. Entomol.16, 10-21.
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Purse, B.V., Brown, H.E., Harrup, L., Mertens, P.P.C., Rogers, D.J., 2008. Invasion of bluetongue and other orbivirus infections into Europe: the role of biological and climatic processes. Revue scientifiqueet technique (International Office of Epizootics) 27, 427-442. Sahnoune, F., Belhamel, M., Zelmat, M., Kerbachi, R., 2013. Climate Change in Algeria: Vulnerability and Strategy of Mitigation and Adaptation. Energy Procedia 36, 1286-1294. Schaffer, A.A., Aravind, L., Madden, T.L., Shavirin, S., Spouge, J.L., Wolf, Y.I., Koonin, E.V., Altschul, S.F., 2001. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 29, 2994-3005. Soren, A.N., Kristensen, M., 2015. Delineation of Culicoides species by morphology and barcode exemplified by three new species of the subgenus Culicoides (Diptera: Ceratopogonidae) from Scandinavia. Parasites Vectors 8, 151. doi:10.1186/s13071-015-0750-4. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. van der Sluijs, M.T.W., Mirjam T.W. van der Sluijs, Schroer-Joosten, D.P.H., Fid-Fourkour, A., Vrijenhoek, M.P., Debyser, I., et al., 2013. Transplacental
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transmission of bluetongue virus serotype 1 and serotype 8 in sheep: Virological and Pathological Findings. Plos One 8, e81429. Wilson, A., Mellor. P., 2008. Bluetongue in Europe: vectors, epidemiology and 460 climate change. Parasitol. Res., 103 (Suppl. 1), pp. S69-S77. Wilson, A.J., Mellor, P.S., 2009. Bluetongue in Europe: past, present and future. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 364, 2669–2681. World Organization for Animal Health (OIE), 2014. Bluetongue (infection with bluetongue virus). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter 2. 1. 3 Zientara, S., Sailleau, C., Viarouge, C., Hopper, D., Beer, M., et al., 2014. Novel bluetongue virus in goats, Corsica, France, 2014. Emerg. Infect. Dis. 20, 21232125. doi: 10.3201/eid2012.140924.
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Figure legends
Figure 1. Geographic distribution of collected samples in Algeria. Collected regions were labeled from A to Q.
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Figure 2. Neighbor-joining analysis of COI sequences of Culicoides species in Algeria. Numbers adjacent to the branches indicate the bootstrap values of 1000 replicates. Each branch was labeled with the species name, GenBank accession number, and country name. Scale bars indicate the number of substitutions per site. Sequences labeled with small circles were most similar sequences in the search of the GenBank database.
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Table 1. Geographic information on collected Culicoides midges in different regions of Algeria. Location
Geographic coordinates
Altitude (m)
Sample number
Mostaganem (A)
35°55.869′ N, 0°5.3508′ E
102
206
Ain Defla (B)
36º16'7.87" N, 1º58'48.92" E
264
21
Algiers (C)
36º44'28.68" N, 3º04'24.22" E
186
15
Medea (D)
36º16'55.7" N, 2º45'37.02" E
910
6
Tizi Ouzou (E)
36º48'10.45" N, 4º03'43.79" E
206
47
Constantine (F)
36°21.9′ N, 6°36.8832′ E
574
114
Skikda (G)
36°52.5702′ N, 6°54.5526′ E
24
3
Annaba (H)
36°54′ N, 7°46.0002′ E
5
5
Guelma (I)
36º31'15.2" N, 7º27'24.98" E
305
2
El Tarf (J)
36º45'0.37" N, 8º16'11.74" E
24
3
Souk Ahras (K)
36º16'23.81" N, 7º58'4.09" E
697
2
Tebessa (L)
35º25'22.26" N, 8º05'58.7" E
867
7
Bayadh-Rogassa (M)
34°01'3.60" N, 0°55'19.79" E
1090
15
Bayadh-Ghassoul (N)
33º23'0.66" N, 1º12'40.36" E
1129
12
Laghouat (O)
33º46'53.95" N, 2º51'13.2" E
764
12
Ouargla(P)
32º12'55.27" N, 4º57'7.4" E
138
13
Adrar (Q)
27º52'15.18" N, 0º15'19.67" W
257
10
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Table 2. Accession numbers of 60 COI sequences of collected Culicoides midges in various regions of Algeria and their identity searched in the GenBank database. Clade
Code name
Collection site
Accession number
Species
Highest sequence identity with GenBank database Identity (%)
Accession number
Country
A
C1.1/O D22/M-1 I12/5.4 L13/8 D17/H1 D16.1/A4 H1/3.1 I12/I1I4 E13/J1J4 C15/O-2 K10/I-6 N14/6 C9.1/E-2 J14.3/4.2
Ain Defla (B) Constantine (F) Ouargla (P) Guelma (I) Constantine (F) Constantine (F) Bayadh-Ghassoul (N) Ouargla (P) Mostaganem (A) Ain Defla (B) Tizi Ouzou (E) Tebessa (L) Ain Defla (B) Adrar (Q)
KX853221 KX853222 KX853223 KX853224 KX853225 KX853226 KX853227 KX853228 KX853229 KX853230 KX853231 KX853232 KX853233 KX853276
C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. obsoletus C. sp. A (S. imicola)*
99.79 99.79 100 99.79 99.36 99.15 99.15 99.79 98.94 99.36 99.15 99.58 99.79 92.37
AJ867226 ⸗ AJ867227 ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ JQ620130 AJ867227
Spain ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ Sweden Morocco
B
J14.2/4.4 J14.2/4.2 I12/5.3 I12/5.2 I11.1/U K9/H.3 N11/1.2 P3-1/X E6.1/M-2 L13/8.1 D13.1/C4 D18.2/C D18.2/3-1 T20/H1
Adrar (Q) Adrar (Q) Ouargla (P) Ouargla (P) Ouargla (P) Tizi Ouzou (E) Tebessa (L) Laghouat (O) Mostaganem (A) Guelma (I) Constantine (F) Constantine (F) Constantine (F) Mostaganem (A)
KX853277 KX853278 KX853234 KX853235 KX853236 KX853237 KX853238 KX853253 KX853254 KX853255 KX853256 KX853257 KX853258 KX853259
C. sp. B (C. enderleini) C. sp. B (C. enderleini) C. kingi C. kingi C. kingi C. kingi C. kingi C. puncticollis C. puncticollis C. puncticollis C. puncticollis C. puncticollis C. puncticollis C. puncticollis
87.66 88.66 99.58 99.79 99.36 98.73 98.29 99.58 98.94 99.58 99.58 99.36 99.36 98.94
KJ833698 ⸗ MF399780⸗ ⸗ ⸗ ⸗ ⸗ KJ624120 ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
Senegal ⸗ Cameroon ⸗ ⸗ ⸗ ⸗ Slovakia ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
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K11.1/G D21.2/F1 E6.3/N-1
Tizi Ouzou (E) Constantine (F) Mostaganem (A)
KX853260 KX853261 KX853262
C. puncticollis C. puncticollis C. puncticollis
99.36 98.30 99.79
⸗ ⸗ ⸗
⸗ ⸗ ⸗
C
J14.1/E1 I12/I2 T19/G K11.2/W-2 E13/J2 B2.1/D D16.1/A
Adrar (Q) Ouargla (P) Mostaganem (A) Tizi Ouzou (E) Mostaganem (A) Annaba (H) Constantine (F)
KX853263 KX853264 KX853265 KX853266 KX853267 KX853268 KX853269
C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus
98.73 98.94 99.15 99.36 98.94 99.15 99.79
KJ624071 ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
Slovakia ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
D
D18/10.3 D18/10.1 D18.1/B K9/H-1 D19.1/D2 K9/H.4 D21.2/F2F3F4 D17/H2H3H4 E5.1/L-2 K14/G N11/1.1 D16.1 /A2 C9.3/P-3 J14.2/4.3
Constantine (F) Constantine (F) Constantine (F) Tizi Ouzou (E) Constantine (F) Tizi Ouzou (E) Constantine (F) Constantine (F) Mostaganem (A) Tizi Ouzou (E) Tebessa (L) Constantine (F) Ain Defla (B) Adrar (Q)
KX853239 KX853240 KX853241 KX853242 KX853243 KX853244 KX853245 KX853279 KX853280 KX853246 KX853247 KX853248 KX853249 KX853250
C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. cataneii C. cataneii C. sp. D (C. longipennis) C. sp. D (C. longipennis) C. sahariensis C. sahariensis C. sahariensis C. sahariensis C. sahariensis
88.46 88.46 89.32 89.32 89.53 97.44 97.01 93.80 93.80 97.22 97.44 97.44 97.44 97.22
KJ729988 ⸗ KJ729978 ⸗ ⸗ KJ729968 ⸗ MF105764 ⸗ KJ730003 ⸗ ⸗ ⸗ ⸗
Tunisia ⸗ Tunisia ⸗ ⸗ Tunisia ⸗ Turkey ⸗ Tunisia ⸗ ⸗ ⸗ ⸗
E
D20/G2 K9/H-2 D10/S-1 E7.1/A3 E8/B2 E5.1/S-2 K3/9.2
Constantine (F) Tizi Ouzou (E) Constantine (F) Mostaganem (A) Mostaganem (A) Mostaganem (A) Tizi Ouzou (E)
KX853251 KX853252 KX853275 KX853270 KX853271 KX853272 KX853273
G4/7.1
Bayadh-Rogassa (M)
KX853274
C. sp. E (C. shaklawensis) C. sp. E (C. shaklawensis) C. langeroni C. halophilus C. halophilus C. halophilus C. halophilus C. newsteadi N3 C. halophilus C. newsteadi N3
92.27 91.86 96.15 98.08 97.87 98.29 98.08 97.87 99.15 98.93
KJ624129 ⸗ KJ729987 JF766298 ⸗ ⸗ ⸗ JQ620119 JF766298 JQ620119
Slovakia ⸗ Tunisia Denmark ⸗ ⸗ ⸗ Sweden Denmark Sweden
*Species within the parenthesis were the most similar species in COI identity in the GenBank database.
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Table 3. List of Culicoides species previously reported in Algeria. No
Species
Subgenus
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14
C. algeriensis Clastrier, 1957: 426. Algeria. C. begueti Clastrier, 1957: 432. Algeria. C. cataneii Clastrier, 1957: 438. Algeria. C. citrinellus Kieffer, 1923a: 674. Algeria (as C. sergenti) C. distigma Kieffer, 1922g: 502. Algeria (as C. parroti) C. donatieni Kieffer, 1922g: 504. Algeria (as C. parroti) C. foleyi Kieffer, 1922g: 503. Algeria. C. griseidorsum Kieffer, 1918a: 46. Algeria. C. kabyliensis Kieffer, 1922g: 505. Algeria (as C. obsoletus) C. marcleti Callot, Kremer and Basset, 1968: 271. Algeria C. montanus (obsoletus group) C. saevus Kieffer, 1922g: 506. Algeria. C. sahariensis Kieffer, 1923a: 678. Algeria. C. semimaculatus Clastrier, 1958a: 55. Algeria
Sensicullicoides Sensicullicoides Unplaced species Oecacta Monoculicoides Monoculicoides Unplaced species Sensicullicoides Avaritia Oecacta Avaritia Pontoculicoides Oecacta Oecacta
Borkent, 2016
15 16 17 18 19 20 21 22 23 24 25 26
C. azerbajdzhanicus Dzhafarov, 1962 - DZ C. faghihi Navai, 1971 C. imicola (imicola group) C. kingi Austen, 1912 - DZ C. langeroni Kieffer, 1921 C. nudipennis Kieffer, 1922g: 507. Algeria C. pallidus Khalaf, 1957 C. pseudolangeroni Kremer, Chaker and Delecolle, 1981 DZ C. pseudopallidus Khalaf, 1961 - DZ C. ravus De Meillon, 1936 C. sejfadinei Dzhafarov, 1958 C. sergenti Kieffer, 1921
Oecacta Wirthomyia Avaritia Oecacta Sensicullicoides Unplaced species Oecacta Unplaced species Sensicullicoides Synhelea Pontoculicoides Oecacta
Mathieu, 2011
Beltranmyia Oecacta Culicoides Sensicullicoides Sensicullicoides Sensicullicoides Sensicullicoides Oecacta Sensicullicoides Culicoides Selfia Sensicullicoides Sensicullicoides Culicoides Monoculicoides Oecacta Avaritia Pontoculicoides Silvaticulicoides
EpiRegMaghreb, 2008
27 C. circumscriptus Kieffer, 1918 28 C. dzhafarovi Remm, 1967 29 C. fagineus Edwards, 1939 30 C. gejgelensis Dzhafarov, 1964 31 C. heteroclitus Kremer & Callot, 1965 32 C. jumineri Callot & Kremer, 1969 33 C. kibunensis Tokunaga, 1937 34 C. longipennis Khalaf, 1957 35 C. maritimus Kieffer, 1924 36 C. newsteadi Austen, 1921 37 C. odiatus Austen, 1921 38 C. pictipennis (Staeger, 1839) 39 C. poperinghensis Goetghebuer, 1953 40 C. pulicaris (Linné, 1758) 41 C. puncticollis (Becker, 1903) 42 C. santonicus Callot, Kremer, Rault & Bach, 1966 43 C. scoticus Downes & Kettle, 1952 44 C. sejfadinei Dzhafarov, 1958 45 C. subfasciipennis Kieffer, 1919 * Alphabetical order of species in each reference has been arranged.
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Graphical abstract
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-3-
Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria Berrayah Hakima , Hwal-Su Hwang , Kyeong-Yeoll Lee PII: DOI: Reference:
S0001-706X(18)31267-1 https://doi.org/10.1016/j.actatropica.2019.105261 ACTROP 105261
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
6 October 2018 7 May 2019 5 November 2019
Please cite this article as: Berrayah Hakima , Hwal-Su Hwang , Kyeong-Yeoll Lee , Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria, Acta Tropica (2019), doi: https://doi.org/10.1016/j.actatropica.2019.105261
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Highlights
Bluetongue virus is transmitted by Culicoides species.
Molecular diagnosis was conducted in Culicoides midges collected in Algeria.
The presence of 14 species were identified in Algeria
Geographic distribution and potential role of bluetongue virus transmission was discussed in identified species.
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Molecular identification of Culicoides (Diptera: Ceratopogonidae) species in Algeria
Berrayah Hakimaa,b, Hwal-Su Hwang b, Kyeong-Yeoll Leeb,c,d
a
Head of General Pathology Department, Internal Audit at the Regional
Veterinary Laboratory of Tlemcen, Algeria b
Division of Applied Biosciences, College of Agriculture and Life Sciences,
Kyungpook National University, Daegu, Republic of Korea c
Institute of Plant Medicine, Kyungpook National University, Daegu, Republic of
Korea d
Agricultural Science and Technology Institute, Kyungpook National University,
Daegu, Republic of Korea
Correspondence: Kyeong-Yeoll Lee E-mail: [email protected] Tel.: 82-53-950-5759 Fax: 82-53-950-6758
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ABSTRACT Bluetongue is a serious vector-borne viral disease that infects wild and domestic ruminants. The causative virus is transmitted by midges of the genus Culicoides, which consists of at least 1,350 species worldwide. Since 1998, bluetongue disease has spread to Europe and northern Africa, including Algeria. To better understand the distribution of Culicoides species in Algeria, adult midges were collected from 17 different regions in Algeria from 2009 to 2015. At first, 492 specimens were grouped into 52 batches by wing patterns and geographic area of Algeria. Analysis of 60 nucleotide sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene showed that the presence of 14 species including five unknown species, which were belonged to seven distinct subgenera. At least five species (C. imicola, C. obsoletus, C. puncticollis, C. kingi, and C. newsteadi) were discussed as potential vectors of bluetongue virus (BTV). The present study provides important insights into the genetic diversity of Culicoides and the potential spread of BTV in Algeria.
Keywords: Bluetongue virus, Culicoides, Genetic diversity, Genotype, Vector
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1. Introduction Bluetongue is a vector-borne viral disease of ruminants, transmitted by certain species of biting midges of the genera Culicoides (Maan et al., 2012; Foxi et al., 2016). The etiological agent is a double-stranded RNA virus belonging to the genus Orbivirus of the family Reoviridae (Borden et al., 1971). Bluetongue disease was first reported in South Africa 120 years ago (Henning, 1956). Since 1998, it has spread to North Africa and Europe (Caporale, 2008). It is a notifiable disease to the World Organization for Animal Health (2014). At least 26 bluetongue virus (BTV) serotypes have been recognized worldwide (Zientara et al., 2014). BTV infects wild and domestic ruminants, mainly sheep, and causes an acute to chronic disease with variable rates of mortality (van der Sluijs et al., 2013). The economic impacts are not only due to direct losses of animals from death but also serious sequelae in surviving animals, which severely restricts trade, affecting farmers’ livelihoods. Culicoides is a highly diversified genus that contains at least 1,350 species within 34 subgenera (Borkent, 2017). However, the vector’s competence to transmit the virus can be influenced by environmental conditions, such as climate change (Paweska et al., 2002; Mellor, 2004; Wilson and Mellor, 2008), and requires that the species feed wholly or largely upon ruminants and occurs in abundance. As a result, only several species are known to be potential or proven vectors of BTV in different regions. Examples include C. imicola and C. bolitinos in Africa, C. imicola and C. fulvus in Asia, C. brevitarsis and C. fulvus in -4-
Australia, C. sonorensis in North America, and C. insignis and C. pusillus in Central and South America (Baldet et al., 2005). The C. obsoletus complex and the C. pulicaris complex transmit BTV in Northern Europe (Bessell et al., 2016). The hematophagous females can transmit a variety of filarial worms, protozoans, and viruses to humans and wild or domestic animals (Borkent, 2004; Meiswinkel et al., 2004b). The expanding distribution of BTV is also facilitated by transplacental transmission and by oral transmission (Wilson and Mellor, 2009; van der Sluijs et al., 2013). In North African countries, BTV was reported in 1999 in Tunisia, and a year later, in Algeria, with 28 outbreaks between July and September 2000 (Hamida, 2000). After the first outbreak in Algeria, the disease clinically affected 2,661 of 21,175 susceptible sheep, in 24 localities in Jijel, which is located in the northeast of the country. The disease continued to spread and reached six districts in the eastern and central parts of the country by the end of the epidemic (Hamida, 2000). Of the several bluetongue outbreaks reported in Algeria, various BTV serotypes have been identified, including serotype 2 (BTV-2) in 2000, and BTV-1 in 2006 and 2008, with new outbreaks in 2009 and 2010 (Madani et al., 2011). In 2014, BTV-1 and BTV-4 were circulating simultaneously in the same temporospatial point (Kardjadj et al., 2016). The increasing prevalence of the disease in Algeria highlights the vector’s competence and persistence and suggests the importance of the identification and surveillance of the vector.
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Identification of Culicoides species in Algeria has been based mostly on the observation of morphological characteristics (EpiReg-Maghreb, 2008; Mathieu, 2011; Borkent, 2014, 2017). However, modern techniques for DNA sequencing are powerful tools for species identification and revealing the genetic variation of small-sized species, including midges (Cêtre-Sossah et al., 2004; Cuellar et al., 2016; Augot et al., 2017; Bakhoum et al., 2018). The present study determined the species identity and geographic distribution of Culicoides midges in Algeria at the morphological and molecular levels and discussed potential vector species of BTV and further transmission of bluetongue in Algeria.
2. Materials and methods
2.1. Insect collection Adult midges were collected from 17 different geographic localities in Algeria from 2009 to 2015 (Table 1; Fig. 1). Midges were collected from areas of either livestock sheds or riversides using 12-V blacklight traps. We did not obtain any records of BTV outbreak in collecting sites in this study. Collected midges were preserved in plastic tubes containing 70% ethanol for further analysis.
2.2. Morphological identification Morphological identification was conducted using the interactive identification keys for female Culicoides (IIKC; Mathieu et al., 2012) available -6-
online at http://www.iikculicoides.net (Fig. 2). The main characteristics, such as veins and colors of the wings of each species, were observed under a light microscope (Olympus, Tokyo, Japan).
2.3. Polymerase chain reaction (PCR) analysis Genomic DNA was extracted from each individual using a PureLink™ Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s recommendations. DNA concentrations were determined using a nanophotometer (Implen, München, Germany). PCR was performed using specific primers for the amplification of COI sequence fragments of Culicoides species
(Mathieu
et
al.,
2012):
forward
primer
(C1J1718)
5′-
GGAGGATTTGGAAATTGATTAGT-3′ and reverse primer (C1N2191) 5′CAGGTAAAATTAAAATATAAACTTCTGG-3′. The amplification reaction mixture) contained 15 µL dNTP premix, 2 µL of each primer, 11 µL of template DNA lysate (30 ng), and a complementary volume of nuclease-free water. A 30µL aliquot of the mixture was amplified in a SimpliAmp® and Veriti® 96-well thermal cycler, under touch-up program for COI amplification: 95 °C for 5 s, 5 cycles (94 °C, 40 s; 45 °C, 40 s; 72 °C, 60 s), followed by 45 cycles (94 °C, 40 s; 50 °C, 40 s; 72 °C, 1 s) and final elongation at 72 °C for 7 min (Mathieu et al., 2012). The PCR products (5 μL) were run on 1% agarose gel and then stained with ethidium bromide for visualization under UV light.
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2.4. DNA sequence analysis PCR products were separated by gel electrophoresis using 1% lowmelting-point agarose, then excised from the gel and purified using a Wizard PCR Preps DNA purification system (Promega, Madison, WI, USA). The purified PCR products were cloned using a T-Blunt vector (Solgent, Daejeon, Korea) and sequenced analyzed at the Solgent Sequencing Facility (Solgent). The GenBank database at the National Center for Biotechnology Information (NCBI) was searched using the BLASTN algorithm (Schaffer et al., 2001), and nucleotide sequences were aligned using ClustalW (Thompson et al., 1994). At least three individuals were analyzed from each group, classified by morphological identification of species. For some species, only 1 or 2 specimens were examined due to the absence of available specimens.
2.5. Clade analysis The determined sequences were manually edited with Chromas® (version 2.31) to produce a consensus sequence of ~400 bp. The sequences were aligned using the multiple sequence comparison by log-expectation (MUSCLE) program in BioEdit© (version 7.2.5). The sequences were compared with publicly available sequences in the NCBI database to establish their relationship. In addition, List of Culicoides species previously reported in Algeria were summarized (Table 3).
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3. Results
3.1. Morphological identification Based on the wing pigmentation patterns of 492 specimens, samples were grouped into 52 batches by wing patterns and geographic area of Algeria.
3.2. Molecular identification Sixty representative specimens from the 52 batches partitioned by species and regions of Algeria were used to amplify 523 bp of partial COI sequences. The 60 sequences identified were submitted to the GenBank database. BLAST analyses showed the highest identity of each sequence (Table 2). The COI sequence variation among all the samples ranged from 0.00% to 22.32% (Table S1). At least 14 species were determined, including nine identified species and five unidentified species. The nine identified species were C. imicola, C. obsoletus, C. kingi, C. puncticollis, C. circumscriptus, C. cataneii, C. sahariensis, C. langeroni, and C. newsteadi. Most of these species were at least 98% identical to specimens reported from other countries in North Africa and Europe. However, C. cataneii was 97.01–97.44% identical to KJ29968 from Tunisia, C. sahariensis was 97.22– 97.44% identical to KJ730003 from Tunisia, and C. langeroni was 96.15% identical to KJ729987 from Tunisia.
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The five unidentified specimens were allocated as Culicoides sp. A, B, C, D, and E (Table 2). A GenBank database analysis showed these species shared the highest identities of 92.37% for Culicoides sp. A to C. imicola (AJ867227) of Morocco; 87.66% for Culicoides sp. B to C. enderleini (KJ833698) of Senegal; 88.46–89.53% for Culicoides sp. C to C. jumineri (KJ729978, KJ729988) of Tunisia; 93.80% for Culicoides sp. D to C. longipennis (MF105764) of Turkey, and 91.86–92.27% for Culicoides sp. E to C. shaklawensis (KT624129) of Slovakia. Intraspecific variation ranges were determined in Algerian populations of some species. For example, the intraspecific variation range of C. imicola was 0.0–1.93% (n = 12), 0.21–1.93% for C. kingi (n = 5), 0.64–2.36% for C. puncticollis (n = 10), 0.21–1.72% for C. circumscriptus (n = 7), 0.0–3.0% for C. sahariensis (n = 5), 0.86–2.36% for C. newsteadi (n = 5), and 0.0–1.72% for Culicoides sp. C (n = 5), as shown in Table S1. While species of C. imicola, C. kingi, C. puncticollis, C. circumscriptus, C. sahariensis, and C. newsteadi were recognized as occurring in various regions of Algeria, C. obsoletus, C. cataneii, C. langeroni, and Culicoides sp. A, B, D, and E were present in only one or two regions of Algeria. Culicoides sp. A and B were even more restricted, occurring only in the Adrar (Q) region, which is a desert area of southern Algeria.
3.3. Phylogenetic relationship of Culicoides species - 10 -
To determine the relationship of the identified species within the genus Culicoides, neighbor-joining analysis of the 409-bp COI sequences (after removing the 5′- and 3′-end regions) was conducted using the 60 sequences identified in this study and the most identical sequences of corresponding Culicoides species obtained from the GenBank database (Table 2; Fig. 2). According to the subgeneric classification of Borkent (2016), 14 species identified in this study were classified into seven subgenera; Avaritia, Remmia, Beltranmyia, Monoculicoides, Sensiculicoides, Oecacta, and Culicoides (Fig. 2). Three species (C. imicola, C. obsoletus, Culicoides sp. A) were grouped into the subgenus Avaritia cluster (Fig. 2). Culicoides imicola exhibited variabilities of 7.94–8.80%, at least 15%, and 16.09–16.31% with Culicoides sp. A (KX853276), C. obsoletus (KX853233), and Culicoides sp. B, respectively (Table S1). Two species (C. kingi, Culicoides sp. B) grouped in the subgenus Remmia and were closely related to the subgenus Avaritia. Two sequences (KX853277, KX853278) of Culicoides sp. B were identical and showed variabilities of 12.66% with C. enderleini (KJ833698), 14–15% with C. kingi, but just 8.37% with Culicoides sp. A. Both C. circumscriptus and C. puncticollis were closely related to each other, with only 16–17% COI sequence variation between them, and belonged to the subgenera Beltranmyia and Monoculicoides, respectively. Five species (C. langeroni, C. cataneii, Culicoides sp. C, Culicoides sp. D, Culicoides sp. E) were - 11 -
closely related to each other and belonged to the subgenus Sensiculicoides. Both C. sahariensis and C. newsteadi were closely related to each other and grouped in the subgenera Oecacta and Culicoides, respectively.
4. Discussion The molecular analysis of Culicoides species in Algeria determined the presence of at least 14 species, including five unidentified species. Both C. imicola and C. obsoletus belong to the subgenus Avaritia and are considered as vectors of BTV. Previous studies have indicated that C. imicola is the major vector of BTV in Africa (Baldet et al., 2005) and the Mediterranean basin (Carpenter et al., 2015), while C. obsoletus is considered to be a putative vector of BTV in northern Europe (Ninio et al., 2011). In the present study, C. imicola was collected from nine different locations (A, B, E, F, H, I, L, N, and P in Table 1) of Algeria. Although they were widely distributed around the country, intraspecific divergence was at least 1.93%. This result suggests that the wide distribution of vector species poses a potentially dangerous situation for the spread of BTV throughout the country. While C. obsoletus has been previously identified in Algeria in 1952 (EpiReg-Maghreb, 2008), the present study revealed that its presence was molecularly identified from Ain Defla (B), and morphologically identified from two additional locations, Tizi Ouzou (E) and Souk Ahras (K). Culicoides kingi was grouped in the subgenus Remmia and closely related to the subgenus Avaritia (Borkent, 2014). Culicoides kingi is one - 12 -
of the C. schultzei species complex (Cornet and Brunhes, 1994). However, this species complex is known to play a minor role in BTV transmission than C. imicola (Meiswinkel et al., 2004a). This species was distributed in northern interior regions (E and L) and the southern region (P) of Algeria. Both C. puncticollis and C. circumscriptus are widely distributed in Algeria but might not be genetically diverse because they had 98–99% similarity to individuals from Tunisia and European countries. Both species have been reported in Algeria previously (EpiReg-Maghreb, 2008). The transmission of BTV by C. puncticollis, but not C. circumscriptus, has been documented (Meiswinkel et al., 2004a; Wilson and Mellor, 2009). One sample collected from Constantine (F) in northeastern Algeria aligned with C. langeroni from Tunisia, sharing 95% identity and belonging to the subgenus Sensiculicoides. It was first reported in Algeria in 1921 (Mathieu, 2011). Both C. sahariensis and C. newsteadi were clustered together but belonged to the subgenera Oecacta and Culicoides, respectively (Borkent, 2014). This study showed that C. sahariensis was distributed in coastal, internal, and southern regions of Algeria, and genetically close to a Tunisian strain. Culicoides newsteadi shares high identity with C. halophilus from northern Europe, including Sweden, Slovakia, and Denmark. Recent molecular evidence has demonstrated that C. halophilus is identical to C. newsteadi N3 within the C. newsteadi species complex (Soren and Kristensen, 2015; Foxi et al., 2016). Culicoides newsteadi was reported in Algeria in 1921 (EpiReg-Maghreb, 2008), and the present study - 13 -
revealed its presence at two coastal locations Mostaganem (A) and Tizi Ouzou (E), and the western highland Bayadh-Rogassa (M). This species is identified as a BTV-competent species in Italy (Foxi et al., 2016). This study determined the COI sequences of five unidentified specimens in Algeria, allocated as Culicoides sp. A, B, C, D, and E. These species identities are uncertain because their COI sequence variations were more than 9% when compared with the most similar sequences in the GenBank database. For example, Culicoides sp. C shared 88.46–89.53% similarity to C. jumineri (KJ729988) of Tunisia. Five specimens were collected from Tizi Ouzou (E) and Constantine (F), which are located in the northern region of Algeria. The similarity of seven COI sequences (KJ729977–KJ729982 and KJ729988) of C. jumineri in the GenBank database was 91.82% (data not shown). Our results showed that the intraspecific variation of five Algerian individuals was 1.72% (Table S1). These results suggest that Culicoides sp. C is outranged to the genetic variation of C. jumineri. Further study is required to determine the identity of this species. Culicoides sp. E shared 91.86–92.27% sequence similarity to C. shaklawensis of Slovakia, which is the only sequence of this species listed in the GenBank database. It has not been reported previously in Algeria, which is in agreement with the distribution map in IIKC. This species was collected from Tizi Ouzou (E) and Constantine (F) in Algeria. Its potential role in the transmission of BTV is not known.
- 14 -
The phylogenetic analysis of 14 species determined from the present study showed clustering of seven subgenera, namely Avaritia, Remmia, Beltranmyia, Monoculicoides, Sensiculicoides, Oecacta, and Culicoides. Five species (C. langeroni, C. cataneii, Culicoides sp. C, Culicoides sp. D, Culicoides sp. E) were closely related to each other and belonged to the subgenus Sensiculicoides. However, this subgenus showed great genetic diversity among species that are grouped into various subclades. All the subgenera were monophyletic, as mentioned previously (Borkent, 2016; Augot et al., 2017). This clustering is consistent with information from the world species catalog of biting midges (Borkent, 2014, 2016). Due to its large geographical area, Algeria might experience a high transboundary and trans-Mediterranean exchange of infected vectors with neighboring countries, such as Morocco and Tunisia. There is 1,622 km of coastline along the Mediterranean basin that is exposed to Europe, which allows spread and invasion between Africa and Europe, especially northern Africa. Simultaneously, there is movement of people and goods between North African and Asian countries. Among the 1,350 known Culicoides species, approximately 50 species are recognized as being involved in BTV transmission (Wilson and Mellor, 2009). Previously, 45 Culicoides species have been identified in Algeria, and 50 and 23 different Culicoides species in Morocco and Tunisia, respectively (EpiRegMaghreb, 2008). The present study confirmed the existence of at least five viruscompetent species in Algeria (C. imicola, C. obsoletus, C. puncticollis, C. kingi, - 15 -
and C. newsteadi). However, the geographical distribution of these five species appears to cover numerous regions in the context of our collected samples. Previous studies have demonstrated the biological sensitivity of Culicoides vectors and BTV to climate is due to the dynamic and climate-mediated vector ability of Culicoides species (Purse et al., 2008). Algeria is affected by temperature increases and an increased frequency of flood events owing to climate change (Sahnoune et al., 2013), creating a favorable environment for midge populations and increasing the chance for BTV to spread to new regions of the country. In conclusion, the present study reaffirmed the identification of at least 14 Culicoides species and their geographic distribution in Algeria, with the existence of at least five BTV-competent species spread over a relatively large distribution area. Thus, the potential impact of BTV dispersal is real. In the midst of climate change in Algeria, further studies are recommended to increase the knowledge regarding the distribution and competence of Culicoides species in the transmission of BTV, considering that bluetongue disease is highly climatesensitive.
Funding This work was supported by the Research Program for Exportation Support of Agricultural Products, Animal and Plant Quarantine Agency, Republic of Korea [grant number Z-1543086-2017-21-01]. - 16 -
Acknowledgments We thank Dr. Jae-Kyoung Shim at Kyungpook National University, Republic of Korea, for her help with DNA sequencing and data analysis.
References
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Figure legends
Figure 1. Geographic distribution of collected samples in Algeria. Collected regions were labeled from A to Q.
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- 25 -
Figure 2. Neighbor-joining analysis of COI sequences of Culicoides species in Algeria. Numbers adjacent to the branches indicate the bootstrap values of 1000 replicates. Each branch was labeled with the species name, GenBank accession number, and country name. Scale bars indicate the number of substitutions per site. Sequences labeled with small circles were most similar sequences in the search of the GenBank database.
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Table 1. Geographic information on collected Culicoides midges in different regions of Algeria. Location
Geographic coordinates
Altitude (m)
Sample number
Mostaganem (A)
35°55.869′ N, 0°5.3508′ E
102
206
Ain Defla (B)
36º16'7.87" N, 1º58'48.92" E
264
21
Algiers (C)
36º44'28.68" N, 3º04'24.22" E
186
15
Medea (D)
36º16'55.7" N, 2º45'37.02" E
910
6
Tizi Ouzou (E)
36º48'10.45" N, 4º03'43.79" E
206
47
Constantine (F)
36°21.9′ N, 6°36.8832′ E
574
114
Skikda (G)
36°52.5702′ N, 6°54.5526′ E
24
3
Annaba (H)
36°54′ N, 7°46.0002′ E
5
5
Guelma (I)
36º31'15.2" N, 7º27'24.98" E
305
2
El Tarf (J)
36º45'0.37" N, 8º16'11.74" E
24
3
Souk Ahras (K)
36º16'23.81" N, 7º58'4.09" E
697
2
Tebessa (L)
35º25'22.26" N, 8º05'58.7" E
867
7
Bayadh-Rogassa (M)
34°01'3.60" N, 0°55'19.79" E
1090
15
Bayadh-Ghassoul (N)
33º23'0.66" N, 1º12'40.36" E
1129
12
Laghouat (O)
33º46'53.95" N, 2º51'13.2" E
764
12
Ouargla(P)
32º12'55.27" N, 4º57'7.4" E
138
13
Adrar (Q)
27º52'15.18" N, 0º15'19.67" W
257
10
- 27 -
Table 2. Accession numbers of 60 COI sequences of collected Culicoides midges in various regions of Algeria and their identity searched in the GenBank database. Clade
Code name
Collection site
Accession number
Species
Highest sequence identity with GenBank database Identity (%)
Accession number
Country
A
C1.1/O D22/M-1 I12/5.4 L13/8 D17/H1 D16.1/A4 H1/3.1 I12/I1I4 E13/J1J4 C15/O-2 K10/I-6 N14/6 C9.1/E-2 J14.3/4.2
Ain Defla (B) Constantine (F) Ouargla (P) Guelma (I) Constantine (F) Constantine (F) Bayadh-Ghassoul (N) Ouargla (P) Mostaganem (A) Ain Defla (B) Tizi Ouzou (E) Tebessa (L) Ain Defla (B) Adrar (Q)
KX853221 KX853222 KX853223 KX853224 KX853225 KX853226 KX853227 KX853228 KX853229 KX853230 KX853231 KX853232 KX853233 KX853276
C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. imicola C. obsoletus C. sp. A (S. imicola)*
99.79 99.79 100 99.79 99.36 99.15 99.15 99.79 98.94 99.36 99.15 99.58 99.79 92.37
AJ867226 ⸗ AJ867227 ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ JQ620130 AJ867227
Spain ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ ⸗ Sweden Morocco
B
J14.2/4.4 J14.2/4.2 I12/5.3 I12/5.2 I11.1/U K9/H.3 N11/1.2 P3-1/X E6.1/M-2 L13/8.1 D13.1/C4 D18.2/C D18.2/3-1 T20/H1
Adrar (Q) Adrar (Q) Ouargla (P) Ouargla (P) Ouargla (P) Tizi Ouzou (E) Tebessa (L) Laghouat (O) Mostaganem (A) Guelma (I) Constantine (F) Constantine (F) Constantine (F) Mostaganem (A)
KX853277 KX853278 KX853234 KX853235 KX853236 KX853237 KX853238 KX853253 KX853254 KX853255 KX853256 KX853257 KX853258 KX853259
C. sp. B (C. enderleini) C. sp. B (C. enderleini) C. kingi C. kingi C. kingi C. kingi C. kingi C. puncticollis C. puncticollis C. puncticollis C. puncticollis C. puncticollis C. puncticollis C. puncticollis
87.66 88.66 99.58 99.79 99.36 98.73 98.29 99.58 98.94 99.58 99.58 99.36 99.36 98.94
KJ833698 ⸗ MF399780⸗ ⸗ ⸗ ⸗ ⸗ KJ624120 ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
Senegal ⸗ Cameroon ⸗ ⸗ ⸗ ⸗ Slovakia ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
- 28 -
K11.1/G D21.2/F1 E6.3/N-1
Tizi Ouzou (E) Constantine (F) Mostaganem (A)
KX853260 KX853261 KX853262
C. puncticollis C. puncticollis C. puncticollis
99.36 98.30 99.79
⸗ ⸗ ⸗
⸗ ⸗ ⸗
C
J14.1/E1 I12/I2 T19/G K11.2/W-2 E13/J2 B2.1/D D16.1/A
Adrar (Q) Ouargla (P) Mostaganem (A) Tizi Ouzou (E) Mostaganem (A) Annaba (H) Constantine (F)
KX853263 KX853264 KX853265 KX853266 KX853267 KX853268 KX853269
C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus C. circumscriptus
98.73 98.94 99.15 99.36 98.94 99.15 99.79
KJ624071 ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
Slovakia ⸗ ⸗ ⸗ ⸗ ⸗ ⸗
D
D18/10.3 D18/10.1 D18.1/B K9/H-1 D19.1/D2 K9/H.4 D21.2/F2F3F4 D17/H2H3H4 E5.1/L-2 K14/G N11/1.1 D16.1 /A2 C9.3/P-3 J14.2/4.3
Constantine (F) Constantine (F) Constantine (F) Tizi Ouzou (E) Constantine (F) Tizi Ouzou (E) Constantine (F) Constantine (F) Mostaganem (A) Tizi Ouzou (E) Tebessa (L) Constantine (F) Ain Defla (B) Adrar (Q)
KX853239 KX853240 KX853241 KX853242 KX853243 KX853244 KX853245 KX853279 KX853280 KX853246 KX853247 KX853248 KX853249 KX853250
C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. sp. C (C. jumineri) C. cataneii C. cataneii C. sp. D (C. longipennis) C. sp. D (C. longipennis) C. sahariensis C. sahariensis C. sahariensis C. sahariensis C. sahariensis
88.46 88.46 89.32 89.32 89.53 97.44 97.01 93.80 93.80 97.22 97.44 97.44 97.44 97.22
KJ729988 ⸗ KJ729978 ⸗ ⸗ KJ729968 ⸗ MF105764 ⸗ KJ730003 ⸗ ⸗ ⸗ ⸗
Tunisia ⸗ Tunisia ⸗ ⸗ Tunisia ⸗ Turkey ⸗ Tunisia ⸗ ⸗ ⸗ ⸗
E
D20/G2 K9/H-2 D10/S-1 E7.1/A3 E8/B2 E5.1/S-2 K3/9.2
Constantine (F) Tizi Ouzou (E) Constantine (F) Mostaganem (A) Mostaganem (A) Mostaganem (A) Tizi Ouzou (E)
KX853251 KX853252 KX853275 KX853270 KX853271 KX853272 KX853273
G4/7.1
Bayadh-Rogassa (M)
KX853274
C. sp. E (C. shaklawensis) C. sp. E (C. shaklawensis) C. langeroni C. halophilus C. halophilus C. halophilus C. halophilus C. newsteadi N3 C. halophilus C. newsteadi N3
92.27 91.86 96.15 98.08 97.87 98.29 98.08 97.87 99.15 98.93
KJ624129 ⸗ KJ729987 JF766298 ⸗ ⸗ ⸗ JQ620119 JF766298 JQ620119
Slovakia ⸗ Tunisia Denmark ⸗ ⸗ ⸗ Sweden Denmark Sweden
*Species within the parenthesis were the most similar species in COI identity in the GenBank database.
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Table 3. List of Culicoides species previously reported in Algeria. No
Species
Subgenus
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14
C. algeriensis Clastrier, 1957: 426. Algeria. C. begueti Clastrier, 1957: 432. Algeria. C. cataneii Clastrier, 1957: 438. Algeria. C. citrinellus Kieffer, 1923a: 674. Algeria (as C. sergenti) C. distigma Kieffer, 1922g: 502. Algeria (as C. parroti) C. donatieni Kieffer, 1922g: 504. Algeria (as C. parroti) C. foleyi Kieffer, 1922g: 503. Algeria. C. griseidorsum Kieffer, 1918a: 46. Algeria. C. kabyliensis Kieffer, 1922g: 505. Algeria (as C. obsoletus) C. marcleti Callot, Kremer and Basset, 1968: 271. Algeria C. montanus (obsoletus group) C. saevus Kieffer, 1922g: 506. Algeria. C. sahariensis Kieffer, 1923a: 678. Algeria. C. semimaculatus Clastrier, 1958a: 55. Algeria
Sensicullicoides Sensicullicoides Unplaced species Oecacta Monoculicoides Monoculicoides Unplaced species Sensicullicoides Avaritia Oecacta Avaritia Pontoculicoides Oecacta Oecacta
Borkent, 2016
15 16 17 18 19 20 21 22 23 24 25 26
C. azerbajdzhanicus Dzhafarov, 1962 - DZ C. faghihi Navai, 1971 C. imicola (imicola group) C. kingi Austen, 1912 - DZ C. langeroni Kieffer, 1921 C. nudipennis Kieffer, 1922g: 507. Algeria C. pallidus Khalaf, 1957 C. pseudolangeroni Kremer, Chaker and Delecolle, 1981 DZ C. pseudopallidus Khalaf, 1961 - DZ C. ravus De Meillon, 1936 C. sejfadinei Dzhafarov, 1958 C. sergenti Kieffer, 1921
Oecacta Wirthomyia Avaritia Oecacta Sensicullicoides Unplaced species Oecacta Unplaced species Sensicullicoides Synhelea Pontoculicoides Oecacta
Mathieu, 2011
Beltranmyia Oecacta Culicoides Sensicullicoides Sensicullicoides Sensicullicoides Sensicullicoides Oecacta Sensicullicoides Culicoides Selfia Sensicullicoides Sensicullicoides Culicoides Monoculicoides Oecacta Avaritia Pontoculicoides Silvaticulicoides
EpiRegMaghreb, 2008
27 C. circumscriptus Kieffer, 1918 28 C. dzhafarovi Remm, 1967 29 C. fagineus Edwards, 1939 30 C. gejgelensis Dzhafarov, 1964 31 C. heteroclitus Kremer & Callot, 1965 32 C. jumineri Callot & Kremer, 1969 33 C. kibunensis Tokunaga, 1937 34 C. longipennis Khalaf, 1957 35 C. maritimus Kieffer, 1924 36 C. newsteadi Austen, 1921 37 C. odiatus Austen, 1921 38 C. pictipennis (Staeger, 1839) 39 C. poperinghensis Goetghebuer, 1953 40 C. pulicaris (Linné, 1758) 41 C. puncticollis (Becker, 1903) 42 C. santonicus Callot, Kremer, Rault & Bach, 1966 43 C. scoticus Downes & Kettle, 1952 44 C. sejfadinei Dzhafarov, 1958 45 C. subfasciipennis Kieffer, 1919 * Alphabetical order of species in each reference has been arranged.
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Graphical abstract
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