Acta Tropica 142 (2015) 5–19

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First overview of the Culicoides Latreille (Diptera: Ceratopogonidae) livestock associated species of Reunion Island, Indian Ocean A. Desvars a,b,1,2 , Y. Grimaud c,1 , H. Guis a,b , O. Esnault c , X. Allène a,b , L. Gardès a,b , T. Balenghien a,b , T. Baldet a,b , J.C. Delécolle d , C. Garros a,b,∗ a

Cirad, UMR15 Contrôle des Maladies Animales Exotiques et Emergentes, Montpellier, France INRA, UMR1309 Contrôle des Maladies Animales Exotiques et Emergentes, Montpellier, France c Groupement de Défense Sanitaire, La Plaine des Cafres, La Réunion, France d Institut de Parasitologie et de Pathologie Tropicale de Strasbourg (IPPTS), Strasbourg, France b

a r t i c l e

i n f o

Article history: Received 1 July 2014 Received in revised form 20 October 2014 Accepted 26 October 2014 Available online 1 November 2014 Keywords: Biting midges Vector species Livestock Species diversity Cytochrome oxidase subunit I Indian Ocean

a b s t r a c t This study establishes the first faunistic inventory of livestock associated Culicoides (Diptera: Ceratopogonidae) species of Reunion Island (Indian Ocean), where bluetongue and epizootic hemorrhagic disease are regularly recorded. Single night-catches were performed at 41 sites using light suction traps at altitudes ranging from 0 to 1525 m, from March to April 2005. Five species were recorded: Culicoides imicola, Culicoides bolitinos, Culicoides enderleini, Culicoides grahamii, and Culicoides kibatiensis, among which at least the first three species are known to be involved in virus transmission to ruminants and equids. This is the first record of C. bolitinos, C. kibatiensis, and C. enderleini on the island. C. imicola was the most abundant species along the sea coast. C. bolitinos was more abundant inland and on two sites on the east coast. C. kibatiensis and C. grahamii were less abundant than the other three species and limited to two foci. Spatial distribution analysis of the different species showed that C. bolitinos, C. enderleini and C. imicola were collected at low altitudes, while the other two species were found at higher altitude. A morphological identification key for adult females and males is given, as well as cytochrome oxydase subunit I sequences. Phylogenetic reconstructions showed a clear divergence between C. bolitinos from Reunion Island and mainland Africa. This monograph will help to identify the Culicoides species in the poorly known entomological fauna of the south-western Indian Ocean region. © 2014 Elsevier B.V. All rights reserved.

1. Introduction More than 50 arboviruses have been isolated from Culicoides Latreille (Diptera: Ceratopogonidae) (Mellor et al., 2000). In certain areas, biting nuisance caused by Culicoides impedes the development of agricultural activities and tourism (Carpenter et al., 2013). Several Culicoides-borne viral diseases of veterinary importance are endemic to the Afrotropical region. Bluetongue (BT), African horse sickness (AHS), and epizootic hemorrhagic disease (EHD) have been reported in the east and south of mainland Africa: South

∗ Corresponding author at: Cirad, UMR15 CMAEE, Campus International de Baillarguet, TA/A15 G, 34398 Montpellier cedex 5, France. Tel.: +0033 4 67 59 39 96; fax: +0033 4 67 59 37 98. E-mail address: [email protected] (C. Garros). 1 Equal contributors. 2 Current address: Department of Clinical Microbiology, Clinical Bacteriology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden. http://dx.doi.org/10.1016/j.actatropica.2014.10.018 0001-706X/© 2014 Elsevier B.V. All rights reserved.

Africa (Barnard, 1998; Coetzee et al., 2012; Mellor and Boorman, 1995), Uganda (Mulabbi et al., 2013), Kenya (Toye et al., 2013), Namibia (Scacchia et al., 2009), and Zimbabwe (Blackburn and Swanepoel, 1998), and in insular territories of the South-West Indian Ocean region (Cêtre-Sossah et al., 2014; Jori et al., 2011; Sailleau et al., 2012). In many sub-Saharan African countries, viruses of BT and EHD (respectively, referred as BTV and EHDV) do not induce severe clinical signs. Indeed, indigenous breeds appear to have achieved a balance with the virus (Gerdes, 2004), unlike European ruminant breeds that develop severe clinical signs. Thus, in the sub-Saharan African endemic region, these two diseases are not considered as a priority for surveillance and control and are therefore greatly under-reported. AHS virus (AHSV) induces high mortality rates in domestic equids, while remains sub-clinical in donkeys and zebras (Mellor, 1993). In the South-West Indian Ocean region, the development of ruminant productions for milk and meat with improved European breeds has renewed the interest in Culicoides and their associated

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viruses (Reunion Island records some horses and donkeys but no wild equids nor ruminants). Yet, the epidemiological situation for Culicoides-borne viruses in the South-West Indian Ocean remains poorly described. The published entomological surveys for the different insular territories of the South-West Indian Ocean region are fragmentary.

Culicoides imicola Kieffer, 1913, (=Culicoides pallidipennis Carter, Ingram and Macfie, 1920) was recorded for the first time from Reunion in 1959 (Clastrier, 1959) (Fig. 1). Its presence was confirmed by Barré et al. (1985) who also recorded Culicoides grahamii Austen, 1909. On Mauritius, an island close to Reunion Island (Fig. 1), C. imicola and Culicoides enderleini Cornet and Brunhes

Fig. 1. Map of the South-West Indian Ocean region localizing Reunion Island and the 41 study sites, and nearby islands.

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(1994) were collected in deer farms (Jori et al., 2011) while Boorman and Mellor (1992) reported the presence of Culicoides brevitarsis Kieffer, 1917. In Madagascar, C. imicola and species of the Schultzei group have been collected, however, the Culicoides species diversity is likely to be underestimated taking into account the size of the island and its unique ecological diversity (Augot et al., 2013; Cornet and Brunhes, 1994; De Meillon, 1961). Two species were recorded for Seychelles: Culicoides kusaiensis Tokunaga, 1940, present in Micronesia and Culicoides leucostictus Kieffer, 1911, considered as an Afrotropical species (Lambrecht, 1970; Wirth and Messersmith, 1977). Cornet and Brunhes (1994) collected two species in Mayotte (within Comoros archipelago), C. enderleini and C. rhizophorensis Khamala and Kettle (1971). The first clinical BT cases in Reunion Island were diagnosed in Lacaune sheep in 1979 (Barré et al., 1985) and were due to serotypes 2 and 4 (BTV-2 and -4). In January 2003, EHDV-2, -6, and -7 caused a severe epizootic in several cattle herds (Bréard et al., 2004). The same year, a clinical outbreak caused by BTV3 was recorded in a flock of Merino sheep (Bréard et al., 2005). During a separate EHD outbreak occurring in 2009, high EHDV-6 seroprevalence (91%) was found in cattle with 4% of the sampled cattle both EDHV-6 and BTV-2 seropositive (Sailleau et al., 2012). In 2011, a survey among 489 cattle and sheep showed a seroprevalence of 58% for BTV and 38% for EHDV, as well EHDV/BTV co-infections, confirming the high exposure of Reunion Island livestock to these two viruses (Cêtre-Sossah et al., 2014). In addition, in 2011 two EHDV outbreaks occurred in the central part of Reunion Island and serotype EHDV-1 was newly identified (Cêtre-Sossah et al., 2014). These results highlight the diversity of Culicoidesborne pathogens affecting livestock on this island and possibly in the whole region. Moreover, the Rusa deer (Cervus timorensis rusa) populations in Reunion and Mauritius could play a role in both BTV and EHDV epidemiology since this species could act as a reservoir with long viremia for both viruses (Savini et al., 2011). Indeed, in Mauritius, Rusa deer were found positive for BTV-2, 17, -10, -21 and EHDV in 2007 (Jori et al., 2011). AHS is absent from the all the insular territories of the South West Indian ocean although regular epizootics are notified from nearby continental African countries. This study aims to (i) describe the diversity of livestock associated Culicoides species on Reunion Island and (ii) establish their geographical and altitudinal distribution, in order to provide a greater understanding of Culicoides-borne disease epidemiology on Reunion Island but also in the wider South-West Indian Ocean region. For the first time, a dichotomous morphological identification key is provided together with cytochrome oxidase subunit I (COI) sequences.

2. Materials and methods 2.1. Study sites and Culicoides collection Reunion Island is a volcanic island of 2510 km2 in the south-west of the Indian Ocean, 800 km east from Madagascar (Fig. 1). It is a mountainous island (highest point 3069 m) with a tropical climate characterized by a rainy season (austral summer) from December to mid-April and a dry season (austral winter) between mid-April and November. Precipitations range from 100 to 300 mm during the dry season. Annual rainfall ranges from 600 to 2000 mm on the western coast and from 2000 to 8000 mm on the eastern coast. Winds come mainly from the eastern coast. Temperatures are closely linked with altitude. The mean annual temperature ranges from 20.5 to 26 ◦ C below 500 m; from 16 to 21 ◦ C at altitude 500–1000 m; from 12 to 17 ◦ C at altitude 1000–2000 m; and is ≤12 ◦ C above 2000 m (Raunet, 1991). Livestock farming is well-developed with several

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Fig. 2. Light suction trap (Rieb type).

European improved breeds of ruminants (Prim’Holstein cattle for milk production, Limousine, Blonde d’Aquitaine cattle, Boer goats, Mérinos and Romane sheep for meat production, data from the French Ministry of Agriculture). Horses are mostly grouped in 30 riding centers spread on the island, and 550 individuals are currently registered. Culicoides collections were conducted at the end of the warm rainy season between 03/05/2005 and 04/21/2005, using light suction traps (4 W, 12 V) as described by Rieb (1982) (Fig. 2), operating from dusk until dawn. Traps were placed outdoors, 1–1.50 m high, sheltered from wind and far from source of light, adjacent to stables, animal sheds or animal manure. For optimal preservation and identification of anatomical structures, all insects were collected in water containing drops of liquid soap. Selected sites (n = 41) included one zoological park, eight horse farms, 13 cattle farms, two sheep farms, and 17 farms with mixed animal production, including two sites with presence of deer (Table 1). Selected sites were distributed throughout livestock farming areas of the island. The south-eastern region was not sampled because it corresponds to the region of the active Piton de la Fournaise volcano, without any farming activity (Fig. 1). Altitude of the study sites varied from 0 to 1525 m above sea level (Fig. 1, Table 1). Each site was sampled once under favorable climatic conditions (absence of heavy rain and/or wind). Insects were kept at 4 ◦ C in 95% alcohol until their identification. All Culicoides were morphologically identified under a stereomicroscope to species level based on identification keys relevant for the Afrotropical region (Cornet and Brunhes, 1994; Glick, 1990; Meiswinkel, 1989). Parts of the specimens were selected and the head, the lower part of the abdomen and one wing were slidemounted to assist species determination and to take pictures. Sex was also recorded.

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Table 1 Locations of the collection made using a light suction trap (Rieb type) operated during March–April 2005 and description of the surrounding environment. The main activity of the farm is specified. Altitude is expressed in meters; latitude and longitude are expressed in decimal degrees; the different animals observed in the area surrounding the trap are described. Site number

Primary activity

Altitude

Latitude

Longitude

Host(s) present at site

Environment surrounding the site

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Zoo Riding center Dairy farm Dairy farm Deer and beef farm Riding center Beef and goat farm Riding center Sheep farm Mixed farm Riding center Beef farm Beef farm Mixed farm Riding center Riding center Mixed farm Beef farm Beef farm Beef farm Beef farm Dairy farm Beef farm Sheep farm Riding center Dairy farm Dairy farm Mixed farm Dairy farm Dairy farm Beef farm Deer farm Dairy farm Riding center Beef farm Dairy farm Dairy farm Mixed farm Beef farm Sheep farm Beef farm

0 358 274 645 299 76 182 60 243 1112 354 770 420 1031 22 482 487 841 1049 1256 1057 1513 1118 836 687 1177 873 1525 1372 1237 515 1362 910 26 10 776 142 125 350 267 89

−20.89 −20.90 −20.93 −20.93 −20.94 −20.95 −20.95 −20.99 −21.03 −21.03 −21.05 −21.06 −21.06 −21.07 −21.07 −21.10 −21.10 −21.11 −21.13 −21.14 −21.15 −21.15 −21.15 −21.16 −21.18 −21.19 −21.20 −21.20 −21.21 −21.23 −21.23 −21.24 −21.26 −21.27 −21.28 −21.31 −21.33 −21.34 −21.36 −21.36 −21.36

55.49 55.40 55.50 55.46 55.52 55.32 55.61 55.68 55.66 55.48 55.27 55.52 55.27 55.32 55.23 55.67 55.67 55.31 55.61 55.49 55.46 55.35 55.44 55.32 55.33 55.36 55.35 55.58 55.56 55.55 55.44 55.57 55.55 55.36 55.37 55.56 55.49 55.51 55.61 55.57 55.54

Equids, poultry, lama, cattle, deer Equids Cattle, poultry Cattle, goat, poultry Deer, cattle Equids Cattle, goat Equids Sheep, equids Cattle, poultry, goat Equids Cattle Cattle Sheep, poultry, cattle Equids Equids Pig, cattle, goat, poultry, sheep Cattle Cattle, goat Cattle Cattle Cattle Cattle Sheep Equids Cattle Cattle Sheep, poultry Cattle Cattle, poultry, pig Cattle Deer, wild boar Cattle, goat, wild boar, poultry Equids Cattle Cattle, goat, poultry, pig Cattle, poultry, pig Sheep, poultry, pig Cattle, sheep, goat, equids, poultry Sheep Cattle

Urban area Forest Urban area Sugar cane fields Sugar cane fields Urban area Sugar cane fields Urban area Sugar cane fields Geranium plantation, meadows, fallow lands Sugar cane fields Geranium plantation, meadows, fallow lands Sugar cane fields Sugar cane fields Urban area Geranium plantation, meadows, fallow lands Geranium plantation, meadows, fallow lands Sugar cane fields Geranium plantation, meadows, fallow lands Geranium plantation, meadows, fallow lands Geranium plantation, meadows, fallow lands Sugar cane fields Geranium plantation, meadows, fallow lands Urban area Sugar cane fields Sugar cane fields Sugar cane fields Geranium plantation, meadows, fallow lands Geranium plantation, meadows, fallow lands Geranium plantation, meadows, fallow lands Sugar cane fields Geranium plantation, meadows, fallow lands Sugar cane fields Savannah Savannah Sugar cane fields Sugar cane fields Sugar cane fields Sugar cane fields Sugar cane fields Sugar cane fields

2.2. DNA extraction, COI amplification, and sequencing Genomic DNA was individually extracted following an extraction protocol with Chelex resin in 5% (Resin Chelex100® , Chelating Ion Exchange Resin) as previously described (Solano et al., 1997; Viennet et al., 2011). A volume of 500 ␮l of Chelex solution was dispensed into each tube of 1.5 ml. Culicoides were individually removed from alcohol and dried on blotting paper. With a sterile toothpick, each Culicoides was then transferred and placed in a tube with Chelex solution and homogenized using a piston. Tubes were then heated at 56 ◦ C for 60 min and then at 95 ◦ C for 30 min (for thermal lyse). Immediately after heating, the tubes were centrifuged at 13,000 r/min for 1 min to pellet the Chelex resin with inhibitor ions and cellular debris. Cytochrome Oxidase subunit I (COI) gene amplification was carried out using primers C1J1718 (forward) and C1N2191 (reverse) to obtain an approximately 600 bp product as described previously (Dallas et al., 2003). Amplification reactions by Polymerase Chain Reaction (PCR) were performed in 25 ␮l of reaction volume with 5 ␮l of buffer 5×, 0.5 ␮l of dNTP (10 mM), 2 ␮l of MgCl2 (25 mM), 0.5 ml of each primer; 0.5 ␮l of Taq polymerase (5 U/␮l), 15 ␮l of sterile water and 1 ␮l of template DNA. PCR amplification in touchdown was used to reduce non-specific amplifications. The cycling profile of the COI gene consisted of an initial denaturation stage of 1 min at 94 ◦ C, followed by 5 cycles of 40 s at 94 ◦ C, 40 s at 45 ◦ C and

1 min at 72 ◦ C, then 35 cycles of 40 s at 94 ◦ C, 40 s at 51 ◦ C, 1 min at 72 ◦ C and a final elongation of 5 min at 72 ◦ C. The PCR products were visualized on 1% agarose gel with a Gel Red staining after migration of 90 min at 100 volts by electrophoresis, before being sent to Cogenics (Grenoble, France) for bidirectional sequencing. 2.3. Phylogenetic reconstructions Alignment of the COI sequences was generated using the CLUSTALW algorithm in BioEdit (Hall, 1999). Summary sequence statistics and molecular evolutionary analyses were conducted using MEGA version 5.2 (Tamura et al., 2011). The phylogenetic reconstructions were performed by Maximum Likelihood (ML). The ML analyses were carried out with MEGA v5.2, incorporating best fit models of sequence evolution determined using the Akaike Information Criterion (AIC) and employing 1000 bootstrap replications to determine node reliabilities. The AIC implemented within jModelTest was used to determine the most suitable evolutionary model(s) for ML analyses (Darriba et al., 2012). Cytochrome Oxidase Subunit 1 sequences from C. enderleini populations from Madagascar (Augot et al., 2013), Senegal and South Africa (Bakhoum et al., 2013) [accession numbers: KF186429–KF186431; KF682478–KF682479; KF682471–KF682477], C. imicola populations from Israel, South Africa (Dallas et al., 2003; Linton et al., 2002) and Spain

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(Calvo et al., 2009) [accession numbers: AF078098–AF078100; AF080531–AF080532, AF080534–AF080535; AF069231, AF069233; DQ868883–DQ868892, DQ868884], C. bolitinos, Meiswinkel (1989), populations from South Africa (Bellis et al., 2014; Linton et al., 2002) [accession numbers: KJ162961–KJ162962; AF071928–AF071931], and C. brevitarsis populations from China, Solomon islands and East Timor (Bellis et al., 2014) [accession numbers: KJ162966–KJ162973] were used in the dataset. No COI sequences for C. grahamii or C. kibatiensis Goetghebuer, 1935 were available in the Genbank database. Culicoides punctatus (Meigen), 1804 was used as outgroup (AM236737) (Nolan et al., 2007).

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2.4. Statistical analysis Several environmental factors (altitude, type of vegetation, Daupi landscape classification, ecological zone, distance to manure, type of soil, hosts present on the site, distance to animals, and geographical position on the island, see Table S1) driving Culicoides midges distribution were investigated using canonical correspondence analysis (Ter Braak, 1986). First, a correspondence analysis was carried out on presence/absence data from the Culicoides collections followed by a multiple correspondence analysis carried out on the environmental parameters of collection sites. Finally, relations between structure of species assemblage and environmental

Table 2 Description of the wing pattern of adult Culicoides present in Reunion Island (available in French upon request to the authors). This atlas is to be used for identification using a stereomicroscope. Species name

Description of the wing pattern

Culicoides bolitinos

Dark wing pattern with well limited pale spots; second radial cell in a pale spot; apex of vein M2 pale or occasionally darkened (morphological variant); usually no pale preapical excision (morphological variant)

Culicoides enderleini

Dark wing pattern with small well limited rounded pale spots; second radial cell entirely dark; cell r5 with 2–4 small pale spots, never attaining the apical wing margin; pale spots at the end of veins M1 , M2 , M3+4 , Cu1

Culicoides grahamii

Gray wing pattern with not well delimited pale spots; second radial cell with pale marking at the apex; post stigmatic pale spot joining the pale marking along vein M1 ; cell r5 with pale spot at the apex

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Table 2 (Continued) Species name

Description of the wing pattern

Culicoides imicola

Dark wing with large pale spots; second radial cell entirely in a pale spot; usually apex of the vein M2 broadly dark preceded by broad pale apical excision but morphological variation exist

Culicoides kibatiensis

Dark wing with large pale spots; second radial cell entirely included in the pale post stigmatic spot; pale spot in cell r5 not reaching the wing margin

drivers were assessed using a permutation test. Statistical analyses were conducted using R version 3.0.2 (R Core Team, 2012).

3. Results A total of 41 sites were sampled during the survey, thirty sites were positive (73.2%) for Culicoides presence. Out of 9920 Ceratopogonidae collected, 3346 (33.7%) were Culicoides (Tables 2 and 3), and 64 of the latter were males (1.9%).

3.1. Morphological identification Five Culicoides species were identified: C. imicola, C. bolitinos, C. grahamii, C. kibatiensis Goetghebuer, 1935, and C. enderleini (Figs. 3 and 4). A dichotomous morphological key for both females and males is presented in Fig. 3, as well as detailed descriptions of wing patterns (Table 2). Culicoides bolitinos, C. enderleini, and C. kibatiensis were described for the first time in Reunion Island, representing new species for the local fauna. Four individuals exhibited wing pattern variations while molecularly identified as C. imicola and C. bolitinos (Figs. 3 and 4). The morphological variant of C. imicola does not have the preapical section excision reaching the vein M2. The morphological variant of C. bolitinos exhibits a dark distal edge of vein M2 (Fig. 3, Table 2).

3.2. Molecular identification Ten females presenting typical wing pattern of the five species were selected and used to produce COI sequences. Sequences were deposited into Genbank database under the following accession numbers: KJ186129–KJ186138. Phylogenetic analyses using the COI sequences generated for typical and variant forms together with published COI sequences revealed six well supported clades (Fig. 4). For each species, populations from the Afrotropical region (Madagascar, Senegal, South Africa) or Mediterranean region (Israel, Spain) nested with the population of Reunion Island except for C. bolitinos. COI analysis suggests that the population of C. bolitinos on Reunion Island may represent a further cryptic species (genetic distance between C. bolitinos from South Africa and Reunion Island, using Jukes Crantor model = 6.4–7.2%). Phylogenetic reconstructions with C. brevitarsis COI sequences (Bellis et al., 2014) do not group with either of the two C. bolitinos clades (Fig. 4), excluding the possibility of misidentifications between these two morphologically close species. 3.3. Species diversity and distribution C. bolitinos and C. imicola were each present at 21 of the 30 positive sites whereas C. kibatiensis, C. enderleini and C. grahamii were found at 11, 8, and 7 sites, respectively. C. imicola represented 78.8% of the Culicoides collected and was the dominant species at 14 of the 30 positive sites, followed by C. bolitinos (11.2%) and C. enderleini

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(6.6%) (Table 3, Fig. 5). None of the sites had the five species in sympatry during the survey. The three most prevalent species (C. imicola, C. bolitinos, and C. enderleini) were sympatric at three sites. C. imicola and C. bolitinos were found in sympatry at 16 sites. C. imicola was collected from sites across the entire island with higher abundance in coastal areas, whereas C. bolitinos was most abundant inland and at three sites in the east part of the island (sites 7, 8, and 9, Fig. 5). C. enderleini and C. grahamii were rarely observed and mostly in low numbers (except at site 13 for C. enderleini and at site 39 for C. grahamii) while C. kibatiensis was the dominant species

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at six sites on the west coast (sites 18, 22, 24, 25, 26, and 27), plus one site inland (site 29). The trap sites were selected to be representative of the different altitudes where farming occurs on the island (10 traps below 0–200 m, 29 traps between 200 m and 1400 m, 2 traps above 1400 m) (Fig. 6). The altitudinal distribution of the species highlighted that C. imicola, C. bolitinos, and C. enderleini are present at low altitude (median altitude 354, 420 and 68 m, respectively) compared to C. kibatiensis and C. grahamii (median altitude 920 and 841 m, respectively). The two latter species were not collected in

Fig. 3. Dichotomous morphological key for adult Culicoides identification present in Reunion Island (available in French upon request to the authors). This key is to be used with slide mounted specimens.

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Fig. 3. (Continued)

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Fig. 3. (Continued)

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Fig. 3. (Continued)

sites below 300 m (Fig. 6). The canonical correspondence analysis demonstrated that altitude was the main driver of species presence. No significant effect of animal host species or environmental conditions around the sites on species presence (type of vegetation, Daupi landscape classification, ecological zone, distance to manure, type of soil, hosts present on the site, distance to animals, and geographical position on the island) was found (data not shown). Nevertheless, animal species present in the surrounding areas were not equally abundant. 4. Discussion This study confirmed the presence of C. imicola and C. grahamii on Reunion Island (Barré et al., 1985; Clastrier, 1959) and reveals the presence of three additional species: C. bolitinos, C. kibatiensis, and C. enderleini. This study provides an updated list of livestock associated Culicoides species present on Reunion Island along with the first morphological keys for these species. This key can be considered as a baseline for further taxonomic work on the Culicoides genus in the South-West Indian Ocean region. However, further field investigations would be required to determine if other species (of veterinary interest or not), especially cryptic species, are present on the island, as well as in the surrounding insular territories. The five Culicoides species recorded during the survey are common and widespread in the Afrotropical region, including Madagascar. C. imicola is the dominant species associated with livestock in Nigeria (Herniman et al., 1983), Bostwana (Mushi et al., 1998), Gambia (Rawlings et al., 1998), South Africa (Labuschagne et al., 2007; Venter et al., 1996), and Zimbabwe (Musuka et al., 2001). It is the second most abundant species in Senegal (Diarra

et al., 2014) and has been recorded in Namibia (Becker et al., 2012, 2013), Central Africa (Itoua and Cornet, 1986; Itoua et al., 1987), and Kenya (Glick, 1990). C. bolitinos is present in Senegal (Bakhoum et al., 2013; Diarra et al., 2014), Zimbabwe (Musuka et al., 2001), and Madagascar (Augot et al., 2013). Thirty-six individuals were identified as C. brevitarsis in Mauritius (Boorman and Mellor, 1992), this species being described as an Oriental and Australasian species. Given its morphological similarity with C. bolitinos, an Afrotropical species (Bellis et al., 2014), this finding should be considered carefully. Our genetic study confirmed the presence of C. bolitinos in Reunion Island. Further field studies would be required to rule on the presence/absence of C. bolitinos in Mauritius. Surprisingly, the molecular analysis suggests that this population is genetically divergent from the mainland populations, while no major morphological differences were observed. This could reflect cryptic diversity or indicate the presence of separate cryptic species. C. enderleini (Schultzei group) is also reported in the Afrotropical region and largely dominant in West Africa (Cornet and Brunhes, 1994). This species was recently presented associated with horses in Senegal (Bakhoum et al., 2013; Diarra et al., 2014), has also been recorded in Gambia (Rawlings et al., 1998), in Sudan (Mellor et al., 1984), and is commonly reported in South Africa (Labuschagne et al., 2007; Venter et al., 1996, 2009), Madagascar (Augot et al., 2013), and Zimbabwe (Musuka et al., 2001). Although not abundant, Culicoides kibatiensis (Imicola group) is mentioned in East Africa (Khamala and Kettle, 1971), Angola (Itoua et al., 1987), Kenya (Glick, 1990) and South Africa (Venter et al., 1996).

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Fig. 4. Maximum likelihood tree inferred from COI sequences using Terminal labels indicate Genbank accession numbers and species. Tree rooted at C. punctatus. Scale bar = 0.02 substitutions per site. Bootstrap values showed are considered as strongly supported.

C. grahamii was collected in East Africa (Khamala and Kettle, 1971), Madagascar (De Meillon, 1961), Gabon (Auriault, 1979), Nigeria (Dipeolu, 1976), Central Africa (Itoua et al., 1987), and Kenya (Davies and Walker, 1974), and Senegal (Cornet, 1969; Diarra et al., 2014). Low species diversity is characteristic of geographically young islands (Whittaker et al., 2008). Recent entomological surveys have also showed limited number of mosquito species (Diptera: Culicidae) on Reunion Island (Boussès et al., 2013), with only 12 species identified. Different factors could explain this low diversity: the young age of this island (3 million years), its relative large distance to the nearest mainland, and its late colonization by livestock and domestic animals (goats were introduced on Reunion Island from Europe in 1629, cattle in 1649, and deer were imported from Mauritius around 1900) (Cheke, 2010). It is probable that the Culicoides species found on Reunion Island were introduced through past animal trade from East Africa or Madagascar (Cheke, 2010). Passive dispersal of Culicoides over long distances is also known to occur (Burgin et al., 2013). Therefore, if other species are present in neighboring territories, there is a risk of introduction and colonization by these species. This highlights the need for faunistic surveys in the neighboring islands, since the eastern coast of Reunion Island is under the wind of Mauritius Island at a distance of 230 km. Microsatellite markers for C. imicola are available (Mardulyn et al., 2013) and genotyping populations in this area could help to understand the genetic structure of the species within the region, and to highlight ongoing gene flows between

the different islands (Kuntner and Agnarsson, 2011; Simard et al., 1999). The present survey revealed an uneven spatial distribution of the collected species which may be explained by local environmental and meteorological conditions. Nearly one third of the midges were collected at one site (site 13). Culicoides abundance is highly variable and may be linked to different meteorological conditions (Diarra et al., 2014; Meiswinkel et al., 2014). C. imicola is the most abundant livestock associated species on Reunion Island and was almost seven times more abundant than C. bolitinos in livestock-associated areas, which supports previous South African studies showing that the estimated abundance C. imicola is ten times greater than that of C. bolitinos in livestock farms (Venter et al., 1996). C. imicola, C. bolitinos, and C. enderleini were collected at low altitude while C. kibatiensis and C. grahamii were collected above 750 m. As previously shown in South Africa (Venter and Meiswinkel, 1994), C. imicola appeared more abundant at low altitude although it was found up to 1200 m in Reunion Island. The major factors explaining Culicoides distribution are temperature, humidity, rainfall (Baylis et al., 1998, 1999, 2001; Calvete et al., 2008; Conte et al., 2003; Purse et al., 2004; Tatem et al., 2003; Wittmann et al., 2001), wind (Baylis et al., 1998) as well as altitude (Baylis et al., 2001; Conte et al., 2003), soil type and moisture (Conte et al., 2007), vegetation indices or land use (Conte et al., 2007), and presence of hosts. Due to its mountainous landscape, Reunion Island exhibits a pronounced temperature gradient. Therefore, the temperature, tightly correlated with altitude, possibly plays a role

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A. Desvars et al. / Acta Tropica 142 (2015) 5–19

Fig. 5. Abundance map and spatial distribution of the females specimens of the five Culicoides species collected on 41 sites using light suction trap (Rieb type) on Reunion Island during March–April 2005.

in the species distribution and abundance. Although, C. imicola is reported not to breed in areas with sandy soils (Meiswinkel, 1997), we reported this species in sandy savannah areas (sites 34 and 35). C. imicola and C. bolitinos are considered as major vectors for economically important viruses. C. imicola is a proven vector of BTV and AHSV, and suspected vector of equine encephalitis virus (EEV) and EHDV. C. bolitinos is a suspected vector of BTV, AHSV, EEV, and EHDV (Diarra et al., 2014; Mellor et al., 2000). C. enderleini was found infected by EHDV in Sudan (Mellor et al., 1984) and BTV in South Africa (Venter et al., 2006) but its vector competence has not been proved although Nevill et al. (1992) rated Culicoides of Schultzei group as having high potential vector capacity. Neither investigations concerning the diagnosis of field-infected specimens nor the vector competence in the laboratory have been conducted for C. grahamii and C. kibatiensis. However, the daily activity and dynamics of C. grahamii has been studied in central Africa (Auriault, 1979). This species was mentioned as a nuisance for humans with a peculiar anthropophilic behavior in this region (Itoua and Cornet, 1986; Itoua et al., 1987). Such nuisance biting has not been reported on Reunion Island.

Although a relative wide geographical area was covered, the survey was done over a short period of time (2 months) and restricted to one season. The results were furthermore based on a single collection per site which could impact the species diversity and abundance if they exhibit spatio-temporal variations. The Rieb trap is less efficient than other light traps in terms of number of Culicoides midges collected (del Río et al., 2013); the potential shortcomings of the trapping material might have influence our results on species diversity and distribution. Moreover, light traps do not estimate correctly biting rate and may underestimate – or overestimate – abundance if one peculiar species is not well – or overly – attracted by the light (Viennet et al., 2011). The present study was conducted at the end of the rainy season, generally considered as the most favorable season to the Culicoides abundance in the tropics (Mellor et al., 2000). Adverse meteorological and light conditions were controlled by operating traps when rain or wind was not expected. Coastal areas were poorly investigated because of limited farming activities. Bites of Culicoides species associated to tropical coastal sandy areas may impact touristic activities (e.g. such as C. furens in the Caribbean, Linley

Table 3 Number of males and females Culicoides collected using light suction trap (Rieb type) on Reunion Island during March–April 2005 at each positive site for the five species described (see Table 1 for site description). Sites where no Culicoides were collected are not shown. Site number

C. bolitinos Male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 4 0 0 0 0 0

Total number of individuals Number of positive sites (% of the total nb of sites)

5 21 (51.2%)

9 0 2 2 6 7 7 0 11 1 5 8 0 59 2 0 1 5 0 0 0 4 91 28 94 18 7 0 0 2 369

C. grahamii

C. imicola

Male

Female

Male

Female

0 0 0 0 0 0 0 0 6 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 19 1 0 0 0 0

2 0 1 2 0 1 0 0 184 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 12 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 2 6 0 0 0 1 1 0 0 0 2 1 0 0 0 0 0 25

38 8 (19.5%)

216

0 7 (17.1%)

38

C. kibatiensis

Male

Female

0 0 0 0 0 0 0 0 0 5 8 0 0 0 0 0 0 1 0 0 0 0 0 0 5 0 0 0 0 0

67 2 259 63 0 0 11 3 884 47 165 403 1 0 2 0 0 10 0 0 0 1 19 1 357 99 0 12 176 4

19 21 (51.2%)

2586

Total

Male

Female

Male

Female

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 5 4 1 8 4 19 3 14 4 0 9 2 0 0 0 0 0 0

0 0 0 0 0 0 0 0 6 17 8 0 0 0 0 0 0 3 0 0 0 0 1 0 28 1 0 0 0 0

78 2 262 67 6 8 18 3 1079 56 170 411 8 69 5 8 5 35 4 14 4 5 121 32 457 129 7 12 176 31

2 11 (26.8%)

73

64 30 (73.2%)

3282

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2 3 5 6 7 8 9 11 13 15 16 17 18 20 21 22 24 25 26 27 29 30 31 33 34 35 36 37 38 39

C. enderleini Female

17

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Fig. 6. Description of the altitudinal distribution and Culicoides species diversity on the 30 positive sites of capture. Specimens were collected using light suction trap (Rieb type) on Reunion Island during March–April 2005.

and Davies, 1971). This is might be of primary interest in the highly touristic islands of the South-West Indian Ocean region. Despite these limitations, this survey reflects the main livestock associated species of the island. To complete these first data, more field work is needed to evaluate (1) the spatial variations and seasonal dynamics in species diversity and abundance, (2) the species diversity in poorly investigated areas of Reunion Island and in the other islands of the Indian Ocean. This survey showed that Culicoides are abundant and widespread on Reunion Island, confirming/explaining a high transmission risk of Culicoides-borne viruses. This study provides the first integrative taxonomy (based on morphological description and molecular identification) of livestock associated Culicoides species on Reunion Island and should be considered as a baseline to additional studies on Culicoides from the Indian Ocean area. Acknowledgements We are grateful to the Cirad Pôle Elevage and P. Lecomte for their support, technical and financial help. We also want to thank the Cirad Pôle 3P, the DDSV974 for assistance, in particular E. Tillard, and A. Tran, B. Mathieu, and J. Gilles for their advice. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica. 2014.10.018. References Augot, D., Randrianambinintsoa, F.J., Gasser, A., Depaquit, J., 2013. Record of two species of Culicoides (Diptera, Ceratopogonidae) new for Madagascar and molecular study showing the paraphylies of the subgenus Oecacta and the Schultzei group. Bull. Soc. Pathol. Exot. 106, 201–205. Auriault, M., 1979. Contribution à l’étude biologique et écologique de Culicoides grahamii (Austen), 1909. V. Rythme d’activité en fôret dense. Cahiers ORSTOM ser Entomol Med Parasitol 17, 77–79.

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