Colonization of the Mediterranean basin by the vector biting midge species Culicoides imicola: an old story.

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Mitochondrial DNA Intraspecific Variability in Sergentomyia minuta (Diptera: Psychodidae).

Recently, there has been growing interest in analysis of the geographical variation between populations of different Phlebotomus spp. and American sand flies by comparing the sequences of various genes. However, little is known about the genetic structure of the genus Sergentomyia França & Parrot. No study has been carried out on Sergentomyia minuta Rondani. Most authors recognize this as a species with a high degree of morphological polymorphism, and some suspect that there are two subspecies: Se. minuta minuta Rondani in Europe, having about 40 horizontal cibarial teeth (sticks aligned along a straight line in the cibarial cavity), and Se. minuta parroti Adler & Theodor in North Africa, having about 70 cibarial teeth. Here we analyzed phylogeographic patterns using cytochrome b (Cytb) and cytochrome C oxidase I mtDNA for 29 populations from 10 countries: Algeria, Cyprus, France (continental and Corsica), Greece (continental and Crete), Malta, Montenegro, Morocco, Portugal (continental and Atlantic Savage Islands), Spain, and Tunisia. We analyzed intra- and interpopulation patterns of genetic diversity. Our results from Bayesian inference showed a complex genetic structure of Se. minuta with four haplogroups including many different haplotypes. One haplogroup includes all the specimens from North Africa. A second haplogroup includes a few specimens from the south of France, Spain, and one from Portugal. The third includes many specimens from southern France, all the specimens from Corsica, one from Spain, and all specimen from Portugal except one. A fourth branch includes specimens from the Balkans, Malta, Crete, Cyprus, and curiously some from the Atlantic Savage Islands; settlement of the latter population remains unexplained. However, our results suggest that the settlement of the Mediterranean basin could have occurred at the same time for Se. minuta and both Phlebotomus perniciosus Newstead and Phlebotomus ariasi Tonnoir. The spatial distribution of haplotypes was congruent with phylogenetic findings.

First overview of the Culicoides Latreille (Diptera: Ceratopogonidae) livestock associated species of Reunion Island, Indian Ocean.

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.

Farms, pastures and woodlands: the fine-scale distribution of Palearctic Culicoides spp. biting midges along an agro-ecological gradient.

The spatial epidemiology of Bluetongue virus (BTV) at the landscape level relates to the fine-scale distribution and dispersal capacities of its vectors, midges belonging to the genus Culicoides Latreille (Diptera: Ceratopogonidae). Although many previous researches have carried out Culicoides sampling on farms, little is known of the fine-scale distribution of Culicoides in the landscape immediately surrounding farms. The aim of this study was to gain a better understanding of Culicoides populations at increasing distances from typical dairy farms in north-west Europe, through the use of eight Onderstepoort-type black-light traps positioned along linear transects departing from farms, going through pastures and entering woodlands. A total of 16 902 Culicoides were collected in autumn 2008 and spring 2009. The majority were females, of which more than 97% were recognized as potential vectors. In pastures, we found decreasing numbers of female Culicoides as a function of the distance to the farm. This pattern was modelled by leptokurtic models, with parameters depending on season and species. By contrast, the low number of male Culicoides caught were homogeneously distributed along the transects. When transects entered woodlands, we found a higher abundance of Culicoides than expected considering the distance of the sampling sites to the farm, although this varied according to species.

Phenology and attraction of potential Culicoides vectors of bluetongue virus in Basque Country (northern Spain).

Bluetongue virus is transmitted by Culicoides biting midges (Diptera: Ceratopogonidae). Culicoides associated with livestock were captured using CDC blacklight traps at three BTV-infected farms in Basque Country between November 2007 and December 2008. Twenty-seven and nineteen Culicoides species were collected in outdoor and indoor habitats respectively. Indoor insect community represented 86.1% of the whole captured individual biting midges. Culicoides obsoletus/Culicoides scoticus (two sibling species of the Obsoletus complex) were dominant throughout all months and sexes with maximum phenological peaks in November 2007 and June-July 2008. Culicoides lupicaris was the second most dominant species followed by Culicoides pulicaris (both species of the Pulicaris complex). Few specimens of Culicoides imicola, the principal Afro-Mediterranean vector of BTV, as well as four new species recorded for the Iberian Peninsula, were also collected. BTV was detected by RT-PCR from pools of C. obsoletus/C. scoticus, C. lupicaris and C. pulicaris parous females. DL-Lactic acid significantly attracted more C. obsoletus/C. scoticus females and males, C. lupicaris females, C. pulicaris females and Culicoides punctatus females and males; whereas acetone increased only the captures of Culicoides achrayi.

An investigation on the Culicoides species composition at seven sites in southern Switzerland.

In the past decade, there have been regular outbreaks of bluetongue (BT) in many parts of Europe. Owing to the presence of BT disease and its vectors in countries adjacent to Switzerland, an initial entomological survey was conducted in 2003, which established the presence of several midges of the genus Culicoides (Diptera: Ceratopogonidae). Subsequently, a sentinel herd monitoring system was established with the primary entomological aim being the determination and further study of Culicoides population compositions. Insects were collected in 2005 and 2006 at seven sentinel herd sites in the south of Switzerland (canton of Ticino) near the border of Italy, using Onderstepoort-type light traps. This region is botanically and zoologically similar to the Mediterranean and is one of the warmest and most humid areas of the country, hence it is considered a potential access path for BT disease into Switzerland. Collections were made at four cattle farms, two equestrian centres and one goat farm. Sites were sampled four times per month from June to October. Traps were operated from dusk until dawn and samples were collected monthly for analysis through microscopy as well as a Culicoides imicola-specific PCR. Results confirmed the absence of C. imicola (Kieffer) and demonstrated that the potential BT virus vectors are highly abundant, notably: Culicoides obsoletus (Meigen), Culicoides scoticus (Downes & Kettle) and Culicoides dewulfi (Goetghebuer) subgenus Avaritia and Culicoides pulicaris (Linnaeus) subgenus Culicoides. These findings expand the current knowledge of Culicoides population composition in the southern part of the Switzerland. Culicoides cataneii (Clastrier), Culicoides flavipulicaris (Dzhafarov), Culicoides indistinctus (Khalaf), Culicoides nubeculosus (Meigen) and species of the Grisescens complex were reported for the first time in Switzerland.

Indoor activity of Culicoides associated with livestock in the bluetongue virus (BTV) affected region of northern France during autumn 2006.

In August 2006, bluetongue virus (BTV) was detected in the Netherlands, Belgium, western Germany, Luxembourg and northern France for the first time. Consequently, a longitudinal entomological study was conducted in the affected region of northern France (Ardennes) throughout the autumn of 2006. Data on the spatio-temporal distribution of Culicoides (Diptera: Ceratopogonidae) associated with livestock were collected and an attempt was made to identify the vector(s) involved in BTV transmission by means of virus detection in wild-caught biting midges. Weekly sampling using standardized Onderstepoort-type blacklight traps were performed simultaneously both outdoors and indoors in one BTV-free and three BTV-affected farms between September and December 2006. Culicoides were sorted according to farm, location (outdoors vs. indoors), time point (in weeks), species and physiological stage. BTV detection was conducted by RT-PCR on monospecific pools of non-bloodfed parous female Culicoides. The principal results showed: (i) the absence of the Mediterranean vector, C. imicola, (ii) the relatively low abundance of C. dewulfi and C. pulicaris, (iii) the widespread occurrence and abundance of C. obsoletus/C. scoticus with longevity and behaviour compatible with BTV transmission, and (iv) all Culicoides pools tested for BTV were negative. In France, the very low levels of BTV-8 circulation were probably due to the limited introduction of the virus from affected neighbouring countries, and not due to the absence of local vector populations. A key finding has been the substantiation, for the first time, that Culicoides, and particularly the potential vectors C. obsoletus/C. scoticus and C. dewulfi, can be active at night inside livestock buildings and not only outside, as originally believed. The endophagic tendencies of members of the Obsoletus group are discussed in light of the prolonged period of BTV transmission during the autumn of 2006 and the risk of BTV overwintering and resurgence in the spring of 2007. Overall, there is an urgent need to improve our knowledge on the ecology of local Culicoides species before any clear, effective and reliable recommendations can be provided to the veterinary authorities in terms of prevention and control.

The 2006 outbreak of bluetongue in northern Europe--the entomological perspective.

After bluetongue (BT) appeared in northern Europe in August 2006 entomological studies were implemented in all five affected Member States (MSs) to establish which species of Culicoides had acted as vectors. The findings can be summarised as follows: (i) C. imicola the principal southern European/African vector of BTV has not penetrated into northern Europe, (ii) three pools of C. obsoletus/C. scoticus and one of C. dewulfi assayed RT-PCR-positive to BTV-8, (iii) in support of these results it was found that both potential vectors had also high parity rates (approximately 40%) indicating increased longevity favouring BTV virogenesis and transmission, (iv) furthermore, C. obsoletus/C. scoticus and C. dewulfi occurred also widely and abundantly on sheep and cattle holdings across the entire affected region, (v) and during the latter part of the season showed strong endophily readily entering livestock buildings in significant numbers to bite the animals inside (endophagy), (vi) which demonstrates that housing at best offers only limited protection to livestock from Culicoides attacks, (vii) in contrast the potential vector C. pulicaris sensu stricto was restricted geographically, was captured rarely, had a low parity rate (10%) and was exophilic indicating it played no role in the outbreak of BT, (viii) the incrimination of C. dewulfi as a novel vector is significant because it breeds in cattle and horse dung this close association raising its vectorial potential, but (ix) problems with its taxonomy (and that of the Obsoletus and Pulicaris species complexes) illustrates the need for morphological and molecular techniques to become more fully integrated to ensure progress in the accurate identification of vector Culicoides, (x) midge densities (as adjudged by light traps) were generally low indicating northern European Culicoides to have a high vector potential and/or that significant numbers of midges are going undetected because they are biting (and transmitting BTV) during the day when light traps are not effective, and (xi) the sporadic capture of Culicoides in the winter of 2007 invites re-examination of the current definition of a vector-free period. The re-emergence of BT over a wide front in 2007 raises anew questions as to precisely how the virus overwinters and asks also that we scrutinise our monitoring systems in terms of their sensitivity and early warning capability.

Culicoides trapping with Rothamsted suction traps before and during the bluetongue epidemic of 2006 in Belgium.

The collection of biting midges was taking place some months before the first bluetongue outbreak in Belgium in August 2006. The Walloon Agricultural Research Centre had been monitoring aphid populations at two sites annually in Belgium (Gembloux and Libramont), using two stationary '12-m' Rothamsted suction traps. For the Gembloux trap, collections of insects captured daily from 11 May 2006 onwards were already available at the time of the outbreak. An examination of these samples revealed the presence of Culicoides, some species of which are considered as potential vectors of the bluetongue virus (BTV). The trapping was therefore extended beyond the normal aphid activity period and the Culicoides captured were identified to species level. From 11 May to 31 December 2006, the Gembloux trap caught 664 Culicoides specimens belonging to 19 species comprising known BTV-vectors. The second trap, at Libramont, was reactivated from 12 September to 13 October and caught 97 specimens belonging to nine species, all of which had been found at the Gembloux site. Among the 19 species identified, four were new to Belgian fauna: Culicoides achrayi, C. deltus, C. lupicaris and C. newsteadi. This paper examines the overall phenology and the physiological status of Culicoides in 2006 before and during the bluetongue epidemic. It discusses the potential of the Rothamsted suction trap to monitor Culicoides.

Occurrence of Culicoides spp. (Diptera: Ceratopogonidae) in Tunisia, with emphasis on the bluetongue vector Culicoides imicola.

Following the bluetongue (BT) outbreaks in Tunisia from 1999 to 2002, BTV (bluetongue virus) serotype 2 was isolated; however, no entomological investigation was performed. In the study presented here, we assessed the Culicoides species populations (particularly C. imicola) in proximity to the BT outbreaks locations, both as a retrospective analysis and to update the list of Culicoides species present in Tunisia. The insects were caught using light traps and the species identification was performed according to the standard entomological methods. This study reveaaled the presence of significant numbers of C. imicola in all the tested locations. In addition, we reported a new Culicoides species for the Tunisian fauna C. punctatus.

Phylogenetic analysis of Culicoides species from France based on nuclear ITS1-rDNA sequences.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play important roles in the transmission of viral diseases affecting wild and domestic ruminants and horses, including Bluetongue (BT) and African horse sickness (AHS) respectively. In southern Europe, BT has been largely transmitted by the classical Afro-Asian vector Culicoides imicola Kieffer. However, other species such as C. obsoletus Meigen, C. scoticus Downs & Kettle and C. pulicaris Linné may also be involved in BTV transmission. As a consequence of the discovery of C. imicola followed by BTV-2 outbreaks on the island of Corsica in October 2000, further studies on these biting midges have been carried out. To better characterize the evolution and phylogenetic relations of Culicoides, molecular analysis in parallel with a morphology-based taxonomic approach were performed. Phylogenetic analyses of French Culicoides species were undertaken using the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) as a molecular target. This region was shown to be useful in understanding evolutionary and genetic relationships between species. Construction of several trees showed that molecular phylogeny within the genus Culicoides correlates not only with morphological-based taxonomy but also with ecological patterns.

Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequences.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages - the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world.

The taxonomy of Culicoides vector complexes - unfinished business.

The thirty species of Culicoides biting midges that play a greater or lesser role in the transmission of bluetongue (BT) disease in the pantropical regions of the world are listed. Where known, each species is assigned to its correct subgenus and species complex. In the Mediterranean region there are four species of Culicoides involved in the transmission of BT and belong in the subgenera Avaritia Fox, 1955 (three species) and Culicoides Latreille, 1809 (one species). Using both morphological and molecular second internal transcribed spacer (ITS2) sequence data, the authors reappraise the taxonomy of these four species and their congeners. A total of 56 populations of Culicoides collected from across Italy and representing 17 species (18 including the outgroup taxon C. imicola Kieffer, 1913) were analysed. The findings revealed the following: C. imicola is the only species of the Imicola Complex (subgenus Avaritia) to occur in the Mediterranean region. In Europe the subgenera Avaritia and Culicoides (usually, but not quite correctly, equated with the C. obsoletus and C. pulicaris groups, respectively) are both polyphyletic, each comprising three or more species complexes (including a hitherto unknown complex). About half the species studied could not be identified with certainty; furthermore, the results indicate that at least three previously described species of Palaearctic Culicoides should be resurrected from synonymy. Finally, a high level of taxonomic congruence occurred between the morphological and the molecular data. One of the 'new' vector species, C. pulicaris, was described by the father of taxonomy, Carl Linnaeus, in 1758, but today, almost 250 years later, no monograph has appeared that treats the Culicoides fauna of the northern hemisphere as a whole. At a time when such economically important livestock diseases as BT are affecting ever larger areas of Europe, it would seem appropriate to commence the production of such a monograph to aid in the field identification of vector Culicoides. This 'unfinished business' might best be achieved through a collaborative network embracing all ceratopogonid specialists currently active in both the Palaearctic and Nearctic faunal realms.

Culicoides (Diptera: Ceratopogonidae) in Albania: results of the 2002 entomological survey for bluetongue.

A survey for Culicoides Latreille, 1809, was made in Albania in 2002 to establish whether Culicoides imicola Kieffer, 1913, the main vector of bluetongue virus in the Mediterranean Basin, or any other suspected vector species, was present. The collections and analyses were performed in accordance with the protocols of the National Reference Centre for Exotic Diseases (CESME: Centro Studi Malattie Esotiche) in Teramo, Italy. A total of 43 catches were made in October and November in 15 districts (Bulqise, Devoll, Dibre, Durres, Fier, Gjirokaster, Has, Kolonje, Korce, Librazhd, Permet, Pogradet, Shkoder, Tirane and Tropoje). Twenty species of Culicoides were identified in the collections; the most abundant species belonged to the Obsoletus Complex (98% of total Culicoides in some catches). Culicoides imicola was never captured during the survey. However, a larger number of Culicoides collections and collection sites are needed to exclude the presence of this species at low abundance levels.

Entomological surveillance of bluetongue in France in 2002.

Bluetongue (BT) is an arboviral disease that appeared in the Mediterranean in 1998. In France, the principal vector, Culicoides imicola, was detected for the first time in Corsica in October 2000, a few weeks before outbreaks of BT virus serotype 2 (BTV-2). Entomological surveillance was implemented in Corsica and on mainland France in 2002. In Corsica, the aim was to study the population dynamics of C. imicola and other potential vectors. On the coastal mainland, the objective was to survey the introduction of C. imicola. One-night catches per site were performed every three weeks at 12 sites on Corsica and every month at 19 sites on the mainland. In Corsica, 84,790 Culicoides belonging to 50 species were collected over 180 nights. C. imicola accounted for 18.3% of the total captured. On the mainland, 16,197 Culicoides (44 species over 109 nights) were collected, none of which were C. imicola. The geographic and seasonal distribution of C. imicola and other species of interest are discussed in relation with their bio-ecology and environment. These datasets are essential for a better understanding of the epidemiology of BT, and to create and validate predictive models based on remote sensing in order to identify areas at risk for BT.

Entomological surveillance for bluetongue on Malta: first report of Culicoides imicola Kieffer.

A survey for Culicoides Latreille, 1809, was made on Malta in 2002 to establish whether Culicoides imicola Kieffer, 1913, the principal vector of bluetongue virus (BTV) in the Mediterranean Basin, or any other suspected vector species, was present. The collections and analyses were performed in accordance with the protocols of the National Reference Centre for Exotic Diseases (CESME Centro Studi Malattie Esotiche) in Teramo, Italy. Eighty-four catches were made between May and October at six permanent sites, namely: Mellieha, Rabat, San Gwann, Zejtun (Malta), Gharb and Sannat (Gozo island). The traps were placed near cattle (four farms), cattle and sheep (one farm: Rabat) and sheep and goats (one farm: Mellieha). Culicoides midges were found in 91.66% (77/84) of the catches and the highest number of midges per catch was 1 726. Culicoides imicola was confirmed on Malta for the first time in October 2002 and was found at four sites (San Gwann, Sannat, Gharb and Mellieha) but at very low abundance levels (<0.1% of the total Culicoides collected). Culicoides paolae Boorman, 1996 was the most widespread and abundant species (more than 80% of total Culicoides). Midges of the Obsoletus Complex were rare, with less than 10 individuals captured. Other species of Culicoides identified in the collections were: C. submaritimus Dzhafarov, 1962, C. cataneii Clastrier, 1957, C. circumscriptus Kieffer, 1918, C. jumineri Callot and Kremer, 1969, C. kingi Austen, 1912, C. maritimus Kieffer, 1924 and C. newsteadi Austen, 1921.

[Morphological variability of Culicoides circumscriptus Kieffer 1918. C. kirovabadicus Dzhafarov 1964 as a probable synonym]. / Variabilité des caractères morphologiques de Culicoides circumscriptus Kieffer 1918. Mise en synonymie de C. kirovabadicus Dzhafarov 1964.

The authors study the variation of the sensilles and the wing pattern of Culicoides circumscriptus Kieffer, 1918, they consider that Culicoides kirovabadicus Dzhafarov 1964, could be synonymous.

[Iconography of the differential characteristics of Fannia larvae (author's transl)]. / Iconographie des caractères différentiels des larves de Fannia canicularis, F. scalaris et F. manicata.

The authors are showing the characteristics of Fannia larvae ventral and dorsal sides. They specify on figures the morphological characters that permit to distinguish the species scalaris, manicata and canicularis.