Afrotropical Culicoides: C. (Avaritia) loxodontis sp. nov., a new member of the Imicola group (Diptera: Ceratopogonidae) associated with the African elephant in the Kruger National Park, South Africa.

Culicoides (Avaritia) loxodontis sp. nov., is described and illustrated from both sexes collected in South Africa. It is the 5th species of the Imicola group of the subgenus Avaritia to be described from the Afrotropical Region, and is presently known only from the Kruger National Park where it has been collected in light-traps and reared from the dung of the African elephant (Loxodonta africana) on various occasions. A number of character states, and statistical analyses of antennal and palpal measurements, are used to separate the new species from its taxonomic congeners C. imicola Kieffer, 1913, C. pseudopallidipennis Clastrier, 1958, C. bolitinos Meiswinkel, 1989 and C. miombo Meiswinkel, 1991. It is suggested that the occurrence of the African elephant is the primary factor that determines the distribution of Culicoides loxodontis sp. nov., and that this close association, coupled with the fact that C. loxodontis sp. nov. can be locally abundant, may result in the cycling of certain arboviruses between this biting midge and the elephant.     

Comparative descriptions of the pupae of five species of the Culicoides imicola complex (Diptera, Ceratopogonidae) from South Africa

The viruses causing the economically important livestock diseases of African horse sickness (AHS) and bluetongue (BT) are transmitted by biting midges of the genus Culicoides (Diptera, Ceratopogonidae). In the Old World the most important vectors of these diseases are Culicoides imicola Kieffer, 1913, Culicoides brevitarsis Kieffer, 1917 and Culicoides bolitinos Meiswinkel, 1989. All three of these vectors belong to the Imicola complex of the subgenus Avaritia Fox, 1955. This species complex now comprises 12 sibling species; ten occur in sub-Saharan Africa and are difficult to identify (based mostly on subtle variations in the wing latterns) and so additional methods of reliable identification are needed. The pupal exuviae of the five commonest sibling species (C. imicola, C. bolitinos, Culicoides loxodontis Meiswinkel, 1992, Culicoides tuttifrutti Meiswinkel, Cornet & Dyce, 2003 and Culicoides sp. # 107) harvested from a variety of large herbivore dung types and from decaying fruits, are described and illustrated in detail. It is shown that they can be differentiated clearly on a number of morphological characters and, furthermore, are separable into two distinct groups based (principally) on the shape of the respiratory organ. A key for identifying and differentiating these five pupae is provided. Also, the pupa of the Oriental-Australasian C. brevitarsis was compared with its allopatric sister taxon, C. bolitinos. Because they share a common larval habitat (cattle and buffalo dung) and are almost inseparable in the adult phenotype, the question of their possible synonymy is raised. However, their respective pupae could not be differentiated on gross morphology and so it is argued that this unresolved problem requires a molecular solution.     

Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe

Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap network for the collection of Culicoides, was implemented in 2002 in southern mainland France. The morphological identification of Culicoides can be both tedious and time-consuming because its size ranges from 1.5 to 3 mm. Therefore, an ITS1 rDNA polymerase chain reaction (PCR)-based diagnostic assay was developed to rapidly and reliably identify Culicoides spp. and C. imicola. The aim of this work was to set up a rapid test for the detection of C. imicola amongst a pool of insects collected in areas at risk for BT. The sequence similarity of the rDNA (nuclear ribosomal DNA), which is greater within species than between species, is the foundation of its utilisation in species-diagnostic assays. The alignment of the 11 ITS1 sequences of Culicoides obtained from Genbank and EMBL databases helped us to identify one region in the 5' end and one in the 3' end that appear highly conserved. PCR primers were designed within these regions to amplify genus-specific fragments. In order to set up a C. imicola-specific PCR, another forward primer was designed and used in combination with the previously designed reverse primer. These primers proved to be highly specific and sensitive and permitted a rapid diagnostic separation of C. imicola from Culicoides spp.     

Climate change: effects on culicoides--transmitted viruses and implications for the UK

Changes in the distribution and abundance of insects are likely to be amongst the most important and immediate effects of climate change. We review here the risk that climate change poses to the UK's livestock industry via effects on Culicoides biting midges, the vectors of several arboviruses, including those that cause bluetongue (BT) and African horse sickness (AHS). The major old-world vector of BT and AHS viruses, C. imicola, occurs in southern Europe and will spread further north as global temperatures increase. It is unlikely, however, that in the foreseeable future it will reach and become established in the UK. As the distribution of C. imicola moves north, however, it may bring BT and AHS viruses into the range of other Culicoides species that are known to be competent vectors and which occur much further north. Once infected via this 'baton effect', these species may be able to spread the viruses over much of Europe, including the UK. Climate change may increase their vector competence further and will also increase the likelihood of viruses surviving from one year to the next. An additional risk is that the predicted increase in the frequency of short periods of hot temperatures may lead to the creation of novel vector species, by removing the barriers that in colder conditions make them refractory to viral infection.     

Evidence for a new field Culicoides vector of African horse sickness in South Africa

Between February and May 1998, approximately 100 horses died of African horse sickness (AHS) in the cooler, mountainous, central region of South Africa. On 14 affected farms, 156,875 Culicoides of 27 species were captured. C. imicola Kieffer, hitherto considered the only field vector for AHS virus (AHSV), constituted <1% of the total Culicoides captured, and was not found on 29% of the farms. In contrast, 65% of the Culicoides were C. bolitinos Meiswinkel, and was found on all farms. Five isolations of AHSV were made from C. bolitinos, and none from 18 other species of Culicoides (including C. imicola).     

A preliminary attempt to use climate data and satellite imagery to model the abundance and distribution of Culicoides imicola (Diptera: Ceratopogonidae) in southern Africa

Abundances of Culicoides imicola, the insect vector of several livestock viruses, including bluetongue and African horse sickness, were recently published for 34 sites in southern Africa, together with associated climate data. Here, these data are analysed statistically in combination with certain satellite-derived variables, with the aim of developing predictive models of C. imicola abundance. Satellite-derived variables were the land surface temperature (LST, a measure of temperature at the earth's surface) and the normalised difference vegetation index (NDVI, a measure of photosynthetic activity). Two models were developed: (1) climatic variables only and (2) satellite-derived and climatic variables. For model I, the best model used a single predictor variable (the mean daily minimum temperature) only, and accounted for nearly 34% of the variance in C. imicola abundance. Two variable climatic models did not perform significantly better. For model II, the best 1-variable model used the annual minimum LST as a predictor of C. imicola abundance, and accounted for nearly 40% of the variance in C. imicola abundance. The best 2-variable model, which gave a significantly better fit than the 1-variable model, combined the minimum LST and minimum NDVI as predictors of C. imicola abundance, and accounted for nearly 67% of variance. A map of predicted C. imicola abundances is produced on the basis of this 2nd model which, despite some anomalies, agrees largely with what is currently known of the prevalence of C. imicola in the region.     

African horse sickness

African horse sickness virus (AHSV) causes a non-contagious, infectious insect-borne disease of equids and is endemic in many areas of sub-Saharan Africa and possibly Yemen in the Arabian Peninsula. However, periodically the virus makes excursions beyond its endemic areas and has at times extended as far as India and Pakistan in the east and Spain and Portugal in the west. The vectors are certain species of Culicoides biting midge the most important of which is the Afro-Asiatic species C. imicola. This paper describes the effects that AHSV has on its equid hosts, aspects of its epidemiology, and present and future prospects for control. The distribution of AHSV seems to be governed by a number of factors including the efficiency of control measures, the presence or absence of a long term vertebrate reservoir and, most importantly, the prevalence and seasonal incidence of the major vector which is controlled by climate. However, with the advent of climate-change the major vector, C. imicola, has now significantly extended its range northwards to include much of Portugal, Spain, Italy and Greece and has even been recorded from southern Switzerland. Furthermore, in many of these new locations the insect is present and active throughout the entire year. With the related bluetongue virus, which utilises the same vector species of Culicoides this has, since 1998, precipitated the worst outbreaks of bluetongue disease ever recorded with the virus extending further north in Europe than ever before and apparently becoming endemic in that continent. The prospects for similar changes in the epidemiology and distribution of AHSV are discussed.     

The use of a membrane feeding technique to determine the infection rate of Culicoides imicola (Diptera, Ceratopogonidae) for 2 bluetongue virus serotypes in South Africa.

Culicoides spp. in the Lowveld of the northern Transvaal, Republic of South Africa, were fed bluetongue virus serotypes 3 and 6 and African horsesickness virus serotype 1 through latex and chicken skin membranes. After an incubation period of 10 days at 25-27 degrees C, the infection rate of C. imicola for bluetongue virus serotypes 3 and 6 was established at 31% and 24% respectively. No African horsesickness virus could be recovered. The membrane feeding technique and handling procedures proved to be suitable for field studies.     

Comparison of black and white light for collecting Culicoides imicola and other livestock-associated Culicoides species in South Africa

Comparison of the effectiveness of 8W fluorescent black and white light sources, in two 4x4 Latin squares (16 replicates) designs under South African conditions, showed black light to be up to three time more effective in collecting Culicoides imicola Kieffer (Diptera, Ceratopogonidae) and other South African Culicoides species. Four Culicoides species, which were collected in low numbers with black light, were not collected in traps equipped with the white light source. No significant difference was found in the parous rate of the C. imicola populations as determined by the two light sources. The study highlighted the superiority of black light as a preferred collection method for C. imicola, considered to be the most widespread and abundant vector of livestock orbiviruses. The results underline the need to develop and adopt standard techniques for measuring the variables of vectorial capacity.     

Seasonal abundance and parity of Culicoides biting midges associated with livestock at Roma, Lesotho (Diptera: Ceratopogonidae)

A light-trap survey was undertaken of the species composition, seasonal abundance and parity of Culicoides at Roma, Lesotho, to establish whether the likely vectors for bluetongue and African horse sickness occur in this area as well as the chance of transmission. A total of 34 catches was made between 21 September 1985 and 24 September 1986; 32,819 Culicoides were caught belonging to 19 species. Culicoides numbers rapidly built up from December to a peak in February which implies that this may also be the optimum time for virus transmission. The number of Culicoides dropped sharply in April with the onset of cooler conditions. C. zuluensis was the dominant species forming 69.6% of the totalled catches, followed by C. pycnostictus with 11.7%. C. imicola, the only proven vector of bluetongue, was never abundant representing only 4.4% of the midges caught. The parous rate for each of the 2 commonest species was low, implying a low vector capacity.     

Prediction of areas around the Mediterranean at risk of bluetongue by modelling the distribution of its vector using satellite imaging.

Bluetongue is an infectious disease of ruminants caused by a virus transmitted by biting midges, one species of which, Culicoides imicola, is the major vector in the Old World. Following an epizootic of African horse sickness,a related disease, in Iberia and Morocco between 1987 and 1991, C imicola was trapped for two years at 44 sites in the affected region and models were developed for predicting the abundance of C imicola at these sites. Discriminant analysis was applied to identify the best model of three levels of abundance from 40 Fourier-processed remotely sensed variables and a digital elevation model. The best model correctly predicted the abundance level at 41 of the 44 sites. The single most important variable was the phase of the annual cycle of the normalised difference vegetation index. The model was used to predict the abundances of C imicola elsewhere around the Mediterranean and predicted high levels of abundance in many areas recently affected by bluetongue, including the Balearics, Sardinia, Sicily, eastern Greece, western Turkey, Tunisia and northern Algeria. The model suggests that eastern Spain, the island of Ibiza, the provinces of Lazio and Puglia in Italy, the Peloponnese and parts of northern Algeria and Libya may be at risk of bluetongue in 2001.     

The effect of climate on the presence of Culicoides imicola in Italy

A model was developed to classify the Italian territories in relation to their suitability to harbour populations of Culicoides imicola and, as a consequence, also able to sustain a bluetongue (BT) epidemic. Italy was subdivided into 3507 10 x 10 km cells. In 546 cells at least one collection was made. The cell was considered the unit for all subsequent analyses. Culicoides were collected using Onderstepoort-type blacklight traps. Some traps were operated weekly at chosen sites; the remainder were moved almost daily to new sites. Only the results obtained during the peak August-November period were used, to exclude bias caused by the seasonality of C. imicola. Climate data for the period 1999-2001 were obtained from 80 weather stations. Multiple logistic regression was performed using the presence or absence of C. imicola in a specific cell as the dependent variable. Annual means of daily values for minimum temperature and minimum relative humidity, and the mean altitude above sea level, were the independent variables. The probability of occurrence of C. imicola in each grid cell was used to create a prediction map for Italy. The model was able to correctly classify 77.5% of the 546 grid cells in which at least one collection had been made. Culicoides imicola was found frequently through much of Sardinia, in parts of southern Italy, and further north along the Tyrrhenian coast, but was absent from along most of the Adriatic coast, and the internal mainland, and from most of Sicily. Six detailed maps are provided. Also mapped are areas where the probability of the occurrence of C. imicola is lower than 5%. This identification of possible mountainous C. imicola-free areas in central Italy could facilitate safer animal trade and transhumance, even if BT infections in traded animals or moving stock, were to go undetected. Needless to say this depends upon no cool-adapted species of Culicoides being involved in the transmission of BT disease.     

The repellent effect of organic fatty acids on Culicoides midges as determined with suction light traps in South Africa

The efficacy of a 15% (w/w) mixture of octanoic, nonanoic and decanoic acids in light mineral oil to repel Culicoides biting midges (Diptera; Ceratopogonidae) was determined in three replicates of a 4 × 4 Latin square design under South African field conditions. The fatty acids were applied to ± 0.07 m(2) polyester meshes with a mesh size 2-3mm fitted to 220 V 8 W Onderstepoort downdraught light traps. To reduce the relatively strong attraction of the light trap, the black light tubes in the Onderstepoort trap were replaced with 8 W 23 cm white light tubes. The traps were operating overnight next to cattle. Two traps treated with the mixture of fatty acids collected 1.7 times fewer midges than two untreated traps. Although this mixture of fatty acids had shown a repellent effect against a number of blood-feeding insects this is the first indication that it also has a significant repellent effect against Culicoides species and especially Culicoides (Avaritia) imicola Kieffer when applied to polyester mesh.     

The distribution of Culicoides imicola in Italy: application and evaluation of current Mediterranean models based on climate

In August 2000 bluetongue (BT) disease appeared amongst sheep on the island of Sardinia spreading later to Sicily and to mainland Italy. The majority of areas affected by BT were surveyed for Culicoides imicola, the only proven vector of the disease known to occur in the Mediterranean region. The data from 1456 light-trap collections, made in months with a mean temperature of 12.5 degrees C, were used to test the accuracy of current models predicting the prevalence and abundance of C. imicola across the region. For Italy, the distribution of C. imicola was found to be very irregular and did not fit the modelled predictions. The possible reasons for this are discussed, and suggestions made as to which variables may improve this fit in the development of future risk models. In Italy, past surveys failed to reveal the presence of C. imicola, and so could be construed as evidence of its recent invasion, and thus rampant spread northwards. Although equivocal, historical records indicate that C. imicola was overlooked in the past. Six recommendations are made as to the possible future course of Culicoides research in southern Europe.     

Bluetongue in Europe and the Mediterranean Basin: history of occurrence prior to 2006

Bluetongue virus (BTV) exists around the world in a broad band covering much of the Americas, Africa, southern Asia and northern Australia. Historically, it also occasionally occurred in the southern fringes of Europe. It is considered to be one of the most important diseases of domestic livestock. Recently BTV has extended its range northwards into areas of Europe never before affected and has persisted in many of these locations causing the greatest epizootic of bluetongue (BT), the disease caused by BTV, on record. Indeed, the most recent outbreaks of BT in Europe are further north than this virus has ever previously occurred anywhere in the world. The reasons for this dramatic change in BT epidemiology are complex but are linked to recent extensions in the distribution of its major vector, Culicoides imicola, to the involvement of novel Culicoides vector(s) and to on-going climate-change. This paper investigates these recent outbreaks in the European theatre, up to the beginning of 2006, highlights prospects for the future and sets the scene for the following papers in this special issue.     

Molecular characterization of Swiss Ceratopogonidae (Diptera) and evaluation of real-time PCR assays for the identification of Culicoides biting midges

Biting midges of the genus Culicoides (Diptera, Ceratopogonidae) are vectors of several viruses of veterinary relevance, and they can cause insect bite hypersensitivity. As the morphological identification of these tiny insects is a difficult task in many cases, alternative approaches are expedient. With the aim to develop real-time PCRs, we determined partial mitochondrial cytochrome oxidase I gene (mt COI) sequences from 380 Culicoides midges representing three regions of Switzerland, namely the Alps, Midland north of the Alps (Atlantic climate), and South of the Alps (Mediterranean climate). The same region was also sequenced from non-biting midges of the genera Atrichopogon, Brachypogon, Dasyhelea, Forcipomyia and Serromyia. A total of 21 Culicoides species were identified by morphology. Sequence variability (haplotypes) was observed in all species. For each of C. grisescens and C. obsoletus, a novel cryptic species was identified. Whereas all individuals of C. grisescens and of the cryptic C. obsoletus species (O2) originated only from Alpine sites, the known C. obsoletus (O1) species was found in all three regions. Further, a sister taxon to C. pulicaris was identified based on the mt COI sequences and named Culicoides sp. Alignments of available mtCOI sequences from Ceratopogonidae (GenBank, this study) were used to design real-time PCR primers and probes to distinguish C. chiopterus, C. deltus, C. dewulfi, C. grisescens (including the cryptic species), C. imicola, C. lupicaris, C. obsoletus O1, C. obsoletus O2, C. pulicaris, C. scoticus and Culicoides sp. Specificities of primers and probes was tested with cloned targets representing 1 to 4 haplotypes of 18 Culicoides spp. and 1 haplotype each from 4 other Ceratopogonidae. No cross-reactivity was observed when plasmid template representing 5 × 10(6) gene copies was tested, but it was evident (Ct values ≤ 30) in few instances when plasmid template representing 5 × 10(9) gene copies was utilized, the latter corresponding to the total gene copy number (as determined in this study) in 20 insects. The sensitivities of two assays (C. imicola, C. grisescens) were tested by spiking single insects into pools of 99 or 999, randomly selected non-target Ceratopogonidae (with approx. 90% Culicoides specimens). In the pools of 100, Ct values were in the range of those obtained with single insects when employing 1% of the isolated DNA, whereas the sensitivity with the pools of 1000 was low, presumably due to the low DNA concentrations obtained with a protocol that seems inadequate for these larger pools. Thus, the assays as described are applicable for the specific identification of biting midges in small pools. Primers and probes of this study were devised to be suitable for multiplexed assays but these evaluations await to be performed.