Afrotropical Culicoides: C (Avaritia) miombo sp. nov., a widespread species closely allied to C. (A.) imicola Kieffer, 1913 (Diptera: Ceratopogonidae)

Culicoides (Avaritia) miombo sp. nov. is described and illustrated from both sexes collected in northern Malawi. Two references in the literature have previously referred to this new species as either C. brosseti Vattier & Adam or C. imicola Kieffer. A further 4 references are discussed that most likely deal with C. miombo sp. nov. and not C. brosseti. C. miombo sp. nov. is apparently widespread in subtropical and tropical Africa and is now recorded from Zimbabwe, Botswana, South Africa, Nigeria and the Ivory Coast. There are also probable records from Angola, Burkina Faso, Zambia and eastern Madagascar. On the African mainland, both north and south of the equator, the pattern of distribution of C. miombo sp. nov. correlates strongly with that of drier Guineo-Congolian rainforest, and Sudanian and Zambezian woodlands, the latter known as miombo in southern Africa. These phytochoria and associated biota are sensitive to frost and experience relatively high temperatures and rainfall-3 factors that appear to limit the distribution of C. miombo sp. nov. to north of the 20-22 degrees C mean annual temperature isotherms in southern Africa. The new species is a member of the Imicola group which consists of 6 species confined to the Afrotropical (including Madagascar), Oriental and eastern Palaearctic regions. One species has in historic times spread to Australia. The worldwide distribution of each species is briefly discussed. It is suggested that the Imicola and Orientalis groups are separate lineages within the subgenus Avaritia. Culicoides miombo sp. nov. is compared with its closest African congeners C. imicola, C. pseudopallidipennis Clastrier and C. bolitinos Meiswinkel; 15 character states are used to separate C. miombo sp. nov. and C. imicola. The female antennal and palpal measurements of C. miombo sp. nov. are subjected to statistical analysis to highlight their taxonomic usefulness. The larval habitat of C. miombo sp. nov. is unknown.     

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.     

An early record of Culicoides species (Diptera: Ceratopogonidae) developing in the dung of game animals in southern Africa

The value of the dung of game animals as a larval habitat for Culicoides species was briefly investigated in 1973/74 in the Kruger National Park, Transvaal, Republic of South Africa. A total of 4 species in the subgenus Avaritia was reared, 3 from elephant dung, 2 from zebra dung and 1 from the dung of buffalo. These early studies have recently led to more intensive rearing and a better understanding of the systematics of this subgenus in the Afrotropical region.     

Afrotropical Culicoides: a redescription of C. (Avaritia) kanagai Khamala & Kettle, 1971, reared from elephant dung in the Kruger National Park, South Africa

The discovery of Culicoides kanagai in South Africa represents a new record for this species. The female is redescribed, and the male is described for the first time. Culicoides (A.) dasyops Clastrier, 1958 is shown to be closely related to it but C. (A.) alticola is only superficially related. Short notes on the larval habitat of C. kanagai, the dung of the African elephant, Loxodonta africana, are given. The dung of both the white rhinoceros, Ceratotherium simum, and the black rhinoceros, Diceros bicornis, is considered to be a possible alternative site for the immatures of C. kanagai.     

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).     

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.     

Three new Chromadoropsis species (Nematoda,Desmodoridae) from Southern Africa and the North Sea

Three new Chromadoropsis species with four pharyngeal bulbi are described from Southern Africa, Namibia (S.W.A.) and the North Sea. C. granulosus sp.nov. is described from a sandy beach near Port Elizabeth. This species is characterized by a very distinct layer of yellow granules just below the cuticle. C. namibiensis sp.nov. is described from a sandy beach at Langstrand, Namibia. This species can be distinguished by the length and shape of the gubernaculum and spicules as well as the size of the capitulum. C. longispiculosa sp.nov. is described from the North Sea and is characterized by the long spicules and the presence of numerous porids. The genus Chromadoropsis Filipjev, 1918 is revised.     

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.     

Culicoides species associated with livestock in the Stellenbosch area of the Western Cape Province, Republic of South Africa (Diptera: Ceratopogonidae)

A total of 33,564 Culicoides midges was collected in 44 light trap collections made at 22 sites in the Stellenbosch area during November 1986. Of the 23 species present in these collections 8 were frequently encountered namely, C. magnus, C. imicola, Culicoides sp. 49, C. zuluensis, C. gulbenkiani, C. pycnostictus, C. distinctipennis and C. nivosus. Although C. magnus was abundant at all trap sites, the prevalence of the other species appeared to be affected by the proximity of the light trap to different host animals and/or larval habitats. Plain-wing species and members of the C. schultzei group were rarely collected. The larval habitats of most of the above species were located by the use of tent-type emergence traps. All these habitats were found on irrigated pastures or where drainage water had accumulated. The difference in the requirements of the various species was associated with certain factors, such as degree of moisture, the type and amount of organic matter present and the particle size of the underlying soil. The identity of the blood-meals of 69 individual Culicoides belonging to 7 species was determined. The 5 commonest species had all fed on cattle and 4 of these on sheep. Two species, C. pycnostictus and C. distinctipennis were positive for bird blood.     

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.     

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.     

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.     

Procamacolaimus tubifer Gerlach, 1953, Procamacolaimus africanus sp.nov. and Eontolaimus capensis gen.nov., sp.nov., (Nematoda,Leptolaimidae) from South Africa

A female and three males of Procamacolaimus tubifer Gerlach, 1953 are described and illustrated from Cape Receife Beach, Port Elizabeth. P. africanus sp.nov. is also described fromCape Receife Beach. It is compared with P. tubifer which it resembles but from which it can be differentiated by length of the buccal cavity, amphid size and the shape of the spicules. Eontolaimus capensis gen.nov., sp.nov. is described, also from the same sandy beach. This new genus is related to Deontolaimus papillatus de Man, 1880 from which it can be distinguished, among other things, by the absence of a tooth-like structure in the buccal cavity and the presence of pre-anal tubuli.     

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.     

A variant of the endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in European zoos.

Newly discovered, lethal elephant endotheliotropic herpesviruses (EEHV) have been identified in both Asian (Elephas maximus) and African (Loxodonta africana) elephants. Carried by otherwise healthy African elephants they can be fatal mainly for young Asian elephants. Since zoos often harbour both elephant species, we conducted a survey on the presence of EEHV in Asian elephants from 12 European zoos, 3 circuses and 1 Israeli zoo. Here, we demonstrate that all EEHV that have affected Asian elephants so far belong to the EEHV1 group. We also describe the detection and the partial sequencing of an endotheliotropic herpesvirus variant (named EEHV1b) in Asian elephants, being either an EEHV endogenous to Asian elephants or indicating different sources (African elephants) of infection.     

Preferred landing sites of Culicoides species (Diptera: Ceratopogonidae) on a horse in Israel and its relevance to summer seasonal recurrent dermatitis (sweet itch)

Six hundred and twenty culicoides of five species were collected from a bait horse at Kannot, Israel, between April and September 1986. Seventy-two per cent of the midges were collected from the belly and 27 per cent from the dorsal aspect of the body, ie, the sweet itch summer seasonal recurrent dermatitis (SSRD) zone. Midges were active mainly from half an hour prior to half an hour after sunset. Only Culicoides puncticollis, C imicola and C schultzei group were collected in considerable numbers. While C imicola was present continuously throughout the season, the appearance of the two other species was intermittent. Ninety-five per cent of C schultzei group and approximately 100 per cent of the C puncticollis prefer to land on and most probably bite the belly. Culicoides imicola showed a clear preference for the dorsal ridge which overlaps the (SSRD) itch zone, and 70 per cent of the midges were collected there, while 28 per cent were collected from the belly. A considerably high proportion of the midges of this species were parous, ie, part of the population lived long enough to bite more than once. Skin temperature measurements showed the belly to be the warmest part of the body. Wind speeds of two to three knots reduced the number of midges collected. The findings of this study, together with a previous one (Braverman et al 1983), incriminate C imicola as the likely principal agent of SSRD in Israel.