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.     

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.     

Culicoides (Diptera: Ceratopogonidae) associated with horses at Mogoditshane,Gaborone, Botswana

Veterinary Research Communications -     

Subsampling of large light trap catches of Culicoides (Diptera: Ceratopogonidae).

Analyses of 2 light trap catches comprising 6,041 and 1,598 Culicoides showed that the reliability of subsampling such catches increased with subsample size, while the subsampling error decreased with an increase in the number of individuals per species present in a subsample. Subsamples comprising approximately 500 Culicoides are deemed sufficient for comparing population densities between sites and sampling occasions and also give an acceptable indication of relative species abundance at a site. It is recommended that species for which the mean number of specimens in subsamples originating from 3 catches at a site is less than 7, should not be included in biometric analyses aimed at comparisons of population densities. For all other species a test level of 1% rather than 5% should be used for such comparisons. When species ratios obtained from subsampled catches are employed as indicators of abundance, the chi-squared test should be utilized at a 1% level if the ratios originate from 3 catches at a site and at a 0.1% level if only one catch per site is made. Due to poor representativeness of small catches, it is suggested that species for which fewer than 7 individuals are present in a single subsample, be excluded from chi-squared tests. A 5-point procedure for subsampling a large light trap collection of Culicoides is given.     

Evaluation of Culicoides insignis (Diptera: Ceratopogonidae) as a vector of bluetongue virus.

Based upon epidemiological evidence, Culicoides insignis Lutz is a probable biological vector of bluetongue viruses (BTV) in South Florida, the Caribbean Region and Central America. The vector potential of this species for BTV was evaluated in the laboratory in a series of experiments using insects caught in the field. Although there was great variation in the percentage of flies that fed from any one catch, it was demonstrated that C. insignis became infected after membrane feeding on a mixture of blood and virus. The infection rates ranged from 20 to 62.5%. Following intrathoracic inoculation, BTV replicated to high titres in C. insignis. Such flies were also shown to be capable of transmitting BTV to susceptible sheep and embryonated chicken eggs. This series of experiments provides the first conclusive evidence that C. insignis is a biological vector of bluetongue virus. This is the first proven vector of BTV in the neotropics.     

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.     

Culicoides spp. (Diptera: Ceratopogonidae) Associated with Farmed Ostriches (Struthio camelus) in Botswana

Veterinary Research Communications -     

Chemical composition of silage residues sustaining the larval development of the Culicoides obsoletus/Culicoides scoticus species (Diptera: Ceratopogonidae)

Culicoides (Diptera: Ceratopogonidae) are biological vectors of bluetongue virus (BTV). Bluetongue is a viral disease that affects domestic and wild ruminants. Since its recent emergence in northern Europe, this disease has caused considerable economic losses to the sheep and cattle industry. The biotopes, and more particularly the chemical characteristics which are suitable for larval development of the main vector species, are still relatively unknown. This study shows that the larvae of biting midges belonging to the species Culicoides obsoletus and Culicoides scoticus are able to breed in different types of silage residue (maize, grass, sugar beet pulp and their combinations). The chemical composition of substrates strongly influences the presence of the immature stages of these biting midges. Higher lignin and insoluble fibre contents seem to favour their presence and could play the role of a physical support for semi-aquatic larvae. In contrast, higher concentrations of magnesium and calcium are negatively correlated with the presence of these two species. These data will help to locate and monitor the breeding sites of these species and could contribute to the control of these insects on farms.     

Rearing of Culicoides nubeculosus (Diptera: Ceratopogonidae) by natural or artificial feeding in the laboratory

A technique for the mass rearing of Culicoides nubeculosus in the laboratory is described. Female midges were fed either on fresh or deep-frozen, defibrinated cattle blood (-70 degrees C) through latex membranes, or on anesthetized white mice. Feeding rates of up to 90% were obtained on the latex membrane, whereas only 41% of the midges fed on mice. The best oviposition rates of greater than 50% were achieved after feeding either on the latex membrane with fresh cattle blood or on mice. An average of greater than 100 eggs per female were deposited. The highest larval hatching rate was observed after feeding with fresh blood; about half of the larvae developed to the adult stage. A reproduction index was defined for the colony based on the feeding rate, oviposition rate, larval hatching rate and development to the adult stage. The highest reproduction index was obtained when the midges were fed on fresh cattle blood through the membrane.     

Seasonal allergic dermatitis in sheep in southern Brazil caused by Culicoides insignis (Diptera: Ceratopogonidae)

The incidence of seasonal dermatitis was studied in a Hampshire Down flock on a farm in southern Brazil. Epidemiological data, clinical signs and macroscopic pathology were obtained by visiting the farm. Histological lesions were studied in skin biopsies of affected sheep. Biting insects were collected from January to April 2005 in an attempt to identify the etiological agent of the disease. Disease prevalence was 40%; the age of the affected animals was variable. Disease occurred from December to March, some animals had lesions for the entire year. Clinical signs include pruritus on the ears, around the eyes and ventral abdomen. Initially erythema and small red papules were seen, followed by alopecia and crust formation. Histologically the lesions were characterized by perivascular eosinophilic dermatitis. Hyperkeratosis and acanthosis were observed in the chronic lesions. Both Anopheles albitarsis and Culicoides insignis were captured during the study. C. insignis bites caused pruritus in sheep. Both types of insects were caught when they approached the sheep baits approximately 30min after sunset. Results suggested that the disease occurred as a result of an immediate hypersensitivity reaction to C. insignis.     

Culicoides arakawae (Diptera: Ceratopogonidae) efficiently blood-fed and infected with Leucocytozoon caulleryi through a natural membrane

Culicoides arakawae, the most common Culicoides sp. on chicken farms in East Asia, is an important blood-sucking insect and Leucocytozoon caulleryi vector. How parasites, in an ingested blood bolus, enter the midgut of insects and deal with this complex and biochemically hostile environment is poorly understood. However, successful blood-feeding through a membrane in C. arakawae is beneficial for studying this phenomenon. Therefore, a membrane-feeding method for C. arakawae was developed in. The blood-feeding success rates of C. arakawae fed through five different membranes were: turkey egg at 43.7+/-11.7%, chicken egg at 45.2+/-12.1%, duck egg at 38.8+/-12.0%, pig gut at 0% and chick skin at 0%. In fertility measurements, the average number of eggs produced for C. arakawae fed through egg-shell membrane, at 77.7+/-15.1 per female, was significantly higher (P<0.01) than the 46.7+/-10.6 found in C. arakawae fed on the breast skin of a live chicken. Meanwhile, in parasite infectivity tests, C. arakawae could be infected by L. caulleryi when the vector was blood-fed with infective blood cells reconstituted with specific pathogen-free (SPF) sera through an egg-shell membrane. The sporozoite average and infection rates of inoculated chicks were 166.8+/-12.5 and 100%, respectively. In conclusion, feeding C. arakawae blood through fowl-egg-shell membranes should be an efficient method for in vitro infection of midges as the engorged midges are infected by parasites and display reproductive potential. Furthermore, the method is practical for feeding a large number of midges.     

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.     

Ingestion of microfilariae from different sources by Culicoides nubeculosus (Diptera: Ceratopogonidae) through an artificial membrane

Laboratory-hatched Culicoides nubeculosus midges were membrane-fed on cattle blood containing various Onchocerca microfilariae which had been isolated from the hides of red deer (Cervus elaphus) and from Australian and native German cattle. All microfilariae were ingested through the membrane except those of O. tarsicola, which were too large. Onchocerca gibsoni and O. lienalis reached the thoracic musculature, but no further development occurred. Microfilariae of O. flexuosa developed to the "sausage" stage. Microfilariae of O. gutturosa developed to the infective larval stage within 9 days at a temperature of 25 degrees C.     

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.     

Models for the dispersal in Australia of the arbovirus vector, Culicoides brevitarsis Kieffer (Diptera: Ceratopogonidae)

Culicoides brevitarsis is the main biting midge responsible for the transmission of bluetongue and Akabane viruses to livestock in Australia. Models are given for its dispersal after winter from endemic areas at the southern limit of its distribution in New South Wales (NSW); the models might also be applicable elsewhere. Model 1 shows that dispersal can be explained by distance from a key point just outside the endemic area in mid-northern/northern coastal NSW. The model provides probability data for times of first occurrence at sites within regions down the southern coastal plain or up the Hunter Valley towards (but rarely reaching) the western slopes and tablelands. Model 2 shows that the movement depends on temperature and wind speed from northerly and easterly directions. Preliminary data also are given to suggest a relationship between density in the endemic area and the maximum distance that C. brevitarsis can travel in a given year. The models can be linked to other information which in combination can provide probabilities for winter survival outside the endemic area, times of occurrence at sites where it cannot survive winter and times when activity ceases naturally at these sites at the end of the season. This information can be used to predict the potential for virus transmission and indicate zones of seasonal freedom from both vector and virus for the export of livestock.     

Bluetongue virus isolations from midges belonging to the Obsoletus complex (Culicoides, Diptera: Ceratopogonidae) in Italy

Between July and September 2002 there were outbreaks of bluetongue on three sheep holdings in the communities of San Gregorio Magno (Salerno, Campania), Laviano (Salerno, Campania) and Carpino (Foggia, Puglia), and the involvement of bluetongue virus (btv) was confirmed serologically and virologically. The mortality rate was at least 11 per cent and involved btv serotype 2 (btv-2) and serotype 9 (btv-9). These holdings were also surveyed for the Culicoides (Diptera: Ceratopogonidae) vectors; approximately 10,000 midges belonging to 15 species were captured, but they did not include a single specimen of the classical Afro-Asiatic bluetongue vector, Culicoides imicola. Species belonging to the Obsoletus complex dominated the light-trap collections, and Culicoides obsoletus Meigen, Culicoides scoticus Downes and Kettle and Culicoides dewulfi Goetghebuer constituted 90 per cent of all the Culicoides species captured. Fifty-six pools of the Obsoletus complex (excluding C dewulfi), each containing 100 individual midges and containing only parous and gravid females, were assayed for virus. btv-2 was isolated from three pools from San Gregorio Magno and Carpino, and btv-9 was isolated from one pool from Laviano. These results indicate that a species other than C imicola is involved in the current re-emergence of bluetongue in the Mediterranean Basin, but whether it is C obsoletus sensu stricto or C scoticus, or both, is uncertain.     

Papular dermatitis induced in guinea pigs by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae).

Histological, ultrastructural, and virological examinations were performed on abdominal skin from guinea pigs after a blood meal by colony-bred biting midges, Culicoides sonorensis. Small, superficial, cutaneous, crateriform ulcers with necrosis of superficial dermis developed at feeding sites and healed within 24-48 hours. Animals developed nonpruritic erythematous papules 5 days after feeding that persisted until the study ended at 12 days after feeding. Papules corresponded histologically to foci of epidermal hyperplasia and superficial interstitial dermatitis with intraepidermal micropustules and scattered intraepidermal polykaryons. The principal ultrastructural changes were spongiosis in germinal epithelium and neutrophilic-histiocytic exocytosis. No viral agents or broken mouthparts were identified in lesions. The dermatitis may represent a host reaction to persisting insect salivary secretion and should be considered as an additional consequence of blood feeding in future studies involving biting midges.