Barcoding of biting midges in the genus Culicoides: a tool for species determination

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.     

Comparing the effect of modeled climatic variables on the distribution of African horse sickness in South Africa and Namibia

Africa horse sickness (AHS) is a lethal disease of horses with a seasonal occurrence that is influenced by environmental conditions that favor the development of Culicoides midges (Diptera: Ceratopogonidae). This study compared and evaluated the relationship of various modeled climatic variables with the distribution and abundance of AHS in South Africa and Namibia. A comprehensive literature review of the historical AHS reported data collected from the Windhoek archives as well as annual reports from the Directorate of Veterinary services in Namibia were conducted. South African AHS reported data were collected from the South African Department of Agriculture, Forestry, and Fisheries. Daily climatic data were extracted for the time period 1993–2011 from the ERA‐interim re‐analysis dataset. The principal component analysis of the complete dataset indicated a significant statistical difference between Namibia and South Africa for the various climate variables and the outbreaks of AHS. The most influential parameters in the distribution of AHS included humidity, precipitation, evaporation, and minimum temperature. In South Africa, temperature had the most significant effect on the outbreaks of AHS, whereas in Namibia, humidity and precipitation were the main drivers. The maximum AHS cases in South Africa occurred at temperatures of 20–22° C and relative humidity between 50–70%. Furthermore, anthropogenic effects must be taken into account when trying to understand the distribution of AHS.     

Evaluation of long-distance dispersal of Culicoides midges into northern Australia using a migration model

The introduction of novel bluetongue serotypes and genotypes into northern Australia is considered possible via the long-distance windborne dispersal of Culicoides (Diptera: Ceratopogonidae) vectors from Southeast Asia. Initial findings from simulation modelling of potential dispersal over a 15-year period revealed that the greatest risk for incursion of windborne Culicoides from the island of Timor into northern Australia occurs during December-March. The regions at greatest risk for incursion include the top end of the Northern Territory and the Kimberley region in Western Australia, but there is potential for more widespread dispersal into northern Australia based on Timor as the putative source. The establishment of a more pathogenic strain of the virus, or of a novel Culicoides vector introduced by such inter-continental dispersal events, could dramatically alter Australia's current bluetongue disease status.     

Phylogenetic analysis of the mitochondrial cytochrome oxidase subunit I gene of five species of the Culicoides imicola species complex

The phylogenetic status of members of the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) species complex of haematophagous midges is unknown, and simple means to identify the members using all life stages are unavailable. In this study, the status of three confirmed (C. imicola s.s., C. bolitinos Meiswinkel and C. loxodontis Meiswinkel) and two provisional (C. tuttifrutti Meiswinkel and C. kwagga Meiswinkel) members of the complex from South Africa was assessed using phylogenetic analysis of partial DNA and amino acid sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The four or five individuals of each species analysed contained one or two haplotypes each. Interspecific divergence was significant and characterized by strong A <--> T transversion bias. Phylogenetic trees constructed using neighbour-joining, parsimony and maximum likelihood showed each species to be distinct. Combinations of sites for two restriction enzymes in the COI sequences were species-specific and could form the basis of a diagnostic PCR assay.     

Effect of different diets on larval growth of Protaetia brevitarsis seulensis (Kolbe) (Coleoptera: Cetoniidae)

The white-spotted flower chafer, Protaetia brevitarsis seulensis (Kolbe), is used as an important source of medicinal properties, such as anti-cancer activity and prevention of liver-related diseases, in Korea. To use this insect species without endangering wild populations, a mass rearing technique is needed. The present study was carried out to determine whether different diets can affect the larval growth of P. brevitarsis seulensis . Six different diets were investigated in the experiment, and the results showed that 20% rice straw with 80% oakwood sawdust was the best diet for larval growth of this beetle.     

A comparison of the susceptibility of the biting midge Culicoides imicola to infection with recent and historical isolates of African horse sickness virus

The susceptibility of Culicoides (Avaritia) imicola Kiefer (Diptera: Ceratopogonidae) to 21 isolates representing all nine known serotypes of African horse sickness virus (AHSV), recovered from clinical cases of the disease in South Africa during 1998–2004, was compared with its susceptibility to approximately 40‐year‐old isolates stored at the Agricultural Research Council‐Onderstepoort Veterinary Institute. Field‐collected C. imicola were fed through a chicken skin membrane on sheep blood spiked with one of the virus isolates to a concentration in the range of 5.6–7.5log ₁₀TCID₅₀/mL. After 10 days incubation at 23.5 °C, five of the nine historical serotypes (AHSV‐1, ‐2, ‐3, ‐7 and ‐9) could not be isolated from C. imicola. All nine serotypes were recovered for the 21 recent isolates, for 16 of which the virus recovery rates were higher than for the corresponding historical isolates. These results emphasize the need to assess the oral susceptibility of local Culicoides populations to viruses in circulation during outbreaks in order to estimate their vector potential.     

Responses of the biting midge Culicoides impunctatus to acetone, CO2 and 1-octen-3-ol in a wind tunnel

Responses of host-seeking female Culicoides impunctatus (Goetghebuer) (Diptera: Ceratopogonidae) to acetone, carbon dioxide and 1-octen-3-ol were measured in a wind tunnel. Carbon dioxide, presented as a filamentous plume, increased upwind flight in a dose-dependent manner, up to 0.09% concentration. A homogenous CO2 plume elicited similar upwind responses at concentrations up to 0.09%, whereas higher plume concentrations (> 0.1%) induced erratic responses with a suppression of upwind flight. Bovine equivalent concentrations of acetone (1.5 x 10(-6)g/l) and 1-octen-3-ol (1.3 x 10(-8)g/) failed to induce any significant upwind response when tested alone. In the presence of CO2, however, 1-octen-3-ol showed highly significant increases in upwind responses at concentrations of 1.3 x 10(-1) - 10(-8)g/l. Mixtures of CO2+ acetone also enhanced upwind flight at 1.5 x 10(-9)g/l. High tunnel concentrations of both 1-octen-3-ol and acetone inhibited upwind responses. These findings are discussed in relation to host finding by C. impunctatus and known mechanisms by which upwind flight is initiated and arrested at high odour concentrations.     

Molecular differentiation of the Old World Culicoides imicola species complex (Diptera, Ceratopogonidae), inferred using random amplified polymorphic DNA markers

Samples of seven of the 10 morphological species of midges of the Culicoides imicola complex were considered. The importance of this species complex is connected to its vectorial capacity for African horse sickness virus (AHSV) and bluetongue virus (BTV). Consequently, the risk of transmission may vary dramatically, depending upon the particular cryptic species present in a given area. The species complex is confined to the Old World and our samples were collected in Southern Africa, Madagascar and the Ivory Coast. Genomic DNA of 350 randomly sampled individual midges from 19 populations was amplified using four 20-mer primers by the random amplified polymorphic DNA (RAPD) technique. One hundred and ninety-six interpretable polymorphic bands were obtained. Species-specific RAPD profiles were defined and for five species diagnostic RAPD fragments were identified. A high degree of polymorphism was detected in the species complex, most of which was observed within populations (from 64 to 76%). Principal coordinate analysis (PCO) and cluster analysis provided an estimate of the degree of variation between and within populations and species. There was substantial concordance between the taxonomies derived from morphological and molecular data. The amount and the different distributions of genetic (RAPD) variation among the taxa can be associated to their life histories, i.e. the abundance and distribution of the larval breeding sites and their seasonality.     

Identification and Characterization of a cDNA Clone Encoding the Heat Shock Protein (Hsp60) from the Biting Midge, Culicoides variipennis sonorensis Wirth and Jones

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67–78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.