Bloodmeal analysis in Culicoides midges collected near horses, donkeys and zebras in the Eastern Cape, South Africa.

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014-2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood-fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood-fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.

Culicoides species as potential vectors of African horse sickness virus in the southern regions of South Africa.

African horse sickness (AHS), a disease of equids caused by the AHS virus, is of major concern in South Africa. With mortality reaching up to 95% in susceptible horses and the apparent reoccurrence of cases in regions deemed non-endemic, most particularly the Eastern Cape, epidemiological research into factors contributing to the increase in the range of this economically important virus became imperative. The vectors, Culicoides (Diptera: Ceratopogonidae), are considered unable to proliferate during the unfavourable climatic conditions experienced in winter in the province, although the annual occurrence of AHS suggests that the virus has become established and that vector activity continues throughout the year. Surveillance of Culicoides within the province is sparse and little was known of the diversity of vector species or the abundance of known vectors, Culicoides imicola and Culicoides bolitinos. Surveillance was performed using light trapping methods at selected sites with varying equid species over two winter and two outbreak seasons, aiming to determine diversity, abundance and vector epidemiology of Culicoides within the province. The research provided an updated checklist of Culicoides species within the Eastern Cape, contributing to an increase in the knowledge of AHS vector epidemiology, as well as prevention and control in southern Africa.

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.

Duration of repellency of N,N-diethyl-3-methylbenzamide, citronella oil and cypermethrin against Culicoides species when applied to polyester mesh.

The repellent efficacy of 15% N,N-diethyl-3-methylbenzamide (DEET), 0.6% citronella oil, and 0.3% alpha-cyano-cypermethrin against Culicoides species was compared in three 5x5 Latin squares (15 replicates) under South African field conditions. DEET, citronella oil or alpha-cyano-cypermethrin were applied to polyester meshes that were fitted to down-draught suction 220V UV light traps which were operated overnight. No significant repellent effect against Culicoides was found for the citronella oil or the alpha-cyano-cypermethrin. DEET had a significant repellent effect against Culicoides species and C. imicola for all catches made from after sunset to before sunrise.

Culicoides biting midges at the National Zoological Gardens of South Africa.

Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the transmission of a large number of pathogens to livestock and wild animals. In this study the presence of the genus, using light traps based at four different sites within the National Zoological Gardens of South Africa, was investigated during 2002-2004. In total, 37 species were recorded, including large numbers of Culicoides imicola Kieffer, 1913, which is responsible for the transmission of economically important arboviruses in South Africa, Europe, Middle and Far East. These results are discussed with reference to the wider Culicoides fauna in the Onderstepoort area of South Africa, their vector competence as well as biosecurity at the National Zoological Gardens.

Efficacy of alphacypermethrin-treated high density polyethylene mesh applied to jet stalls housing horses against Culicoides biting midges in South Africa.

The efficacy of alphacypermethrin-treated high density polyethylene (HDPE) mesh applied to jet stalls against Culicoides biting midges (Diptera: Ceratopogonidae) was determined by mechanical aspiration of midges from horses and using Onderstepoort 220 V downdraught black light traps in four blocks of a 3 × 2 randomised design under South African field conditions. The alphacypermethrin-treated HDPE mesh applied to the stall significantly (P = 0.008) reduced the number of Culicoides midges, predominantly Culicoides (Avaritia) imicola Kieffer, mechanically aspirated from horses housed in the stall. The mesh reduced the Culicoides midge attack rate in the treated stall compared to the untreated stall and a sentinel horse by 6 times and 14 times, respectively. The number of Culicoides midges and C. imicola collected in light traps from the untreated and alphacypermethrin HDPE mesh-treated stalls did not differ significantly (P = 0.82). Alphacypermethrin-treated HDPE mesh could be used to reduce exposure of horses in jet stalls to Culicoides midges, specifically C. imicola, and the risk of midge-borne Orbivirus transmission.

Field and in vitro insecticidal efficacy of alphacypermethrin-treated high density polyethylene mesh against Culicoides biting midges in South Africa.

The efficacy of untreated and alphacypermethrin-treated high density polyethylene (HDPE) mesh against Culicoides biting midges (Diptera: Ceratopogonidae) was determined using Onderstepoort downdraught black light traps and a contact bioassay. Three traps were operated overnight in four replicates of a 3×3 randomised Latin square design near horses under South African field conditions. Both the untreated and alphacypermethrin-treated HDPE mesh significantly (P<0.05) reduced the numbers of Culicoides midges, predominantly Culicoides (Avaritia) imicola Kieffer, collected in the light traps by 4.2 and 7.2 times, respectively. A repellent effect of the alphacypermethrin-treated mesh was not confirmed because the number of midges collected in the light traps with untreated and alphacypermethrin-treated HDPE mesh was not significantly different (P=0.656). Bioassay of the insecticidal contact efficacy indicated median C. imicola mortality of 100% from 30 and 10 min following exposure to the alphacypermethrin-treated HDPE mesh for 1 or 3 min, respectively. In the bioassay, mortality was significantly higher (P=0.016) at 5 min post exposure in the midges exposed to the alphacypermethrin-treated mesh for 3 min (74.8%) compared to the 1 min exposure group (59.5%). The HDPE mesh could be used to reduce exposure of housed animals to Culicoides midges, specifically C. imicola, and viruses transmitted by these midges. Mesh treated with alphacypermethrin had the additional benefit of a rapid insecticidal effect on C. imicola.

The attraction range of the Onderstepoort 220V light trap for Culicoides biting midges as determined under South African field conditions.

Despite some limitations suction light traps are the primary tools used for the collection of Culicoides species (Diptera: Ceratopogonidae). The range of attraction of the Onderstepoort light trap is not known but an insight into the range of a trap will determine where the trap must be positioned relative to the hosts present, possible breeding sites and environmental structures in the trapping vicinity. It will therefore contribute to a more meaningful interpretation and comparison of results between trapping events. In the present study the number of Culicoides midges collected in a single trap was compared to those of traps made with an additional trap respectively 1m, 4m and 8.5m away from the first. Treatments between sites were rotated in three replicates of a 4×4 Latin square design. While interactions were found in traps 4m apart no statistically significant interactions were found when they were 8.5m apart. The range of attraction, indicated by the interaction between two traps, will be between 2m and 4m. In interpreting light trap results the limitations of this collection method needs to be taken into consideration.

The effect of 1-octen-3-ol and 4-methylphenol on Culicoides midge numbers collected with suction light traps in South Africa.

Despite some shortcomings, suction light traps are the primary monitoring tool for the collection of Culicoides species (Diptera: Ceratopogonidae). Factors that may increase the efficiency of these traps need to be investigated. In the present study the numbers of Culicoides midges collected with two Onderstepoort black light traps baited with a mixture of 1-octen-3-ol and 4-methylphenol, as a potential olfactory cue, were compared to those of two unbaited traps. Comparisons were done in two and three replicates of a 4 × 4 randomized Latin square design in the presence and absence of cattle. The addition of 1-octen-3-ol and 4-methylphenol, released at 9.1 and 15.5mg/h, respectively, did not influence species richness, numbers collected, sex ratios or age-grading results. Comparisons of Culicoides numbers and especially the abundance of Culicoides imicola Kieffer in collections done in the presence and absence of cattle confirm previous findings that show that host animals will be the primary attraction for Culicoides midges and that light traps mostly sample midges already in the near vicinity of the host.

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.

Life-history parameters of Culicoides (Avaritia) imicola Kieffer in the laboratory at different rearing temperatures.

This laboratory study investigates the sub-adult developmental cycle of field collected Culicoides (Avaritia) imicola Kieffer (Diptera; Ceratopogonidae). The period required from blood-feeding field-collected females to the production of progeny adults occupied 34-56 days at 20 degrees C, 15-21 days at 25 degrees C and 11-16 days at 28 degrees C, demonstrating clear temperature dependence. When reared at 28 degrees C, C. imicola demonstrated higher variability in fecundity (between 2.4 and 20.6 eggs/female) and lower hatching rates (50.0-62.2%), although larval survival rates to pupation were low at all temperatures (20-30%). Similarly, the mean emergence rate from pupae was the highest at lower temperatures. These results highlight the difficulty in establishing and maintaining a laboratory colony of this species from field-collected material and results are discussed in reference to future research directions that may aid this process.

Comparison of the efficiency of five suction light traps under field conditions in South Africa for the collection of Culicoides species.

Culicoides biting midges (Diptera: Ceratopogonidae) are involved in the transmission of a variety of pathogens, the economically most important being the orbiviruses that cause bluetongue and African horse sickness; both of which have been shown to be multi-vector diseases. The identification of all potential vectors will be crucial for the implementation of integrated control measures and disease risk analysis. The primary monitoring tools used for the collection of Culicoides midges are various models of suction light traps. In order to facilitate comparison of data between laboratories the efficiency of five traps (Onderstepoort, Rieb, mini-CDC, Pirbright, BG-sentinel), used at present and in the past in Europe, was compared in the field in South Africa. Comparisons were done either in three replicates of a 4x4 or two replicates of a 5x5 randomized Latin square design. The Onderstepoort trap collected significantly more Culicoides midges than the other traps. Relatively small but statistically significant differences were found in the species composition, parous rates, sex ratios as well as the ratio of Culicoides midges to other insects, as determined by the different traps. It will be important to determine the significance and underlying causes for these differences.

Stabling and the protection of horses from Culicoides bolitinos (Diptera: Ceratopogonidae), a recently identified vector of African horse sickness.

The stabling of horses at night reportedly offers protection from African horse sickness and the most significant vector of the disease, Culicoides imicola Kieffer, has been shown to be exophilic. In certain high-lying regions of South Africa, however, C. bolitinos Meiswinkel, may be the major vector of the disease but its entry behaviour into stables is unknown. Accordingly, in the eastern Free State province of South Africa, light trap catches of C. bolitinos inside stables and outside, were compared. Two horse-baited stables, one traditional, and one modern, were used and combinations of stable (old/new), ceiling fans (on/off) and accessibility to Culicoides (stable doors open/closed or windows gauzed/ungauzed) were investigated as treatments. A total of 111,452 Culicoides of 26 species was collected on 60 trap nights; C. bolitinos was dominant (89.1% overall) with C. imicola second in abundance (2.9%). Outside catches were greater on warmer, drier, evenings but were suppressed by high wind speeds. Catches of C. imicola inside stables with doors open, or with windows ungauzed, were less than the numbers captured outside. In contrast, more C. bolitinos were caught in open stables than outside, i.e. open structures may protect horses from the exophilic C. imicola, but may increase attack rates from the endophilic C. bolitinos. The closing of doors and the gauzing of windows, however, led to a 14-fold reduction in numbers of C. bolitinos and C. imicola entering stables. A well-gauzed 'traditional' stable was as effective as a closed 'modern' stable. Ceiling fans had no suppressant effect.

Christopher Columbus and Culicoides: was C. jamaicensis Edwards, 1922 introduced into the Mediterranean 500 years ago and later re-named C. paolae Boorman 1996?

The biting midge, Culicoides paolae Boorman, described from specimens collected in the extreme south of Italy in 1996, belongs in the subgenus Drymodesmyia. This subgenus was erected by Vargas in 1960 for the so-called Copiosus species group, an assemblage of 22 species endemic to the tropical regions of the New World and, where known, breed in vegetative materials including the decaying leaves (cladodes) and fruits of Central American cacti. The Mexican peoples have utilised these cacti for over 9,000 years; one of these, Opuntia ficus-indica Linnaeus, was brought to Europe by Christopher Columbus following his voyages of discovery. As a taxon C. paolae is very similar to the Central American C. jamaicensis Edwards, 1922 raising the possibility that it (or a closely related species of Drymodesmyia) was introduced into the Mediterranean Region at the time of Columbus, but was (perplexingly) discovered only 500 years later and named C. paolae. The comparison of Sardinian specimens of C. paolae with Panamanian material of C. jamaicensis (housed in the Natural History Museum in London) confirmed the two species to be very similar but unusual differences were noted around the precise distribution of the sensilla coeloconica on the female flagellum. Until it is understood whether these differences represent either intra- or interspecific variation, the question of the possible synonymy of C. paolae must be held in abeyance.

Multiple vectors and their differing ecologies: observations on two bluetongue and African horse sickness vector Culicoides species in South Africa.

Blacklight traps were used to collect Culicoides biting midges weekly between September 1996 and August 1998 at 40 sites distributed equidistantly across South Africa. The seasonal and geographic prevalences of 86 species of Culicoides were elucidated simultaneously, and included C. imicola Kieffer and C. bolitinos Meiswinkel the principal vectors of bluetongue (BT) and African horse sickness (AHS) in the region. These two species were amongst the most prevalent Culicoides to be found and, together, comprised >50% of the more than three million biting midges captured. The data are presented as coloured matrices, and are transformed also into inverse distance weighting (IDW) interpolative maps. The data reveal that the prevalence of each vector is somewhat fractured and it is posited that this is (in part) due to significant differences in their respective breeding habitats. The results illustrate also that the presence of multiple vectors (in any region of the world) will complicate both the epidemiology of the orbiviral diseases they transmit and the formulation of rational livestock movement and disease control strategies. This is especially true for southern Europe where the recent devastating cycle of BT has been shown to involve at least three vectors. Finally, the influence that man has on the development of large foci of vector Culicoides around livestock may be less important than previously suggested but must be investigated further.