Compositional turnover in ecto- and endoparasite assemblages of an African bat, Miniopterus natalensis (Chiroptera, Miniopteridae): effects of hierarchical scale and host sex.

We studied the compositional turnover in infracommunities and component communities of ecto- and endoparasites infesting a bat, Miniopterus natalensis (Chiroptera, Miniopteridae), across seven sampling sites using the zeta diversity metric (measuring similarity between multiple communities) and calculating zeta decline and retention rate (both scales) and zeta decay (component communities). We asked whether the patterns of zeta diversity differ between (a) infracommunities and component communities; (b) ecto- and endoparasites and (c) subsets of communities infecting male and female bats. The pattern of compositional turnover differed between infracommunities and component communities in endoparasites only. The shape of zeta decline for infracommunities indicated that there were approximately equal probabilities of ecto- and endoparasitic species to occur on/in any bat individual within a site. The shape of zeta decline for component communities suggested the stochasticity of ectoparasite turnover, whereas the turnover of endoparasites was driven by niche-based processes. Compositional turnover in component communities of ectoparasites was more spatially dependent than that of endoparasites. Spatial independence of compositional turnover in endoparasites was due to subcommunities harboured by female bats. We conclude that the patterns of compositional turnover in infracommunities were similar in ecto- and endoparasites, whereas the patterns of turnover in component communities differed between these groups.

African Horse Sickness Virus: History, Transmission, and Current Status.

African horse sickness virus (AHSV) is a lethal arbovirus of equids that is transmitted between hosts primarily by biting midges of the genus Culicoides (Diptera: Ceratopogonidae). AHSV affects draft, thoroughbred, and companion horses and donkeys in Africa, Asia, and Europe. In this review, we examine the impact of AHSV critically and discuss entomological studies that have been conducted to improve understanding of its epidemiology and control. The transmission of AHSV remains a major research focus and we critically review studies that have implicated both Culicoides and other blood-feeding arthropods in this process. We explore AHSV both as an epidemic pathogen and within its endemic range as a barrier to development, an area of interest that has been underrepresented in studies of the virus to date. By discussing AHSV transmission in the African republics of South Africa and Senegal, we provide a more balanced view of the virus as a threat to equids in a diverse range of settings, thus leading to a discussion of key areas in which our knowledge of transmission could be improved. The use of entomological data to detect, predict and control AHSV is also examined, including reference to existing studies carried out during unprecedented outbreaks of bluetongue virus in Europe, an arbovirus of wild and domestic ruminants also transmitted by Culicoides.

Structural insight into African horsesickness virus infection.

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T=13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines.

An Investigation of Culicoides (Diptera: Ceratopogonidae) as Potential Vectors of Medically and Veterinary Important Arboviruses in South Africa.

Culicoides-borne viruses such as bluetongue, African horse sickness, and Schmallenberg virus cause major economic burdens due to animal outbreaks in Africa and their emergence in Europe and Asia. However, little is known about the role of Culicoides as vectors for zoonotic arboviruses. In this study, we identify both veterinary and zoonotic arboviruses in pools of Culicoides biting midges in South Africa, during 2012-2017. Midges were collected at six surveillance sites in three provinces and screened for Alphavirs, Flavivirus, Orthobunyavirus, and Phlebovirus genera; equine encephalosis virus (EEV); and Rhaboviridae, by reverse transcription polymerase chain reaction. In total, 66/331 (minimum infection rate (MIR) = 0.4) pools tested positive for one or more arbovirus. Orthobunyaviruses, including Shuni virus (MIR = 0.1) and EEV (MIR = 0.2) were more readily detected, while only 2/66 (MIR = 0.1) Middelburg virus and 4/66 unknown Rhabdoviridae viruses (MIR = 0.0) were detected. This study suggests Culicoides as potential vectors of both veterinary and zoonotic arboviruses detected in disease outbreaks in Africa, which may contribute to the emergence of these viruses to new regions.

The utilisation of CytB and COI barcodes for the identification of bloodmeals and Culicoides species (Diptera: Ceratopogonidae) reveals a variety of novel wildlife hosts in South Africa.

Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) are vectors of pathogens that can cause diseases of major economic importance in humans and animals. Identifying host ranges of these biting midges might aid in understanding the complex epidemiology of such diseases, often involving reservoir hosts and multiple species. In this study, we aim to identify bloodmeal origin from engorged female Culicoides biting midges. All bloodfed females were opportunistically collected as part of an ongoing surveillance programme using Onderstepoort light traps in two provinces in South Africa. DNA of individuals was extracted and subjected to PCR targeting the cytochrome B (CytB) gene region of mammals and avians as well as cytochrome oxidase I (COI) for species identification. In total, 21 new reference barcodes were generated for C. bedfordi, C imicola, C. leucosticus, C. magnus, and C. pycnostictus. Seventy-four blood meals were identified, originating from 12 mammal and three avian species. COI sequence data performed well for species delimitation and 54 Culicoides specimens were identified with C. imicola the predominant species identified (41.8%). Generally, Culicoides species feed on a variety of hosts and host availability might be an important factor when selecting a host. Culicoides species thus appear to be opportunistic feeders rather than specialists. This implicates Culicoides as transfer vectors and demonstrates possible transmission routes of arboviruses and other pathogens from wildlife onwards to domestic animals and humans.

A distribution model for Glossina brevipalpis and Glossina austeni in Southern Mozambique, Eswatini and South Africa for enhanced area-wide integrated pest management approaches.

BACKGROUND: Glossina austeni and Glossina brevipalpis (Diptera: Glossinidae) are the sole cyclical vectors of African trypanosomes in South Africa, Eswatini and southern Mozambique. These populations represent the southernmost distribution of tsetse flies on the African continent. Accurate knowledge of infested areas is a prerequisite to develop and implement efficient and cost-effective control strategies, and distribution models may reduce large-scale, extensive entomological surveys that are time consuming and expensive. The objective was to develop a MaxEnt species distribution model and habitat suitability maps for the southern tsetse belt of South Africa, Eswatini and southern Mozambique. METHODOLOGY/PRINCIPAL FINDINGS: The present study used existing entomological survey data of G. austeni and G. brevipalpis to develop a MaxEnt species distribution model and habitat suitability maps. Distribution models and a checkerboard analysis indicated an overlapping presence of the two species and the most suitable habitat for both species were protected areas and the coastal strip in KwaZulu-Natal Province, South Africa and Maputo Province, Mozambique. The predicted presence extents, to a small degree, into communal farming areas adjacent to the protected areas and coastline, especially in the Matutuíne District of Mozambique. The quality of the MaxEnt model was assessed using an independent data set and indicated good performance with high predictive power (AUC > 0.80 for both species). CONCLUSIONS/SIGNIFICANCE: The models indicated that cattle density, land surface temperature and protected areas, in relation with vegetation are the main factors contributing to the distribution of the two tsetse species in the area. Changes in the climate, agricultural practices and land-use have had a significant and rapid impact on tsetse abundance in the area. The model predicted low habitat suitability in the Gaza and Inhambane Provinces of Mozambique, i.e., the area north of the Matutuíne District. This might indicate that the southern tsetse population is isolated from the main tsetse belt in the north of Mozambique. The updated distribution models will be useful for planning tsetse and trypanosomosis interventions in the area.

Gamma Irradiation and Male Glossina austeni Mating Performance.

An area-wide integrated pest management (AW-IPM) strategy with a sterile insect technique (SIT) component has been proposed for the management of African animal trypanosomosis (AAT) in South Africa. In preparation for the SIT, the mating performance of colony reared Glossina austeni males under influencing factors such as radiation dose and the development stage that is exposed to radiation, was assessed under laboratory and semi-field conditions. The radiation sensitivity of G. austeni colonized 37 years ago when treated as adults and late-stage pupae was determined. Radiation doses of 80 Gy and 100 Gy induced 97-99% sterility in colony females that mated with colony males treated as adults or pupae. Males irradiated either as adults or pupae with a radiation dose of 100 Gy showed similar insemination ability and survival as untreated males. Walk-in field cage assessments indicated that a dose of up to 100 Gy did not adversely affect the mating performance of males irradiated as adults or late stage pupae. Males irradiated as adults formed mating pairs faster than fertile males and males irradiated as pupae. The mating performance studies indicated that the colonized G. austeni males irradiated as adults or late stage pupae will still be suited for SIT.

The influence of temperature and humidity on the flight activity of Culicoides imicola both under laboratory and field conditions.

BACKGROUND: Insight into the factors that regulate the circadian host-seeking flight activity of Culicoides vectors (Diptera: Ceratopogonidae) will be of importance to assess the risk of transmission of Culicoides-borne pathogens. This study aimed to determine the impact of temperature and humidity on the flight activity of Culicoides imicola Kieffer, and other livestock associated Culicoides species, under both laboratory and field conditions. METHODS: Batches of 500 field-collected C. imicola females were acclimatized at a predetermined range of temperatures (10-29 °C) and relative humidity (34-85%). After acclimatization, these females, prompted by a light source, were allowed to escape through a transparent plastic funnel into a paper cup, where they were counted after an hour. Flight activity under field conditions was determined seasonally by hourly light trap collections done overnight at four sites near cattle. RESULTS: Experiments conducted at various test conditions in the laboratory indicated that flight activity started at 13 °C. Peak in activity was observed between 16 °C to 18 °C, and temperatures above 20 °C seemingly inhibit flight. Under field conditions, a peak in numbers collected was observed immediately after sunset. With mean nocturnal temperatures below 19 °C, more than 74% of the Culicoides were collected within two to three hours after sunset. With mean nocturnal temperature above 19 °C, the peak in numbers at sunset was sustained until after midnight, with somewhat higher numbers collected after midnight once temperatures dropped below 20 °C. No peak in numbers was observed at dawn. Although very low numbers were collected during the day, which partly may have been a result of the collecting method, Culicoides were present throughout periods of 24 hours. Humidity seemed to play a minor role in the regulation of flight activity. CONCLUSIONS: Abundance and species diversity results as obtained in this study indicated a high level of risk of virus transmission in the first hours following sunset. A strong relationship was found between host-seeking activity, and hence trap efficiency, and within limits, temperature. Light traps primarily measure flight activity and may as such underestimate adult abundance of C. imicola if deployed at temperatures outside thresholds of 16-20 °C.

Using genetic and phenetic markers to assess population isolation within the southernmost tsetse fly belt in Africa.

The effective control of tsetse flies (Diptera; Glossinidae), the biological vectors of trypanosome parasites that cause human African trypanosomosis and African animal trypanosomosis throughout sub-Saharan Africa, is crucial for the development of productive livestock systems. The degree of genetic isolation of the targeted populations, which indicate reinvasion potential from uncontrolled areas, will be critical to establish a control strategy. Molecular and morphometrics markers were used to assess the degree of genetic isolation between seemingly fragmented populations of Glossina brevipalpis Newstead and Glossina austeni Newstead present in South Africa. These populations were also compared with flies from adjacent areas in Mozambique and Eswatini. For the molecular markers, deoxyribonucleic acid was extracted, a r16S2 Polymerase chain reaction (PCR) was performed and the PCR product sequenced. Nine landmarks were used for the morphometrics study as defined by vein intersections in the right wings of female flies. Generalised Procrustes analyses and regression on centroid size were used to determine the Cartesian coordinates for comparison between populations. Both methods indicated an absence of significant barriers to gene flow between the G. brevipalpis and G. austeni populations of South Africa and southern Mozambique. Sustainable control can only be achieved if implemented following an area-wide management approach against the entire G. brevipalpis and G. austeni populations of South Africa and southern Mozambique. Limited gene flow detected between the G. austeni population from Eswatini and that of South Africa or Mozambique may imply that these two populations are in the proses of becoming isolated.

Flight and swarming behaviour of Culicoides species (Diptera: Ceratopogonidae) on a livestock farm in Northern Spain.

The efficacy of sweep nets and a CDC white light-suction trap for the sampling of Culicoides species (Diptera: Ceratopogonidae) were compared on a livestock farm in Northern Spain during the Summer of 2013. A total of 6,082 specimens representing 26 species were collected with sweep nets in 4 areas at di erent heights (ground level, 1.5 m, and 3 m), and 8,463 specimens representing 28 species with a single white light trap. Eight species - Culicoides brunnicans, Culicoides punctatus, Culicoides obsoletus/Culicoides scoticus, Culicoides lupicaris, Culcoides picturatus, Culicoides achrayi, and Culicoides simulator - were dominant and accounted for 97.4% and 97.2% of the total specimens collected with both methods, sweep nets, and light traps, respectively. The sex ratios with sweep netting and light trapping were strongly female biased (78.4% and 97.1%, respectively). Nulliparous and parous females were predominantly captured with both methods. A high percentage (17%) of gravid females was, however, captured on manure at ground level while sweeping. Searches for male swarms revealed the presence of several C. punctatus swarms consisting of 26 to 196 males and 3 swarms of C. obsoletus that ranged from 1 to 12 males in size. This study suggested that both methods are suitable and complementary tools for Culicoides sampling.

Evaluation of radiation sensitivity and mating performance of Glossina brevipalpis males.

BACKGROUND: Area-wide integrated pest management strategies that include a sterile insect technique component have been successfully used to eradicate tsetse fly populations in the past. To ensure the success of the sterile insect technique, the released males must be adequately sterile and be able to compete with their native counterparts in the wild. METHODOLOGY/PRINCIPAL FINDINGS: In the present study the radiation sensitivity of colonised Glossina brevipalpis Newstead (Diptera; Glossinidae) males, treated either as adults or pupae, was assessed. The mating performance of the irradiated G. brevipalpis males was assessed in walk-in field cages. Glossina brevipalpis adults and pupae were highly sensitive to irradiation, and a dose of 40 Gy and 80 Gy induced 93% and 99% sterility respectively in untreated females that mated with males irradiated as adults. When 37 to 41 day old pupae were exposed to a dose of 40 Gy, more than 97% sterility was induced in untreated females that mated with males derived from irradiated pupae. Males treated as adults with a dose up to 80 Gy were able to compete successfully with untreated fertile males for untreated females in walk-in field cages. CONCLUSIONS/SIGNIFICANCE: The data emanating from this field cage study indicates that, sterile male flies derived from the colony of G. brevipalpis maintained at the Agricultural Research Council-Onderstepoort Veterinary Institute in South Africa are potential good candidates for a campaign that includes a sterile insect technique component. This would need to be confirmed by open field studies.

Oral susceptibility to bluetongue virus of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom.

Oral susceptibility to infection with bluetongue virus (family Resviridae, genus Orbivirus, BTV) serotype 9 was characterized in three Palaearctic species of Culicoides (Diptera: Ceratopogonidae). Variation in susceptibility to infection by using a recently described feeding technique was shown to occur between populations of Culicoides obsoletus Meigen complex midges from different geographic regions of the United Kingdom with virus infection rates varying from 0.4 to 7.4% of those tested. Susceptibility to infection was consistent on an annual basis at selected sites. Prevalence of infection in the most susceptible populations of both the C. obsoletus and Culicoides pulicaris L. complexes was comparable with that of Culicoides imicola Kieffer, the major vector of BTV in southern Europe and throughout Africa, when using the same feeding method and virus. These results are discussed with reference to the potential threat of the virus to susceptible livestock in northern Europe.

Improving the Diet for the Rearing of Glossina brevipalpis Newstead and Glossina austeni Newstead: Blood Source and Collection - Processing - Feeding Procedures.

One of the challenges to maintain tsetse fly (Diptera: Glossinidae) colonies is the sustainable supply of high quality blood meals. The effect of using anticoagulants during collection of the blood, the addition of phagostimulants to the blood meals as well as using mixtures of bovine and porcine blood in different proportions for feeding on colony productivity was assessed. Defibrinated bovine blood was found to be suitable to maintain both the Glossina brevipalpis Newstead and Glossina austeni Newstead colonies. Blood collected with the anticoagulants sodium citrate, citric sodium combination, citrate phosphate dextrose adenine and citric acid did not affect colony performance of both species. Defibrinated bovine and porcine blood in a 1:1 ratio or the feeding of either bovine or porcine blood on alternating days improved pupae production of G. austeni and can be used to enhance colony growth. Bovine blood is appropriate to maintain G. brevipalpis colonies, however, feeding either bovine or porcine blood on alternating days did improve productivity. Adding the phagostimulants inosine tri-phosphate, cytosine mono-phosphate and guanosine mono-phosphate to the blood at a concentration of 10-4 M improved pupae production of the G. brevipalpis colony. The addition of adenosine tri-phosphate and inosine tri-phosphate improved the performance of the G. austeni colony. Decisions on the most suitable rearing diet and feeding protocols will not only depend on the biological requirements of the species but also on the continuous supply of a suitable blood source that can be collected and processed in a cost-effective way.

Detection of African horse sickness virus in Culicoides imicola pools using RT-qPCR.

African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub-Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT-qPCR. Midges were fed on AHSV-infected blood. A single blood-engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepared for RT-qPCR. The virus was successfully detected and the optimal pool size for the limit of detection of the virus was determined at a range between 1 to 25. Results from this investigation highlight the need for a standardized protocol for AHSV investigation in Culicoides midges especially for comparison among different studies and for the determination of infection rate.

Possible over-wintering of bluetongue virus in Culicoides populations in the Onderstepoort area, Gauteng, South Africa.

Several studies have demonstrated the ability of certain viruses to overwinter in arthropod vectors. The over-wintering mechanism of bluetongue virus (BTV) is unknown. One hypothesis is over-wintering within adult Culicoides midges (Diptera; Ceratopogonidae) that survive mild winters where temperatures seldom drop below 10 °C. The reduced activity of midges and the absence of outbreaks during winter may create the impression that the virus has disappeared from an area. Light traps were used in close association with horses to collect Culicoides midges from July 2010 to September 2011 in the Onderstepoort area, in Gauteng Province, South Africa. More than 500 000 Culicoides midges were collected from 88 collections and sorted to species level, revealing 26 different Culicoides species. Culicoides midges were present throughout the 15 month study. Nine Culicoides species potentially capable of transmitting BTV were present during the winter months. Midges were screened for the presence of BTV ribonucleic acid (RNA) with the aid of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. In total 91.2% of midge pools tested positive for BTV RNA. PCR results were compared with previous virus isolation results (VI) that demonstrated the presence of viruses in summer and autumn months. The results indicate that BTV-infected Culicoides vectors are present throughout the year in the study area. Viral RNA-positive midges were also found throughout the year with VI positive midge pools only in summer and early autumn. Midges that survive mild winter temperatures could therefore harbour BTV but with a decreased vector capacity. When the population size, biting rate and viral replication decrease, it could stop BTV transmission. Over-wintering of BTV in the Onderstepoort region could therefore result in re-emergence because of increased vector activity rather than reintroduction from outside the region.

An update of the tsetse fly (Diptera: Glossinidae) distribution and African animal trypanosomosis prevalence in north-eastern KwaZulu-Natal, South Africa.

An unpredicted outbreak of African animal trypanosomosis or nagana in 1990 in north-eastern KwaZulu-Natal necessitated an emergency control programme, utilising the extensive cattledipping system in the area, as well as a reassessment of the tsetse and trypanosomosis problem in the province. Since 1990, sporadic blood sampling of cattle at the dip tanks in the naganainfested areas were undertaken to identify trypanosome species involved and to determine the infection prevalence in cattle. The distribution and species composition of the tsetse populations in the area were also investigated. From November 2005 to November 2007 selected dip tanks were surveyed for trypanosome infection prevalence. During April 2005 to August 2009 the distribution and abundance of tsetse populations were assessed with odour-baited H traps. The tsetse and trypanosome distribution maps were updated and potential correlations between tsetse apparent densities (ADs) and the prevalence of trypanosomosis were assessed. Glossina brevipalpis Newstead and Glossina austeni Newstead were recorded in locations where they have not previously been collected. No significant correlation between tsetse relative abundance and nagana prevalence was found, which indicated complex interactions between tsetse fly presence and disease prevalence. This was epitomised by data that indicated that despite large differences in the ADs of G. austeni and G. brevipalpis, trypanosome infection prevalence was similar in all three districts in the area. This study clearly indicated that both tsetse species play significant roles in trypanosome transmission and that it will be essential that any control strategy, which aims at sustainable management of the disease, should target both species.

Determination of the optimal mating age of colonised Glossina brevipalpis and Glossina austeni using walk-in field cages in South Africa.

BACKGROUND: For the control of Glossina brevipalpis and Glossina austeni that occur in South Africa an area-wide integrated pest management (AW-IPM) program with a sterile insect technique (SIT) component has been proposed. The quality of the released sterile male tsetse flies will greatly determine the success of the SIT component of the programme. Sterile males need to be able to compete with wild males immediately after their release in the affected area. The mating competitiveness can be affected by many factors including the optimal mating age of the fly which can have an impact on the timing of the release. METHODS: To assess the optimal mating age for G. brevipalpis and G. austeni, mating competitiveness studies were carried out in a walk-in field cage. First, the time of peak fly activity was determined by performing the experiment in the morning and then again in the afternoon. Thereafter, 3, 6 and 9-day-old male flies competed for 3-day-old virgin females. RESULTS: There were no significant differences in mating performance when the field cage experiments were done in the morning or in the afternoon. However, the mating latency was shorter in the afternoon than in the morning. For both species 9-day-old males mated significantly more often than 6 or 3-day-old males. Age did not affect the males' ability to transfer sperm, mating duration or the mating latency. All females that mated were inseminated. CONCLUSIONS: Age did influence the mating competitiveness of G. brevipalpis and G. austeni and it is recommended that sterile males are not released before the age of 9 days. Keeping the male flies in the rearing facility for 8 days will have economic and logistic consequences for AW-IPM programmes that have a SIT component.

Delineation of the population genetic structure of Culicoides imicola in East and South Africa.

BACKGROUND: Culicoides imicola Kieffer, 1913 is the main vector of bluetongue virus (BTV) and African horse sickness virus (AHSV) in Sub-Saharan Africa. Understanding the population genetic structure of this midge and the nature of barriers to gene flow will lead to a deeper understanding of bluetongue epidemiology and more effective vector control in this region. METHODS: A panel of 12 DNA microsatellite markers isolated de novo and mitochondrial DNA were utilized in a study of C. imicola populations from Africa and an outlier population from the Balearic Islands. The DNA microsatellite markers and mitochondrial DNA were also used to examine a population of closely related C. bolitinos Meiswinkel midges. RESULTS: The microsatellite data suggest gene flow between Kenya and south-west Indian Ocean Islands exist while a restricted gene flow between Kenya and South Africa C. imicola populations occurs. Genetic distance correlated with geographic distance by Mantel test. The mitochondrial DNA analysis results imply that the C. imicola populations from Kenya and south-west Indian Ocean Islands (Madagascar and Mauritius) shared haplotypes while C. imicola population from South Africa possessed private haplotypes and the highest nucleotide diversity among the African populations. The Bayesian skyline plot suggested a population growth. CONCLUSIONS: The gene flow demonstrated by this study indicates a potential risk of introduction of new BTV serotypes by wind-borne infected Culicoides into the Islands. Genetic similarity between Mauritius and South Africa may be due to translocation as a result of human-induced activities; this could impact negatively on the livestock industry. The microsatellite markers isolated in this study may be utilised to study C. bolitinos, an important vector of BTV and AHSV in Africa and identify sources of future incursions.

Assessment of the repellent effect of citronella and lemon eucalyptus oil against South African Culicoides species.

The use of insect repellents to reduce the attack rate of Culicoides species (Diptera:Ceratopogonidae) should form part of an integrated control programme to combat African horse sickness and other diseases transmitted by these blood-feeding midges. In the present study the repellent effects of a commercially available mosquito repellent, a combination of citronella and lemon eucalyptus oils, on Culicoides midges was determined. The number of midges collected with two 220 V Onderstepoort traps fitted with 8 W 23 cm white light tubes and baited with peel-stick patches, each containing 40 mg of active ingredient, was compared with that of two unbaited traps. Two trials were conducted and in each trial the four traps were rotated in two replicates of a 4 x 4 randomised Latin square design. Although more midges were collected in the baited traps, the mean number in the baited and unbaited traps was not significantly different. This mosquito repellent did not influence either the species composition or the physiological groups of Culicoides imicola Kieffer. The higher mean numbers in the baited traps, although not statistically significant, may indicate that this mosquito repellent might even attract Culicoides midges under certain conditions.

Thermal limits of two biting midges, Culicoides imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae).

BACKGROUND: Culicoides imicola Kieffer and Culicoides bolitinos Meiswinkel (Diptera: Ceratopogonidae) are both of veterinary importance, being vectors of Schmallenberg, bluetongue and African horse sickness (AHS) viruses. Within South Africa, these Culicoides species show a marked difference in their abundances according to altitude, with C. imicola highly abundant in lower altitudes, but being replaced as the dominant species by C. bolitinos in cooler, high-altitude regions. METHODS: The thermal physiology of field collected adults of each species was determined to evaluate whether it could account for differences in their distribution and abundance. Critical thermal maxima (CTmax) and minima (CTmin), as well as upper and lower lethal temperatures (ULT and LLT) were assessed after acclimation temperatures of 19'C, 24'C and 29'C. Critical thermal limits were determined using an ecologically relevant rate of temperature change of 0.06'C x min(-1). RESULTS: Significant differences in CTmin and CTmax were found between acclimation temperatures for C. imicola and C. bolitinos. In C. bolitinos, the LLT of individuals acclimated at 24'C was significantly improved (LLT50 = -6.01'C) compared with those acclimated at the other temperatures (LLT50 = -4'C). Acclimation had a weak (difference in LLT50 of only 1'C) but significant effect on the LLT of C. imicola. When CTmin, CTmax, LLT and ULT were superimposed on daily maximum and minimum temperature records from locations where each tested Culicoides species is dominant, it was found that temperatures frequently declined below the CTmin and LLT of C. imicola at the location where C. bolitinos was dominant. CONCLUSIONS: The distribution and abundance of C. imicola is likely directly constrained by their relatively poor tolerance of lower temperatures. Results for C. bolitinos suggest that the adult phase is hardy, and it is hypothesised that the thermal biology of other life stages could determine their range.