Vector competence of Culicoides species and the seroprevalence of homologous neutralizing antibody in horses for six serotypes of equine encephalosis virus (EEV) in South Africa

Abstract. Field‐collected Culicoides species (Diptera: Ceratopogonidae) were fed on horse blood–virus mixtures containing one of the six serotypes of equine encephalosis virus (EEV1 to EEV6). The virus mean titres in the bloodmeals varied between 6.1 and 7.0 log₁₀TCID₅₀/mL. Of 19 Culicoides species assayed after 10 days extrinsic incubation at 23.5°C, five yielded the challenge virus, namely Culicoides (Avaritia) imicola Kieffer (EEV1–6), C. (A.) bolitinos Meiswinkel (EEV1, 2, 4, 6), C. (Meijerehelea) leucostictus Kiefer (EEV1, 2), C. (Culicoides) magnus Colaço (EEV1) and C. (Hoffmania) zuluensis de Meillon (EEV2). Virus recovery rates ranged from 0.5 to 13%. The mean levels of viral replication differed between serotypes and Culicoides species and ranged from 1.0 to 2.3 log₁₀TCID₅₀/midge. Culicoides midges shown in this study to be susceptible to oral infection with EEV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites. Of 1456 horses tested, 1144 (77%) had antibody to EEV. Homologous virus‐neutralizing antibodies to all six serotypes were detected in individual horses from all eight geographical provinces of South Africa. The distribution, prevalence, and the rate of exposure to individual serotypes varied significantly between regions. The potential for vectoring of EEV in the field by several Culicoides species with unique ecologies and lack of cross‐protection to re‐infection with multiple serotypes highlights some of the mechanisms that are likely to play a role in the virus' natural maintenance cycle and the highly efficient level of countrywide transmission amongst South African horses.     

Oral susceptibility of South African Culicoides species to live-attenuated serotype-specific vaccine strains of African horse sickness virus (AHSV)

Abstract. The oral susceptibility of livestock‐associated South African Culicoides midges (Diptera: Ceratopogonidae) to infection with the tissue culture‐attenuated vaccine strains of African horse sickness virus (AHSV) currently in use is reported. Field‐collected Culicoides were fed on horse blood‐virus mixtures each containing one of the seven serotype‐specific vaccine strains of AHSV, namely serotypes 1, 2, 3, 4, 6, 7 and 8. The mean titres of virus in the bloodmeals for the seven vaccine strains were between 6.8 and 7.6 log₁₀TCID50/mL. All females (n = 3262) that survived 10 days extrinsic incubation (10 dEI) at 23.5°C were individually assayed in microplate BHK‐21 cell cultures. In midges tested immediately after feeding, AHSV was detected in 96.1% individuals; mean virus titre was 2.0 log₁₀TCID50/midge. After 10 dEI virus recovery rates varied in Culicoides (Avaritia) imicola Kieffer from 1% (AHSV‐2) to 11% (AHSV‐7) and in Culicoides (A.) bolitinos Meiswinkel from 0% (AHSV‐3) to 14.6% (AHSV‐2). Although our results indicate that two major field vectors C. imicola and C. bolitinos are susceptible to oral infection with vaccine strains of AHSV, the level of viral replication for most of the vaccine strains tested was below the postulated threshold (=2.5 log₁₀TCID50/midge) for fully disseminated orbivirus infection. In this study, for the first time AHSV has been recovered after 10 dEI from six non‐Avaritia livestock‐associated Old World species: C. engubandei de Meillon (AHSV‐4), C. magnus Colaço (AHSV‐3, ‐4), C. zuluensis de Meillon (AHSV‐2, ‐4), C. pycnostictus Ingram & Macfie (AHSV‐2), C. bedfordi Ingram & Macfie (AHSV‐7), and C. dutoiti de Meillon (AHSV‐7). As little is known about the virogenesis of AHSV in the southern African species of Culicoides, the epidemiological significance of our findings in relation to the potential for transmission of current AHSV vaccine strains by Culicoides requires further assessment.     

Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos

Abstract. The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV‐1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field‐collected Culicoides were fed on sheep blood containing 7.5 log₁₀TCID₅₀/mL of BTV‐1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK‐21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30°C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge ≥ 3 log₁₀ TCID₅₀) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4–10 post‐infection (dpi), at 15–30°C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0–13.7% and 0–46.8%. In both species the highest IR was recorded at 25°C and the highest TP at 30°C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25°C to 8 dpi at 15°C. In C. imicola it ranged from 4 dpi at 30°C to 10 dpi at 23.5°C; no individuals with TP were detected at 15°C. There was no evidence of virus replication in flies held at 10°C. When, at various points of incubation, individual flies were transferred from 10°C to 23.5°C and then assayed 4–10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaço was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV‐1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa.     

A comparison of the vector competence of the biting midges,Culicoides (Avaritia) bolitinos and C. (A.) imicola,for the Bryanston serotype of equine encephalosis virus

Abstract Equine encephalosis virus (EEV) is widespread and prevalent in southern Africa. In this study, the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) to EEV was confirmed. In addition, C. (A.) bolitinos Meiswinkel, collected in the high‐lying eastern Free State, South Africa, was systemically infected with the Bryanston serotype of EEV after feeding through a membrane on artificially infected equine blood containing 4.7 log₁₀ PFU/mL of EEV. The mean infectivity of Bryanston virus in C. bolitinos increased from 1.2 log₁₀ PFU/midge, in midges assayed for virus immediately after feeding on the blood‐virus mixture, to 3.1 log₁₀ PFU/midge in midges assayed after 10 days' incubation at 23.5°C. Elevated virus infectivity titres, found in individual infected C. bolitinos, suggested that this Culicoides species is a vector of EEV. This bovine dung‐breeding Culicoides species may play an important role in transmitting EEV in the cooler parts of southern Africa, where it can be the most abundant Culicoides species collected near livestock. In the present study the prevalence of infection obtained for C. bolitinos (2.2%) with the Bryanston serotype of EEV was significantly lower than that of C. imicola (18.4%). After incubation, the Bryanston serotype of EEV was also isolated from one of 110 C. onderstepoortensis Fiedler assayed. However, the virus titre in this midge was 1.2 log₁₀ PFU/midge, which is not different from the titre that would be expected immediately after feeding on the blood‐virus mixture. Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. magnus Colaço (96), C. bedfordi Ingram & Macfie (95) and C. pycnostictus Ingram & Macfie (45).     

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.     

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.     

Oral susceptibility of South African stock-associated Culicoides species to bluetongue virus

Field-collected South African Culicoides species (Diptera, Ceratopogonidae) were fed on sheep blood containing bluetongue virus (BTV) represented by 13 low-passage reference serotypes: -1, -2, -4, -6, -7, -8, -9, -10, -11, -12, -13, -16 and -19. After 10 days of extrinsic incubation at 23.5 degrees C, of the 13 serotypes used, seven were recovered from C. (Avaritia) imicola Kieffer and 11 from C. (A.) bolitinos Meiswinkel. Virus recovery rates and the mean titres for most serotypes were significantly higher in C. bolitinos than in C. imicola. In addition, BTV was recovered from three non-Avaritia Culicoides species, namely C. (Remmia) enderleini Cornet & Brunhes (BTV-9), C. (Hoffmania) milnei Austen (BTV-4) and C. (H.) zuluensis de Meillon (BTV-16). No virus could be recovered from 316 individuals representing a further 14 Culicoides species. In Culicoides species fed on blood containing similar or identical virus titres of distinct BTV serotypes, significant differences were found in virus recovery rates. The results of this study confirm the higher vector competence of C. bolitinos compared with C. imicola.     

The oral susceptibility of South African field populations of Culicoides to African horse sickness virus

Twenty‐two isolates of African horse sickness virus (AHSV), representing its distinct serotypes, geographical and historical origins, were fed to three populations of South African livestock‐associated Culicoides spp. (Diptera, Ceratopogonidae). Infective blood meals included 12 recent isolates, nine historical reference strains and one live attenuated vaccine strain serotype 7 (AHSV‐7) of the virus. Field‐collected midges were fed through a chicken‐skin membrane on sheep blood spiked with one of the viruses, which concentrations ranged from 5.4 to 8.8 log₁₀TCID₅₀/mL of blood. After 10 days incubation at 23.5°C, AHSV was isolated from 11 Culicoides species. Standard in vitro passaging of AHSV‐7, used for the preparation of live attenuated vaccine, did not reduce its ability to infect Culicoides species. Virus recovery rates in orally infected Culicoides midges differed significantly between species and populations, serotypes, isolates and seasons. Significant variations in oral susceptibility recorded in this study emphasize a complex inter‐relationship between virus and vector, which is further influenced by multiple intrinsic and extrinsic factors. As it is not possible to standardize all these factors under laboratory conditions, conclusive assessment of the role of field‐collected Culicoides midges in the transmission of orbiviruses remains problematic. Nevertheless, results of this study suggest the potential for multi‐vector transmission of AHSV virus in South Africa.     

Protection of livestock against bluetongue virus vector Culicoides imicola using insecticide‐treated netting in open areas

The protection of livestock against Culicoides species (Diptera: Ceratopogonidae) using physical barriers or chemically treated barriers is difficult owing to the small size of these biting midges and animal welfare concerns associated with the reduction of air flow. Culicoides imicola Kieffer is the main bluetongue virus vector in the Mediterranean basin, including the southern Iberian peninsula, where livestock is mainly housed in open pens or sheds which offer no physical protection against C. imicola. In this study we assessed the efficacy of surrounding yearling ewe pens with a canvas barrier or a cypermethrin‐treated canvas barrier in reducing the entry of Culicoides spp. and C. imicola. Analyses were based on comparisons of Culicoides catches in traps in pens with and without barriers, and in traps located outside pens. Although there was no clear reduction in the abundance of Culicoides other than C. imicola in pens with either barrier, the C. imicola presence was markedly reduced by the insecticide‐treated barrier compared with the untreated barrier; the latter did not reduce the abundance of this species in pens. Estimates of the protection conferred against C. imicola by the treated barrier differed depending on whether catch comparisons were based on outside traps or on traps located inside no‐barrier pens. The results suggest that the use of insecticide‐treated barriers may reduce contact between livestock and C. imicola in open areas or sheds. More research is necessary to assess the degree of protection as a function of barrier height, C. imicola abundance, and the size of the area to be protected.     

First molecular identification of the vertebrate hosts of Culicoides imicola in Europe and a review of its blood‐feeding patterns worldwide: implications for the transmission of bluetongue disease and African horse sickness

Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife, livestock and, occasionally, humans. Culicoides imicola (Kieffer, 1913) is considered to be the main vector of the pathogens that cause bluetongue disease (BT) and African horse sickness (AHS) in southern Europe. The study of blood‐feeding patterns in Culicoides is an essential step towards understanding the epidemiology of these pathogens. Molecular tools that increase the accuracy and sensitivity of traditional methods have been developed to identify the hosts of potential insect vectors. However, to the present group's knowledge, molecular studies that identify the hosts of C. imicola in Europe are lacking. The present study genetically characterizes the barcoding region of C. imicola trapped on farms in southern Spain and identifies its vertebrate hosts in the area. The report also reviews available information on the blood‐feeding patterns of C. imicola worldwide. Culicoides imicola from Spain feed on blood of six mammals that include species known to be hosts of the BT and AHS viruses. This study provides evidence of the importance of livestock as sources of bloodmeals for C. imicola and the relevance of this species in the transmission of BT and AHS viruses in Europe.     

Influence of carbon dioxide on numbers of Culicoides midges collected with suction light traps in South Africa

To implement risk management against diseases transmitted by species of Culicoides Latreille, 1809 (Diptera: Ceratopogonidae), it is essential to identify all potential vectors. Light traps are the most commonly used tool for the collection of Culicoides midges. Given the indiscriminate artificial attraction of light, traps will collect all night‐flying insects rather than only livestock‐associated Culicoides midges. Factors that may increase the efficacy of traps, especially for livestock‐associated Culicoides midges, require investigation. In the present study, results obtained with Centers for Disease Control (CDC) and Onderstepoort light traps baited with carbon dioxide (CO₂) were compared with those of unbaited controls. Comparisons were made using two replicates of a 4 × 4 randomized Latin square design. With both trap types, the mean numbers of Culicoides midges collected in 16 baited traps were higher than those caught in 16 unbaited traps. Although exceptionally low numbers were collected with the CDC traps, the increases in the numbers and frequency of collection of Culicoides imicola Kieffer, 1913 were more pronounced in the CDC traps compared with the Onderstepoort traps. These results indicate that the addition of CO₂ may increase the efficiency of these traps for the collection of C. imicola and other livestock‐associated Culicoides species.     

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.     

Spatial and Temporal Distribution of Culicoides Species in Mainland Portugal (2005–2010). Results of the Portuguese Entomological Surveillance Programme

Bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes an infectious, non-contagious disease of ruminants. It has been rapidly emerging in southern Europe since 1998. In mainland Portugal, strains of BTV belonging to three serotypes have been detected: BTV-10 (1956-1960), BTV-4 (2004-2006 and 2013) and BTV-1 (2007-2012). This paper describes the design, implementation and results of the Entomological Surveillance Programme covering mainland Portugal, between 2005 and 2010, including 5,650 caches. Culicoides imicola Kieffer was mostly found in central and southern regions of Portugal, although it was sporadically detected in northern latitudes. Its peak activity occurred in the autumn and it was active during the winter months in limited areas of the country. Obsoletus group was present at the highest densities in the north although they were found throughout the country in substantial numbers. Culicoides activity occurred all year round but peaked in the spring. A generalized linear mixed model was developed for the analysis of the environmental factors associated with activity of the species of Culicoides suspected vectors of BTV in the country. For C. imicola Kieffer, the most important variables were month, diurnal temperature range (DTR), the number of frost days (FRS) and median monthly temperature (TMP). For the Obsoletus group, the most important factors were month, diurnal temperature range (DTR), and linear and quadratic terms for median monthly temperature (TMP). The results reported can improve our understanding of climatic factors in Culicoides activity influencing their distribution and seasonal pattern.     

Distribution of Culicoides in Greece

Culicoides biting midges (Diptera: Ceratopogonidae) were trapped between 1999 and 2004 at 122 locations in mainland Greece and on most of the larger Aegean and Ionian islands, using OVI light traps, in order to determine the distribution and seasonal activity of bluetongue virus vectors and other Culicoides species. Thirty‐nine Culicoides species were identified, six of which (C. furcillatus, C. impunctatus, C. paolae, C. pictipennis, C. riethi, and C. scoticus) were identified for the first time in Greece. Two of these (C. impunctatus and C. scoticus) may be of veterinary importance due to their role as vectors of bluetongue virus and related orbiviruses. In addition, C. imicola was detected for the first time in mainland Greece.     

The effect of ambient temperature fluctuations on Culicoides biting midges population dynamics and activity in dairy farms: a longitudinal study

The effect of climatic factors on the presence of Culicoides Latreille (Diptera: Ceratopogonidae) was previously studied. Nevertheless, lack of laboratory rearing data hampers species‐specific prediction of weather fluctuations effect on population size. To determine fluctuations in population size in the field, we recorded Culicoides and other Nematocerans in seven Israeli dairy farms over two‐years (2011–2012) and analysed the association of their dynamics with fluctuations in ambient temperature and total rainfall. In six farms, the most abundant species were Culicoides imicola Kieffer and Culicoides schultzei (Enderlein) gp., primarily composed of parous females, and in one farm Culicoides obsoletus (Meigen) gp., mostly nulliparous females, were dominant. While the total number of insects was similar in both years, Culicoides numbers were significantly higher in 2012, but appeared later in the season and reached a higher peak. A multi‐variable linear regression model demonstrated positive association of C. imicola and C. schultzei numbers with the monthly multi‐annual ambient temperature and its specific deviation, but not with monthly rainfall. C. obsoletus populations peaked at spring and sharply decreased when temperature exceeded 20 °C, and were best modelled by adding quadratic terms. Weather‐specific estimation of population size under field conditions may enable to predict outbreaks intensity of Culicoides‐borne viruses.     

Recovery rates of bluetongue virus serotypes 1, 2, 4 and 8 Spanish strains from orally infected Culicoides imicola in South Africa

Bluetongue (BT) is an infectious disease of ruminants that has spread northwards in Europe during the last decade. The aetiological agent of the disease is an arbovirus [bluetongue virus (BTV)] that belongs to the genus Orbivirus (family Reoviridae). The virus is transmitted by certain species of biting midge within the genus Culicoides (Diptera: Ceratopogonidae). Information on the vector status of the Culicoides species in a specific area will be essential to predict the risk for BTV incursion. Field-collected Culicoides (Avaritia) imicola Kieffer from South Africa were fed on blood containing several Spanish isolates of BTV. Despite the high virus concentrations in the bloodmeal (5.1-6.4 log(10) TCID(50) /mL of blood), virus was recovered from <1% of midges assayed after incubation. Virus concentrations >2.5 log(10) TCID(50) /midge in individual infected C. imicola suggest virus replication with possible risk for transmission to susceptible vertebrate hosts in the field for at least two of the serotypes assayed (BTV-1 and BTV-2). A third serotype (BTV-4) was very close to the estimated threshold for transmission. The relatively low to near refractory status of C. imicola compared with other vector species such as Culicoides bolitinos supports previous results, indicating that Culicoides species other than C. imicola may play a more important role in the epidemiology of BTV.     

The Mondrian matrix: Culicoides biting midge abundance and seasonal incidence during the 2006–2008 epidemic of bluetongue in the Netherlands

During the northern Europe epidemic of bluetongue (BT), Onderstepoort‐type blacklight traps were used to capture Culicoides Latreille (Diptera: Ceratopogonidae) biting midges weekly between November 2006 and December 2008 on 21 livestock farms in the Netherlands. Proven and potential vectors for the bluetongue virus (BTV) comprised almost 80% of the midges collected: the Obsoletus complex, constituting C. obsoletus (Meigen) and C. scoticus Downes & Kettle (44.2%), C. dewulfi Goetghebuer (16.4%), C. chiopterus (Meigen) (16.3%) and C. pulicaris (Linnaeus) (0.1%). Half of the 24 commonest species of Culicoides captured completed only one (univoltine) or two (bivoltine) generations annually, whereas multivoltine species (including all BTV vectors) cycled through five to six generations (exceeding the one to four generations calculated in earlier decades). Whether this increment signals a change in the phenology of northern Europe Culicoides or simply is an adaptive response that manifests during warmer episodes, thus heightening periodically the incursive potential of midge‐borne arboviruses, remains to be clarified. Culicoides duddingstoni Kettle & Lawson, C. grisescens Edwards, C. maritimus Kieffer, C. pallidicornis Kieffer and C. riethi Kieffer are new records for the biting midge fauna of the Netherlands. It is suggested that C. punctatus (Meigen) be added to the European list of vector Culicoides.     

Phylogenetic relationships of the subgenus AvaritiaFox, 1955 including Culicoides obsoletus (Diptera, Ceratopogonidae) in Italy based on internal transcribed spacer 2 ribosomal DNA sequences

Abstract. Biting midges of the genus Culicoides include vector species for orbiviral diseases, such as bluetongue and African horse sickness. Although the Afro-Asiatic species C. imicola is the major vector of bluetongue in the Mediterranean basin, recent outbreaks in regions where C. imicola is absent has incriminated other Culicoides, including those belonging to the Obsoletus Complex of the subgenus Avaritia Fox, 1955. The classical taxonomy of this species complex is unclear and this stimulated the molecular analysis of twenty Culicoides populations sampled from eighteen localities across Italy. Ribosomal internal transcribed spacer 2 sequences were used to characterize the intra- and interspecific variation between Italian members of the Obsoletus Complex and related species, by means of an analysis of molecular variance and phylogenetic analyses. Although morphological differentiation is often extremely difficult, the molecular analysis clearly demonstrated a high degree of divergence between most species. The study showed that at least seven species of the subgenus Avaritia occur in Italy; these are C. obsoletus, C. scoticus, C. montanus, C. dewulfi, C. imicola and two species that could not be identified with certainty, but one of which is similar to C. chiopterus. Finally, a simple polymerase chain reaction assay was developed that rapidly discriminates between four members of the Obsoletus Complex in Italy, a prerequisite for vector identification.     

Refugia and habitat partitioning among midges (Diptera: Chironomidae) in rain-pools

ABSTRACT.

• 1 Rain-pools on granite exposures are common in tropical Africa and they are inhabited by the larvae of two midge species, Chironomus imicola Kieffer and Chironomus pulcher Wiedemann.
• 2 These pools differ from other ephemeral island habitats such as carrion, fallen fruit, sap flows, mushrooms and dung in their high degree of spatial predictability. Most pools are predictably inhabited by either C.imicola or C.pulcher. This finding is considered in the light of contemporary competition theory.
• 3 An arrangement whereby the same species repeatedly invades the same temporary habitat indicates that there is something about the habitat itself causing the discrimination. I suggest that degree of exposure to sunlight may provide a cue allowing females to discriminate between pools as oviposition sites.
• 4 Both species survive the long dry season by retreating to pools of river water left by receding rivers. Dispersal to rain-pools by emerging adults occurs during the following season with imicola occupying rain-pools remote from shaded rivers. Appropriately, measured against pulcher, the biology of imicola is that of a colonizing species.
• 5 The possibility is discussed that the chironomids in rock-pools do not survive to breed. Instead, these populations may be maintained externally, for example by invasions from breeding populations in the Great Lakes of Africa, where larval densities may be too low to be detected by sampling. The evolutionary implications of such a situation are considered.