A study of the composition of the Obsoletus complex and genetic diversity of Culicoides obsoletus populations in Spain

Background The Culicoides obsoletus species complex (henceforth ‘Obsoletus complex’) is implicated in the transmission of several arboviruses that can cause severe disease in livestock, such as bluetongue, African horse sickness, epizootic hemorrhagic disease and Schmallenberg disease. Thus, this study aimed to increase our knowledge of the composition and genetic diversity of the Obsoletus complex by partial sequencing of the cytochrome c oxidase I (cox1) gene in poorly studied areas of Spain. Methods A study of C. obsoletus populations was carried out using a single-tube multiplex polymerase chain reaction (PCR) assay that was designed to differentiate the Obsoletus complex sibling species Culicoides obsoletus and Culicoides scoticus, based on the partial amplification of the cox1 gene, as well as cox1 georeferenced sequences from Spain available at GenBank. We sampled 117 insects of the Obsoletus complex from six locations and used a total of 238 sequences of C. obsoletus (ss) individuals (sampled here, and from GenBank) from 14 sites in mainland Spain, the Balearic Islands and the Canary Islands for genetic diversity and phylogenetic analyses. Results We identified 90 C. obsoletus (ss), 19 Culicoides scoticus and five Culicoides montanus midges from the six collection sites sampled, and found that the genetic diversity of C. obsoletus (ss) were higher in mainland Spain than in the Canary Islands. The multiplex PCR had limitations in terms of specificity, and no cryptic species within the Obsoletus complex were identified. Conclusions Within the Obsoletus complex, C. obsoletus (ss) was the predominant species in the analyzed sites of mainland Spain. Information about the species composition of the Obsoletus complex could be of relevance for future epidemiological studies when specific aspects of the vector competence and capacity of each species have been identified. Our results indicate that the intraspecific divergence is higher in C. obsoletus (ss) northern populations, and demonstrate the isolation of C. obsoletus (ss) populations of the Canary Islands. Graphical abstract

Temperature and food sources influence subadult development and blood-feeding response of Culicoides obsoletus (sensu lato) under laboratory conditions

Background Culicoides obsoletus (s.l.) is the most abundant Culicoides species in northern Europe and an important vector of bluetongue virus and Schmallenberg virus. Nevertheless, information on its subadult life stages remains scarce and no laboratory-reared colony exists. Methods C. obsoletus (s.l.) adults were collected in Belgium and transferred to the laboratory in an attempt to establish a laboratory-reared colony. C. obsoletus (s.l.) were reared from eggs to adults at different temperatures (28 °C, 24 °C, 20/16 °C) and under different food regimes. Results The most suitable temperature for rearing seemed to be 24 °C for most developmental parameters, but resulted in a biased 3:1 male/female sex ratio. The latter could be optimized to a 1:1 sex ratio when a 20/16 °C day/night temperature gradient was applied, but rearing at these low temperature conditions resulted in significantly lower egg hatching and pupation rates and a longer subadult development time. Independent of the rearing temperature, adding dung as an additional food source during larval development resulted in a significantly higher adult emergence rate and a decrease in subadult development time. Furthermore, blood-feeding rates of field-collected C. obsoletus (s.l.) were compared for different blood sources and feeding systems. The overall blood-feeding success was low and only successful with cotton pledgets (2.7% blood-fed midges) and through a membrane system with chicken skin (3.5% blood-fed midges). Higher feeding rates were obtained on cattle blood compared to sheep blood. Conclusions These results will help us to determine the necessary conditions to rear a viable laboratory colony of this important vector species, although further optimization is still required.

High dispersal capacity of Culicoides obsoletus (Diptera: Ceratopogonidae), vector of bluetongue and Schmallenberg viruses, revealed by landscape genetic analyses

Background In the last two decades, recurrent epizootics of bluetongue virus and Schmallenberg virus have been reported in the western Palearctic region. These viruses affect domestic cattle, sheep, goats and wild ruminants and are transmitted by native hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae). Culicoides dispersal is known to be stratified, i.e. due to a combination of dispersal processes occurring actively at short distances and passively or semi-actively at long distances, allowing individuals to jump hundreds of kilometers. Methods Here, we aim to identify the environmental factors that promote or limit gene flow of Culicoides obsoletus, an abundant and widespread vector species in Europe, using an innovative framework integrating spatial, population genetics and statistical approaches. A total of 348 individuals were sampled in 46 sites in France and were genotyped using 13 newly designed microsatellite markers. Results We found low genetic differentiation and a weak population structure for C. obsoletus across the country. Using three complementary inter-individual genetic distances, we did not detect any significant isolation by distance, but did detect significant anisotropic isolation by distance on a north-south axis. We employed a multiple regression on distance matrices approach to investigate the correlation between genetic and environmental distances. Among all the environmental factors that were tested, only cattle density seems to have an impact on C. obsoletus gene flow. Conclusions The high dispersal capacity of C. obsoletus over land found in the present study calls for a re-evaluation of the impact of Culicoides on virus dispersal, and highlights the urgent need to better integrate molecular, spatial and statistical information to guide vector-borne disease control.

Culicoides biting midges involved in transmission of haemoproteids

Background Culicoides biting midges (Diptera, Ceratopogonidae) are known vectors of avian Haemoproteus parasites. These parasites cause diseases, pathology and even mortality in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in the transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites. Methods Biting midges were collected using a UV trap as well as sticky traps installed in bird nest boxes. Individual parous females were diagnosed for the presence of haemoproteids using both PCR-based and microscopic methods. Results We collected and dissected 232 parous Culicoides females from 9 species using a UV trap and 293 females from 11 species from bird nest boxes. Culicoides obsoletus was the dominant species collected using a UV trap, and Culicoides kibunensis dominated among midges collected in nest boxes. PCR-based screening showed that 5.2% of parous biting midges collected using a UV trap and 4.4% of midges collected from nest boxes were infected with avian haemosporidian parasites. Haemoproteid DNA was detected in C. kibunensis, Culicoides pictipennis, Culicoides punctatus, Culicoides segnis and Culicoides impunctatus females. The sporozoites of Haemoproteus minutus (genetic lineages hTURDUS2 and hTUPHI01) were detected in the salivary glands of two C. kibunensis females using microscopy, and this finding was confirmed by PCR. Conclusions Culicoides kibunensis was detected as a new natural vector of Haemoproteus minutus (hTURDUS2 and hTUPHI01). Haemoproteid DNA was detected in females from five Culicoides species. This study contributes to the epizootiology of avian Haemoproteus infections by specifying Culicoides species as vectors and species that are likely to be responsible for the transmission of haemoproteids in Europe.

Blood meal analysis: host-feeding patterns of biting midges (Diptera, Ceratopogonidae, Culicoides Latreille) in Slovakia

Biting midges of the genus Culicoides are vectors of important pathogens affecting domestic and wild animals and have played a major role in the re-emergence of new outbreaks of bluetongue (BTV) and Schmallenberg (SBV) viruses in Europe. To determine vector-host specificity, trophic preference from blood meal analysis is of major importance in the surveillance of arthropod-borne diseases. Of 28,752 specimens collected, we identified 17 Culicoides species and investigated a total of 48 host sequences from the blood meals. Culicoides obsoletus/C. scoticus, C. dewulfi, C. pulicaris, C. lupicaris, C. punctatus, C. newsteadi, C. riethi, and C. furcillatus were found to feed on mammals (cattle, horses, and humans), birds (domestic chickens), small rodents (Apodemus flavicollis), and hares (Lepus europaeus). To our knowledge, this is the first study investigating trophic preferences of Culicoides spp. in Slovakia. This study demonstrated that Culicoides species are able to feed on domesticated host vertebrates as well as birds, rodents, and humans.

Diversity, Abundance and Host Preference of Culicoides (Diptera: Ceratopogonidae), Potential Vectors and Nuisance Insect in Zoo Park Košice, Slovakia

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) were captured in the course of three seasons (2017–2020) in the Zoo Košice. The total number of captured individuals was 5,667 biting midges, and 12 species of Culicoides were identified, while the most frequently captured and the most populous species were C. obsoletus/C. scoticus, C. furcillatus, and C. festivipennis. The most frequent hosts on which the biting midges were blood-feeding were humans (Homo sapiens), Burchell's zebra (Equus quagga burchellii), and domestic cow (Bos taurus). This is the first study to confirm a wide range of biting midges species in a zoo which are capable of using not only animals as their hosts, but also humans, in whom a bite may induce an allergic reaction. More than 80% of the fauna of biting midges in the Zoo Košice represented Culicoides obsoletus/C. scoticus, which are in the Central Europe regarded as the potential vectors of arboviruses, to which primarily ruminants are highly susceptible.

Diversity, Abundance and Host Preference of Culicoides (Diptera: Ceratopogonidae), Potential Vectors and Nuisance Insect in Zoo Park Košice, Slovakia

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) were captured in the course of three seasons (2017–2020) in the Zoo Košice. The total number of captured individuals was 5,667 biting midges, and 12 species of Culicoides were identified, while the most frequently captured and the most populous species were C. obsoletus/C. scoticus, C. furcillatus, and C. festivipennis. The most frequent hosts on which the biting midges were blood-feeding were humans (Homo sapiens), Burchell's zebra (Equus quagga burchellii), and domestic cow (Bos taurus). This is the first study to confirm a wide range of biting midges species in a zoo which are capable of using not only animals as their hosts, but also humans, in whom a bite may induce an allergic reaction. More than 80% of the fauna of biting midges in the Zoo Košice represented Culicoides obsoletus/C. scoticus, which are in the Central Europe regarded as the potential vectors of arboviruses, to which primarily ruminants are highly susceptible.

Post-epidemic Occurrence of Bluetongue Virus Vectors (Culicoides Species) în Iași County

Culicoides is a genus of biting midges in the family Ceratopogonidae, representing a big threat, giving that several species serve as biological vectors of pathogens of veterinary importance. BTV distribution and persistence is thought to be almost entirely dependent upon the presence or absence of Culicoides adults vector. In this study, biting midges were collected during 2015-2016, once a week, from April to December. In the first year, 2617 BTV vector specimens were collected and identified as Culicoides obsoletus (41.2%; 95% CI: 39-43), Culicoides pulicaris (58%; 95% CI: 56-59) and Culicoides nubeculosus (0.8%; 95% CI: 0.4-1.1). In the second year, 165 BTV vector specimens were collected and identified as Culicoides obsoletus (44.25%; 95% CI: 36-51), Culicoides pulicaris (50.3%; 95% CI: 42-57), Culicoides dewulfi (4.84%; 95% CI: 1.5-8.1) and Culicoides nubeculosus (0.61%; 95% CI: 0.5-1.7). Culicoides pulicaris and Culicoides obsoletus complex were especially abundant within the two collection sites in Iași County. Our data on post-epidemic occurrence and composition of BTV vector species in the studied area will facilitate a better understanding of Culicoides vectors distribution and the possible role in bluetongue virus overwintering.