Equine Culicoides hypersensitivity in Florida: biting midges aspirated from horses

Nine species of Culicoides were aspirated from horses in Florida during the second phase of a study of equine Culicoides hypersensitivity (CHS). Approximately 90% of the 2933 midges were Culicoides insignis Lutz, 4% were C.stellifer (Coquillett), 3% were C.niger Root and Hoffman, 2% were C.alachua Jamnback and Wirth and the remaining 1% included C.venustus Hoffman, C.scanloni Wirth and Hubert, C.lahillei Iches (= C.debilipalpis Lutz), C.pusillus Lutz, and C.edeni Wirth and Blanton. Culicoides were sampled on seven farms located throughout Florida. The location and seasonality of lesion development on horses with CHS correlated with the Culicoides spp. collected directly from the horses as to their sites of attack, their seasonality and geographical distribution.     

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus,important Palaearctic vectors of bluetongue virus

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.     

The influence of host number on the attraction of biting midges,Culicoides spp., to light traps

A preliminary study was undertaken to investigate how the number of sheep below a light‐suction trap affects the number of female Culicoides obsoletus Meigen (Diptera: Ceratopogonidae) caught. As the number of sheep increased from zero to three, the number of midges caught increased, but there appeared to be no further increase when six sheep were used. The lack of increase between three and six sheep is attributable to different activity rates on certain nights, perhaps in response to weather, and suggests, therefore, that catches in light traps increase linearly with sheep numbers, at least for small host numbers.     

Molecular identification of bloodmeals and species composition in Culicoides biting midges

Investigations of host preferences in haematophagous insects, including Culicoides biting midges (Diptera: Ceratopogonidae), are critical in order to assess transmission routes of vector‐borne diseases. In this study, we collected and morphologically identified 164 blood‐engorged Culicoides females caught in both light traps and permanent 12‐m high suction traps during 2008–2010 in Sweden. Molecular analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the biting midges was performed to verify species classification, discern phylogenetic relationships and uncover possible cryptic species. Bloodmeal analysis using universal vertebrate cytochrome b primers revealed a clear distinction in host selection between mammalophilic and ornithophilic Culicoides species. Host sequences found matches in horse (n = 59), sheep (n = 39), cattle (n = 26), Eurasian elk (n = 1) and 10 different bird species (n = 18). We identified 15 Culicoides species previously recorded in Scandinavia and four additional species haplotypes that were distinctly different from the described species. All ornithophilic individuals (n = 23) were caught exclusively in the suction traps, as were, interestingly, almost all mammalophilic species (n = 41), indicating that many biting midge species may be able to cover long distances after completing a bloodmeal. These results add new information on the composition of Culicoides species and their host preferences and their potential long‐distance dispersal while blood‐engorged.     

Evaluation of matrix-assisted laser desorption/ionization time of flight mass spectrometry for characterization of Culicoides nubeculosus biting midges

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has shown promise in species identification of insect species. We evaluated its potential to consistently characterize laboratory-reared biting midges of the species Culicoides nubeculosus (Meigen) (Diptera: Ceratopogonidae). Twenty-one reproducible potential biomarker masses for C. nubeculosus were identified under different experimental treatments. These treatments included the homogenization of insects in either water or known concentrations of formic acid. The biomarker masses were present independent of age, gender and different periods of storage of individuals in 70% ethanol (a standard preservation method). It was found that the presence of blood in females reduced the intensity of the MALDI-TOF pattern, necessitating the removal of the abdomen before analysis. The protein profiles of a related non-biting midge, Forcipomyia sp. (Diptera: Ceratopogonidae), and of Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) mosquitoes were also examined and were distinctly different. These findings provide preliminary data to optimize future studies in differentiation of species within the Culicoides genus using MALDI-TOF MS which is a rapid, simple, reliable and cost-effective technique.     

Genetic structure and population dynamics of the biting midges Culicoides obsoletus and Culicoides scoticus: implications for the transmission and maintenance of bluetongue

Culicoides species belonging to the Obsoletus complex (Diptera: Ceratopogonidae) have been indicated as primary bluetongue (BT) vectors in many European countries and their possible involvement in the maintenance and overwintering of BT viruses has been suggested, even in regions where Culicoides imicola Keiffer is the main vector. The Obsoletus complex includes two predominant taxa, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle. However, the role played by each species in the epidemiology of BT is still unknown. Taxonomic identification is mainly based on the morphology of male genitalia and the lack of other reliable diagnostic features makes the screening of trap‐collected vector populations, mainly females, particularly difficult. Although molecular markers have facilitated species identification, little information is yet available on the biology, abundance and population dynamics of the two taxa. The aim of this work was to investigate the genetic profile and temporal distribution of C. obsoletus and C. scoticus by using isozyme electrophoresis applied to adult midges, collected weekly at two selected farms in southern Sardinia. A total of nine enzyme loci were analysed and five of them provided diagnostic allozyme markers (Hk, Mdh, Pgi, Idh‐1 and Idh‐2). Nei's genetic distance between the two taxa was in the range of other well‐separated taxa (D = 1.792), supporting their status as true species. Culicoides scoticus represented almost 61% of the 562 specimens analysed; its genetic structure was characterized by a very low level of intra‐population variation (mean heterozygosity H_e = 0.019) and higher genetic divergence between populations (F_ST = 0.0016) than in C. obsoletus. The latter species had significantly more heterozygotes (H_e = 0.123), a higher percentage of polymorphic loci, and no inter‐population differentiation (F_ST?0). We suggest that different biological and ecological constraints, such as breeding habitat requirements, may contribute to shaping the genetic profiles of C. scoticus and C. obsoletus. However, enough gene flow was maintained between populations of each species as no spatial and temporal structuring was sustained by Fisher's exact probability test (P > 0.5). The seasonal distributions of C. scoticus and C. obsoletus only partially overlapped: both species were mainly found early in the year, when the main vector, C. imicola, was present in low numbers, and peaked in abundance in April and May. Culicoides scoticus was predominant until May, decreased rapidly in the following months and increased again in winter, whereas C. obsoletus decreased more slowly and was still present in early summer. Consequently, C. scoticus may be a good candidate for playing a role in the transmission and maintenance of BT virus in Sardinia, as well as in other Mediterranean countries, during the months of late winter and early spring when the seroconversion of sentinel animals is still occurring in the absence of the main vector.     

Revision of the lygaeid genus Nysius (Heteroptera: Lygaeidae: Orsillinae) of Japan,with description of a new species

Japanese species of the lygaeid–orsilline genus Nysius were revised, and five species including a new species, N. hidakai, were recognized. Male and female genitalia of all the species were illustrated, and a key to species of Japanese Nysius was provided. The DNA barcoding information of each species except N. expressus were recorded.     

Light and suction trap catches of Culicoides midges in southern England

1. The seasonal flight periods are given for eleven species or species groups of Culicoides midges at Pirbright, Surrey, U.K., from 1979 to 1981 and in 1983. 2. Most species were relatively uniformly distributed both within the study site and between years; only C.clintoni Boorman was limited in its spatial distribution and only C.segnis Campbell and Pelham-Clinton varied greatly in numbers between years. 3. Species fall into one of three patterns of seasonal abundance: spring emergers, summer emergers, or spring to autumn emergers. 4. Only the C.obsoletus group showed a pattern of emergence that could correspond to distinct generations, of one or more species in the group. Three distinct emergences were observed, and possible explanations for these discussed. 5. For all species except C.impunctatus Goetghebuer the catches from suction/light and suction only traps are poorly correlated whereas for C.impunctatus they are highly correlated. This may suggest that this species is not greatly attracted to light.     

Efficacy of the Entomopathogenic Fungus, Culicinomyces clavisporus against Larvae of the Biting Midge, Culicoides nubeculosus (Diptera: Ceratopogonidae)

Culicinomyces clavisporus, a fungal pathogen of a wide range of mosquito species, was investigated in relation to potential pathogenicity against Culicoides nubeculosus biting midge larvae. Seven different C. clavisporus strains were assayed. Each showed some degree of activity against C. nubeculosus larvae with LC₅₀ values of between 3.2×10^-5 and 1.1×10^-6 spores/mL; these effects occurred in dose-dependent manners and tended to be delayed until 72-96 h post treatment. The results are discussed in relation to incorporation of C. clavisporus into biocontrol programmes for Culicoides spp.     

Species diversity and seasonal abundance of Culicoides biting midges in northwestern Argentina

The species diversity and seasonal abundance of biting midges of the genus Culicoides (Diptera: Ceratopogonidae) were studied in northwestern Argentina during the period 2003–2005. A total of 5437 Culicoides specimens were collected using CDC light traps in three areas of the mountainous rainforest area. The most common species were Culicoides paraensis (Goeldi) and C . insignis Lutz, Culicoides lahillei (Iches), C . venezuelensis Ortiz & Mirsa, C . debilipalpis Lutz and C . crescentis Wirth & Blanton were also collected. Culicoides paraensis was abundant during the summer, and C . insignis and C . lahillei during late summer and early fall. Accumulated rainfall was the climatic variable most related to fluctuation in abundance of C . paraensis .     

Host preferences of Palaearctic Culicoides biting midges: implications for transmission of orbiviruses

Feeding success depends on host availability, host defensive reactions and host preferences. Host choice is a critical determinant of the intensity at which pathogens are transmitted. The aim of the current study was to describe host preferences of Palaearctic Culicoides species (Diptera: Ceratopogonidae) Latreille using traps baited with the five different host species of poultry, horse, cattle, sheep and goat. Collections were carried out nightly in July and August 2009 in western France with three replicates of a 5 × 5 randomized Latin square (five sites, five hosts). Moreover, an ultraviolet (UV) light/suction trap was operated during host‐baited collections to correlate Culicoides biting rates and UV light/suction trap catches. A total of 660 Culicoides belonging to 12 species, but comprised mainly of Culicoides scoticus Downes and Kettle, Culicoides dewulfi Goetghebuer and Culicoides obsoletus Meigen, were collected on animal baits. Abundance was highest for the horse, which accounted for 95% of all Culicoides caught, representing 10 species. The horse, the largest bait, was the most attractive host, even when abundance data were corrected by weight, body surface or Kleiber's scaling factor. Culicoides obsoletus was the only dominant species attracted by birds. Both C. scoticus and C. dewulfi were collected mainly from the upper body of the horse. Finally, the quantification of host preferences allows for discussion of implications for the transmission of Culicoides‐borne pathogens such as bluetongue virus.     

DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match

DNA metabarcoding enables efficient characterization of species composition in environmental DNA or bulk biodiversity samples, and this approach is making significant and unique contributions in the field of ecology. In metabarcoding of animals, the cytochrome c oxidase subunit I (COI) gene is frequently used as the marker of choice because no other genetic region can be found in taxonomically verified databases with sequences covering so many taxa. However, the accuracy of metabarcoding datasets is dependent on recovery of the targeted taxa using conserved amplification primers. We argue that COI does not contain suitably conserved regions for most amplicon-based metabarcoding applications. Marker selection deserves increased scrutiny and available marker choices should be broadened in order to maximize potential in this exciting field of research.     

The swarming behaviour of the Scottish biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae)

Abstract.

• 1 Swarms of Culicoides impunctatus males were examined in the field in Scotland. Most swarms were close to midge emergence/breeding grounds over a variety of vegetation, some of which clearly acted as swarm markers. Low light (0–1000 lux) and still/humid conditions favoured swarming.
• 2 Swarm size ranged between less than 10 and more than one thousand midges. The modal size was 200 individuals. The smallest swarms were usually columnar in shape and the larger swarms ovoid.
• 3 Midges behaved individually within swarms, moving in an elliptical manner characteristic of other dipterans in swarms.
• 4 Swarms were classified as either ‘persistent’ or ‘transient’ in terms of their shape, size and stability. Wind was the most influential factor in disrupting swarms.
• 5 Swarms were confirmed as mating sites for C.impunctatus.

     

Toward development of neem-based repellents against the Scottish Highland biting midge Culicoides impunctatus

Oil of neem, from the tree Azadirachta indica A. Juss (Meliaceae), was evaluated for repellent and antifeedant activity against Culicoides biting midges (Diptera: Ceratopogonidae), by three complementary methods with serial dilutions. Electroantennograms revealed the sensitivity of Culicoides nubeculosus (Meigen) females to neem > or = 0.10%. Culicoides impunctatus Goetghebuer females were repelled by > or = 1% in a Y-tube olfactometer, Using a membrane feeder for wild-caught parous females of C. impunctatus, the proportion blood-feeding was significantly reduced by topical applications of neem oil > or = 0.10% concentrations, with blood-feeding completely prevented by > or =1%. On the basis of these response data, we developed 2% neem-based formulations for personal protection against biting midges.     

Species‐specific mitochondrial gene rearrangements in biting midges and vector species identification

Abstract Partial mitochondrial gene sequences of 16 Culicoides species were determined to elucidate phylogenetic relations among species and to develop a molecular identification method for important virus vector species. In addition, the analysis found mitochondrial gene rearrangement in several species. Sequences of the mitochondrial genome region, cox1–trnL2–cox2 (1940–3785 bp) of 16 Culicoides and additional sequences were determined in some species, including whole mitochondrial genome sequences of Culicoides arakawae. Nine species showed common organization in this region, with three genes cox1–trnL2–cox2 and a small or no intergenic region (0–30 bp) between them. The other seven species showed translocation of tRNA and protein‐coding genes and/or insertion of AT‐rich non‐coding sequences (65–1846 bp) between the genes. The varied gene rearrangements among species within a genus is very rare for mitochondrial genome organization. Phylogenetic analyses based on the sequences of cox1+cox2 suggest a few clades among Japanese Culicoides species. No relationships between phylogenetic closeness and mitochondrial gene rearrangements were observed. Sequence data were used to establish a polymerase chain reaction tool to distinguish three important vector species from other Culicoides species, for which classification during larval stages is not advanced and identification is difficult.     

Determination of the oral susceptibility of South African livestock-associated biting midges,Culicoides species,to bovine ephemeral fever virus

A total of 10 607 Culicoides midges (Diptera: Ceratopogonidae) were fed on either sheep or horse blood containing not less than 6.5 log10 TCID50/ml of bovine ephemeral fever virus (BEFV). Insects were collected during two consecutive summers from two distinct climatic areas. Two seed viruses, originating from either South Africa or Australia, were used separately in the feeding trials. Blood-engorged females were incubated at 23.5 degrees C for 10 days and then individually assayed in microplate BHK-21 cell cultures. Of the 4110 Culicoides that survived, 43% were C. (Avaritia) imicola Kieffer and 27% were C. (A.) bolitinos Meiswinkel. The remainder represented 18 other livestock-associated Culicoides species. Although BEFV was detected in 18.9% of midges assayed immediately after feeding, no virus could be detected after incubation. The absence of evidence of either virus maintenance or measurable replication suggests that most of the abundant livestock-associated Culicoides species found in South Africa are refractory to oral infection with BEFV. Future studies should be carried out using species of mosquitoes that are associated with cattle in the BEF endemic areas.