Søren Achim Nielsen

Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) in Denmark

BackgroundHost preference studies in haematophagous insects e.g. Culicoides biting midges are pivotal to assess transmission routes of vector-borne diseases and critical for the development of veterinary contingency plans to identify which species should be included due to their risk potential. Species of Culicoides have been found in almost all parts of the world and known to live in a variety of habitats. Several parasites and viruses are transmitted by Culicoides biting midges including Bluetongue virus and Schmallenberg virus. The aim of the present study was to determine the identity and diversity of blood meals taken from vertebrate hosts in wild-caught Culicoides biting midges near livestock farms.MethodsBiting midges were collected at weekly intervals for 20 weeks from May to October 2009 using light traps at four collection sites on the island Sealand, Denmark. Blood-fed female biting midges were sorted and head and wings were removed for morphological species identification. The thoraxes and abdomens including the blood meals of the individual females were subsequently subjected to DNA isolation. The molecular marker cytochrome oxidase I (COI barcode) was applied to identify the species of the collected biting midges (GenBank accessions JQ683259-JQ683374). The blood meals were first screened with a species-specific cytochrome b primer pair for cow and if negative with a universal cytochrome b primer pair followed by sequencing to identify mammal or avian blood meal hosts.ResultsTwenty-four species of biting midges were identified from the four study sites. A total of 111,356 Culicoides biting midges were collected, of which 2,164 were blood-fed. Specimens of twenty species were identified with blood in their abdomens. Blood meal sources were successfully identified by DNA sequencing from 242 (76%) out of 320 Culicoides specimens. Eight species of mammals and seven species of birds were identified as blood meal hosts. The most common host species was the cow, which constituted 77% of the identified blood meals. The second most numerous host species was the common wood pigeon, which constituted 6% of the identified blood meals.ConclusionsOur results suggest that some Culicoides species are opportunistic and readily feed on a variety of mammals and birds, while others seems to be strictly mammalophilic or ornithophilic. Based on their number, dispersal potential and blood feeding behaviour, we conclude that Culicoides biting midges are potential vectors for many pathogens not yet introduced to Denmark.

Molecular identification of bloodmeals from biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) in Denmark

Culicoides vectors are critical to the survival and transmission of bluetongue virus as infection only occurs in areas or regions where competent vectors are present. The success of Culicoides biting midges as vectors is mainly related to their vast population sizes and to their means of dispersal. Their choice of host for blood feeding is sparsely described. The aim of the present study was to establish methods for the identification of bloodmeal hosts and determine the identity and diversity of bloodmeals of vertebrate hosts from wild-caught biting midges near livestock farms. The study includes some of the most common and abundant species of biting midges in Denmark: Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus. We collected 8,378 biting midges including nine species of Culicoides of which blood-fed specimens were found from six species. We identified 251 blood engorged biting midges, and hosts were identified in 115 of 125 analysed specimens (90%). Cow, roe deer, horse, mallard and wood pigeon were identified as hosts. The most abundant host species was cow, which constituted 73.9% of the total identified bloodmeals, but the common wood pigeon was found with a frequency as high as 18.3%. In conclusion, the molecular methods applied were proven useful in identifying bloodmeal hosts from different Culicoides species. The results indicate that Culicoides species are opportunistic in their choice of bloodmeal host with a preference for cattle when present, which is important to have in mind for epidemiologist when making predictive models. Accordingly, the results of this study will add useful parameters for modelling bluetongue virus transmission and in the development of veterinary contingency plans.

Monitoring of biting midges (Diptera: Ceratopogonidae: Culicoides Latreille) on farms in Sweden during the emergence of the 2008 epidemic of bluetongue.

In light of the emergence of bluetongue in northern Europe, populations of Culicoides species were monitored in 2007–2008 by means of Onderstepoort blacklight suction traps operating at livestock farms in Sweden. The location of the 22 sampling sites ranged from about latitude 55°N to about 68°N. A total of 61,669 male and female Culicoides were captured, of which, 52,319 were trapped outside the farms and 9,350 in byres or livestock sheds. Thirty-three Culicoides species were recorded, of which, 30 were new to Sweden. The species and their relative abundance and spatial distribution on sites are presented. Two species incriminated as vectors of bluetongue virus, viz. Culicoides obsoletus (about 38%) and Culicoides scoticus (about 36%), were predominant and common in the environment of livestock farms practically all over the Swedish mainland, penetrating far north to at least 65°N. The two species were also recorded from the island of Gotland. Culicoides pulicaris, another potential vector species, accounted for about 9% of all Culicoides captured.

Establishing a community‐wide DNA barcode library as a new tool for arctic research

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to‐date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology‐based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology‐based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species‐level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community.

Morphological and molecular identification of species of the Obsoletus group (Diptera: Ceratopogonidae) in Scandinavia

After the introduction of bluetongue in northern Europe in 2006, populations of Culicoides have been monitored in many European countries. Large quantities of Culicoides specimens shall be determined to species, and it is thus important to find reliable morphological characters that are visualized in a stereomicroscope. Culicoides obsoletus, Culicoides scoticus, Culicoides chiopterus, and Culicoides dewulfi all belonging to subgenus Avaritia are common in collections in northern Europe. C. obsoletus and C. scoticus often make up more than 50% of the total catch of biting midges. Separation of the females of the four species by morphological characters has frequently been questioned, and in many cases, female specimens are grouped as an entity in veterinary and ecological studies. We show how it is possible using a stereomicroscope to separate the females of the four species by combining the shape of the third segment of the maxillary palp and the number and location of hairs on the first abdominal tergit. Validation of the quick stereomicroscope identification method was achieved by morphometric measurements and a molecular marker. In all cases, both methods verified the quick morphological species identification of the Obsoletus group females. In conclusion, the females of all four species of the Obsoletus group can be separated by a quick morphological method under the stereomicroscope.

Delineation of Culicoides species by morphology and barcode exemplified by three new species of the subgenus Culicoides (Diptera: Ceratopogonidae) from Scandinavia

BackgroundCulicoides biting midges (Diptera: Ceratopogonidae) cause biting nuisance to livestock and humans and are vectors of a range of pathogens of medical and veterinary importance. Despite their economic significance, the delineation and identification of species where only morphology is considered, as well as the evolutionary relationships between species within this genus remains problematic. In recent years molecular barcoding has assisted substantially in the identification of biting midges in the multiple entomological survey projects which were initiated in many European countries following the bluetongue outbreak in 2006–2009. These studies revealed potentially new species and “species-complexes” with large genetic and morphological variability. Here we use molecular barcoding, together with morphological analysis, to study subgenus Culicoides Latreille from Scandinavia with focus on three potentially new species.MethodsBiting midges were collected at various sites in Denmark and Sweden. Culicoides specimens were described by variation of a fragment of their cytochrome c oxidase subunit 1 (COI) gene sequence and wing, palp and antennal characters.ResultsIt is shown that three new species initially separated by DNA barcoding with mitochondrial COI can be distinguished by morphological characters. In this context a key to Scandinavian subgenus Culicoides using wing and maxillary palp characters is presented. The key is including the three new species Culicoides boyi, Culicoides selandicus and Culicoides kalix.ConclusionThree new species of Culicoides biting midges were identified and could be identified by both molecular and morphological differences. Evaluation of differences between and within taxa of biting midges using COI barcode yielded a rough estimate of species delineation; interspecies differences across Culicoides subgenera approaches 20%, whereas intraspecies differences are below 4% and in most cases below 1%.

Energetic Cost of Subacute Chlorpyrifos Intoxication in the German Cockroach (Dictyoptera: Blattellidae)

Abstract The energetic cost of a sublethal treatment with chlorpyrifos was estimated by use of direct microcalorimetry to measure metabolic heat in susceptible and resistant strains of the German cockroach Blattella germanica L. Moreover, one of the detoxification enzyme systems known to be involved in detoxification of chlorpyrifos, glutathione-S-transferase, was measured. Individual cockroaches were exposed for 20 min on a glass-surfaces treated with 1.14 μg/cm2 of chlorpyrifos. There was no difference in glutathione-S-transferase activity of susceptible or resistant strains after the treatment. The heat production increased in the susceptible strain ≈30 min after exposure and declined again after ≈120 min to the basal level. The energetic cost of the exposure to the insecticide corresponds ≈5 h of normal metabolism. There were no significant differences in heat production after toxic treatment in any of the resistant strains. It was shown that measurement of heat production is a sensitive method to prove toxic reactions after exposure to a low dose of insecticide. The use of microcalorimetry as a promising biomarker technology was shown.

Three new Scandinavian species of Culicoides (Culicoides): C. boyi sp. nov., C. selandicus sp. nov. and C. kalix sp. nov. (Diptera: Ceratopogonidae)

Abstract Background In the context of a major monitoring program of Culicoides in Denmark and Sweden due to the appearance of bluetongue disease in 2007–2008, a large number of specimens were collected by light traps and sorted morphologically, with COI barcodes generated for selected specimens. New information Three species are described as new to science based on both morphological and molecular data: Culicoides (Culicoides) boyi sp. nov. (Denmark: Jutland), C. (C.) selandicus sp. nov. (Denmark: Zealand) and C. (C.) kalix sp. nov. (Sweden: Norrbotten). All are diagnosed morphologically as well as by molecular barcoding. A key to slide-mounted females of all Scandinavian species of Culicoides (Culicoides) is presented.

The Occurrence of Summer Mastitis Related to the Foraging Activities of Hydrotaea Irritans (Fall.)

The incidence of summer mastitis was studied as a function of climatic fluctuations and fluctuations in the foraging activity of Hydrotaea irritans in order to evaluate the possible role of the fly as a vector of the disease. The results obtained supported the assumption that Hydrotaea irritans was a causative link in the transmission of summer mastitis.

Chromosome and Enzyme Variation of Hydrotaea Irritans (Fallen) (Diptera: Muscidae)

The analysis of metaphases from the brain of third instar larvae from flies caught in the Netherlands revealed numbers of chromosomes ranging from 12 to 15. Of these, five pairs of large metacentric and submetacentric chromosomes were common to all individuals, so the number of supernumerous chromosomes varied from 2 to 5. In an ongoing study of variation in chromosome number of flies caught in Denmark, we found two common types each with 12 and one rare type with 11 chromosomes in forest flies. In the majority of flies from nearby pastures, we counted 13 or 14 chromosomes, but larvae with 12 or 11 chromosomes were also found.