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Acetylcholinesterase (AChE) insensitive to organophosphate and carbamate insecticides has been identified as a major resistance mechanism in numerous arthropod species. However, the associated genetic changes have been reported in the AChE genes from only three insect species; their role in conferring insecticide insensitivity has been confirmed, using functional expression, only for those in Drosophila melanogaster. The housefly, Musca domestica, was one of the first insects shown to have this mechanism; here we report the occurrence of five mutations (Val-180-->Leu, Gly-262-->Ala, Gly-262-->Val, Phe-327-->Tyr and Gly-365-->Ala) in the AChE gene of this species that, either singly or in combination, confer different spectra of insecticide resistance. The baculovirus expression of wild-type and mutated housefly AChE proteins has confirmed that the mutations each confer relatively modest levels of insecticide insensitivity except the novel Gly-262-->Val mutation, which results in much stronger resistance (up to 100-fold) to certain compounds. In all cases the effects of mutation combinations are additive. The mutations introduce amino acid substitutions that are larger than the corresponding wild-type residues and are located within the active site of the enzyme, close to the catalytic triad. The likely influence of these substitutions on the accessibility of the different types of inhibitor and the orientation of key catalytic residues are discussed in the light of the three-dimensional structures of the AChE protein from Torpedo californica and D. melanogaster.

Abstract:Simulation of soil moisture content requires effective soil hydraulic parameters that are valid at the modelling scale. This study investigates how these parameters can be estimated by inverse modelling using soil moisture measurements at 25 locations at three different depths (at the surface, at 30 and 60 cm depth) on an 80 by 20 m hillslope. The study presents two global sensitivity analyses to investigate the sensitivity in simulated soil moisture content of the different hydraulic parameters used in a one-dimensional unsaturated zone model based on Richards' equation. For estimation of the effective parameters the shuffled complex evolution algorithm is applied. These estimated parameters are compared to their measured laboratory and in situ equivalents. Soil hydraulic functions were estimated in the laboratory on 100 cm 3 undisturbed soil cores collected at 115 locations situated in two horizons in three profile pits along the hillslope. Furthermore, in situ field saturated hydraulic conductivity was estimated at 120 locations using single-ring pressure infiltrometer measurements. The sensitivity analysis of 13 soil physical parameters (saturated hydraulic conductivity (K s ), saturated moisture content ( s ), residual moisture content ( r ), inverse of the air-entry value (˛), van Genuchten shape parameter (n), Averjanov shape parameter (N) for both horizons, and depth (d) from surface to B horizon) in a two-layer single column model showed that the parameter N is the least sensitive parameter. K s of both horizons,  s of the A horizon and d were found to be the most sensitive parameters. Distributions over all locations of the effective parameters and the distributions of the estimated soil physical parameters from the undisturbed soil samples and the single-ring pressure infiltrometer estimates were found significantly different at a 5% level for all parameters except for˛of the A horizon and K s and  s of the B horizon. Different reasons are discussed to explain these large differences.

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.

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.