Successful Blood Feeding Research Articles

An assessment of Culicoides surveillance techniques in northern Europe: have we underestimated a potential bluetongue virus vector?

Summary 1. Bluetongue virus (BTV), a pathogen of ruminants transmitted by Culicoides midges, has emerged dramatically across Europe since 1998. Surveillance of Culicoides is currently carried out in every European country affected by BTV, most commonly using standardized light-suction traps that sample the population of adult Culicoides present. To date, however, it is not clear whether these trap catches accurately reflect the biting population of Culicoides . 2. In our study, we carried out 192 drop-trap catches at dusk on Poll Dorset sheep, drawing comparisons with surveillance samples taken using standard trapping protocol at the same site. A multiplex polymerase chain reaction technique was optimized for high-throughput processing of cryptic species to allow Culicoides collected to be identified to species level, the first time this has been achieved in this region for an ecological study. A mathematical model was then constructed to describe rates of midge biting under the meteorological conditions recorded during trials. 3. Light-trapping surveillance was found to substantially underestimate the numbers of Culicoides chiopterus present on sheep. This finding is extremely significant given that, due to the low numbers of this species caught at light traps across northern Europe, C. chiopterus had not been seriously considered as a potential vector of BTV. 4. The models constructed of successful blood feeding predict that while biting rates on sheep are significantly reduced under conditions adverse to midge flight (wind speeds that exceeded 3 mps, wind turbulence of greater than 40 degrees change in direction during the trial and solar intensity exceeding 200 Wm ‐2 ), low levels of biting can also occur under sub-optimal meteorological conditions, that have the potential to span entire days in the later part of the adult season. 5. Synthesis and applications. It is clear from these results that light-trapping surveillance does not provide an accurate reflection of the biting population of Culicoides present. Given that the surveillance systems are already in wide employment across Europe, but provide misleading results as we have demonstrated, and considering the huge economic and animal welfare impact of BTV incursions, it is vital that alternative/additional methods of surveillance are explored. Additionally, the vector competence of C. chiopterus as a potential vector of arboviruses requires urgent assessment, along with a clearer understanding of diel periodicity of Culicoides attacks throughout the adult season.

Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections

Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Blood-fed adult females digest large amounts of protein, and xanthine dehydrogenase is thought to be a key enzyme involved in protein catabolism through the production of urate. Large amounts of heme are also released during digestion with potentially damaging consequences, as heme can generate oxygen radicals that damage lipids, proteins and nucleic acids. However, urate is an antioxidant that may prevent such oxidative damage produced by heme. We investigated xanthine dehydrogenase by developing the RNAi technique for sand flies and used this technique to knock down the Lu. longipalpis xanthine dehydrogenase gene to evaluate its role in survival of adult females after blood feeding. The gene sequence of Lu. longipalpis xanthine dehydrogenase is described together with expression in different life cycle stages and RNAi knock down. Semi-quantitative RT-PCR of xanthine dehydrogenase expression showed a significant increase in expression after bloodmeal ingestion. Microinjection of dsRNA via the thorax of 1-day-old adult female sand flies resulted in approximately 40% reduction of xanthine dehydrogenase gene expression in comparison to flies injected with a control dsRNA. A significant reduction of urate in the whole body and excretions of Lu. longipalpis was observed after dsRNA xanthine dehydrogenase microinjection and feeding 96 h later on rabbit blood. Sand flies injected with XDH dsRNA also exhibit significantly reduced life span in comparison with the mock-injected group when fed on sucrose or when rabbit blood fed, showing that urate could be indeed an important free radical scavenger in Lu. longipalpis. The demonstration of xanthine dehydrogenase knock down by dsRNA microinjection, low mortality of microinjected insects and the successful bloodfeeding of injected insects demonstrated the utility of RNAi as a tool for functional analysis of genes in phlebotomine sand flies.

Toward an Understanding of the Molecular Mechanism for Successful Blood Feeding by Coupling Proteomics Analysis with Pharmacological Testing of Horsefly Salivary Glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-gamma production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

Age-Related Adult Activity and Sugar Feeding by Culicoides Variipennis (Diptera: Ceratopogonidae) in Southern California, USA

Adults of Culicoides variipennis were collected with 6 CO2-baited traps placed in an east–west transect across a breeding site in southern California, USA. These insects were active over the entire 12-month sampling period but were less abundant in late winter. Gravid females also could be collected year-round near the breeding area by sweep net, which indicated some level of successful blood feeding. Ovarian dissections revealed some females with as many as 5 dilatations in August and November. Anthrone tests detected simple sugars in males (11%), nullipars (12%), and pars (24%); sugar positivity increased during winter and spring. Traps placed directly adjacent to the breeding pond generally collected fewer flies with a higher level of parity than did those placed 50 or 100 m away. Nullipars and males showed a tendency to disperse upwind in the vicinity of the breeding site. Trap location thus is a potential source of sampling bias for survivorship studies in this species.