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Title: Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae)

Author
item LANGNER, KATHRIN - UNIVERSITY OF WYOMING
item DARPEL, KARIN - PIRBRIGHT LAB-UK
item DENISON, ERIC - PIRBRIGHT LAB-UK
item Drolet, Barbara
item LEIBOLD, WOLFGANG - HANNOVER SCHOOL VET MED
item MELLOR, PHILIP - PIRBRIGHT LAB-UK
item MERTENS, PETER - PIRBRIGHT LAB-UK
item NIMTZ, MANFRED - HELMHOLTZ CENTRE-GERMANY
item GREISER-WILKE, IRENE - HANNOVER SCHOOL VET MED

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/19/2006
Publication Date: 3/1/2007
Citation: Langner, K.F.A., K.E. Darpel, E. Denison, B.S. Drolet, W. Leibold, P.S. Mellor, P.P.C. Mertens, M. Nimtz, I. Greiser-Wilke. 2007. Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). Journal of Medical Entomology 44(2):238-248.

Interpretive Summary: Salivary proteins of blood feeding Culicoides midges are thought to play an important role in pathogen transmission and skin hypersensitivity. Analysis of these proteins, however, has been problematic due to the difficulty in obtaining adequate amounts of secreted saliva. In the current study, a collection method for midge saliva was developed. Analysis of 55 salivary proteins from Culicoides nubeculosus revealed that they were very similar to salivary proteins of another Culicoides species, Culicoides sonorensis. These included proteins involved in sugar meal digestion, defense, and coagulation inhibition as well as allergens. Intradermal injection of the saliva in human skin produced edema, vasodilatation and significantly prolonged clotting times for human platelets. The potential role of the identified salivary proteins in the transmission of pathogens and the induction of allergies is discussed.

Technical Abstract: Salivary proteins of hematophagous Culicoides spp. are thought to play an important role in pathogen transmission and skin hypersensitivity. Analysis of these proteins, however, has been problematic due to the difficulty in obtaining adequate amounts of secreted Culicoides saliva. In the current study, a collection method for midge saliva was developed. Over a three day period, three to five day old male and female Culicoides nubeculosus were repeatedly placed onto the collection system and allowed to deposit saliva into a filter. Salivary products were eluted from the filters and evaluated by gel electrophoresis and mass spectrometry as well as by intradermal testing and determination of clotting time. Gel electrophoresis revealed approximately 55 protein spots displaying relative molecular masses from 5 to 67 kDa and isoelectric points ranging from 4.5 to 9.8. The majority of molecular species analyzed by mass-spectrometry showed high convergence with salivary proteins recently obtained from a cDNA-library of Culicoides sonorensis. These included proteins involved in sugar meal digestion, defense, and coagulation inhibition as well as members of the D7 family and unclassified salivary proteins. In addition, the proteome analysis revealed evidence for a number of yet unknown Culicoides proteins. Intradermal injection of the saliva in human skin produced edema, vasodilatation and pruritus. The anticoagulant activity of the saliva was demonstrated by significantly prolonged clotting times for human platelets. The potential role of the identified salivary proteins in the transmission of pathogens and the induction of allergies is discussed.