Anopheles gambiae, the major vector of malaria, uses ~2% of its protein-coding genes for structural cuticular proteins. These genes have been annotated, their expression patterns determined, and the regions of the animal where many are expressed have been established. Other laboratories have implicated the cuticular proteins of Anopheles in insecticide and desiccation resistance, in mate recognition, and in being synthesized in response to a blood meal. We propose to determine whether cuticle is a dynamic structure by verifying the increased levels of transcripts of its component proteins after a blood meal and whether expression of cuticular protein genes can change in response to desiccation. Quantitative real-time RT-PCR will verify that genes already implicated in responding to a blood meal are upregulated. Simple physiological experiments will demonstrate whether Anopheles adapts to a desiccation challenge;if it does, genome-wide analyses of transcripts will show whether cuticular protein genes are involved. As a first step toward better understanding of cuticle assembly and structure, the location within the cuticle of selected proteins will be determined by visualizing secondary antibodies labeled with colloidal gold that have bound to primary antibodies on EM sections. The next step toward better understanding assembly and structure will be to determine the affinity of all cuticular proteins for chitin and for each other in their natural environment by extracting pulverized whole animals first with buffer to remove soluble proteins and then with increasing concentrations of agents that interfere with chitin binding. The complement of cuticular proteins in the various fractions will be determined by quantitative mass spectrometry on trypsinized fractions. If well represented proteins have peptides that are not detected, these missing peptides may be those that are participating in catechol-based crosslinking. From the studies described above, genes for cuticular proteins will be selected and RNAi will be used to reduce their transcript levels to establish whether this treatment compromises form and function of the mosquito, possibly revealing features that could be exploited in controlling malaria.

Public Health Relevance

The Anopheles mosquito is the carrier of malaria. As with all insects the exterior of the mosquito - the cuticle - is both its skeleton and its skin and contains many proteins, most of which have been identified in our previous study and constitute about 2% of its protein diversity. We now propose to extend our studies into determining the specific role of these proteins in the life of the mosquito, hoping that this knowledge will contribute to the control of this deadly insect.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI055624-08
Application #
8490666
Study Section
Vector Biology Study Section (VB)
Program Officer
Costero, Adriana
Project Start
2004-05-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
8
Fiscal Year
2013
Total Cost
$310,937
Indirect Cost
$101,552
Name
University of Georgia
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Vannini, Laura; Willis, Judith H (2016) Immunolocalization of cuticular proteins in Johnston's organ and the corneal lens of Anopheles gambiae. Arthropod Struct Dev 45:519-535
Vannini, Laura; Willis, Judith H (2016) Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae. Arthropod Struct Dev :
Zhou, Yihong; Badgett, Majors J; Bowen, John Hunter et al. (2016) Distribution of cuticular proteins in different structures of adult Anopheles gambiae. Insect Biochem Mol Biol 75:45-57
Vannini, Laura; Bowen, John Hunter; Reed, Tyler W et al. (2015) The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea. Insect Biochem Mol Biol 65:57-67
Chipman, Ariel D; Ferrier, David E K; Brena, Carlo et al. (2014) The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima. PLoS Biol 12:e1002005
Vannini, Laura; Reed, Tyler W; Willis, Judith H (2014) Temporal and spatial expression of cuticular proteins of Anopheles gambiae implicated in insecticide resistance or differentiation of M/S incipient species. Parasit Vectors 7:24
Vannini, Laura; Augustine Dunn, W; Reed, Tyler W et al. (2014) Changes in transcript abundance for cuticular proteins and other genes three hours after a blood meal in Anopheles gambiae. Insect Biochem Mol Biol 44:33-43
Ioannidou, Zoi S; Theodoropoulou, Margarita C; Papandreou, Nikos C et al. (2014) CutProtFam-Pred: detection and classification of putative structural cuticular proteins from sequence alone, based on profile hidden Markov models. Insect Biochem Mol Biol 52:51-9
Werren, John H; Richards, Stephen; Desjardins, Christopher A et al. (2010) Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science 327:343-8
Willis, Judith H (2010) Structural cuticular proteins from arthropods: annotation, nomenclature, and sequence characteristics in the genomics era. Insect Biochem Mol Biol 40:189-204

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