For many years I studied insect cuticular proteins (CPs) as molecular markers of metamorphic stage. We now focus the precise role CPs play in constructing insects. We began by annotating the CP genes of Anopheles gambiae, the major vector of malaria. Unexpectedly, we learned that Anopheles devotes almost 2% of its protein coding genes to CPs. A shot-gun proteomics analysis confirmed that over 90% of the genes we called CPs actually code for proteins found in the cuticle. Some of these are present in the genome as clusters of highly similar genes. These sequence clusters, found in genomes of two other mosquitoes, are not present in Drosophila or most other insects whose genomes have now been sequenced. Once we had identified the Anopheles CP genes, we turned to learning when and where they are expressed. A comprehensive analysis with quantitative real-time RT-PCR revealed that most mRNAs for CPs are found in brief periods immediately before or after a molt. Spatial expression was a total surprise. Some CP genes are used only to build limited structures, a subset of bristles, Johnston’s organ, parts of the eye, while others are expressed in most cells that form cuticle.
Annotation has been expanded to other species with the discovery that the largest family, represented by 156 genes in Anopheles only has 28 members in the honeybee. We have analyzed the evolution of these genes, within Anopheles and comparatively.
Other laboratories have implicated the CPs of Anopheles in insecticide and desiccation resistance, in mate recognition, and in being synthesized in response to a blood meal. We propose to learn whether the adult cuticle is a dynamic structure by verifying increased levels of transcripts after appropriate challenges. We want to learn where in the cuticle secreted proteins are located using secondary antibodies labeled with colloidal gold on EM sections. We hope to employ RNAi to reduce transcript levels of selected CP genes to learn whether this treatment compromises form and function of mosquitoes.
Dr. Willis is no longer training graduate students in her lab.
Dotson, E.M., A. J. Cornel, J. H. Willis and F. H. Collins. 1998. A family of pupal-specific cuticular protein genes in the mosquito Anopheles gambiae. Insect. Biochem. Molec. Biol. 28: 459-472.
Magkrioti, C. K., I. C. Spyropoulos, V. A. Iconomidou, J. H. Willis and S. J. Hamodrakas. 2004. cuticleDB: a relational database of Arthropod cuticular proteins. BMC Bioinformatics 5:138
Iconomidou, V. A., J. H. Willis and S. J. Hamodrakas. 2005. Unique features of the structural model of ‘hard’ cuticle proteins: implications for chitin-protein interactions and cross-linking in cuticle. Insect Biochem. Molec. Biol. 35:553-560.
The Honeybee Genome Sequencing Consortium 2006. Insights into social insects from the genome of the honeybee Apis mellifera Nature 443:931-949.
He, N., J. M. C. Botelho, R. J. McNall, V. Belozerov, W. A. Dunn, T. Mize, R. Orlando and J. H. Willis. 2007. Proteomic analysis of cast cuticles from Anopheles gambiae by Tandem Mass Spectrometry. Insect Biochem. Molec. Biol. 37:135-146.
Karouzou, M.V., Y. Spyropoulos, V. A. Iconomidou, R. S. Cornman, S. J. Hamodrakas and J. H. Willis. 2007 Drosophila cuticular proteins with the R&R Consensus: annotation and classification with a new tool for discriminating RR-1 and RR-2 sequences. Insect Biochem. Molec. Biol. 37:754-760.
Togawa, T, W. A. Dunn, A. C. Emmons, and J. H. Willis. 2007. CPF and CPFL, two related gene families encoding cuticular proteins ofAnopheles gambiae and other insects. Insect Biochem. Molec. Biol. 37:675-688.
Cornman, R.S., T. Togawa, W. A. Dunn. N. He, A. C. Emmons, J. H. Willis. 2008. Annotation and analysis of a large cuticular protein family with the R&R Consensus in Anopheles gambiae. BMC Genomics 9:22.
Togawa, T., W. A. Dunn, A. C. Emmons, J. Nagao, and J. H. Willis. 2008. Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae. Insect Biochem. Molec. Biol. 38:508-519.
Cornman, R.S. and J. H. Willis. 2008. Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes. Insect Biochem. Molec. Biol. 38:661-676.
Cornman, R.S. and J. H. Willis 2009. Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle. Insect Molec. Biol.18:607-622.
Willis, J. H. 2010. Structural cuticular proteins from arthropods: Annotation, nomenclature, and sequence characterization in the genomics era. Insect Biochem. Molec. Biol. 40:189-204.
Papandreou, N. C., V. A. Iconomidou, J. H. Willis and S. J. Hamodrakas (2010). A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin. J. Insect Physiol. 56:1420-1426.
Willis, J. H., D. L. Cox-Foster (2010) Letter to the Editor Insect metamorphosis via hybridogenesis: An evidentiary rebuttal. J. Insect Physiol. 56:333-335.
Willis, J.H., N. C. Papandreou, V.A. Iconomidou, S. J. Hamodrakas (2012) Cuticular proteins. Chapter 5 in Insect Molecular Biology and Biochemistry (ed. L. I. Gilbert) Academic Press pp. 134-166. [Invited extensive revision of chapter in Comprehensive Insect Molecular Science (2005)].