The Identification of candidate genes for developing transgenic resistance to banana weevil in East African Highland banana. Major outputs inlude:

• A system for the dissection of adult and larval banana weevil mid guts and subsequent extraction of their active proteinases has been developed. This has been followed by the development of a fluorimetric in-vitro assay to evaluate cysteine proteinase activity, optimum pH and inhibition of the gut extracts. The assay uses a synthetic peptide which releases a highly fluorescent compound on breakdown by proteolytic enzymes present in the gut extracts. Activity is measured by a fluorescence spectrophotometer or can be scored visually under UV light.

• Cysteine proteinase activity has been detected in both adult and larval banana weevils as has been reported in many other coleopteran insects.

• Cysteine proteinase activity in weevil gut extracts has been highly inhibited, in-vitro, by a synthetic cysteine proteinase specific inhibitor E-64 and also by two plant cystatins; oryzacystatin I (OC-I) from rice (Oryza sativa) and papaya cystatin from papaya (Carica papaya). The two phytocystatins were obtained in large purified amounts by cloning, expression in E. coli and purification by affinity chromatography.

• An in-vivo bio assay system based on vacuum infiltration of banana stems with cystatin solutions, and subsequent rearing of weevil larvae on the stems has been developed. Preliminary experiments with this system have shown that larvae reared on cystatin infiltrated stems experienced up to 76% reduction in early larval growth and development. This assay system is a significant achievement since there is no known artificial diet for banana weevil. A publication is in preparation.

• During the period from February 2002 when the project started, and April 2003, various aspects of this project were communicated in form of scientific presentations at two international meetings; the 3^rd International Symposium on Cellular and Molecular Biology of Banana and Plantain held at Leuven, Belgium on 8^th September 2002 and the joint International Conference of the South African Association of Botanists and the International Society for Ethnopharmacology, held in Pretoria on 14^th January 2003. Other local presentations included one at the Rand Afrikans University, Johannesburg annual postgraduate symposium in Plant Science (30^th October 2002), the “South African Banana Growers Association Annual Symposium” at Pretoria (17^th October 2002) and the Uganda banana biotechnology project 3^rd planning meeting on the 27^th November 2002 in Kampala, Uganda.

• A combined advanced molecular biology and bio-informatics course was attended and the knowledge gained in sequence analysis, and protein design through structural modeling has been implemented in the current research.