Blood-sucking insects transmit a number of important diseases. Control programs based on the use of insecticides had early success, but the recent development of resistant insects has made such measures problematic. It is now clear that control measures based on the biology of the insect, so- called biorational control, should be developed. Relatively little work using modern biochemical and molecular biological approaches has been done on insect vectors. The ingestion of the blood meal induces many morphological and biochemical changes in the midgut, which are required for digestion of the blood meal. The long-range goal of this research is to understand this differentiation of the midgut and to identify potential steps in this process which might serve as targets for controlling these insects. In this application we propose to use two important disease vectors: Aedes aegypti, a mosquito which transmits yellow fever and dengue fever, and Rhodinus prolixus, a hemipteran which transmits Chagas disease. We propose to study how the blood meal regulates the expression of the genes encoding the major midgut digestive proteases as a paradigm for understanding the differentiation of the midgut. A. aegypti uses a trypsin-based digestive system, whereas R. prolixus uses a cathepsin-based digestive system. In A. aegypti there is evidence for the existence of two forms of trypsin, an """"""""early"""""""" form, which appears immediately after the blood meal, and a """"""""late"""""""" form, which is responsible for most of the digestion of the protein in the blood meal. In R. prolixus digestion in the lumen of the midgut is carried out by enzymes which are normally found in lysosomes.
The specific aims of this proposal are as follows: 1) clone and sequence the cDNA for the """"""""early"""""""" trypsin from the midgut of the mosquito Aedes aegypti; 2) clone and sequence the genes for the """"""""early"""""""" and """"""""late"""""""" trypsin from Aedes aegypti; 3) clone and sequence the cDNAs and genes for the cathepsin B and D from the midgut of Rhodinus prolixus; 4) Use cDNA and antibody probes to analyze factors in the blood meal, or factors produced in the insects in response to the blood meal, which affect the expression of the genes encoding the digestive proteases; 5) Identify cis-acting elements in the protease genes and use these DNA sequences to isolate cDNAs for the trans-acting factors which bind to the cis-acting elements. Sequence these cDNAs and use them to investigate how the blood meal affects the amount or activity of the trans-acting factors.
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