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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI031951-01
Application #
3146957
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1992-02-01
Project End
1996-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Isoe, Jun; Stover, Weston; Miesfeld, R Barrett et al. (2013) COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities. Insect Biochem Mol Biol 43:732-9
Mack, Daniel J; Isoe, Jun; Miesfeld, Roger L et al. (2012) Distinct biological effects of golgicide a derivatives on larval and adult mosquitoes. Bioorg Med Chem Lett 22:5177-81
Alabaster, Amy; Isoe, Jun; Zhou, Guoli et al. (2011) Deficiencies in acetyl-CoA carboxylase and fatty acid synthase 1 differentially affect eggshell formation and blood meal digestion in Aedes aegypti. Insect Biochem Mol Biol 41:946-55
Isoe, Jun; Collins, Jennifer; Badgandi, Hemant et al. (2011) Defects in coatomer protein I (COPI) transport cause blood feeding-induced mortality in Yellow Fever mosquitoes. Proc Natl Acad Sci U S A 108:E211-7
Zhou, Guoli; Isoe, Jun; Day, W Antony et al. (2011) Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells. PLoS One 6:e18150
Rascon Jr, Alberto A; Gearin, Johnathon; Isoe, Jun et al. (2011) In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti. BMC Biochem 12:43
Brackney, Doug E; Isoe, Jun; W C 4th, Black et al. (2010) Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito, Aedes aegypti. J Insect Physiol 56:736-44
Isoe, Jun; Rascon Jr, Alberto A; Kunz, Susan et al. (2009) Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes. Insect Biochem Mol Biol 39:903-12
Isoe, Jun; Zamora, Jorge; Miesfeld, Roger L (2009) Molecular analysis of the Aedes aegypti carboxypeptidase gene family. Insect Biochem Mol Biol 39:68-73
Kelleher, Erin S; Pennington, James E (2009) Protease gene duplication and proteolytic activity in Drosophila female reproductive tracts. Mol Biol Evol 26:2125-34

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