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Project description

The alimentary track of glassy-winged sharpshooter as a target for control of Pierce's disease. (02XA008)
Program Exotic Pests and Diseases Research Program
B.A. Federici, Entomology, UC Riverside
Host/habitat Grapes
Pest Glassy-Winged Sharpshooter Homalodisca coagulata; Pierce's Disease Xylella fastidiosa
Discipline Entomology
Agricultural Systems
Start year (duration)  2002 (Two Years)
Objectives Determine the structure and cell types in the midgut epithelium and salivary glands of the glassy-winged sharpshooter (GWSS), Homalodisca coagulata.

Prepare a normalized cDNA microarray of GWSS using pooled cDNAs isolated from each developmental stage.

Screen the microarray using cDNA probes derived from midgut and salivary gland tissue-specific probes to determine the tissue-specific expression of key midgut microvillar and saliva proteins.

Clone and sequence genes encoding one or more key midgut microvillar and saliva proteins and determine their suitability as targets for a molecular biological approach to GWSS and Pierce's disease control.

In situ hybridizations have been performed using paraffin-embedded thick sections of dissected glassy-winged sharpshooter (GWSS) guts. These demonstrate the expected expression of the V-ATPase subunits and membrane transporter throughout the alimentary tract and salivary glands. Polymerase chain reaction (PCR) products of each of the 10,848 clone inserts have been purified and are in the process of being quantified for normalization before microarray spotting.

We have begun microarray screening with a limited array of 1,536 clones and targets prepared from sharpshooters exposed to sublethal and LD50 levels of the insecticide esfenvalerate. These control experiments were initially performed at the Custom Microarray Facility at the University of Arizona during a microarray training course and are being repeated using the new Hyb4 hybridization station purchased from Genomic Solutions by our laboratory. RNA has been extracted from GWSS dissected guts and salivary glands for target preparations. A single GWSS gut yields sufficient RNA for the preparation of a single target.

We have pooled several dissected gut and salivary gland samples for extraction so we have sufficient RNA for dye swap experiments. A minimum of three pooled samples have been prepared for each target type. Antibodies are being prepared by Orbigen, Inc. to the GWSS V-ATPase c subunit and a novel amino acid transporter recovered from our cDNA library using the clone capture technique. The clone capture technique allows isolation of the full-length cDNA of any gene in the library using complementary oligo probes that have been biotinylated. After hybridization, the captured clones are removed by association with streptavidin-coated magnetic beads. Once the antibodies to these two proteins have been prepared, we will clone the variable regions into the EZnetTM Display cDNA Library Screening Kit from Maxim Biotech, Inc. We should be able to conduct feeding screenings for efficacious antibody peptides by the end of summer.

We have performed ultra structural analyses of GWSS gut components and salivary glands. We are now performing in situ hybridization studies, which will confirm where each of the genes we have cloned is expressed in these tissues. A normalized cDNA library containing approximately 200,000 full-length clones has been prepared. This library should contain 100 copies of all of the expressed genes in GWSS. The cloned cDNAs are being amplified from 10,080 library clones by polymerase chain reaction (PCR). The PCR products will be used to spot microarrays of the expressed GWSS genes. Microarrays will be screened by hybridization to labeled RNAs prepared from the tissues we are targeting in GWSS. These experiments will allow us to identify novel GWSS genes that can be used as targets of mimetic peptides, which will exclusively kill or prevent feeding of GWSS. In addition, portions of five GWSS genes have been cloned using reverse transcription PCR (RT-PCR) and degenerate primers based on genes characterized in other insects. Two of these encode parts of vacuolar ATPase subunits A and c, which establishes the essential salt balance across the membrane and maintains the pH of the insect gut. The third is a membrane transport protein, which moves amino acids from inside the gut into cells where they are used as essential nutrients. The remaining two genes encode saliva proteins, a maltase-like protein, involved in sugar degradation, and a trypsin-like protein involved in amino acid degradation. Each of these gene products are being evaluated for mimetic peptide production.

GWSS gut and salivary gland dissections have been performed, and samples are being prepared for ultrastructural analysis. These studies will determine the tissue and cellular organization of these organs. We have accumulated samples of GWSS for RNA extraction and cDNA library synthesis. In addition, portions of two GWSS genes have been cloned. The first encodes part of a vacuolar ATPase A (V-ATPase A) subunit, similar to that isolated from the yellow fever mosquito, Aedes aegypti. The second encodes a protein similar to the potassium-coupled amino acid transporter isolated from the tobacco hornworm, Manduca Sexta. Both of these genes encode proteins essential to insect feeding. Gut V-ATPases are responsible for the generation of energy required to move ions across cell membranes. This ion transport establishes salt balance across the membrane and maintains the pH of the insect gut. Gut amino acid transporters move required amino acids from inside the gut into cells where they act as essential nutrients and building blocks for insect protein synthesis. Both genes were cloned by genomic DNA- and RT-PCR amplifications. We are now ready to move to the next step in developing a mimetics-based insecticide for GWSS. Through the identification of antigenic epitopes on the cloned gene encoded proteins, we are determining the structure of peptides we will use for antibody production. Antibody peptides which strongly bind these epitopes will be assessed in GWSS feeding studies to identify those with the ability to block protein function leading to GWSS death and/or feeding deterrence.

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