Source: Insect Biotechnology, Inc. submitted to NRP
NOVEL PESTICIDE DELIVERY SYSTEM FOR INSECTS: NEXT GENERATION PROTEIN INSECTICIDE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
0199654
Grant No.
2004-33610-14310
Cumulative Award Amt.
(N/A)
Proposal No.
2004-00092
Multistate No.
(N/A)
Project Start Date
May 15, 2004
Project End Date
Mar 31, 2007
Grant Year
2004
Program Code
[8.2]- (N/A)
Recipient Organization
Insect Biotechnology, Inc.
(N/A)
Chapel Hill,NC 27515
Performing Department
(N/A)
Non Technical Summary
Insects pose several challenges to mankind. They consume the food crops that we grow, they transmit diseases to plants, man and animals, and cause infrastructure damage and destruction. Current methods to control insects are largely based on the use of toxic chemicals, principally neurotoxins, which also pose health risks to the users, consumers, and other nontarget organisms. There are numerous examples of safer, naturally occurring biocompatible materials that could be utilized to control insect populations if the material could be applied in such a manner that it was taken up by the target insect and reached its site of action. The technology developed in this project will facilitate the translocation of new biocompatible insecticidal agents (peptides, proteins, or nucleic acids) into the target insect to provide control of insect populations while reducing the risk associated with nontarget exosure. The purpose of this project is to prepare derivatives of Trypsin Modulating Oostatic Factor (TMOF) to enhance its transport across the gut into the hemolymph (blood) of tobacco budworm (Heliothis virescens) and evaluate the potential effectiveness of TMOF as a safer, new insect control technology for application to an agriculturally significant insect pest.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2115220100060%
2116110115040%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
6110 - Economy of the United States and sectors thereof; 5220 - Pesticides;

Field Of Science
1000 - Biochemistry and biophysics; 1150 - Toxicology;
Goals / Objectives
The tasks to be completed in Phase I are the following: Task 1, Synthesize TMOF-RO(ethyleneglycol).sub.7-O-hexane and -hexanoic acid conjugated polymers (TMOF-MCP); Task 2, Demonstrate that our current ELISA for native TMOF will also detect TMOFK (TMOF with an added carboxy terminal lysine residue) and TMOF-MCP formulated with yeast in meal pads and in the feces and the hemolymph of last stadium tobacco budworms, Heliothis virescens; Task 3, Determine the 24 h stability of TMOFK and TMOF-MCP in insect meal pads; Task 4, Feed fifth stadium larvae ad libitum meal pads containing TMOFK and TMOF-MCP and develop a washing protocol that will remove both TMOFK and TMOF-MCP from the external cuticle surface; Task 5, Feed fifth stadium larvae ad libitum meal pads containing TMOFK or TMOF-MCP, wash the larvae, obtain hemolymph and feces, and measure TMOF levels in each over a 24 h period; Task 6, Apply TMOFK and TMOF-MCP topically and determine the levels of each in hemolymph over a 24 h period; Task 7, Feed TMOFK and TMOF-MCP to budworm neonates and determine the dose for 50% inhibition of gut protease activity and growth; and the median lethal dose for both compounds; Task 8, Evaluate results and commercial potential; and Task 9, Prepare final report.
Project Methods
Bt is the first successful example of the use of a protein for the control of agricultural insect pests. Many polypeptide hormones and insect-specific toxins have been identified as alternative protein insecticides to that of Bt. However, the limiting factors in their use are that they are digested when eaten and they do not act on the gut wall like Bt. Insect Biotechnology Inc. (IBI) has licensed from North Carolina State University (NCSU), a novel protein delivery system for insects using methyl(ethyleneglycol).sub.7-O-hexanoic acid conjugated polymers. Preliminary studies conducted at NCSU with an insulin-polymer developed as the first oral insulin, suggests that these polymers also work in the delivery of proteins into insect hemolymph. In this proposal we will synthesize TMOF-RO(ethyleneglycol).sub.7-O-hexane and -hexanoic acid conjugated polymers. TMOF (trypsin modulating oostatic factor) is an insect peptide hormone that has been exclusively commercialized by IBI for the control of mosquito larvae but which also has activity against the tobacco budworm. Its mode of action is the inhibition of digestive enzyme biosynthesis and the cessation of feeding by binding to receptors on the hemolymph side of the gut ventricular epithelium. Feasibility will be demonstrated when we can show enhanced accumulation in the hemolymph and disruption of development. If successful, our discoveries will have high commercial value not just for the delivery of TMOF but as an enabling technology for most companies that are developing insecticides. One goal of IBI will be to license our technology or co-develop other applications for insect control, which will greatly broaden the commercial significance of this USDA SBIR proposal.

Progress 05/15/04 to 03/31/07

Outputs
The goal of this project is to develop an enhanced peptide/protein delivery system for insects to facilitate and accelerate the transport of peptide/protein insecticides across the midgut epithelium of insects thereby improving their efficacy. As a model system, the decapeptide hormone Trypsin Modulating Oostatic Factor (TMOF, YDPAP6), originally identified in mosquitoes, was selected. TMOF functions as an effective mosquito larvicide through down-regulating the expression of trypsin-like digestive proteases in the mosquito; unable to digest its food, the mosquito larva succumbs to starvation. TMOF also has activity in other lepidopteran larvae. Polyethylene glycol (PEG) was proposed as a derivativzation agent. PEG is well known to enhance the uptake of exogenous protein by mammalian cells and to protect orally administered proteins such as insulin in the mammalian digestive tract with uptake of physiologically effective quantities. A similar approach was proposed for enhancing the uptake of TMOF in mosquito larvae and other significant lepidopteran insect pests. The active portion of the TMOF molecule is found at the amino terminus. Derivativzation of the one available amino functional group would be expected to stereochemically block biological activity. Thus, TMOF was synthesized containing an added carboxyl-terminal lysine residue (TMOFK) with the amino terminus blocked as the trifluoroacetamide. This yielded a peptide with an amino group distal to the expected active portion of the TMOF molecule. TMOFK-PEG4 was successfully synthesized by reacting the blocked TMOFK with N-hydroxysuccinamido-12-methoxy-4,7,10-trioxadodecanoate (NHS-m-dPEG4), deblocking of the peptide and isolation of TMOFK-PEG4 in high yield following HPLC (purity, 97%). TMOFK-PEG4 was evaluated for biological activity against 1-day old Aedes aegypti larvae and newly hatched Heliothis virescens and Helicoverpa zea with no treatment (water) as the control; treatment of larvae with TMOF and TMOFK was also tested. Growth of H. zea reared for 5 days on a diet containing TMOFK-PEG4 attained a weight 57% of the weight in comparison to no treatment controls, statistically significant at a 99.99 % confidence level. H. zea raised on diets supplemented with TMOF and TMOFK were statistically significantly smaller than the no treatment controls but were larger than TMOFK-PEG4 treated larvae. H. virescens larvae were unaffected by TMOF, TMOFK or TMOFK-PEG4. A. aegypti larvae reared in the presence of the highest concentration of TMOFK-PEG4 tested (2 mM) showed an enhanced death rate compared to untreated controls and TMOF-fed larvae. A. aegypti raised in the presence of TMOF did not show the enhanced death as compared to untreated control larvae. TMOFK had no effect on A. aegypti larvae. Experimental evidence suggests that derivativzation of TMOF with PEG is effective for enhancing the biological activity of TMOF. Unfortunately, time and resources available for this project did not permit a more detailed evaluation of the biological activity. Further testing is necessary to establish the efficacy of utilizing PEG derivativzation to enhance uptake of protein and peptide insecticides.

Impacts
This study provided a successful way to prepare derivatives of peptide/protein insecticides that enhances their delivery into the hemolymph of the target insect through the gut epithelium. The exemplary peptide chosen was Trypsin Modulating Oostatic Factor (TMOF). The derivatives are readily prepared, stable and effective agains mosquito larvae and Helicoverpa zea. No activity was found agains Heliothesis virescens. The derivativzation technology is readily applicable to other peptide/protein insecticides and may also be applicable to nucleic acid-based insecticidal agents such as small interfering RNA (siRNA)or micro-RNA.

Publications

  • No publications reported this period


Progress 10/01/03 to 09/30/04

Outputs
This project is being transfered to a new business entity (EpiFeX) under the direction of the Principal Investigator. Until this process is completed and approved, no activity will be undertaken.

Impacts
None entered.

Publications

  • No publications reported this period