Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Biological & Environmental Engineering
Non Technical Summary
Insects are major pests of crops and act as vectors transporting diseases to animals and humans causing billions of dollars' worth of losses. In the US, the armyworm, diamondback moth, and red flour beetle, each cause > $1 billion in damage annually. In extreme cases, entire fields are intentionally destroyed or abandoned. Infestation of crop commodities can also significantly reduce the storage life and nutrition quality. Furthermore, mosquito-borne diseases such as equine encephalitis, dog heartworm, and West Nile virus continue to be a growing problem each year in US and worldwide. Some of the most effective control measures have been insecticides. Unfortunately, insecticides have non-target effects that are often compounded by environmental persistence. Unlike most insecticides, dsRNA's are biocompatible, biodegradable, environmentally benign, and has the potential of being a selective insecticide that interferes with genes critical to insect survival. More importantly, the phenomenal potential of RNAi is that it is both specific for the gene of interest, and for the organism being targeted. Thus, dsRNA would offer extremely high pest specificity without the downsides of human and environmental risk. Encapsulation of dsRNA in biodegradable polymers (e.g. PLA) will not only prevent premature degradation of the dsRNA, but will also provide a stable release platform allowing a gradual and steady release of the insecticide. By incorporating UV-blockers, such as titanium dioxide, we will be able to further enhance the stability of the dsRNA allowing it to effectively deliver the insecticide for a greater duration compared to current commercially available pesticides. Upon translation to market, the technology will benefit farmers and non-farmers alike because it will offer a pest-specific insecticide with virtually no adverse human or environmental effects. It will also benefit the economy, including the Biopesticide Industry Alliance, a consortium of businesses dedicated to fostering adoption of biopesticide technology. Upon demonstrating the technology, several national and international research programs could further develop it to benefit a broader audience. These programs include USDA's new Research and Education for Improved Pest Control which encourages development and implementation of IPM while reducing human and environmental risks. Additionally, organizations such as NIH, WHO and the Gates Foundation could be interested to help globally reduce the spread of insect-born human diseases.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our goal is to engineer an effective and economic double-stranded RNA (dsRNA) bio-insecticide formulation that is specific to a target pest and can be designed to provide sustained and controlled released of an environmentally friendly pesticide alternative to replace conventional broad-spectrum insecticides. The process of maintaining populations of the target insects in check offers the potential to produce more commodities that can supply our ever growing need for food at national and world levels. In addition, the ability to control the release rate would reduce the treatment costs since fewer pesticide applications would be required. The encapsulated dsRNA would have a slow and constant release, in contrast to an initial release spike followed by a greatly diminished release that is typically seen from conventional pesticide applications. Unlike most insecticides, dsRNA's, which operate by interfering with gene transcription (termed RNA interference or RNAi), are biocompatible, biodegradable and environmentally benign. More importantly, the phenomenal potential of RNAi is that it is both specific for the gene of interest, and for the organism being targeted. Furthermore, by developing methods to target pathogen carriers such as mosquitoes, we can reduce the potential spread of disease and at the same time have an environmentally friendly reduced-risk insecticide that will not adversely affect higher members of the food chain, such as fish, birds, and humans. The following objectives have been proposed to achieve our overall goal: 1) Use drug delivery technologies to encapsulate dsRNA. 2) Determine whether biopolymer encapsulated dsRNA can deliver a toxic dose to insects. 3) Optimize biopolymer formulations to impart robust stability to dsRNA. 4) Identify other dsRNAs that target Colorado potato beetle critical genes for suppression. 5) Test new candidate UV-stabilized biopolymer formulations and dsRNA's under a wide range of environmental conditions. Upon the conclusion of this project we envision having developed a novel, economic, environmentally friendly, and target specific insecticide and delivery system. This system will be able to be mass marketed with a wide range of applications for targeting insects that are responsible for crop destruction to insects that are capable of transporting pathogens to humans and animals.
Project Methods
The methods in which we will implement our project design are as follows: 1) Since dsRNA's are highly unstable in the environment and need to be protected before being applied, we will encapsulate the dsRNA in biodegradable polymers, e.g. poly-lactic acid (PLA), using a double emulsion (water/oil/water) method. PLA, which is bulk-produced from renewable resources and commercially available at reasonable cost, is UV-stable, biodegradable, and have been employed as an FDA-approved medical drug delivery vehicle for human consumption. The biopolymer will be dissolved in methylene chloride first and dsRNA added to the solution drop-wise; the mixture is then cooled and sonicated. After a double-emulsion forms, it will be purified by centrifuging and freeze-dried. The formulations will then be exposed to UV radiation for varying durations before bioassays with Colorado Potato Beetles (CPB) to determine the amount of dsRNA stabilization. dsRNA that is specific for CPB, vATPaseE (vacuolar ATP synthase subunit E) and Sec23 (a transport protein) will be encapsulated into PLA in a sprayable microsphere format. 2) We will conduct bioassays by feeding PLA-encapsulated dsRNA to CPB larvae. Larval mortality will be assessed daily and compared to controls. We will also compare the relative amount of vATPaseE and Sec23 transcripts using quantitative RT-PCR in larvae that were fed leaves + water (control), compared to leaves treated with dsRNA and held for 0, 2 and 4 days before being presented to larvae. 3) Concentrations of dsRNA will be varied to test for dose response and, if needed, formulations will be further engineered for UV stability by the addition of UV-protectant compounds, e.g. titanium dioxide. 4) Novel dsRNA's will be identified for efficacy of beetle gene suppression. 5) Testing of novel formulations and dsRNA's under diverse environmental conditions. The primary metric is dsRNA stabilized for up to 3 days UV exposure. We will measure beetle mortality and quantify gene suppression as a function of irradiation duration. Secondary metrics are dissemination and publication of results so that interested researchers are stimulated to consider this technique for stabilizing other unstable pesticides. Initially, the outcomes will be extensively quantified in a controlled laboratory environment, followed by wider environmental conditions.