Recipient Organization
FORESIGHT SCIENCE & TECHNOLOGY, INC.
11750 DOCKER HILL RD
COMPTCHE,CA 95427
Performing Department
(N/A)
Non Technical Summary
This project will test and prove the efficacy of an entirely new method of controlling red imported fire ants (RIFA) using a USDA developed and patented peptides as the active ingredient for a new bait formulation. The technology utilizes novel bioactive peptides that act as antagonists to the RIFA neurohormone receptor found in all fire ant life stages. The technology also known as "RECEPTOR-I" technology was invented by USDA scientists, Dr. Robert Vander Meer and Dr. Man-Yeon Choi and patented by the USDA. The Phase I research showed promising results as a proof of concept for the technology. The project has been developed as a continuation of the previous basic and applied research performed at USDA-ARS that resulted in the issuance of two patents: a) a) US Patent 9,771,393 B2. Bioactive Peptides Having Insecticide Activity, Choi and Vander Meer, issued Sep. 26, 2017; and b) US Patent 10,017,538. Bioactive Peptides Having Insecticide Activity, Choi and Vander Meer, issued Jul. 10, 2018); and PCT WO2016/0262388 A1 and WO 2018/0086790 A1. The Phase II SBIR grant will further develop the bait formulation and delivery mechanism, which will then be tested in field trials. The goal will be to demonstrate a prototype bait formulation with sufficient technology readiness level to engage in licensing discussions with major manufacturers. The peptide bait formulation is biodegradable and species specific, making it an environmentally neutral alternative to chemical pesticides.
Animal Health Component
15%
Research Effort Categories
Basic
70%
Applied
15%
Developmental
15%
Goals / Objectives
Objective 1. Expand the small queenright colony feeding experiments to large mature fieldcollected monogyne (70,000 workers) colonies, under controlled laboratory conditions. Estimatecolony size and determine minimum AI concentration needed to consistently achieve colonycontrol.Preliminary studies demonstrated the effectiveness of the peptides -CMARYMSAC and CIQQGHSFC to cause worker mortality, queen death, and negatively affectthe normal functioning of small fire ant colonies (see results from Phase 1, Fig. 4 & 5). We needto evaluate freshly caught mature queenright field colonies in the laboratory to get a betterestimate of the amount of active ingredient is necessary to consistently achieve control. Theseresults will give us the effective lethal dose information needed for planning field trials inObjective 3.Extrapolation of results from tests with field colonies in small scale laboratoryconditions can accurately reflect the requirements for successful treatment of mature fieldcollected queenright monogyne colonies.Objective 2. Determine the effects of peptide mixture CMARYMSAC and CIQQGHSFC onsmall queen right colonies (up to 15k worker population brood and female sexuals).The preliminary feeding experiments with these peptides have shown workermortality. Experiments combining the two peptides had negative effects of fire ant colonies areneeded to make decisions about which of these peptides and/or their combination should beevaluated in large scale, more labor intensive, and costly field experiments.Combinations of peptides will provide greater efficacy against fire ants than thecompounds by themselves. Will the effects of the mixture be additive or synergistic?Objective 3. Simulate homeowner backyard conditions with 10-15 fire ant colonies and developbest practices under these conditions.Background: The target market for the bait formulation is household-related fire ant treatments,as the largest economic sector related to fire ant control. Demonstrating the effectiveness of anAI-Peptide in these conditions is critical for success in finding a commercialization partner tolicense the technology and to produce a commercial product. Optimization of our formulation forefficacy and ease of use is extremely important for successful commercialization and is aprimary goal for these field tests. The results under Objective 3 for one or more bait formulationswill drive treatment protocols for more extensive field trials.Conclusions from Objectives 1&2 will define successful treatment parametersbased on testing in a simulated homeowner situation including control and treatment colonies.Objective 4. Nano particle delivery of peptides: determine mortality effects of selected peptideson brood, workers, and queen if fed in combination with nano particles.Nanoparticle (NP)-mediated delivery through feeding has become a promisingapproach for sustainable pest management.Nanoparticles (1-100 nm in diameter) can be used as effective pesticides against weeds, plantpathogens, and insect pests and may also be incorporated into new formulations of insecticidesand insect repellents. Unlikeconventional hydrophobic pesticides, nanocides may be water-soluble (hydrophilic), whichenhances bioactivity and coverage uniformity . Among nanomaterials, silica(SiO2) nanoparticles (NPs) have received considerable attention as a possible alternative toconventional insecticides. The insecticidal properties of silica NPs are thought to be due to directabrasion of the insect cuticle or sorption through the cuticular layers.The synthetic peptides will have better transport across the insect midgut anddelivery into the fire ant target site when combined with nano particles.Objective 5. Peptides combined with surfactants in the development of a suitable formulation.Solubility of peptides like CMARYMSAC which has shown high potential for anegative impact on fire ant colonies goes down with increasing concentration because of itshydrophobic nature. A suitable formulation of peptides can be developed by use of non-ionicsurfactants which previously shown to improve the solubility behavior, permeability, and theefficiency of AI's used against pests. Tween 80, Triton X, and PEG6000 were more suitable tobe used as surfactant in improving insecticide solubility and reducing the cytotoxicity.Surfactants are a fundamental component of nano-emulsions, and their proper choice isessential to obtain a stable formulation over time. Tween (20, 40, 60, or 80) (MERCK,Darmstadt, Germany), modified starch, soy lecithin, sodium alginate, sodium caseinate,maltodextrin, acacia gum, and whey protein are the mostcommon surfactants. In the case of essential oil formulations, polysorbates (Tween 80 and 20)are two of the most commonly usedsurfactants because they form stable formulations withoutadditional co-surfactants. Tween 80 has been referred to as one of theprincipal surfactants used to prepare aqueous nano-formulations of essential oils (Eos) at a ratioof 1:1 or 1:2 (oil: surfactant). Dissolving the peptide active ingredient (individually in combination) with suitableemulsifier will enhance the solubility and efficacy of AI's and therefore intake by field RIFA.
Project Methods
Objective 1. Expand the small queenright colony feeding experiments to large mature fieldcollected monogyne (70,000 workers) colonies, under controlled laboratory conditions. Estimatecolony size and determine minimum AI concentration needed to consistently achieve colonycontrol.Experimental Design: Queenright (with queen) monogyne colonies are readily collected in theGainesville, FL area and standardprocedures are available for isolating them from soil andmaintaining them in the laboratory. The objective here is to get the coloniesinto the treatment protocol as soon after collection as possible (within 48 hours), because afterbeing in the laboratory for 2-3 months, worker behavior becomes less robust . After separation from the soil, colony size will be estimated by dividing total weightof isolated workers and brood by 0.8 mg (average worker weight). A treatment (syntheticpeptide) and control feeding regime will start at 0.1% AI concentration with in 10% sucrosesolution, presented in Eppendorf tubes topped with a cotton plug. Several of these treatment andcontrol reservoirs will be placed in their respective colonies. Colonies will also have crickets andwater tubes for food and moisture. The colonies will be fed ad libitum until they stop producingworker brood, the queen(s) dies, or worker numbers reach < 95% of the original workerestimate. Because colonies from the field represent a variety of colony sizes, we will be able tocorrelate colony size with the amount of AI needed for control. This information will go into theplanning and execution of Objective 3. Health of the queen will be monitored daily. Queenweights will be measured from both controls and treated colonies once a week, without causingsignificant disturbance to the colonies.Accumulated piles of dead workers will be counted everyday and brood production from the queen will also be monitored. A separate set of colonies willbe given AI treatments just once during the entire experiment and observations will be made inparallel with the colonies to which continuous supply of AI material is given. This will inform usas to the type of treatment needed for worker and queen death.Objective 2. Determine the effects of peptide mixture CMARYMSAC and CIQQGHSFC onsmall queen right colonies (up to 15k worker population brood and female sexuals).Experimental Design: Freshly collected fire ant colony units will be set up with known amountsof workers, brood low weight female sexuals and monogyne queen. The concentrations of thecompounds tested will be range from 0.01. 0.1, 0.5, and 0.1% of peptides in 10% sucrosesolutions in two peptides in combinations. Controls will be single compounds (10% sucrosesolutions). This will allow us to determine if the results are additive suppressed, or synergistic. A2-component peptide (one of each from CVKLGHSFC and CERVGHSFC will be tested)mixture will also be formulated at 0.33% of each and the controls will be each component at0.33% and at 1%. All treatments and controls will be replicated at least three times. Experimentalunits will be monitored for worker mortality, female sexual behavior, brood mortality, and queenmortality. Results will be analyzed statistically. At the time of the experiment, we will determineif it would be advantageous to periodically sample brood, workers, and female sexuals for futureanalysis.Objective 3. Simulate homeowner backyard conditions with 10-15 fire ant colonies and developbest practices under these conditions.Experimental Design: A field site will be located with moderate to low fire ant populationlevels that can be segregated into discrete 10 to 15 colony treatment/control units. All colonies ata treatment site will be located by GPS, creating an accurate mound map of the site. The volumeof each colony will be determined (L x W x D), as well as their Population Index (PI)Mound groups of 10-15 colonies that are readily defined willbe identified and treatments and control groups will be matched, as well as possible for totalmound volume and PI. At least 3 replicates will be defined for treatments (individual andcombined peptides) and control. Comparing treatments requires careful attention to the time ofapplication, since foraging activity changes as the daytime temperature changes. The number andplacement of bait stations/bait-matrix (bait stations will either be one that is commerciallyavailable or centrifuge tubes with a cotton plug at the end placed in bait station made of PVCpipes) will be determined after the mound volume and PI data are analyzed. The treatments andcontrols will be checked for changes in PI and mound volumes at least once a week. Bait stationreservoirs will be replenished until 3 replicates of a treatment results in control.Objective 4. Nano particle delivery of peptides: determine mortality effects of selected peptideson brood, workers, and queen if fed in combination with nano particles.Experimental Design: NPs are customized to carry a specific biomolecule to the organ, tissue,or even cell, and using a nanocarrier can easily penetrate the insect or plant cell and accuratelydeliver the products. For delivery and tracking purposes, various inorganicnanomaterials with distinctive chemical and physical properties, viz., silica, metal oxides, metals,semiconductors, and carbon-based materials, have been prepared.Forthe delivery of pesticides, nanocarriers like synthetic silica, polymer, titania, silver, alumina, andcopper are mostly used. For successful formation of nano bio-pesticides, the biologicalcompounds having pesticidal properties serve as capping and reducing agents and are blendedwith silver salt.Due to the large surface area present in NPs, they can bondother compounds quickly and can circulate easily in the lepidopteran insect system. Mortality bioassays will be initially developed for peptide and nano particle formulationsagainst fire ants. We will eventually scale up those bioassays, to get more information from onrequired concentration of peptides treatments blended in nano particles which can be effectivelyused to mimic a fully functional field colony under controlled laboratory conditions. This isnecessary to better identify potential problems in preparation for Phase 2 field evaluations. Smalltest colony units will be used to evaluate optimal formulations of potential peptides thatmaximizes a) bait acceptability; b) bait uptake; and c) distribution to workers, queen, sexuals,and immatures. This includes optimization of AI concentration and nano particle carriers.Knowing the AI distribution in the colony will help determine the optimal formulation.Objective 5. Peptides combined with surfactants in the development of a suitable formulation.Experimental Design: Optimal formulation will be developed by weighing the AI (1%-0.000001% wt/v), dissolving in a suitable phagostimulant (sucrose or oils or etc.,) and therequired tween emulsifier will be added, to achieve the desired AI concentration. Higher AIsolubility will help us determine more precisely the amount of AI needed for field colony control(up to 200,000 workers/colony, monogyne). Delayed action after initial feeding is critical for thisobjective to be successful. Initial experiments will be monitored to see how long the ants need toconsume and distribute the AI. Once consumed, the health of the ants will be monitored on aregular basis. The success of this objective will be expanded into Objectives 1 and 3.