NOVEL PEPTIDES FOR CONTROLLING INVASIVE PEST ANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1022885
• Grant No.: 2020-33610-31986
• Cumulative Award Amt.: $101,321.00
• Proposal No.: 2020-00586
• Project Start Date: Sep 1, 2020
• Project End Date: Mar 31, 2023
• Grant Year: 2020
• Program Code: [8.2]- Plant Production and Protection-Biology

Recipient Organization
FORESIGHT SCIENCE & TECHNOLOGY, INC.
11750 DOCKER HILL RD
COMPTCHE,CA 95427

Performing Department
(N/A)

Non Technical Summary
This Project addresses National Institute of Food and Agriculture, United States Department of Agriculture (USDA) SBIR Topic Area 8.2, Plant Production and Protection (Biology). Imported fire ants cause expenditures of over $6 billion annually in the United States. The Project will raise the technology readiness level and demonstrate commercial potential for a control method for imported fire ants using novel bioactive peptides that have shown promise in preliminary studies. These peptides act as antagonists to a fire ant receptor that is found in all fire ant life stages. The peptides are covered in (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 c) PCT WO 2016/0262388 A1 and WO 2018/0086790 A1. The research will be performed under a CRADA with the USDA, Agricultural Research Service.The Project will build on previous basic and applied research to further evaluate the potential of these peptides to control fire ants under laboratory conditions and to show proof of concept. If these evaluations are successful, the results will be used to support an SBIR Phase II grant proposal for field trials.The objective of the Project is to develop an effective, environmentally neutral bait formulation for bait stations, targeted primarily for household and small garden applications, to control invasive destructive ant infestation. Commercialization will be through licensing of the intellectual property and know-how to a private commercial entity for product development and for introduction into mass market distribution.

Animal Health Component

65%

Research Effort Categories

Basic

15%

Applied

65%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
721	3110	1060	100%

Knowledge Area
721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1060 - Biology (whole systems);

Keywords

red invasive fire ants

synthetic peptides

rifa

bait technology

pest ants

Goals / Objectives
The goal of the project is to develop an effective, environmentally neutral bait formulation for control of fire ants, targeting household and small garden applications.The project will build on previous basic and applied research that has revealed that peptides act as antagonists to a fire ant receptor found in all fire ant life stages. The discoveries are covered in US Patent 9,771,393 B2 and US Patent 10,017,538, both - Bioactive Peptides Having Insecticide Activity, Choi and Vander Meer, issued in 2017 and 2018, respectively. Foreign patent protection is partially covered in PCTWO 2016/0262388 A1 and WO 2018/0086790 A1. The research proposed here will be carried out through a CRADA with the USDA Agricultural Research Service to demonstrate proof of concept for use of novel bioactivepeptides to control fire ants under laboratory conditions.The specific goals and objectives are as follows:1. Evaluate the role of antagonist peptides:a) Determine mortality effects of selected peptides on brood, workers, and queen. Is there stage/caste specificity?b) Determine the effects of sucrose solutions of peptide mixtures on small "queenright" (queen, workers, brood, and female sexuals). Question: Will the effects of the mixture be additive or synergistic? This objective will provide decision making data on whether or not there are advantages to continued work with mixtures.c) Field Simulation.The ultimate objective in Phase 1 is to mimic a fully functional field colony under controlled laboratory conditions.2. Determine optimal formulations of potential peptides that maximizes:a) bait acceptability;b) bait uptake; andc) distribution to workers, queen, sexuals, and immatures.3. Expansion of the embodiment of active compound:EvaluateS. invictaderived peptide bait formulations for use against otherSolenopsisspecies, e.g.,S. geminataandS. richteri. The same experimental procedures will be used with the target ant species changed to determine where the peptides are effective only against S. invicta or whether they have broader application.Evaluate S. invicta derived peptide bait formulations against other pest ant species, e.g., the little fire ant, and the tawny crazy ants, under laboratory conditions. The same experimental procedures will be used with the target ant species changed to determine where the peptides are effective only against S. invicta or whether they have broader application.Any positive results against other species will broaden the scope of the intellectual property and increase commercial interest.If these evaluations are successful, the results will be used to support an SBIR Phase II grant proposal for working prototype bait formulations through field trials.

Project Methods
Objective 1. Evaluate the role of antagonist peptides:1a. Determine mortality effects of selected peptides on brood, workers, and queen. Is there stage/caste specificity?Experimental Design: Mortality bioassays are well developed for fire ants. We will scale current bioassays, so as to get more information from the purchased peptides, as exemplified below: Synthetic peptide (1% in 10% sucrose solution) treatments were provided to laboratory maintained ant colonies (collected from Gainesville, FL). Ants were starved overnight, then weighed to give about 500 workers) and placed in a fluon coated container (Versatainer brand restaurant, 160mmx40mmx100mm) for 1hr before the start of the experiment. The treatment was provided in test tubes (10ml) that allowed the ants to feed on the treatment. Worker ant mortality was counted every 24hrs. Control set-ups consist of a similar group of ants in a container and provided with a water tube (20ml test tube with water (10ml), plugged with a cotton ball). This general set-up can be expanded or downsized to accommodate specific experiments. For example, the ultimate objective in Phase 1 is to mimic a fully functional field colony under controlled laboratory conditions. This is necessary in order to better identify potential problems in preparation for Phase 2 field evaluations.1b. Determine the effects of sucrose solutions of peptide mixtures on small "queenright" (queen, workers, brood, and female sexuals). Will the effects of the mixture be additive or synergistic?Experimental Design: Experimental laboratory fire ant colony units will be set up with known amounts of workers, brood, low weight female sexuals and a polygyne queen. The concentrations of compounds tested will be 0.5% in 10% sucrose solutions in two peptides in combinations. Controls will be the single compounds at 1.0% (10ug/uL) and at 0.5% concentration. This will allow us to determine if the results are additive suppressed, or synergistic. A three-component mixture will also be formulated at 0.33% of each and the controls will be each component at 0.33% and at 1.0%. All treatments and controls will be replicated at least three times. Experimental units will be monitored for worker mortality, female sexual behavior, brood mortality, and queen mortality. Results will be analyzed by methods described in the statistics section. At the time of the experiment we will determine if it would be advantageous to periodically sample brood, workers, and female sexuals for future analysis. We will monitor with more precision the phenotypic effects of the peptides on the queen, e.g., queen weight, mobility, queen worker attraction pheromone.Objective 2: Field Simulation:2a. The ultimate objective in Phase 1 is to mimic a fully functional field colony under controlled laboratory conditions. This is necessary in order to better identify potential problems in preparation for Phase 2 field evaluations.Experimental Design: Queenright (with queen) monogyne and polygyne colonies are readily collected in the Gainesville, FL area and standard procedures are available for isolating them from soil and maintaining them in the laboratory (Banks et al. 1981). The objective here is to get the colonies into the treatment protocol as soon after collection as possible, because after being in the laboratory for 2-3 months worker behavior becomes less robust (Obin and Vander Meer 1988). After separation from the soil colony size will be estimated by dividing total weight of isolated workers and brood by 0.8 mg (average worker weight). A treatment and control feeding regime will start at 1% concentrations of the treatments in 10% sucrose solution presented in Eppendorf tubes topped with a cotton plug. Several of these treatment and control reservoirs will be placed in their respective colonies. Colonies will also have crickets and water tubes for food and moisture. The colonies will be fed ad libitum until they stop producing worker brood, the queen(s) dies, or worker numbers reach < 95% of the original worker estimate. Because colonies from the field represent a variety of colony sizes, we will be able to correlate colony size with the amount of AI need for control.2b. Determine optimal formulations of potential peptides that maximizes: 1) bait acceptability; 2) bait uptake; and 3) distribution to workers, queen, sexuals, and immatures. This includes optimal AI concentration, AI solvents (water/oils), phagostimulants (currently sucrose), and the design of bait stations targeting above and/or below ground fire ant foraging activity. Knowing the flow of AI in the colony context will guide the search for the optimal formulation.Experimental Design: We have experience in evaluation of potential fire ant phagostimulants that are soluble in water (Vander Meer et al. 1995). We will first evaluate the distribution of AI formulated in aqueous sucrose solutions to determine the effect of sucrose concentration on phagostimulation using the method of Vander Meer et al, (1995). High concentrations of sucrose can be advantageous, since high osmotic pressure deters the growth of microorganisms; therefore, acting as a preservative and phagostimulant. However, we do not know the effects of high sucrose concentration and fire ant phagostimulation. In another series of experiments, we will use a probe style sonicator to form aqueous emulsions of sucrose solutions with soybean oil and/or proteins. The phagostimulation bioassay will be used to evaluate the ants feeding response to the emulsions. Formulations with the best phagostimulation results will also be subject to periodic sampling for peptide analyses to monitor distribution of the AI within the test colony sub-units. These experiments will be conducted concurrently with experiments associated with other Objectives.Objective 3. Expansion and Embodiment of active compound:3a. Evaluate S. invicta derived peptides against other Solenopsis species, e.g., S. geminata and S. richteri. Are the peptides specific to S. invicta?Experimental Design: We have a number of pest ant species from Formicinae sub-family available locally by collection as needed, as well as a few already maintained in laboratory colonies. The initial experiments will be similar to the S. invicta worker feeding experiments, where peptides are dissolved in 10% sucrose solution. The treatments and controls are presented to groups of test ants as droplets on silicon treated filter paper. This allows observation of the ants' feeding activity. Worker mortality/behavior will be monitored for up to one week. This procedure is not cast in stone as observations during the experiments may generate a need for modifications. These experiments can be conducted simultaneously with fire ant related experiments, as time and resources permits. Any positive results have the potential to broaden the scope of the intellectual property.3b. Evaluate S. invicta derived peptides against other pest ant species, e.g., the little fire ant, and the tawny crazy ants under laboratory conditions. How broad or specific are the peptides? Any positive results will broaden the scope of the intellectual property and increase commercial interest.Experimental Design: We have a number of pest ant species in both ant sub-families available locally by collection as needed, as well as a few already maintained in laboratory colonies. The treatments and controls are presented to groups of test ants as droplets on silicon treated filter paper. This allows observation of the ants' feeding activity. Worker mortality/behavior will be monitored for up to one week. This procedure is not cast in stone as observations during the experiments may generate a need for modifications. These experiments can be conducted simultaneously with fire ant related experiments, as time and resources permits. Any positive results have the potential to broaden the scope of the intellectual property.

Progress 09/01/20 to 03/31/23

Outputs
Target Audience:The target market for our initial stage bait station product is homeowners and small-scale organic gardeners/growers in the 14 quarantined states that currently use chemical pesticides to control RIFA, many of whom would prefer an organic or environmentally neutral alternative.The specific market demand niche that our technology will address is for an environmentally neutral bait method of fire ant control primarily applicable for home gardens, home lawns, pick-your-own farms, green houses, andchicken raising facilities. This does not include the commercial organic farming market. It is just home and garden uses. The study also notes that the expansion of the market is being driven by consumers' interest in environmentally safe alternatives to traditional chemical pesticides. This technology will evolve into an effective organically neutral alternative for invasive fire ant control primarily for an increasingly organic-oriented consumer base and for appropriate sectors of commercial organic farming and animal production. Our customers will be those companies who supply pesticides and home garden supplies to both the individual consumer market and to the commercial organic farming market. In preparing our commercialization plan, we differentiate between end users and our customer. Foresight plans to license the technology to one or more major manufacturers who will be our customer. The most prominent companies operating in the global ant control market are Syngenta AG, BASF SE, Sumitomo Chemical, Bayer CropScience, Ensystex, United Phosphorus Limited, FMC Corporation, Nufarm Limited, Rentokil Initial plc. Control solution, ADAMA Agricultural Solutions, and others. These same companies are potential licensees of our technology as an environmentally neutral addition to their product line. Our business model is to license the technology to a company or companies who want to provide a new organic biological treatment to prevent the spread of fire ants to their existing product line. We will be looking for a company or companies that markets to environmentally conscious individual consumers and to organic commercial farmers or greenhouses that currently have few options to control fire ants. Bothhome and commercial markets for organic products are rapidly growing. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Choice Test Bioassay: Two bioassays were developed with all the seven-cysteine flanked peptidesto get a clear idea and better understanding of the significance of peptide purity on ant mortality and ant acceptance. A choice-test bioassay was used to determine the ants' preference (feeding on) or repellency towards the test peptides.The test materials were compared against a positive control (10% sucrose) and a negative control (water) to then calculate a corresponding ranking. Scores equivalent to the ranking of sucrose were classified as not repellent or preferred if the ranking is higher than the sucrose control. The basic methodology was as follows. An ant cell (petri dish) housing about 1,000 ants is taken from their rearing colony and placed in an arena with ten, equidistant marked locations. Each selected peptide, e.g., CMARYMSAC was available at three levels of purity (88, 92 and 95%), 100μl of treatments (4ug/ul) and controls (water and 10% sucrose) were randomly assigned to locations around the ant cell. The ant response was quantified (number of workers feeding on treatments and controls at a series of time periods). Among the tested peptides 'CMARYMSAC-95% purity' showed the most promising results supporting previous positive tests. The CMARYMSAC peptide at lower purities were less effective in the bioassays. Mortality Study: In this bioassay, ants could continuously feed on the test peptide (e.g., the MARYMSAC peptide with a purity of 88, 92 and 95% and at a concentration of 4ug/ul). The bioassay systems were monitored for mortality every day. Three sets of twenty worker ants are taken from three separate monogyne colonies and are placed in cups with access to moist cotton (for water and humidity) and the test or control material (10% sucrose) provided in 1ml centrifuge tubes with cotton caps; test/control material is refilled as needed over the time of the experiment. Worker mortality was recorded daily. At the end of three weeks, the cumulative mortality for test materials and controls were compared and analyzed for differences. The bioassay with the CMARYMSAC showed 27% of mortality with the 95% purity peptide compared to 13% mortality in the control. The other CMARYMSAC purified forms showed 18% (88% pure material) and 11% mortality (92% pure material). The mortality experiment conducted with the 'CQKIGSHFC' peptide showed 35% mortality when ants were provided with 95% pure material compared to 13% mortality with control. The 92% purity peptide gave 22% mortality, and the 88% pure peptide gave 10% worker mortality. Mortality experiment by feeding conducted with the 'CRGATPMSC' peptide showed a 25% mortality when ants were provided with 95% pure material compared to 15% mortality with control. A mortality of 23% was observed when ants were fed with 92% pure material and 15% mortality when provided with 88% of pure material. Feeding Bioassay The five peptides CQKIGSHFC, CRGATPMSC, CERVGSHFC, CVKLGSHFC, and CMARYMSAC were used at a purity of 95% and concertation of 1%, dissolved in a 10% sucrose solution. With the five different treatment peptides and 10% sucrose control, three different colonies were used and allocated with 100 ants from each colony in separate trays. For the first ten days of the experiment, ants were fed 0.1ml of treatment in microcentrifuge tubes (4 tubes per tray) and water provided via two fully soaked cotton wicks, refilled daily. From days 11-21, the feeding method was changed to ensure full availability of treatment for the ants. The new method consisted of starving ants for 48 hours between application of 30μl of treatment or control liquid on phase separation paper. Water was provided via cotton wicks one hour after treatment/control were administered. After ten days, percent mortality only reached above 5% for one treatment (VKL, 7%). However, by the end of the experiment, (28 days post initial treatment) total mortality was averaging above 20% for all treatments with the peptides VKL, RGA, and ERV averaging 40%, 35% and 32%, respectively and the control remaining under 10% mortality. A second experiment was carried out utilizing the second feeding method over 28 days using peptides MARY, VKL, RGA, and ERV which resulted in mortality rate not significantly higher than control. High variability across all treatments made statistically analysis impossible. Mortality Bioassay-Field Ants: Under Controlled Laboratory Conditions In a different set of experiments field caught ants (Newberry Road, Gainesville, FL) were used and fed 1% concentrations of peptides in 10% sucrose solution (treatment) after overnight starving (replicates =9, n=20 ants per replicate) via a droplet initially (25µl) on a phase separation filter paper (2x2cm) over 4hrs followed by providing the material in 1ml centrifuge tubes overnight and monitoring mortality every 24hrs showed 73% of mortality with 'CLKIGHSFC' (88% pure) compared to 23% mortality in control. Same experiment with 'CERVGHSFC' (88% pure) showed 73% of mortality with treatment and 45% mortality with controls. Another experiment where 0.1% concentration of peptides (in 10% sucrose solution) CERVGSHFC, CVKLGSHFC, CIQQGHSFCand CMARYMSAC (Biomatik >95% purity)along with 10% sucrose solution (control) was provided to field ants collected from the Gainesville, FL area. Test ants were starved overnight (n~200) and placed 1hr before the start of the experiment in Fluon coated containers(VERSAtainer brand restaurant, 160mmx40mmx100mm). The treatment and control diets were continuously provided via test tubes (10ml)that allowed access to the ants as needed. Dead ants were counted daily (a moist cell was provided with 20ml test tubefilled with water (10ml) and cotton ball at the top of the water). Theresults from the above described experimentclearly showed that CMARYMSAC and CIQQGHSFC had significantly higher negative impact (mortality) than controls and at lower peptide concentrations (0.1%, >95% purity) after 2 weeks of continuous feeding. These results prompted us to execute an experiment with field collected Queen Right (QR) RIFA colonies.Results with CMARYMSAC and CIQQGHSFC at 0.1% showed significant mortality compared to the controls and other peptides evaluated after two weeks. Feeding experiments with QR RIFA field caught colonies. Queen right colonies (QR, n=4 treatment, n=3 control) with approximately 2000-8000 worker ants and brood were treated with 0.1% CMARYMSAC (n=4) and 0.1% CIQQGHSFC (n=4) in 10% sucrose, along with controls (n=3,10% sucrose)over a period of 15 weeks with continuous supply of peptides. Mortality was recorded every other day. Resultsshowed that formulations resulted ~ 80% mortality (in MARY, P<0.0001)and above 60% mortality (in CIQQ) in workers and the death of 5 colony queens in all (3 from CIQQ and 2 from MARY each treatment). Worker mortality remained close to 15% in control. Queen death occurred during the 7th week of feeding. This suggests that peptide molecules have the potential to kill the colony queen, which would be the demise of the colony. The control colonies remained healthy, and the queens were unaffected throughout the experiment. Reduction of queen weight with peptide feeding: Queen weights were noted periodically during the entire feeding experiment. After the experiment entered 5thweek, queens significantly started losing weight (~35% weight loss)before they were found dead, which clearly showed a negative impact from the peptide feeding. Weights were measured on day 33 (and day 73 for CIQQ) after which the four queens were found dead before the next scheduled reading. On the other hand, the control queens remained healthy throughout the experiment.

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The specific market demand niche that our technology will address is for an organic bait method of fire ant control primarily applicable for home gardens, home lawns, pick-your-own farms, green houses, and chicken raising facilities.This does not include thecommercial organic farming market. It is just home and garden uses. The study also notes that theexpansion of the market is being driven by consumers' interest in environmentally safe alternatives to traditional chemical pesticides.This technology will evolve into an effective organically neutral alternative for invasive fire ant control primarily foran increasingly organic-oriented consumer base and for appropriate sectors of commercial organic farming and animal production.Our customers will be those companies who supply pesticides and home garden supplies to both the individual consumer market and to the commercial organic farming market. Our business model is tolicense the technology to a company or companies who want to provide a new organic biologicaltreatment to prevent the spread of fire ants to their existing product line. We will be looking for acompany or companies that markets to environmentally conscious individual consumers and to organiccommercial farmers or greenhouses that currently have few options to control fire ants. Both home andcommercial markets for organic products are rapidly growing. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Objectives 1a and 1b will be performed on a field caught queen right monogyne fire ant colonies with worker numbers ranging anywhere between 8000-45000 along with brood. This will start field simulation experiments that covers objective of simulating home owners backyard. In addition, we will investigate the species-specificity of the peptides by evaluating the effects of the peptides on another Solenopsis species, S. geminata and a non-Solenopsis, species, the little fire ant (but same ant sub- family), and a species from a different ant sub-family, the tawny crazy ant (objective 3)

Impacts
What was accomplished under these goals? ?Choice Test Bioassay: Experiments with seven different high purity form (>95%, Biomatik) cysteine flanked peptides with fresh wild caught fireant colonies (Gainesville, FL) evaluated under laboratory conditions showed promising results. Theprobing studies were aimed atant mortality and ant acceptance, via different bioassay methodologies. A choice-test bioassay was used to determine the fireants preference (feeding on) or repellency towards the test peptides. The test materials are compared against a positive control (10% sucrose) and a negative control (water) to then calculate a corresponding ranking. Anything scoring at the sucrose ranking is scored as not repellent or preferred if the ranking is higher than the sucrose control. Since ant colonies' preferences towards certain materials can be variable, replicate choice tests need to be done with the same test materials, but different colonies to secure a more realistic understanding of the activity the evaluated peptides. The basic methodology follows. An ant cell (petri dish) housing 2,000 ants is taken from their rearing colony and placed in an arena with nine, equidistant spots and 7 test materials consisting of a selected peptide e.g.,CQKIGSHFC,CLKIGSHFC,CIQQGSHFC,CERVGSHFC,CVKLGSHFC,CMARYMSAC,CRGATPMSCpeptide highest levelof purity (> 95%), 100μl of treatments (4ug/ul) and controls (water and 10% sucrose) were randomly assigned to 9 locations around the ant cell. The ant response was quantified (number of workers feeding on treatments and controls at a series of time periods). Among the tested peptides 'CIQQGSHFC,CERVGSHFC,CVKLGSHFC,CMARYMSAC-95% purity' showed the most promising results supporting previous tests as being consumed and pass successfully through the ant gut while remaining active. These 4peptide at higherpurities were further tested in the bioassays evaluating ant mortality at a minor scale. Ant Mortality Bioassay: Ant mortality bioassay was performed directly by feeding the peptidesCIQQGSHFC,CERVGSHFC,CVKLGSHFC,CMARYMSAC-95% purityand checking the mortality every 24hrs.1% concentration of peptides (biomatik >95% purity)in 10% sucrose solution (treatment) was provided to wild ants (Gainesville, FL). Ants starved overnight were weighed (~n=200 ) and placed 1hr before the start of the experiment in fluon coated containers(VERSAtainer brand restaurant, 160mmx40mmx100mm)(11/28/2022 9:30 am). The treatment was provided in test tubes (10ml)let the ants feed on it followed by counting the dead ants every 24hrs. (a moisture cell was provided with 20ml test tubefilled with water (10ml) and cotton ball)1% concentration of peptides (biomatik >95% purity)in 10% sucrose solution (treatment) was provided to wild ants. Perfroming the feeding experiments over 2weeks showed that peptide 'CMARYMSAC' showed close to 70% mortality whileCIQQGSHFC,CERVGSHFC,CVKLGSHFC showed 60%, 505 and 40% mortality respectively. The control experiment reported mortality close to 5% and it remained healthy.?

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience is ultimately the residential or small garden owning consumer seeking an organic pesticide in areas afflicted by red invasive fire ants. However, the means of reaching this audience will be through the commercial activities of one or more companies that will license the intellectual property created by this research to market a commercially available bait station for use in those states afflicted by the red invasive fire ant and possibly for application internationally. Therefore our immediate audience is established companies active in providing household and landscape pesticides. Changes/Problems:As a result of the COVID pandemic, access to the ARS lab in Gainesville was for a long period of time limited in allowing research and continues in this status at present. Our understanding is that there will be a re-evaluation of lab operating procedure on or before October 1. As a result of very limited access to the lab fro a limited amount of time, it has been impossible to complete the experiments planned under this grant as expected. The lab continues to have extremely limited access, allowing only very limited experiments to be conducted. The timeline for completing the experimental work is totally dependent upon the course of the pandemic and upon the USDA regulations for use of the ARS lab facilities. In addition, the pandemic prevented the use of any undergraduate student employees as originally proposed. The only alternative was to enlist lab technical employees to assist with the limited experiments. These employess were compensated at the same rate as included in the original proposal for students. We estimate that about 35% of the experimental work has been completed. This percentage was used in calculating the FTEs for this period. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?As of now USDA-ARS labs remain in maximum telework mode, with strictly limited lab access due to Covid-19 pandemic. Once the lab fully opens, objectives 1a and 1b will be performed on a field caught queen right monogyne fire ant colonies with worker numbers ranging anywhere between 8000-45000 along with brood. This will start field simulation experiments that covers objective 2. In addition, we will investigate the species-specificity of the peptides by evaluating the effects of the peptides on another Solenopsis species, S. geminata and a non-Solenopsis, species, the little fire ant (but same ant sub-family), and a species from a different ant sub-family, the tawny crazy ant.

Impacts
What was accomplished under these goals? Choice Test Bioassay: All the seven-cysteine flanked peptides that showed promising results in preliminary studies were obtained at three different purities from Biomatik to further test their efficacy as a possible active ingredient(s) for fire ant (Solenopsis invicta) control. In order to get a clear idea and better understanding of the significance of peptide purity (at various purities) in terms of ant mortality and ant acceptance, two bioassays were developed. A choice-test bioassay was used to determine the ants' preference (feeding on) or repellency towards the test peptides. The test materials are compared against a positive control (10% sucrose) and a negative control (water) to then calculate a corresponding ranking. Anything scoring at the sucrose ranking is scored as not repellent or preferred if the ranking is higher than the sucrose control. Since ant colonies' preferences towards certain materials can be variable, replicate choice tests need to be done with the same test materials, but different colonies to secure a more realistic understanding of the activity the evaluated peptides. The basic methodology follows. An ant cell (petri dish) housing 1,000 ants is taken from their rearing colony and placed in an arena with ten, equidistant spots and 3 test materials consisting of a selected peptide, e.g., CMARYMSAC peptide at three levels of purity (88, 92 and 95%), 100μl of treatments (4ug/ul) and controls (water and 10% sucrose) were randomly assigned to 10 locations around the ant cell. The ant response was quantified (number of workers feeding on treatments and controls at a series of time periods). Among the tested peptides 'CMARYMSAC-95% purity' showed the most promising results supporting previous tests as being consumed and pass successfully through the ant gut while remaining active. The CMARYMSAC peptide at lower purities were less effective in the bioassays. Mortality Study: In this bioassay, ants could continuously feed on test peptide (e.g., the MARYMSAC peptide with a purity of 88, 92 and 95% and at a concentration of 4ug/ul). The bioassay systems were monitored for mortality every day. Three sets of twenty worker ants are taken from three separate monogyne colonies and are placed in cups with access to moist cotton (for water and humidity) and the test or control material (10% sucrose) provided in 1ml centrifuge tubes with cotton caps; test/control material is refilled as needed over the time of the experiment. The experiments were evaluated daily. At the end of three weeks, the cumulative mortality for test materials and controls were compared and analyzed for differences. The bioassay with the CMARYMSAC showed 27% of mortality with the 95% purity peptide compared to 13% mortality in the control. The other CMARYMSAC purified forms showed 18% (88% pure material) and 11% mortality (92% pure material). The mortality experiment conducted with the 'CQKIGSHFC' peptide showed 35% mortality when ants were provided with 95% pure material compared to 13% mortality with control. The 92% purity peptide gave 22% mortality and the 88% pure peptide gave 10% worker mortality. Mortality experiment by feeding conducted with the 'CRGATPMSC' peptide showed a 25% mortality when ants were provided with 95% pure material compared to 15% mortality with control. A mortality of 23% was observed when ants were fed with 92% pure material and 15% mortality when provided with 88% of pure material. The probing experiments conducted using both the bioassays above suggested higher purity materials might have highest intake possibility by the ants and at the same time bring the maximum desired mortality. Peptides- CIQQGSHFC, CERVGSHFC, CVKLGSHFC, CMARYMSAC are currently being tested for acceptability and mortality by the ants. Mortality Bioassay-Field Ants: In a different set of experiments, field caught ants were used and fed 1% concentrations of peptides in 10% sucrose solution (treatment) after overnight starving (replicates =9, n=20 ants per replicate) via a droplet initially (25µl) on a phase separation filter paper (2x2cm) over 4hrs followed by providing the material in 1ml centrifuge tubes overnight and monitoring mortality every 24hrs showed 73% of mortality with 'CLKIGHSFC' (88% pure) compared to 23% mortality in control. Same experiment with 'CERVGHSFC' (88% pure) showed 73% of mortality with treatment and 45% mortality with controls.

Publications