Progress 09/01/17 to 08/31/19
Outputs
Target Audience:The target audience of users is the residential owner or small-scale gardener seeking an organic pesticide in areas afflicted by red imported fire ants. The means of reaching these consumers is through the actual target audience of large scall manufacturers of household and garden insecticides. The path to market will be to license the intellectual property created by this research to one or more of such companies for creation of a commercially available bait station to control the spread of red imported fire ants. Therefore the commercial outreach conducted during this project has been to established, large-scale companies active in or interested in providing an environmentally neutral pesticide that could be certified as organic once passing through OMRI and EPA review. Outreach to such companies has shown definite interest, pending demonstration of the effectiveness and cost-competitiveness of our technology. Changes/Problems:As indicated in our previous Progress Report, we examined the potential impact of the bait formulation at different levels of concentration on honeybees in laboratory tests and found significant mortality risk. As a result, we needed to pay extra attention to bait station design in our field tests to isolate the bait from honeybees. Also as described in the Accomplishments section of this report, we found in our field testing that the RIFA had a "taste" aversion to the AI in our bait formulation. After experimenting with various types of formulation of the bait to overcome this aversion, we discovered that using a masking agent used in human and animal medications showed promise in overcoming the aversion. Our intention is to continue to experiment in the laboratory to include the appropriate masking agent and then to move into another round of field trials with the revised bait. What opportunities for training and professional development has the project provided?During the course of the project, Dr. Chinta has pursued additional professional development through paticipating in significant and relevant professional meetings, including: Entomological Society of America annual meeting in Denver, November 2017 Entomological Society of America Southeast Branch conference in Orlando, March 2018 Entomological Society of America annual meeting in Vancouver, Canada, November 2019 International Society of Chemical Ecology annual meeting, Atlanta, June 2019 How have the results been disseminated to communities of interest?Given our commercialization goals and the necessity of protecting our intellectual propoperty, there has been no publication of the results of this research. There have been discussions with companies who might be commercialization partners with exchange of general, non-proprietary information. We anticipate beginning more detailed exchange of research information with some of these companies in the near future under non-disclosure agreements. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The initial research during SBIR Phase II grant period focused on formulation of a new bait material active ingredient (AI) implementing the discovery in the SBIR Phase I grant that tyramine fed to RIFA workers causes worker mortality. This discovery has been submitted for patent protection as Serial No. 62/465,503, PCT/US 18/20396, Vander Meer and Chinta, "Biologically-Based Methods for Insect Pests." The objective of SBIR Phase II first year activities was to continue to evaluate the effects of tyramine, as well as tyramine derivatives on RIFA life forms and to develop the first bait station prototypes for field applications. Bait Formulation Treatment of laboratory-reared queen right colonies with 1% N-Me tyramine in 10% sucrose showed steep worker mortality and queen death of 90% within 10 to 40 days, depending on the size of the colonies. After completing this phase of the work, we began field tests that simulated homeowner backyard conditions. For field evaluation we selected a small grassy area of less than 1 acre in Gainesville, FL that contained more than 35 monogyne fire ant colonies. The total number of mounds surveyed in total were 61. Mounds were randomly sampled for Monogyne/Polygyne social form using a molecular marker. Only monogyne colonies were selected for the experiment. Bait Station Design The water-soluble nature of the active ingredients dictates the use of a bait station method of presenting the material to the target ants. The bait station was constructed of PVC pipes to prevent rodent and other animal access to the bait formulation. Foraging ants could readily enter the bait station. The bait station design is such that bees are unable to access the AI. Field experiment protocol Three sets of paired colonies were selected (3 replicates). For each pair the control and treatment colonies were randomly selected (Control 10% sucrose and treatment 1% N-Me tyramine in 10% sucrose). Three bait stations were placed 3 feet from the center of the colony at 120-degree intervals\ with 18mL per tube, 5 tubes per station. Every week (for 4 weeks) colonies were measured for height, width, length, and PI. Bait stations were checked weekly for ant presence, amount of bait taken, and possible bait replacement (AI and control). Results of Field Trials: Observations from field experiments showed that the fire ant workers in the wild did not feed on the treatment containing 1% N-methyl tyramine, but they fed on the control bait (10% sucrose). Our conclusion was that the wild ants are sensitive to the smell or taste of the active ingredient, N-methyl tyramine. Previous experiments with lab reared fire ant colonies (collected from the field and maintained in the lab) did not show a feeding problem, indicating that lab reared colonies are less sensitive compared to field colonies, which rejected the AI. The field experiment was terminated, because of zero feeding on the treatment. Next Steps: We then went back to the laboratory to see if we could discover a method or bait ingredient that would overcome the resistance to feeding on the 1% N-Me tyramine in 10% sucrose bait. Various laboratory experiments were conducted to confirm the field results and to identify a path to overcome the resistance to feeding on the bait. Choice test bioassay: A. Laboratory Colonies. Fire ant workers from laboratory reared colonies were not sensitive to the AI and actively fed on several concentrations of AI (1%, 0.66%, 0.33%, 0.1%) in 10% sucrose. The results were not significantly different from the 10% sucrose control (p>0.05). B. Field Colonies. Fire ant workers from field collected colonies did not feed on the AI at 1%, 0.66%, or 0.33% in 10% sucrose; however, better acceptability was shown for the 0.1% bait formulation. C. Increased Sucrose Concentration. Choice test bioassays with 1% AI dissolved in increasing sucrose concentrations (10%, 20%, 30%, 40%) did not show improved feeding from freshly caught field ants (p<0.0001) compared to control (10% sucrose). D. Aqueous based phagostimulants. Choice tests were conducted with freshly caught monogyne field ants and 1% AI dissolved in blue agave nectar or almond milk, as novel phagostimulants. Neither phagostimulant overcame the distasteful effects of the AI. E. Vegetable oil phagostimulants. Laboratory reared monogyne fire ant workers were challenged with the AI (1%) dissolved in the following vegetable oil phagostimulants: 1. extra virgin olive oil, 2. corn oil, 3. olive oil, and 4. coconut oil. There was insignificant feeding observed on the vegetable oil phagostimulants when compared to AI(1%) in 10% sucrose after one week. Success with a taste masking agent. Experiments were performed with a taste masking agent previously used with human and animal medications. The fire ant bait AI was formulated with the masking agent in 1% sucrose and fed to field collected fire ants. The experiment was first performed as probing experiment with 20 worker ants and then after apparent success with 500 worker ants monitoring worker mortality every 24 hours over 15 days. All concentrations of masking agent utilized in the bait formulation showed significantly higher feeding than the control. Summary We have discovered that a bait formulation of N-Me-tyramine in 10% sucrose solution is highly effective in causing the mortality of RIFA workers and queens under laboratory conditions. This indicated potential for an effective commercial bait formulation to control RIFA in residential and small garden applications. However, field ants would not consume the bait due to negative taste or smell. Commercialization of our novel AI technology will depend upon being able to overcome the negative "taste" of the AI to RIFA in the field. As noted above, a taste masking agent used in human and animal medications showed promise in masking the negative effects of our AI. There are several classes of taste masking agents currently being used in the pharmaceutical industry which to our knowledge have never been implemented against insects and success in masking the new RIFA AI bait formulations will most likely generate new, patentable IP, as well as making our current IP attractive for licensing as a new, environmentally neutral (possibly certified organic) means of controlling the spread of RIFA. We are continuing to evaluate the use of taste masking agents through an extension of our CRADA with the USDA and are looking for additional funding sources for laboratory work to select the optimally effective taste masking agent and then to conduct a new round of field tests. In the meantime, we continue to maintain communications with companies that have expressed interest in our project, including Bayer, SC Johnson, MGK, Corteva and BASF.
Publications
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Progress 09/01/17 to 08/31/18
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 organic pesticides. Changes/Problems:Based on the research conducted under Phase I and Phase II (year one) of this project, the focus of the bait formulation will be on the use of a tyramine derivative. Further, the research has examined the potential impact of the bait formulation at different levels of concentration on honeybees. The percent mortality results from allowing honeybees to feed on 1% alternate formulations 1 or 2 showed significant mortality compared to the 10% sugar water control (4 replicates). The two bait formulations were not significantly different from each other. We also compared concentrations of active material of 1% versus 0.1%. Higher concentrations (1%) of either bait formulation had a stronger negative effect on honeybees, killing them 10 days earlier than the treatments with lower concentrations (0.1%). Honeybee mortality for the 0.1% formulations (10% sucrose) were not significantly different from the 10% sucrose controls. Since we expect that a 1% concentration of the tyramine active material will be required in the field to control larger colonies, we will need to pay extra attention to bait station design to isolate the bait from honeybees. There are bee-proof bait stations available commercially that we will evaluate. These may require some modification or even redesign. This is a task that we expect to address jointly with a commercialization partner later in the second year of the Phase II research. What opportunities for training and professional development has the project provided?During the course of the first year, Dr. Chinta participated in the Entomological Society of America annual meeting in November 2017 and in the Entomological Society of America Southeast Branch conference in March 2018. How have the results been disseminated to communities of interest?Given our commercialization goals and the necessity of protecting intellectual property, there has been no publication of the results of the research. There have been discussions with companies who might be commercialization partners with exchange of general, non-proprietary information. These contacts have been made primarily at Entomological Society of America meetings with preliminary follow up discussions. What do you plan to do during the next reporting period to accomplish the goals?Early in the next reporting period, we will begin field tests to simulate homeowner backyard conditions. A treatment site that the USDA has used previously had been identified and is being prepared for the field tests. Initial small scale field tests (< 1 acre) will be carried out on USDA-ARS property. This small area does not need an EUP (Experimental Use Permit) or an EUP exemption. Expansion of the field tests will still be on a small scale (<10 acres) for which we will request a State of Florida, EUP exemption. The field site will be located with moderate to low fire ant population levels. All colonies at a treatments site will be located by GPS, creating an accurate mound map of the site. The volume of each colony will be determined (LxWxD), as well as their Population Index (PI). Mounds will be paired as well as possible for mound volume and PI. At least 3 paired replicates will identified. Comparing treatments requires careful attention to the time of application, since foraging activity changes as the daytime temperature changes. The number and placement of bait stations will be determined after the mound volume and PI data are analyzed. The bait station design is currently under investigation. The treatments and controls will be monitored for changes in PI and mound volumes at least once a week. Bait station reservoirs will be replenished as needed until 3 replicates of a treatment results in control. Our principal objective for the initial field tests and others that will follow with refined bait formulations is to determine the effectiveness of treatments formulated in bait stations compared with controls, under field conditions. Special attention will be paid in bait station design to prevent access to bees. Our strategy is to license the technology to one or more established companies active in providing household and landscape pesticides and agricultural pesticides or organic farm supplies. We have learned from our preliminary discussions with such companies that successful field test data is the prerequisite for discussions about becoming a commercial development partner and potential licensing. We plan to broaden our outreach during Phase II through participating in professional meetings and through direct outreach to potential partners. Our implementation goals include beginning first round field tests by the end of September. We then expect to have sufficient preliminary data to take next steps with commercial outreach in conjunction with the Entomological Society of America annual meeting in Vancouver, November 11-14, 2018. Next steps for further research will depend upon the outcome of the first round of field tests. The pace and timing of commercialization discussions will similarly depend upon the results of field tests and related company response.
Impacts
What was accomplished under these goals?
The research carried out in SBIR Phase I resulted in a provisional patent application, Serial No. 62/465,503, PCT/US 18/20396, Vander Meer and Chinta, entitled: Biologically-Based Control Methods For Insect Pests, that relates the discovery that tyramine fed to RIFA workers causes worker mortality. The objective of SBIR Phase II first year activities was to continued to evaluate the effects of tyramine, as well as tyramine derivatives on RIFA life forms, and develop the first bait station prototypes for field applications. These compounds are water soluble and are typically presented to the target RIFA in aqueous solution with 10% sucrose (phagostimulant) in a test tube closed with an absorbent cotton ball. The worker ants are induced by the sucrose to suck the solution from the cotton ball. This works well for laboratory studies, but for field studies we will develop a bait station that will protect the liquid formulation from environmental conditions, but provide access to fire ant workers. We expect several iterations of bait stations for field applications. Laboratory experiments evaluated derivatives of tyramine for their effects on RIFA mortality. Tyramine and derivatives that showed worker mortality were further evaluated at several concentrations against colonies of various sizes. Because these experiments lasted several weeks a natural food preservative was added to the bait formulation. Details of the experiments are shown below. In addition, two concentrations of active ingredients were evaluated for their potential impact on honey bees. This topic will be covered under the section of this report on Major Problems. Bait Formulation - Mortality from Alternative Materials and Concentrations Tests were conducted on fire ant worker mortality with 1.0, 0.1, and 0.01% concentrations of the active ingredient in 10% sucrose solution. The fire ant test units were composed of monogyne colonies collected from SW 75th Road, Gainesville, FL. The experiment was carried out in enamel trays (28/15/15cm) sprayed with fluon (ICI, Wilmington, DE) on the sides. Worker ants (N=300) and female alates (N=5) were placed in the tray. Treatments were placed in 20ml glass tubes closed with cotton and experiment was carried out for 14 days counting the mortality every 24hrs with a continuous supply of material. All active ingredient concentrations were significantly different from the 10% sucrose control. Significant fire ant worker mortality over a wide range of concentrations (100-fold dilution) is a key characteristic of an effective fire ant bait insecticide. Interestingly, the two lower concentrations (0.1 and 0.01%) showed significantly higher mortality than the 1.0% concentration. One possible explanation for these results is that the 1.0% active ingredient is not as palatable as the lower concentrations. The results for 0.1 and 0.01 concentrations are not significantly different (p=0.11) from each other. The tests also examined a comparison of the effects of 1.0% concentrations of two different analogous forms of tyramine versus the 10% sucrose control. Mortality results for both treatments are significantly different from the sucrose control. These tests showed clear differences between the two alternative forms of tyramine. Monogyne queen right colonies were treated with 1%, 2%, 3%, and 4% of alternative form 1 and alternative form 2 (both contain 0.1% sodium propionate preservative to prevent fungal growth) respectively with a larger colony size than previously tested ranging from 10,000 -68,000 workers and brood. Results indicate that worker mortality in colonies treated with 1% alternative form 1 was consistently higher compared to 1% alternative form 2. In contrast colonies treated with 1% alternative form 2 resulted in 100% queen death compared to 80% queen death in colonies treated with 1% alternative form 1. All the queens in control groups survived and no death was observed. Bait Formulation for Larger Colony Size - Mortality from Alternative Materials and Concentrations Queen right colonies were treated with 1%, 2%, 3%, and 4% of tyramine alternative form 1 and alternative form 2 (both contain 0.1% sodium propionate preservative) respectively with a larger colony size than previously tested, ranging from 10,000 -68,000 workers and brood. The rate of death in treatments was a steep curve compared to controls (10% sugar water and 0.1% sodium propionate) in colony sizes varying from small (<35,000), medium (35,000-45,000) and large (>45,000). Timing of queen death varied from day 20 -day 37. Worker mortality in treatments that include 1%-4% alternative form 2 showed a steep curve compared to controls (10% sugar water and 0.1% sodium propionate) in colony size varying from small (<35,000), medium (35,000-45,000) and large (>45,000). Treatments with alternative form 2 showed worker mortality double that of the controls and higher than treatments involving alternative form 2. Treatment with 1% alternative form 2 showed steep worker mortality and queen death of 90% within 20 days for all replicates. Treatment with lesser concentrations of alternative form 2 showed important levels of effectiveness, but not equal to the 1% concentration. Alternative form 2 treatments with 0.67%, 0.33% and 0.1% respectively show similar worker mortality curves with most of the replicates reaching close to 75% mortality or in some cases higher. Concentrations involving 0.67% and 0.33% of alternative form 2 did not show 100% queen death, although the timing for all different concentrations remained close to 20 days. In contrast the 1% concentration of alternative form 2 did result in 100% queen death in all replicates. There were also tests of a combination of 1% alternative form 1 and 1% alternative form 2 to rule out possibility of synergistic effects, but this also failed to bring 100% queen deaths although all the replicates achieved close to 80% or higher worker mortality. Bait Formulation - Preservatives. As the queen death with treatments can take up to 4 weeks, the material provided in glass tubes needed to be preserved from the growth of fungi/mold. Common food preservatives were identified and sodium propionate (Na(C2H5COO), a natural food preservative used in many human food products was selected. For our purposes here, the sodium propionate was formulated at 0.1% concentration with 10% sugar water and the treatments as appropriate. The preservative successfully prevented fungal/mold growth for the duration of our laboratory feeding experiments. Impact on Honey Bees As will be discussed in section 5, the bait materials had a negative effect on honey bees in laboratory tests that allowed the bees to feed on the sucrose/treatment formulations, indicating the necessity of using bait stations that are accessible to the fire ants, but not to bees. Summary In summary, experiments conducted in year one of Phase 2 showed that a 1% concentration of alternative form 2 had the best results (with sodium propionate as a preservative). Experiments with honeybees highlighted the importance of bait station design for field tests.
Publications
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