LEVERAGING NEMATODE SIGNALS TO ENHANCE ENTOMOPATHOGENIC NEMATODE EFFICACY FOR PEST CONTROL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1012599
• Grant No.: 2017-33610-26808
• Cumulative Award Amt.: $100,000.00
• Proposal No.: 2017-00120
• Project Start Date: Jul 15, 2017
• Project End Date: Mar 14, 2018
• Grant Year: 2017
• Program Code: [8.2]- Plant Production and Protection-Biology

Recipient Organization
KAPLAN SCHILLER RESEARCH, LLC
747 SW 2ND AVE STE 347
GAINESVILLE,FL 32601

Performing Department
(N/A)

Non Technical Summary
The proposed project will test the feasibility of using nematode released factors to improve entomopathogenic nematodes' (EPNs) efficacy (ability to kill) in biological control. Broad-spectrum synthetic pesticides have adverse effects on the environment and human health. For example, approximately 26 million human poisonings and 220,000 deaths occur annually due to synthetic pesticides worldwide. We need environmentally friendly non-toxic agricultural pest control. One of the solutions is to use environmentally friendly biopesticides such as EPNs. EPNs, natural enemies of insects, are used as biological control agents for many economically important insect pests such as the black vine weevil, Japanese beetle, Diaprepes root weevil, and fungus gnats. Furthermore, they are so safe to the environment and humans that EPNs are not regulated as pesticides in the US. One of the major problems with EPNs is their variable efficacy in the field. This is a major hindrance to EPNs' wider adoption for insect pest control. One of the reasons for this variability is that EPNs clump together and do not disperse sufficiently in commercial aqueous applications. Recently, our group found that nematode released factors can disperse EPNs under laboratory conditions. The overall goal of this project is to demonstrate the feasibility of nematode factors to improve dispersal leading to the improved EPN efficacy. We will test the effects of adding nematode factors in laboratory and greenhouse assays with three different types of soil, and optimize exposure time to the nematode factors for consistent dispersal and infectivity. We will use two commercially important EPNs as our model organisms. First, exposure time to nematode factors will be optimized for consistent dispersal. Efficacy will be determined by measuring two behaviors; 1- EPN dispersal and 2- infectivity in the presence and absence of the nematode factors in laboratory and greenhouse assays. In the greenhouse experiment, commercially important insect pests will be tested. The laboratory and greenhouse experiments together will provide proof of concept that nematode factors improve EPNs' efficacy to suppress insect pests. These experiments will lay the groundwork for optimizing formulation and application parameters, expanded field testing, and scaling-up production of nematode factors for Phase II and III.

Animal Health Component

100%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
215	3130	1120	100%

Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
3130 - Nematodes;

Field Of Science
1120 - Nematology;

Keywords

epn

biopesticide

entomopathagenic

extract

insect

natural product

nematode

soil

Goals / Objectives
Our overall goal is to demonstrate the feasibility of pheromones to enhance the efficacy of EPNs to kill insects in the soil. Specific objectives:Optimize pheromone activation and concentration of S. feltiae and S. carpocapsae for dispersal at4 different temperatures (15, 20, 25 and 30C)Determine whether pheromones enhance S. feltiae and S. carpocapsae dispersal in laboratory soil assays in3 different types of soil4 different temperatures (15, 20, 25 and 30C)Determine whether enhanced dispersal leads to increased infectivity of S. feltiae and S. carpocapsae in response to pheromones in laboratory soil assays in3 different types of soil4 different temperatures (15, 20, 25 and 30C)Determine whether enhanced dispersal leads to improved pest control efficacy of S. feltiae and S. carpocapsae in response to pheromones in greenhouse soil experiments with4 agronomically important insect pests

Project Methods
Objective 1. Optimize pheromone activation time of S. feltiae and S. carpocapsae for dispersalMethods and activities: We will focus on two commercially available EPN species (S. feltiae and S. carpocapsae). EPN IJs will be used for testing pheromone effect. To stimulate a measureable pheromone response, IJs need to be deconditioned to the pheromones prior to testing. The experiments will also provide information on how long the initial pheromone effect from host cadaver lasts (the effect when EPNs first emerge from host cadaver). This will be measured by clumping behavior where EPNs stay in the application spot and do not leave. The deconditioning time is already determined to be 36 h for S. feltiae. Pheromone deconditioning time will be determined for S. carpocapsae IJs. Then the pheromone will be added to disperse the EPNs and the duration of dispersal after pheromone application will be observed in a visual assay in agar plates. The timing of pheromone exposure for optimum dispersal will be determined to estimate how long the activated EPNs will be effectively dispersing in the field. Considering the target insect is mobile, the natural enemy, EPN, needs to disperse and seek insect hosts for efficient insect death. The activities for objective 1 include 1- generate crude pheromone extract, 2- produce IJs for assays, 3- determine the time for pheromone deconditioning, 4- determine duration of the dispersal pheromone effect using agar plate dispersal assay and 5- test whether pheromone exposure time has any effect on the duration of the pheromone's effect on nematode dispersal.Objective 2. Determine whether pheromones enhance S. feltiae and S. carpocapsae dispersal in laboratory soil assays in three different types of soil in 4 different temperatures.Methods and activities: First, we will test whether S. feltiae disperse in laboratory soil assays when exposed to crude pheromone extracts because preliminary data show that pheromones at physiologically relevant concentration can disperse S. feltiae in laboratory plate assays. Then, we will test S. carpocapsae to rule out the possibility that the effect is specific to S. feltiae. We will test sandy soil first Then we will test two other soil types (sandy loam and clay loam) to determine whether dispersal is affected by different soil types. The activities for objective 2 include 1- generate pheromone extract and 2- test dispersal in laboratory soil assays in three different types of soils.Objective 3. Determine whether enhanced dispersal in response to pheromones leads to increased infectivity of S. feltiae and S. carpocapsae in laboratory soil assays in3 different soil types in 4 different temepratureMethods and activities: Under conditions where S. feltiae and S. carpocapsae show enhanced dispersal, they will be tested further to determine whether improved dispersal results in improved infectivity in laboratory soil assays. We will conduct the test with G. mellonella larvae using sandy soil first. After testing sandy soil, we will test two other soil types (sandy loam and clay loam) to determine whether infectivity is affected by different soil types. The activities for objective 3 include 1- generate pheromone extract and 2- test infectivity in laboratory assays in three different soil types.Objective 4. Determine whether enhanced dispersal leads to improved pest control efficacy of S. feltiae and S. carpocapsae in response to pheromones in greenhouse soil experiments withthree agronomically important insect pests Methods and activities: After demonstrating enhanced infectivity in S. feltiae and S. carpocapsae under laboratory conditions, we will move one step further to greenhouse experiments and this time use agronomically important plant pest species such as pecan weevil, citrus weevil and plum curculio. The soil type will be selected based on the target pest's natural soil environment. The activities for objective 4 include 1- generate pheromone extract and 2- test infectivity on three different types of agronomically important insect larvae in greenhouse soil assays.

Progress 07/15/17 to 03/14/18

Outputs
Target Audience:Final report SBIR Phase I technical objectives Our overall goal was to demonstrate the feasibility of pheromone extracts to enhance the efficacy of EPNs to kill insects in the soil. Specific objectives: 1- Optimize pheromone activation and concentration of S. feltiae and S. carpocapsae for dispersal at 4 different temperatures (15, 20, 25 and 30°C) 2- Determine whether pheromone extracts enhance S. feltiae and S. carpocapsae dispersal in laboratory sandy soil assays at 25°C 3- Determine whether enhanced dispersal leads to increased infectivity of S. feltiae and S. carpocapsae in response to pheromone extracts in sandy soil assays at 25°C 4- Determine whether enhanced dispersal leads to improved pest control efficacy of S. feltiae and S. carpocapsae in response to pheromone extracts in greenhouse soil experiments with 3 agronomically important insect pests; pecan weevil, citrus weevil and black soldier fly (Objectives 2, 3 and 4 were updated in last year's report to speed up proof of concept trials.) Phase I Results' Summary Through the activities in Phase I objectives, we demonstrated the technical and commercial feasibility of pheromone extracts to improve EPN efficacy in soil. We focused on two commercially important and available EPN species (S. feltiae and S. carpocapsae) because they control a wider range of insect pests than other commercially available EPNs. Our 1st objective we showed that in the absence of pheromone, S. feltiae IJs deconditioned to pheromones (meaning reduced their dispersal significantly). Particularly this affect is stronger at low temperatures. Pheromones stimulated their dispersal at optimum, high and low temperatures (15, 20, 25, and 30°C). For S. carpocapsae IJs, their dispersal was more complex. Part of their dispersal was controlled by pheromones at low temperature (15 and 20°C), where they show a dispersal lag time. This lag time was overcome by pheromones. Our results showed that 15 min pheromone exposure was enough for both EPN species to disperse, suggesting a min of 15 pheromone treatment for the following objectives. Our 2nd and 3rd objectives. Soil column data showed that 20 min pheromone pre-treatment increased dispersal, meaning we had more IJs further away from the application site and more IJs inside the bait, T. molitor. We went one step further to determine whether the increased number of IJs inside the host is due to increased insect host encounter or induction of infection. We provided equal opportunity to IJs (pheromone treated, or control water treated) to access the host. We found that dispersal pheromone mixture induced infectivity behavior measured by host invasion. These two results (stimulation of dispersal and induction of infectivity) indicated we should move forward with greenhouse efficacy trials. Our 4th objective. Consistent with data in objective 1, 2 and 3, the pheromone treatment (20 min), increased EPN efficacy against pecan weevil, citrus weevil, and black soldier fly, demonstrating a successful proof of concept. This Phase I feasibility research produced 1 new provisional patent on infectivity behavior by USDA-ARS and Pheronym (USDA Docket No. 0138.18) for a second function and 4 manuscripts (one published and another is in review at J. Invertebr. Pathol., one submitted to PLoS ONE and one in preparation with a submission date of May 2019 to PLoS ONE). Furthermore, our findings were presented in our USDA SBIR Phase II application. At the same time, we are disseminating our proof of concept finding as poster and oral presentations. Patent and 4 manuscripts (published, in review or in preparation with a submission date) Wu S, Kaplan F, Lewis EE, Alborn H, Shapiro-Ilan DI (2018) Infected host macerate enhances entomopathogenic nematode movement towards hosts and infectivity in a soil profile. J. Invertebr. Pathol. 159:141-144 Oliviera-Hofman C, Kaplan F, Stevens G, Lewis EE, Wu S, Alborn HT, Perret-Gentil A, Shapiro-Ilan DI (2019) Pheromone extracts act as boosters for entomopathogenic nematodes efficacy. J. Invertebr. Pathol. In review. Perret-Gentil A, Shapiro-Ilan D, Sun J, Mirti A, Sampson E, Schiller KC, Lewis EE, Kaplan F (2019a) Absence of pheromone signals shifts behavior to a non-dispersal phase in Steinernema feltiae PLoS ONE Submitted April 2019 Perret-Gentil A, Giurintano J, Torres C, Sun J, Lewis EE, Shapiro-Ilan DI, Kaplan F (2019b) Steinernema carpocapsae shows a dispersal lag at low temperatures in the absence of pheromones PLoS ONE To be submitted in May 2019 Shapiro Ilan DI, Kaplan F (2019) Methods and compositions for increasing infectivity of entomopathogenic nematodes USDA Docket No.0138.18 Conferences and professional meetings Poster presentations: Bay Area Worm Meeting April 27, 2019, Berkley, CA. Perret-Gentil A, Shapiro-Ilan D, Sun J, Mirti A, Sampson E, Schiller KC, Lewis EE, Kaplan F (2019) "Absence of pheromone signals shifts behavior to a non-dispersal phase in Steinernema feltiae" Society of Nematologist Annual Meeting July 7-10, 2019 Raleigh. NC. Perret-Gentil A, Shapiro-Ilan D, Sun J, Mirti A, Sampson E, Schiller KC, Lewis EE, Kaplan F (2019) "Absence of pheromone signals shifts behavior to a non-dispersal phase in Steinernema feltiae" Society of Invertebrate Pathology Meeting, Valencia Spain July 28-August 1, Camila Oliveira-Hofman, Shaohui Wu, Fatma Kaplan, Edwin Lewis, and David Shapiro-Ilan "Pheromones as drivers of entomopathogenic nematodes movement and infectivity". Oral presentations: World Agritech Innovation Summit, March 19-20, 2019 San Francisco, CA. Plant Protection and Nutrition: Innovation and Commercialization, Biocontrol session, May 21-22, 2019, Raleigh, NC. Entomological Society of America-Southeastern Branch, March 3-6 2019, Mobile Alabama, Camila Oliveira-Hofman, Shaohui Wu, Fatma Kaplan, Edwin Lewis, Paul Schliekelman and David Shapiro-Ilan "Leveraging Entomopathogenic Nematode Movement For Improved Biocontrol". Georgia Entomological Society, April 11-12, 2019, Lake Blackshear, GA, Camila Oliveira-Hofman, Shaohui Wu, Fatma Kaplan, Edwin Lewis, Paul Schliekelman and David Shapiro-Ilan Leveraging Entomopathogenic Nematode Movement For Improved Biocontrol Changes/Problems:Our major challenges and how we solved them: 1- Objective 1: Applying bioassays from S. feltiae to S.carpocapsae. We did not anticipate that nematodes with different foraging style would also respond to pheromone deconditioning differently. So it took longer than we anticipated. 2- Obj 2 and 3: We did not realize that having 3 different soil type and 4 different temepratures in Objectives 2 and 3 would take more than 2 years. So we made a request last year to use only one type of soil and one temperature. 3- Objective 4: One of our collaborators moved to University of Idaho and did not have access to naval orangeworms which made the experiment impossible. We requested to replace navel orangeworm with black soldier fly or wireworm which is an important pest for Idaho farmers who grow sugarbeets, potatos and carrots and onions. The winter was harsh and wireworms died in the field so we did the trials with the alternative insect, the black soldier fly. Also, objective 4 mentions 4 insect pests, which is a typo. It was supposed to be 3 insect pests as can be seen in the milestone page. What opportunities for training and professional development has the project provided? Talking to customers to determine how we can integrate our technology to the market need Presenting at professional meetings and growers meetings for potentail customers Learning about biopesticide industry's need and farmers need Prototyping Commercilization and customer acquisition Manufacturing How have the results been disseminated to communities of interest? Peer reviewed manuscrip preparation Conference poster and oral presentations Talking to customers Investor training how our technology works What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objectives 2, 3 and 4 were updated in last year's report to speed up proof of concept trials. Phase I Summary of our accomplishments Through the activities in Phase I objectives, we demonstrated the technical and commercial feasibility of pheromone extracts to improve EPN efficacy in soil. We focused on two commercially important and available EPN species (S. feltiae and S. carpocapsae) because they control a wider range of insect pests than other commercially available EPNs. Our 1st objective we showed that in the absence of pheromone, S. feltiae IJs deconditioned to pheromones (meaning reduced their dispersal significantly). Particularly this affect is stronger at low temperatures. Pheromones stimulated their dispersal at optimum, high and low temperatures (15, 20, 25, and 30°C). For S. carpocapsae IJs, their dispersal was more complex. Part of their dispersal was controlled by pheromones at low temperature (15 and 20°C), where they show a dispersal lag time. This lag time was overcome by pheromones. Our results showed that 15 min pheromone exposure was enough for both EPN species to disperse, suggesting a min of 15 pheromone treatment for the following objectives. Our 2nd and 3rd objectives. Soil column data showed that 20 min pheromone pre-treatment increased dispersal, meaning we had more IJs further away from the application site and more IJs inside the bait, T. molitor. We went one step further to determine whether the increased number of IJs inside the host is due to increased insect host encounter or induction of infection. We provided equal opportunity to IJs (pheromone treated, or control water treated) to access the host. We found that dispersal pheromone mixture induced infectivity behavior measured by host invasion. These two results (stimulation of dispersal and induction of infectivity) indicated we should move forward with greenhouse efficacy trials. Our 4th objective. Consistent with data in objective 1, 2 and 3, the pheromone treatment (20 min), increased EPN efficacy against pecan weevil, citrus weevil, and black soldier fly, demonstrating a successful proof of concept.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Perret-Gentil A, Shapiro-Ilan D, Sun J, Mirti A, Sampson E, Schiller KC, Lewis EE, Kaplan F (2019a) Absence of pheromone signals shifts behavior to a non-dispersal phase in Steinernema feltiae PLoS ONE
• Type: Journal Articles Status: Other Year Published: 2019 Citation: Perret-Gentil A, Giurintano J, Torres C, Sun J, Lewis EE, Shapiro-Ilan DI, Kaplan F (2019b) Steinernema carpocapsae shows a dispersal lag at low temperatures in the absence of pheromones PLoS ONE To be submitted
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wu S, Kaplan F, Lewis EE, Alborn H, Shapiro-Ilan DI (2018) Infected host macerate enhances entomopathogenic nematode movement towards hosts and infectivity in a soil profile. J. Invertebr. Pathol. 159:141-144
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Oliviera-Hofman C, Kaplan F, Stevens G, Lewis EE, Wu S, Alborn HT, Perret-Gentil A, Shapiro-Ilan DI (2019) Pheromone extracts act as boosters for entomopathogenic nematodes efficacy. J. Invertebr. Pathol.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Bay Area Worm Meeting April 27, 2019, Berkley, CA. Perret-Gentil A, Shapiro-Ilan D, Sun J, Mirti A, Sampson E, Schiller KC, Lewis EE, Kaplan F (2019) Absence of pheromone signals shifts behavior to a non-dispersal phase in Steinernema feltiae
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Society of Nematologist Annual Meeting July 7-10, 2019 Raleigh. NC. Perret-Gentil A, Shapiro-Ilan D, Sun J, Mirti A, Sampson E, Schiller KC, Lewis EE, Kaplan F (2019) Absence of pheromone signals shifts behavior to a non-dispersal phase in Steinernema feltiae
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Society of Invertebrate Pathology Meeting, Valencia Spain July 28-August 1, Camila Oliveira-Hofman, Shaohui Wu, Fatma Kaplan, Edwin Lewis, and David Shapiro-Ilan Pheromones as drivers of entomopathogenic nematodes movement and infectivity
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: World Agritech Innovation Summit, March 19-20, 2019 San Francisco, CA
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Plant Protection and Nutrition: Innovation and Commercialization, Biocontrol session, May 21-22, 2019, Raleigh, NC
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Entomological Society of America-Southeastern Branch, March 3-6 2019, Mobile Alabama, Camila Oliveira-Hofman, Shaohui Wu, Fatma Kaplan, Edwin Lewis, Paul Schliekelman and David Shapiro-Ilan Leveraging Entomopathogenic Nematode Movement For Improved Biocontrol
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Georgia Entomological Society, April 11-12, 2019, Lake Blackshear, GA, Camila Oliveira-Hofman, Shaohui Wu, Fatma Kaplan, Edwin Lewis, Paul Schliekelman and David Shapiro-Ilan Leveraging Entomopathogenic Nematode Movement For Improved Biocontrol

Progress 07/15/17 to 03/14/18

Outputs
Target Audience:Our target audiences are biopesticide companies, greenhouse growers, and farmers. We contacted representatives from each of these groups. Changes/Problems:Scaling up pheromone production was more time consuming than we expected. Each step up in quantity of pheromones produced required new method development. S. carpocapsae:Adapting the pheromone deconditioning assay fromS. feltiaetoS. carpocapsaewas more challenging than we expected.S. carpocapsaebehavior was quite different at different temperatures. For example,at high temperature,S. carpocasaeIJs run away from the plates. At low temperature, they did not move for a period of time and then dispersed. We finally optimized high temperature experiments. Currently, we are finalizing optimization for low temperature experiments. What opportunities for training and professional development has the project provided?The company was accepted into IndieBio seed biotech accelerator to pursue this project. During the accelerator program, the team learned about entrepreneurship including branding, trademarks, marketing, sales, and fundraising from investors for Phase III commercialization. We also provided scientific training to a postdoctoral scientist and two undergraduatescientists. 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?We plan to complete greenhousesoil efficacy trials with S. feltiae. We also plan to write and submit a Phase II grant to scale up production and conduct field trials.

Impacts
What was accomplished under these goals? Our overall goal is to demonstrate the feasibility of pheromone extracts to enhance the efficacy of EPNs to kill insects in the soil. Specific objectives: 1- Optimize pheromone activation and concentration of S. feltiae and S. carpocapsae for dispersal at 4 different temperatures (15, 20, 25 and 30°C) Determine the time for pheromone deconditioning Determine the duration of pheromone extract effect Determine optimum pheromone extract exposure time S. feltiae: we have determined pheromone deconditioning time for S. feltiae at 15, 20, 25 and 30°C. This ranged from 4- 6 days. Low temperature reduced dispersal. Figure 1. shows S. feltiae pheromone deconditioning time over a period of 6 days at room temperature (RT), which is 20°C. This experiment was repeated at low (15) and at high temperatures. Each experiment was repeated in time at least 3 times and contained at least 3 independent replications. Figure 1: S. feltiae IJ pheromone deconditioning over a period of 6 days. Pheromone deconditioning is expressed as reduced dispersal. The graph is a representative experiment done at room temperature (RT) which is 20°C. The experiments were repeated 3 times with at least 3 independent replications. We also determined that pheromone effect on IJ dispersal lasts 3-4 days at RT. This depends on the pheromone exposure time. As little as 15 min exposure to pheromones is enough to see increased dispersal compared to control (water) at RT (20°C) Currently, we are testing whether pheromones improve dispersal at low (15°C) and high temperature at 25°C and 30°C. S. carpocapsae: Adapting the pheromone deconditioning assay from S. feltiae to S. carpocapsae was more challenging than we expected. S. carpocapsae behavior was quite different at different temperatures. For example, at high temperature, S. carpocasae IJs run away from the plates. At low temperature, they did not move for a period of time and then dispersed. We finally optimized high temperature experiments. Currently, we are finalizing optimization for low temperature experiments. 2- Determine whether pheromone extracts enhance S. feltiae and S. carpocapsae dispersal in laboratory soil assays at 4 different temperatures (15, 20, 25 and 30°C) We have tested both S. feltiae and S. carpocapsae in sandy soil at 25°C after 15 min of pheromones. We were able to observe increased dispersal. The high and low temperature experiment will be done based on the result in Objective 1. 3- Determine whether enhanced dispersal leads to increased infectivity of S. feltiae and S. carpocapsae in response to pheromone extracts in laboratory soil assays in 3 different types of soil 4 different temperatures (15, 20, 25 and 30°C) We have tested both S. feltiae and S. carpocapsae in sandy soil at 25°C after 15 min of pheromones. We observed increased dispersal led to 3 times increased infectivity compared to water control. Fig. 2: Number of nematodes that infected T. molitor in the bottom (furthest) column section. The overall number of nematodes that dispersed to the bottom half of the column was also assessed and showed the same outcome (data not shown). The high and low temperature experiment will be done based on the result in Objective 1. 4- Determine whether enhanced dispersal leads to improved pest control efficacy of S. feltiae and S. carpocapsae in response to pheromone extracts in greenhouse soil experiments with 4 agronomically important insect pests We have produced pheromones for the greenhouse efficacy tests and scheduled greenhouse efficacy tests with S. feltiae at 25°C. Overall production of pheromones took a lot more time than we anticipated. We had a method that produced enough for laboratory plate assays but not enough for soil efficacy assays. We purchased equipment and developed new protocols to increase production.

Publications