Source: MICHIGAN STATE UNIV submitted to NRP
DEVELOPING YEAST-BASED INTERFERING RNA BIOPESTICIDES AGAINST SPOTTED-WING DROSOPHILA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1032512
Grant No.
2024-70006-43410
Cumulative Award Amt.
$325,000.00
Proposal No.
2024-03400
Multistate No.
(N/A)
Project Start Date
Sep 1, 2024
Project End Date
Aug 31, 2026
Grant Year
2024
Program Code
[ARDP]- Applied Research and Development Program
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
ENTOMOLOGY
Non Technical Summary
This is an ARDP applied research (single function) project addressing the CPPM focus area of Plant Protection Tools and Tactics; it is aligned with the program goals of using system approaches in the "discovery, development, and introduction of new pest management tactics for use in IPM systems". Spotted-wing drosophila (SWD), Drosophila suzukii, are invasive vinegar flies of East Asian origin that have wreaked global havoc in cherry and berry crops with both economic and environmental consquences. SWD management necessitates weekly applications of insecticides to prevent infestation, increasing the cost to produce these crops and increasing the risk of creating pesticide resistant SWD populations. In addition, these prophylactic insecticides can result in unwanted environmental impacts, including disruptions to natural enemies. It is therefore critical to identify new biorational pesticides for controlling SWD. The proposed research will use RNA interference technology combined with the attractive properties of baker's yeast to develop yeast-based RNAi pesticides that target SWD. This type of species-specific, yeast-based biopesticide allows for cost-effective scaled production, can be readily shipped and stored, and has been demonstrated to have excellent residual activity in trials with mosquitoes. Preliminary research by the project team has identified promising RNAi targets in SWD. It is anticipated that our proposal will lead to the development of a new class of stakeholder-accepted, species-specific insecticides that can be deployed as either yeast-sugar baits or sprayables and can be integrated with other management techniques for the prevention of SWD infestation in susceptible crops in North America and beyond.
Animal Health Component
(N/A)
Research Effort Categories
Basic
25%
Applied
(N/A)
Developmental
75%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21611991130100%
Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
1199 - Deciduous and small fruits, general/other;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
In the proposed research, the Wilson Tree Fruit Entomology laboratory at Michigan State University (MSU), with expertise in applied, on-farm research and Extension related to Drosophila suzukii in tart cherry orchards, will collaborate with the Scheel laboratory at Indiana University (IU), which recently developed cost-effective, scalable, stakeholder-accepted, user-friendly, species-specific yeast ribonucleic acid (RNA) pesticides that target mosquitoes (Duman-Scheel, 2019) and who has now identified double-stranded RNA (dsRNA) targets specific to D. suzukii. This interdisciplinary collaboration synergizes the strength of MSU's D. suzukii applied chemical ecology and behavioral science expertise with that of IU's insect genetics expertise, to develop interfering RNA (RNAi) attractive targeted sugar bait (ATSB) technology against D. suzukii. We propose to exploit the attractive properties of yeast to lure these economically destructive agricultural pests to feed on ATSBs containing yeast that has been designed to express biorational RNA insecticides that target D. suzukii-specific neural genes required for fly survival. It is hypothesized that this strategy can be used for species-specific D. suzukii control, and this hypothesis will be tested through pursuit of the following objectives.Objective 1. Scaled production of D. suzukii-specific heat-killed yeast RNAi insecticides (IU). We have identified a yeast strain expressing short hairpin RNA (shRNA) corresponding to the D. suzukii Shaker gene. Preliminary data show that this yeast will kill D. suzukii but is not toxic to non-target insects. Using established methodology, we will pursue scaled production of the insecticidal yeast for further assessment of this hypothesis in laboratory and field trials.Objective 2. Evaluation of yeast-based RNAi ATSBs for competitive efficacy, longevity, and non-target effects to relevant biological control agents (MSU). We hypothesize that yeast RNAi pesticides 1) can be designed to outcompete other attractants including ovipositional substrates, 2) will maintain their attractiveness for the duration of a growing season, and 3) will not affect parasitoids of D. suzukii.Objective 3: Evaluation of yeast-based RNAi ATSBs to prevent fruit infestation by D. suzukii (MSU). We hypothesize that RNAi baits will be able to prevent fruit infestation below detectible levels and that the best deployment strategy will be to use ATSBs along orchard margins adjacent to habitat out of which D. suzukii are expected to emigrate. Objective 4. Extension of project results to stakeholders. We hypothesize that when presented with the results of this study, 1) growers of susceptible fruit crops will be receptive to the idea of using RNAi technology to manage D. suzukii on their farms, and 2) pesticide manufacturers will be convinced of this technology as being valuable and marketable.
Project Methods
Objective 1. Scaled production of D. suzukii-specific heat-killed yeast RNAi insecticides.1.1. Generation of robust Sh.706 yeast strain for scaled yeast production. To produce a more robust yeast strain, we will use our recently described methodology to express high levels of the insecticidal Sh.706 hairpin in the S. cerevisiae FL100 strain. Control and insecticidal yeast strains will be mixed with 10% sucrose solution with a tracer dye and presented to individuals starved for 4-5 h, allowing the flies to feed for 5 h. Feeding will be confirmed by the presence of the tracer dye and by engorgement of the individuals fed on the mixture, and flies monitored for survival for one week. Seven replicate trials (n=20 adults per replicate) will be conducted. Flight and locomotion defects will be noted in D. suzukii following treatments.1.2. Confirmation of the RNAi insecticide activity of the newly-generated robust Sh.706 yeast strain. Lab-reared D. suzukii will be used in trials replicated at least 3 times with 20 adults and compared to an untreated yeast control. Following treatment, D. suzukii will be reared on standard diet and monitored every 24 hrs for up to one week to assess morbidity. The results will be statistically analyzed using Student's T test.Confirmation of Shaker target gene silencing will be pursued in D. suzukii using qRT-PCR assays with data standardized to the D. suzukii Actin reference gene. Dose-response curves will also be generated.1.3. Scaled yeast fermentation. Following confirmation of Sh.706 yeast activity and optimized dose determined above, yeast will be produced in large scale using Chemglass bioreactors with yeast prepared in 2x5 L volumes per run. The yeast will subsequently be heat-killed and dried. For quality control purposes, the insecticidal activity of a sample of yeast from each run will be confirmed in D. suzukii as described above. ATSB treated yeast will be produced in this manner throughout the project period and shipped to MSU for pursuit of the Objective 2 and 3 studies.Objective 2. Evaluation of yeast-based RNAi ATSBs for competitive efficacy and longevity.2.1. Optimize the competitive attraction of yeast-based RNAi ATSBs in cage studies to develop a field prototype (lab study). Attraction of heat-killed ATSB yeast-sugar baits will be tested against an untreated control and unmodified baker's yeast-sugar bait in 60 x 60 x 60 cm white mesh cages in the laboratory. Each cage will receive 50 pairs of D. suzukii flies, and photos will be taken every 5 min in the first hour after the start of the experiment to capture the number of flies visiting each treatment. This experiment will be replicated 5 times. Additionally, the attractiveness of this yeast-sugar bait will be compared to that of fresh fruit and the commercial standard lure used for D. suzukii monitoring. Based on these results, we will refine the ATSB formulation so that we bring the most attractive ATSBs to the field study. We may combine ATSBs with live unmodified yeast strains or fresh fruit or the combination of the three to improve and/or enhance its attractiveness to D. suzukii while maintaining its insecticidal efficacy.2.2 Evaluate the use of a yeast-based RNAi ATSB prototype for early-season suppression of D. suzukii (field study). Adjacent to cherry orchards and blueberry fields maintained at a research farm, a replicated trapping study using deli-cup style traps will be conducted at the crop edge from where early-season D. suzukii is expected to migrate as the crop ripens. The following 4 different bait/lure combinations will be tested: 1) RNAi yeast-sugar bait prototype developed in Exp. 2.1 alone, 2) standard unmodified baker's yeast-sugar bait, 3) the current commercial standard lure alone, and 4) #1 and 3 together. The number of male and female D. suzukii and total number of non-target Drosophilids will be enumerated and recorded for each trap. Attractiveness and selectivity of each trap will be compared before, during, and after fruit harvest. Data will be analyzed using a generalized linear mixed effect model, with treatment and cropping system used as fixed effects.2.3. Evaluate non-target effects of yeast-based RNAi ATSB on the Samba wasp (lab study). Ganaspis brasiliensis is a specialist parasitoid wasp that attacks D. suzukii; we will evaluate potential non-target effects of the RNAi yeast-sugar bait in a replicated experiment.Objective 3. Evaluation of yeast-based RNAi ATSBs to prevent fruit infestation by D. suzukii.3.1. Compare performance of bait-station style and sprayable ATSBs to prevent fruit infestation. We will explore whether ATSBs can prevent fruit infestation by D. suzukii in a replicated lab study. Ten female and ten male D. suzukii will be added to each arena set up an a 2 (A,B) x 3 (X,Y,Z) factorial design with ripe fruit either sprayed with the A) modified yeast prepared in solution or B) water and with fruit adjacent to either to X) an ATSB bait station, Y) an unmodified yeast-sugar bait (positive control), or Z) no bait. After 2 days, fly mortality will be counted and fruit will be tested for infestation using the water extraction method. The number of larvae and dead flies will be compared across treatments using a generalized linear mixed effect model.3.2. Evaluate the ability of ATSBs to prevent fruit infestation in field conditions. This experiment will be conducted on single tree plots in tart cherry orchards with a wooded edge in a 2 x 2 factorial design, where each tree will receive one of the following treatments: 1) sprayable ATSBs applied 1 week prior to harvest; 2) bait stations containing ATSBs deployed around the base of the trees at bloom time; 3) treatments 1 and 2 combined; and 4) untreated control. This will be repeated 6 times across the cherry orchard, which will not be sprayed with any other insecticides before or during this experiment. The sprayable formulation will be applied using a backpack sprayer with an adjuvant such as Nu-Film P® to help yeasts adhere better to leaf and fruit surfaces as well as improve its rainfastness. RNAi yeast will be deployed in two bait stations surrounding the base of the tree, elevated from the ground using metal t-posts. Standard deli-cup traps will be used to monitor D. suzukii in each plot. The number of male and female D. suzukii, and total number of non-target Drosophilids will be recorded for each trap; D. suzukii numbers will be compared across the 4 treatments. D. suzukii infestation prior to harvest will be evaluated by collecting 2 pounds of fruit from each treated tree and counting larvae using the water-extraction method. Data from this objective will be tested for normality and subsequently analyzed using appropriate parametric or non-parametric statistical tests.Objective 4. Extension of project results to stakeholders.4.1. Results presented to stakeholders and potential industry partners. We will provide research results to both grower and private sector pest management industries through a variety of mediums including publications, presentations, field days, and through direct conversations with potential industry partners. We will provide our data and methodology to private sector companies interested in mass production of this biopesticide so that beyond the scope of this project, testing can be conducted in scaled efficacy trials, an important step before these tools become available to growers.4.2. Grower perceptions about and receptivity towards use of RNAi technology collected via pre- and post-presentation surveys. We will utilize anonymous real time surveys geared toward growers, to evaluate clientele knowledge and opinions of yeast-based RNAi biopesticides.

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:The project is in its early development phase, but we expect that our findings this past year will eventually be beneficial to berry and cherry crop growers and consumers of berries and cherries who will benefit when ecologically-friendly insecticides are used instead of broad-spectrum insecticides for control of SWD. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the Scheel Lab, postdoctoral fellow Akilah Stewart was trained under the project. Notre Dame undergraduates Jackson Graham, Saisuhas Nelaturi, and Carolina Dille, who received Notre Dame course credits for their research efforts, were trained. Saisuhas wrote an undergraduate thesis for departmental honors in the Chemistry and Biochemistry Department at the University of Notre Dame. Graham also graduated and plans to apply to a PhD program in related science this fall after gaining field experience during the upcoming gap year. Dille will return to the lab this fall for another year of instruction. Mysore's attendance at the Entomology Society of America meeting allowed him to pursue professional development and presentation of the work to advance his upcoming application for promotion to Associate Research Professor. In the Wilson Lab, senior research associate, Dr. Juan Huang, has overseen formulation development and laboratory testing at MSU. An undergraduate student, Barman Nasirpour, has been assisting her with laboratory bioassays. How have the results been disseminated to communities of interest?Dr. Wilson spoke about the technology as part of a presentation on current and future SWD management intwo separate extension events: 1) the Great Lakes Fruit, Vegetable & Farm Market EXPO in Grand Rapids, Michigan in December 2024 during the cherry education session attended by 100+ growers and crop advisors, and 2) the Northwest Orchard and Vineyard Show in Acme, Michigan in January 2025 attended by 75+ growers and crop advisors. Dr. Mysore (Scheel Lab) presented results from Objective 1 during a session at the Annual Meeting of the Entomological Society of America in Phoenix, AZ in November 2024. What do you plan to do during the next reporting period to accomplish the goals?The Scheel Lab will continue to produce yeast for analysis in the Wilson lab and are assisting with transfer of this technology to their research program. The Wilson Lab will be evaluating their gel formulation + attractant against ripening fruit in choice test assays to see whether the bait station results in suppression of fruit infestation in cage studies. In addition, they are continuing to work on a prototype bait station that could be tested during the 2026 field season.

Impacts
What was accomplished under these goals? Objective 1. Scaled production of D. suzukii-specific heat-killed yeast RNAi insecticides (IU). The Scheel Lab modified Saccharomyces cerevisiae, baker's yeast, to express an RNA interference (RNAi) pesticide that specifically targets the SWD Shaker (Sh) gene when flies feed on the yeast. This was delivered in a feeder as a component of an "attractive targeted sugar bait" (ATSB). The yeast, which was heat killed prior to preparation of the ATSB, silenced the Sh gene, resulting in severe neural defects and 96±9% fly mortality in laboratory trials. Despite this toxicity observed in SWD, consumption of the yeast had no impact on the survival of other dipteran insects tested in preliminary trials. The RNAi yeast was fed to the flies in an easily assembled soda bottle feeder that continuously rewetted the yeast formulation with soda, which lured and killed the flies in no-choice tests in semi-field trials. Residual activity of this formulation and delivery system was confirmed over one week in outdoor semi-field trials. Long-term residual storage experiments of this formulation are ongoing. The Scheel lab scaled production of the yeast in 5 L cultures grown in their bioreactor. Further details regarding the outcomes of this objective are included in a preprint publication that has been submitted for review: https://www.biorxiv.org/content/10.1101/2025.03.26.645612v1. Objective 2. Evaluation of yeast-based RNAi ATSBs for competitive efficacy, longevity, and non-target effects to relevant biological control agents (MSU). The effectiveness of yeast-based RNAi relies on bait consumption by D. suzukii which uses its proboscis to suck up liquid as a way of feeding. This feeding behavior requires the bait to remain hydrated for flies to feed upon whenever they are ready. The original ATSB recipe contained heat-kill yeast RNAi mixed with sugar water. However, this formulation desiccated within several hours after preparation under laboratory conditions without the bottle feeder, preventing flies from feeding on the ATSB. The Wilson Lab tried several modifications to create a gel formulation, adding humectants such as corn starch, corn meal, glycerin, honey, two surfactants (tween® 20 and span® 20), sodium polyacrylate, water storing polyacrylamide, and some of their combinations. We found that the gel formulation containing yeast-based RNAi mixed with water storing polyacrylamide and honey retained water the best. This gel formulation stays hydrated for at least a week under laboratory conditions without additional hydration. Whether the life and therefore efficacy of the gel formulation could be extended season-long with a rewetting apparatus such as a bottle feeder containing water, still needs to be tested. The ultimate goal of any management program targeting D. suzukii is to prevent egg-laying in ripening fruit, so a feeding station needs to be more attractive than adjacent fruit and must provide rapid knock-down of mated females. Similar to other Drosophila, D. suzukii is known to be attracted to yeast and its fermentation products, however, since the RNAi yeast has been heat-killed, no yeast fermentation products are produced by the material itself, thereby decreasing the attractiveness of the modified yeast to the flies compared with active yeast. Thus, additions to the yeast formulation are needed to improve their attractiveness beyond simple sugar baits, which are not more attractive than volatiles derived from fruit or fermentation processes. The Wilson Lab tested the relative attractiveness of their gel formulation to D. suzukiiwith the addition of wine-vinegar powder, blueberry fruit powder, raspberry fruit powder, or Combi-Protec, a commercial bait formula. The gel formulation with wine-vinegar powder increased its attractiveness by at least 2-fold over the RNAi-yeast alone. The attractiveness of this new formulation against ovipositional substrates such as cherry, blueberry or raspberry fruits (i.e., where SWD are likely to want to lay their eggs) as well as its longevity and non-target impacts on D. melanogaster and SWD parasitoids are ongoing. If necessary, additional D. suzukii attractants such as a 4-component blend currently used in commercial lues could be used to make a "super bait". Objective 3: Evaluation of yeast-based RNAi ATSBs to prevent fruit infestation by D. suzukii. The Wilson Lab is currently in the develop stage of a prototype bait station for field deployment of the gel formulation that includes the wine-vinegar powder attractant or another "super attractant" and a means for continuous rehydration. This prototype will be tested during the field season in 2026. Objective 4. Extension of project results to stakeholders. Dr. Wilson spoke about the technology at the Great Lakes Fruit, Vegetable & Farm Market EXPO in December 2024 as part of a presentation on current and future SWD management during the cherry education session; growers in attendance were interested in affordable and effective technologies that will prevent fruit infestation by SWD. We are still a long way off from knowing exactly how best to implement this technology and what the cost will be when we do; however, over the past year, we were contacted by two private companies who are interested in developing and/or testing RNAi-based pesticides against SWD; these conversations are on-going.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Keshava Mysore, Teresia Njoroge, Akilah Stweart, Majidah Hamid-Adiamoh, David S. Kang, David Severson, and Molly Duman-Scheel. Advances in yeast RNAi insecticide technology for control of insect pests of medical and agricultural importance. November 10, 2024, Annual Meeting of the Entomological Society of America, Phoenix, AZ