Progress report for LNC23-486
Project Information
Drosophila suzukii, or spotted wing Drosophila (SWD), are invasive vinegar flies of East Asian origin that have wreaked havoc on the small fruit industry in the North Central Region (NCR). In locations where SWD is well established, weekly insecticide applications are necessary, resulting in increased economic costs, as well as unwanted environmental impacts resulting from loss of non-targeted organisms. With increased use of insecticides, populations that are resistant to these insecticides will inevitably emerge, and it is therefore critical that new classes of biorational pesticides and cost-effective technologies for controlling SWD are identified. The proposed research will utilize the attractive properties of baker’s yeast, which has been designed to express an RNA interference (RNAi) pesticide that specifically targets an SWD gene, to lure and kill flies that feed on the yeast which will be delivered as a component of an attractive targeted sugar bait (ATSB). This yeast, which permits cost-effective scaled pesticide production, can be readily shipped and stored and has significant residual activity despite the fact that the yeast is heat-killed prior to deployment. The proposed research program will evaluate the hypothesis that stakeholder-accepted RNAi yeast ATSBs will enable species-specific SWD control through pursuit of the following aims: 1) Generation and characterization of SWD-specific RNAi yeast ATSBs, 2) Assessing stakeholder acceptance of yeast ATSBs, and 3) Field evaluation of yeast ATSBs. These studies will promote trust and cooperation between NCR fruit farmers and scientists, thereby building a community united to combat SWD. It is anticipated that these studies will identify a new class of stakeholder-accepted, species-specific insecticides that can be deployed as ATSBs for SWD control in the NCR and beyond.
Objectives include generation, optimization, and evaluation of the acceptance of yeast RNAi-based ATSBs. Stakeholder engagement will permit researchers to educate stakeholders and gain feedback about SWD control and yeast-ATSB technology. This will help build trust, which may increase SWD surveillance and enhance farmers’ willingness to evaluate and potentially adopt yeast-based interventions, reducing broad-based chemical pesticide use. The scientists will gauge interest in the technology and learn how to best optimize, deploy, and distribute it. These interactions will promote advancement toward the long-term goals of widespread yeast ATSB deployment and SWD control in the NCR and globally.
SWD are vinegar flies of East Asian origin that have wreaked havoc on the small fruit industry worldwide, including Europe and the Americas. SWD impact most berry crops, cherries, grapes and other tree fruits. The flies oviposit in ripe fruits, laying eggs in a variety of wild and cultivated plants, and compromising fruit integrity. When not adequately controlled, the flies, which complete multiple generations in a single year, generate upward of 80% crop loss and an estimated $700 million economic loss for U.S. producers annually. In locations where SWD is well established, weekly insecticide applications are necessary, resulting in increased economic costs, as well as unwanted environmental impacts due to loss of non-targeted organisms. With increased insecticide treatments, it was recently demonstrated that there is potential for the emergence of resistance to organophosphates and pyrethroids in this species (https://fruit.cornell.edu/spottedwing/). It is therefore critical that new classes of pesticides and technologies for controlling SWD are identified.
RNAi is often used to characterize gene function in the laboratory. Although it has generated interest in the insect control realm, few have attempted to translate it from the bench to the field. RNAi pesticides targeting neural genes in multiple species of vector mosquitoes were recently generated. These mosquito-specific RNAi insecticides were designed to target nucleotide stretches conserved in mosquitoes, but not in any other organisms, including humans. For example, an RNAi yeast insecticide was designed to target a conserved site in the mosquito Rbfox1 gene. Rbfox1 genes encode an evolutionarily conserved RNA binding protein, which functions in many biological processes. In the mosquito Aedes aegypti, silencing of the Rbfox1 gene resulted in high levels of mosquito mortality, which correlated with severe defects in neural activity within the mosquito brain. The insecticidal impacts of Rbfox1 silencing were subsequently confirmed in trials conducted on multiple species of mosquitoes.
The insecticides characterized in mosquitoes consisted of short hairpin RNA (shRNA) molecules that were expressed in baker’s yeast, Saccharomyces cerevisiae, which is consumed by mosquitoes, resulting in silencing of the target neural genes. The use of S. cerevisiae enables inexpensive and scalable preparation of the insecticides. Moreover, the yeast can be delivered to mosquitoes as the active ingredient in attractive targeted sugar baits (ATSBs). ATSBs capitalize on the natural sugar feeding behavior of insects that are attracted to feed on a sugar source laced with a poison. Characterization of the RNAi yeast ATSBs against mosquitoes in laboratory and semi-field trials has demonstrated the efficacy and biorationality of these pesticides. Several of the targeted genes, which function in the nervous system, have orthologues in SWD. Recent studies have shown that baker’s yeast, S. cerevisiae, is an effective attractant of major agricultural pests. Combined, these findings support the hypothesis that we can create species-specific RNAi yeast ATSBs to target SWD. In this project, we characterize an RNAi yeast ATSB targeting the SWD Rbfox1 gene. Additionally, we will seek feedback from local farmers regarding interest in and perceptions of this technology. We will also education them about our new class of insecticides.
Cooperators
- - Technical Advisor
- - Technical Advisor
Research
We hypothesize that stakeholder-accepted RNAi yeast ATSBs will enable species-specific SWD control
Our interdisciplinary team of Indiana University, USDA-ARS, and Lincoln University Cooperative Extension scientists worked together, in consultation with State Extension scientists and NCR fruit farmers, to develop the proposed project plan. Pursuit of the following aims will facilitate evaluation of the hypothesis that stakeholder-accepted RNAi yeast ATSBs will enable species-specific SWD control:
I. AIM 1: Generation and characterization of SWD-specific RNAi yeast ATSBs:
Overview: SWD-Rb.1 yeast will be suspended in different lures and provided to flies in ATSB sachets composed of a variety of different membranes. The attractiveness, efficacy, and residual activity of the sachets will be evaluated in laboratory trials. Following down-selection of top ATSB systems, dose-response assays will be performed, and the mode of action will be confirmed in SWD.
Bait preparation: Dried heat-inactivated yeast will be prepared from the experimental or control yeast strains as described (23) and used to prepare ATSB bait suspensions that will be evaluated in simulated field trials in the insectary. In addition to evaluating attractive sugar baits that have worked well in mosquitoes, additional baits will be developed using FDA-approved non-toxic food-grade reagents, including:
- Attractants, for example sucrose, yeast, grape juice, wine or apple cider vinegar.
- Sustained release agents, i.e. glycerine, or polyethylene glycol.
- Preservatives, such as potassium sorbate, citric or benzoic acid.
- Stabilizers, such as gellan or xanthum gum.
Sachet optimization: 5x5 cm white nylon membrane sachets, which have worked well for mosquitoes, will be evaluated, and sachets of different materials, mesh sizes, surface areas, and colors will also be assessed.
Laboratory ATSB trials: Lab trials will be performed in cages placed in the insectary as described (14,15) using an SWD strain established from locally-captured flies. Briefly, sugar bait solution, sugar bait+control RNAi yeast (lacking an SWD target site, 14,15), or sugar bait+ SWD-Rb1 yeast will prepared in a sachet and provided to SWD sugar-starved for 4-5 h. Feeding (marked by presence of a marker dye) and mortality rates will be assessed in at least four replicate trials (n=20 adults/treatment) analyzed using ANOVA as described (14,15). Behavioral phenotypes related to flight or locomotion defects will be noted. A variety of baits and membranes will be evaluated in this manner, with the most inexpensive ATSB sachets through which the highest efficacy is achieved selected for further characterization.
Evaluation of storage capacity and residual activity: Shelf life will be evaluated with the EPA accelerated storage protocol (24). To evaluate residual activity, 20 new adults will be added to each cage at the conclusion of each trial until no insecticidal activity is detected.
Down-selection: Top-performing formulations will be down-selected on the basis of materials cost, efficacy, storage, and residual activity (see further details in the evaluation plan). Top formulations will then be characterized in greater detail:
Confirmation of target gene silencing and mode of action: Target gene silencing will be evaluated in SWD-Rb.1 vs. control-treated flies using qRT-PCR as described (25). Immunohistochemical analysis of neural activity will be performed and statistically evaluated with Student’s t-test as described (14,15). Based on mosquito studies (14), target gene silencing and loss of neural activity, as marked by reduction of nc82 (DSHBank, Iowa City, Iowa) levels, is expected.
Dose-response assays: LC50 and LC90 concentrations of down-selected formulations will be evaluated through pursuing dose-response curves as described (14,15), with data analyzed through Probit analysis with the SPSS program (14,15).
Confirmation of non-toxicity in select non-target arthropods. Toxicity assays will be conducted on select non-target arthropods such as Drosophila melanogaster, Tribolium castaneum, Pogonomyrmex barbatus, Oncopeltus fasciatus, and Vanessa cardui (Carolina Biologicals). Insects will be exposed to the ATSB sachets or alternatively fed from droplets supplied on a petri dish as described (14,15). Data from at least three replicate trials will be analyzed through ANOVA. It is anticipated, on the basis of in silico sequence data that has revealed no conservation of the SWD-Rb.1 target site in other organisms, that no significant toxicity will be observed.
II. AIM 2. Assessing stakeholder acceptance of yeast ATSBs:
Overview: Given that early stakeholder involvement is central to the successful development of new mosquito control interventions (30), we have successfully adapted the Responsible Research and Innovation (RRI) (31) approach as a critical component of our RNAi insecticide programs (28,29). RRI is a transparent, interactive process by which societal actors and innovators become mutually responsive to each other with a view to the acceptability, sustainability, and societal desirability of new innovations. Effective engagement involves early initiation of activities that serve to: i) ensure that the purpose and goals of the research are clear to the community, ii) establish relationships and commitments to build trust, iii) allow researchers to understand the community, its diversity, changing needs, and assets, iv) maximize opportunities for stewardship, ownership, and shared control by the community, v) provide a platform for expression of dissenting opinions or in extreme cases, prohibition of the research, and vi) give the researchers an opportunity to modify the proposed research strategies, as needed (30). To this end, we have developed the following robust engagement strategy for our SWD research program:
Farmer consultants: We have included commitment letters from five farmers who have agreed to serve as consultants in the proposed Farmer Consultant Engagement Study, including one female Latina farmer from a Latino-owned blueberry farm and an organic farmer. Each participating farm will be compensated $500 for participation in the following activities:
Visits to farms: We will visit each farm twice, for ~2-3 hours per visit, during which time we will discuss how SWD is impacting the farm. During the visit, we will also tell the farmers and their staff about the new tools we are developing to control this pest and get feedback, both positive and negative, regarding our research and seek suggestions for improvement of ATSB technology.
Ongoing Consultation: We will occasionally contact the farmers by phone or email to seek their input on questions that emerge during the course of our research. We anticipate that this would consist of two or three 15 minute interactions during the course of the research program.
Webinar: At the conclusion of the study, we will ask participating farms to attend an online webinar (~1 hour) that will involve other farmers throughout the NCR. Farmers will be contacted with the assistance of relevant state extensions/educators. We have begun to develop a list of historically under-represented farmers in the NCR region (for example Latino and Native American farmers), which will be expanded during the project period; these farmers will be sent multiple advertisements or in person-invitations to encourage their attendance. During the webinar, the research team will present the outcomes of the study, answer participant questions, and give attendees the opportunity to share final thoughts about the new technology.
Program outreach survey: Webinar participants will be prompted to complete a short Post-Webinar Survey Study that will be offered electronically at the conclusion of the webinar. The survey, offered through a Qualtrics (32) link, will be analyzed using Qualtrics Stats IQ software (32).
IRB Approval: Deemed exempt by Indiana University; amendment files for SWD studies: Farmer Consultant Engagement; Post-Webinar Survey.
III. AIM 3: Field evaluation of yeast ATSBs.
Overview: Down-selected ATSB sachets will be will be attached to SWD-lure sticky traps, and the attractiveness/specificity of the system will be monitored in the field at a USDA test plot and at local Missouri grape farms. The efficacy of the top-performing formulations will also be evaluated in semi-field trials. In these assays, insecticidal yeast sachets will be provided to SWD that are contained in an insect cage placed in a tent enclosure at grape farms in Missouri.
Evaluation of sachet attractiveness and selectivity: The attractiveness and specificity of the yeast sachets will be assessed in field studies, which will be conducted at Missouri grape farms (see letters from consultants 4-5). Control yeast sachets mixed with top performing lures identified in Aim 1 will be attached to sticky card or SWD-specific Trécé-lure-baited red sticky card traps (Great Lakes, IPM, Vestaburg, MI). The SWD lure will be positioned above the sticky card ~1.5 m off the ground within the fruit zone in a shaded area. Monitoring will begin when the daily temperature is above 10o C for several days. The sticky traps will be collected for evaluation, and SWD will be identified through morphological characters (33).
Semi-field assessment of ATSB sachets: Semi-field performance of the top yeast ATSB sachets will be assessed in outdoor cage trials. Two of the top performing adulticide formulations will be assessed in these semi-field trials, which will include sugar bait alone (negative control), control yeast-sugar bait (negative control), and a spinosad reference pesticide ATSB positive control. The sugar baits will be marked with a red dye to track sugar ingestion. The experiments will be conducted in Sansbug tents (Amazon.com) in which insect rearing cages will be placed as described (14,15). Two sachets (of each specific control or experimental treatment) will be placed inside each cage. 10 male and 10 female SWD from a local SWD strain will be deprived of sugar for ~4-5 h prior to placement of the ATSBs. Morbidity rates will be assessed 1-6 days after initiation of the trials. At least six replicate trials will be performed for each formulation, and data will be analyzed with ANOVA as described (14,15). Upon completion of each assay, new flies will be evaluated until no significant adulticidal activity is detected, permitting assessment of residual activity.
IV. Alternative strategies: Our success in mosquitoes suggests that this approach can be applied to SWD. However, the bait and sachet membranes may need to be optimized for SWD, and we have therefore outlined a strategy that will facilitate this process. Likewise, ATSB deployment in the field studies will likely need to be optimized, and we will rely heavily on conversations with the farmers to pursue this. We are currently experimenting with higher RNA expression yeast strains, and such strains could likely be developed for SWD as needed. If the SWD-Rb.1 insecticide results are not satisfactory, it would also be possible to target alternate genes. Finally, in the event that the yeast is not successful, the study could also be completed with naked siRNA per Mysore et al. (27), which works well but is more expensive. We hope that the stakeholders will embrace this technology, but in the event that they do not, we will try to engage them in further conversations and continue to educate them regarding the technology, listening to their feedback to try to design a stake-holder approved intervention.
V. Multi-State Involvement and Partnerships in the NCR Region:
Molly Duman Scheel and Keshava Mysore will lead the laboratory (Indiana) and community engagement (Indiana-Michigan-Missouri; NCR-wide webinar) studies, while David Kang will direct the field studies in consultation with Clement Akotsen-Mensah (Missouri). Each will be responsible for the direct supervision of personnel in their own respective research groups. Each will be involved in decision-making regarding key experiments and approaches for the proposed aims. However, it is expected that primary responsibility will lie with each scientist in their particular area of expertise. Scheel, the designated contact PI, will be responsible for communication with NCR-SARE and reporting. Indiana University will be responsible for fiscal administration of the grant.
The Indiana and Missouri researchers will communicate no less than monthly via video conferencing and weekly via email. Joint group meetings involving all key personnel will be held at least six times annually to review results and discuss plans. Members of the research team will visit each collaborating farm at least twice during the course of the investigation. Between visits, farmers will be contacted as needed for advice regarding the project (roughly 2x15 min annually). During field trials, Kang and Akotsen-Mensah will correspond with participating farms at least weekly, but as often as needed.
The Scheel lab has a data management plan on file with Indiana University and will adhere to it. Data shared between institutions will be stored within a secure shared folder that is password-protected and accessible only to the researchers. Electronic human subjects data will be de-identified and stored per the Scheel lab IRB protocol.
A recently constructed yeast strain corresponding to the D. suzukii Rbfox1 gene is hereafter referred to as Rbfox.687. The shRNA expressed in Rbfox.687 yeast corresponds to a target sequence present in the D. suzukii Rbfox1 gene, but NCBI blast searches failed to identify other organisms with an identical 25 bp target site. Heat-inactivated dried Rbfox.687 or control yeast was mixed with sucrose and fed to SWD in assays conducted in petri dishes. Significant fly mortality was observed in flies that had consumed Rbfox.687 (P<0.001 vs. control-treated flies, which survived, Fig. 1 Rbfox-Lab Assay) Dose-response assays revealed an LD50 of 1.669 ug/ul for Rbfox1 (Fig. 2. Rbfox-dose response curve). Moreover, as predicted by the lack of a target site in other insects, no significant mortality was observed in A. aegypti, A. stephensi, C. quinquefasciatus, and D. melanogaster that consumed Rbfox1 yeast (P>0.05;Fig. 3 Rbfox-non-target experiments). Combined, these results suggest that Rbfox.687 could facilitate precision SWD control.
Next, we explored the mode of action for the Rbfox.687 yeast. In the mosquito A. aegypti, consumption of yeast targeting the Rbfox1 gene resulted in loss of Rbfox1 transcripts. Rbfox1 transcript levels were therefore assessed in Rbfox.687-treated adult SWD flies. Silencing of Rbfox1 expression was verified through qRTPCR assays (Fig. 4. qRT-PCR), which revealed significant (P<0.001) silencing of Rbfox1 transcript in flies that had consumed Rbfox.687 vs. control yeast. In adult mosquitoes, consumption of yeast corresponding to the Rbfox1 gene resulted in loss of neural activity in the adult brain. Based on these results, it was hypothesized that silencing of Rbfox1 in D. suzukii would result in comparable neural defects. Treatments with Rbfox1 yeast resulted in reduction in nc82 levels in the adult SWD brain. Although levels of Bruchpilot, a marker of active synapses, were significantly reduced in flies that consumed the insecticidal yeast, no significant difference in TO-PRO nuclear staining levels was observed in the fly brain (P>0.05). These results, which were similar to those observed in A. aegypti, suggested that loss of nc82 signal likely results from a reduction in neural activity rather than loss of neural density.
Next we explored potential systems for RNAi yeast delivery to SWD flies in the field. Coca-Cola, which is an excellent attractive sugar bait for many insects, was recently identified as one of the most attractive sugar baits for mosquitoes. When Coca-Cola was mixed with Rbfox.687 yeast or control yeast and evaluated in SWD, significant mortality was observed in the Rbfox.687 flies (P<0.001 vs control-treated flies). In our mosquito studies we have been exploring the use of a soda bottle feeder that delivers RNAi ATSBs to mosquitoes. The system, which is easy to contruct with a 12 oz. soda bottle, was used in the development of a soda feeder that delivered a constant supply of soda sugar bait that constantly hydrated the yeast. This feeder was assessed in simulated field trials conducted with SWD. Although control-treated flies did not die in these assays, significant death was observed in Rbf.687-treated flies (Rbfox-CokeFeederAssay; P<0.001 vs. control-treated flies). Alternative beverages were assessed in the feeder system, (including cherry juice and apple juice), but the performance of Coca-Cola was superior.
Although the RNAi yeast + Coca-Cola ATSB performed well in lab trials, we wanted to explore its ability to attract SWD in a natural environment. Preliminary studies suggested that this mixture alone did not attract many SWD to sticky traps, which were placed adjacent to the ATSB. However, the addition of an SWD-specific pheromone to the soda resulted in excellent SWD attraction. This will be further investigated next summer.
In addition to these laboratory assays, we also visited three blueberry farms, where we met and discussed the farmers' experiences with SWD. We also described our RNAi yeast ATSB system, which was generally of interest to them, particularly for John Nelson, an organic farmer who was intrigued by the species-specific technology. AJ Walters discussed his farm's signifiant concerns about SWD, which causes them to spray throughout the season. We look forward to continuing to engage the farmers as the project progresses, and they have agreed to participate in the webinar that we will host toward the end of the project.
This research demonstrates that a species-specific RNAi yeast insecticide can serve as a highly toxic component of an ATSB that effectively kills D. suzukii, an invasive fruit and berry pest. The yeast ATSB, which silences the Rbfox1 gene resulting in severe neural phenotypes, can be prepared using soda delivered in an inexpensive easily constructed soda bottle feeder which effectively killed SWD under simulated field conditions. RNAi yeast insecticides may represent a new class of effective, yet biorational insecticides that can be used in integrated pest management programs for control of this destructive insect pest. Feedback from local farmers is helping us to consider if the RNAi yeast is something that would benefit their farms. Our interactions to date have been very positive and have also helped us think about when and where the insecticides might be deployed.
Education
Project Activities
Educational & Outreach Activities
Participation Summary:
Learning Outcomes
- A webinar in which these learning outcomes can be assessed will be held near the conclusion of the project.
Project Outcomes
0 (not expected until the end of the project)