Non Technical Summary
The European spongy moth (Lymantria dispar dispar ) (ESM) is a non-native insect pest with larvae that feed on leaves of over 300 broad-leaved species, including forest, shade, ornamental, and fruit trees as well as shrubs. The spongy moth defoliated over 98 million acres of U.S. forests from 1924 to 2018. Current biological insecticides used in spongy moth control have not been efficacious enough and chemical insecticides cause ecological damage. The ranking of the spongy moth as the third most expensive invasive insect in the world, with an estimated annual 3.2 billion dollars cost just in North America, is indicative of the impact of this lepidopteran pest. NanoSUR's demonstrated RNAi-based insecticides technology will result in the development of a selective, affordable, safe, and effective insecticide suitable for the large-scale control of the ESM. In anticipation of a possible break-out spread of the more devastating Asian Spongy Moth (ASM), we will also propose RNAi treatments designed to control it. The successful execution of this grant will provide an RNAi trigger formulation ready to initiate field testing in the forest. Thus, we will accomplish our overall goal of creating a product for broad use in controlling ESM and eventually ASM, that is effective, environmentally friendly, and safe to non-target organisms including other lepidoptera present in the forest such as the monarch butterfly. To accomplish this technical challenge, we will build on prior research funded by NSF and USDA work, in collaboration with the Beltsville USDA Agricultural Research Station.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	3110	1130	100%

Knowledge Area
123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
3110 - Insects;

Field Of Science
1130 - Entomology and acarology;

Goals / Objectives
In Phase I NIFA Award 2021-33610-34700 we demonstrated that dsRNA targeting two different Spongy moth (ESM) genes modified with NanoSUR's technology resulted in efficacious RNAi triggers against the ESM larva. In Phase II, we will build on these results with the goal of developing an efficacious and selective formulation for commercial spraying to control ESM in forests. In anticipation of a possible break-out spread of the more devastating Asian Spongy Moth, we will also propose RNAi treatments designed to control it. To that end, we will take advantage of both ESM and ASM genomes which have been recently sequenced by USDA ARS IIBBL. (Spark, 2021) (Hebert, 2019) All this will be accomplished through three objectives. First, we have identified 6 additional ESM genes that regulate larval development to be studied together with EGM locus 365 and EGM P450. We will select 8 sequence segments predicted by our algorithms to result in MdsRNAs with the highest gene depression translated into efficacy in the artificial diet (AD) assay. Second, we will optimize the efficacy of the best 2 MdsRNAs compositions from Objective 1 in a leaf assay to the point where the technology is economically attractive to control ESM. Third, we will focus on the scale-up and formulation of the optimized MdsRNA, aiming at setting specifications required for their large-scale manufacturing and conducting greenhouse testing on the formulations. Achievingthese objectives will provide an RNAi trigger formulation ready to initiate field testing in the forest and we will accomplish our overall goal of creating a product for broad use in controlling ESM and eventually ASM, that is effective, environmentally friendly, and safe to non-target organisms including other lepidopteran insects present in the forest such as the monarch butterfly. The Technical Objectives are as follows:Objective 1. Prepare MdsRNA targeting insect fecundity and/or larval development in ESM -and also ASM- that result in the highest ESM control and gene knockout consistent with an RNAi mechanism, in an artificial diet (AD) assay. Here we plan to select a set of 16 ESM/ASM sequences from 8 genes targeting insect fecundity and/or larval development. We will prepare the corresponding MdsRNAs using the most efficacious modifier group found in Phase I and will test the effect of the MdsRNAs on ESM larva mortality in an artificial diet assay and for gene expression knockdown using qPCR procedures. Objective 2. Optimize the efficacy of the best 2 MdsRNAs compositions from Objective 1 to obtain the highest efficacy against ESM in a leaf bioassay. In objective 2 we will evaluate the top 2 MdsRNAs in a leaf bioassay, a more predictive test of field performance. We plan to study the effect of different modifying groups, their %modification, and adjuvants on the efficacy of the MdsRNAs in the leaf disc assay. In vivo, evaluation using ASM will not be performed within this Phase II Project. Objective 3. Scaleup and testing of the best MdsRNA from Objective 2 to control ESM in the greenhouse. We will use the preparation route developed during our NSF Phase II award to scale up the MdsRNAs from Objective 2 in enough quantity to allow their formulation and testing for ESM control in the greenhouse. We anticipate using the learnings from Objective 2 on efficacious dosage and stability in the insect gut to design focused greenhouse studies that will allow the rapid identification of the best formulation for the control of ESM. The greenhouse testing will be done with oak or poplar saplings in plant growth chambers or in a small (8' x10') standalone greenhouse.

Project Methods
Objective 1. Prepare MdsRNA targeting insect fecundity and/or larval development in ESM -and also ASM- that result in the highest ESM control and gene knockout consistent with an RNAi mechanism, in an artificial diet (AD) assay.Selection of ESM and ASM sequences and Synthesis of MdsRNAs.Approach: Our approach for the selection of new additional sequences for the RNAi trigger candidates will be based on the functional orthologs which have been successfully used as efficient RNAi targets in other lepidopteran species by us and by others as judged from the literature data. Some of the genes suggested here are expressed in insect midgut. Silencing midgut genes usually has a very profound effect on larva growth and development (Saxena, 2021). We have selected six additional EGM genes. The corresponding MdsRNAs will be synthesized and purified using the same synthetic and analytic methods as in Phase I in our laboratory in Madison, WI.Expected Outcomes: Given our Phase I results and our experience in preparing MdsRNAs, we anticipate that we will successfully prepare the proposed 16 MdsRNAs in enough quantity and with >90% purity to be tested in all of Objective 1 biological assays.Biological testing of the MdsRNAs Approach: The insecticidal activity of MdsRNAs 1-16 will be tested in an artificial diet assay to measure combined mortality and stunting for the exposed ESM larvae (Doane, 1981). ESM larvae will be hatched from eggs obtained from a colony at USDA/APHIS, OTIS, MA, USA. Bioassays will be conducted with freeze-dried artificial diet pellets in wells of a plastic bioassay tray.Biological testing of the MdsRNAs Approach: The insecticidal activity of MdsRNAs 1-16 will be tested in an artificial diet assay to measure combined mortality and stunting for the exposed ESM larvae (Doane, 1981). ESM larvae will be hatched from eggs obtained from a colony at USDA/APHIS, OTIS, MA, USA.A cohort of 8 larvae will be fed each individual dsRNA treatment or control and mortality will be determined for each cohort to provide 8 data points daily for each experimental sample or control. Cumulative %mortality plus stunting will be calculated for the duration of the experiment. We will also run the same protocol starting with 2 nd or 3rd instar larva. The successcriteria for MdsRNAs in the screening assay is a %mortality greater than 50%. The MdsRNAs that achieve greater than 50% mortality will be then tested for the corresponding gene expression on 1st instar larvae alive using droplet qPCR.Expected Outcome:We are confident that we will find at least 2 treatments out of the core set of 16 proposed MdsRNAswith a %mortality greater than 50%Milestone 1: Two MdsRNAs from the AD screening advanceto in planta studies.Objective 2. Optimize the efficacy of the top 2 MdsRNAs compositions from Objective 1 to obtain the highest efficacy against ESM in a leaf bioassay.Approach: We will optimize the efficacy of the top 2 MdsRNA compositions using a standard leaf disc assay. This in planta assay will provide us with a first look into the requirements for the development of formulation prototypes. We will identify a modifying group and adjuvants that will produce a MdsRNA formulation with the highest ESM efficacy, i.e., %mortality and egg mass reduction. We will learn about the acceptance of the formulations by ESM. We will also learn about the delivery of the formulation by spraying and make a first estimation of the predicted product rate and volume per acre needed to control ESM.To evaluate the insecticidal activity of MdsRNAs we will perform a standard leaf disc larva assay bioassay (Asano et al., 1993).Experiments will be run until pupation. Cumulative %mortality plus stunting will be calculated for the duration of the experiment. For triggers affecting fecundity, we will also dissect the pupal stage females of controls versus treated for comparison of the egg mass.Expected Outcomes: Given the criteria used to advance the candidates from Objective 1 (mortality greater than 50% in the AD assay) we are very confident that we will find at least one MdsRNA formulation with mortality greater than 60% in the leaf assay. If we see less than 60% mortality in the leaf assay, we will vary the length of the dsRNA target sequence.Milestone 2. Identify a MdsRNA formulation that produces greater than 60% mortality when sprayed in the leaf assay.This level of activity will ensure that the candidate formulation can control ESM in the greenhouse at commercially relevant product rates.Objective 3. Scaleup and testing of the best MdsRNA from Objective 2 to control ESM in the greenhouse. Small greenhouse studies will be carried out to assess the potential of MdsRNA(s) to reduce feeding damage on hardwood trees. The most effective (i.e., %mortality, developmental delay, and/or egg mass reduction) MdsRNA candidate(s) as defined by laboratory leaf disk bioassays outcome will be scaled up to optimize the efficacy of MdsRNA, modification, and formulation composition in small greenhouse assays. Healthy deciduous hardwood Quercus species oak seedlings will initially be grown from acorns within laboratory plant growth chambers. If Quercus species are unavailable, then local Poplar (Populus species) hardwood saplings will be used instead. For the scale-up of the MdsRNAs, we will use the method developed by NanoSUR during NSF Phase II Award 1853008 to produce materials for small plot pesticide field tests. Once the most active formulation in the greenhouse is identified NanoSUR will meet again with the BPPD group at EPA to discuss the studies requiredto obtain the registration for the product.Expected Outcomes.The implementation of thedevelopment and pre-commercialization plan includingpilot plant production, aerial spraying of the formulation on forests,andthestudies for Regulatory will be initiated for the MdsRNA formulation candidate.Milestone 3. Deliver a detailed development and pre-commercialization plan including Regulatory activities for the selected MdsRNA candidate to control ESM in forests by spraying.