Progress 07/01/20 to 06/30/22

Outputs
Target Audience:The work completed by Frontier Scientific was designed to advance reasearch in the crop protection field by provideinga novel solution for the laboratory rearing and bioassay requirements of the western corn rootworm (WCR) pest complex. Prior to our work, no commercially available media existed to consistently and effectively rear WCR larvae without the addition of difficult to source host plant material, maize roots. Broadly speaking, the audience for our work is any researcher--whether they be at a small college or a major agchem company--that is working with WCR. In terms of market share, our main target are those larger companies that are screening thousands of compounds and proteins annually to determine both the insecticidal propoerties and relative safety of a given product. Our work allows for a standard media to be commercially available to any researcher and provides a previously not attainable oppurtunity to compare data sets from different groups--an advancement that we hope aids in the continuing battle against resistance development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The goal of this SBIR was to internally optimize the WCRMO-2 diet for commercializtion. This formulation is currently available for purchase through the Frontier online catalogue, www.insectrearing.com. Outreach has been accomplished through our normal communication channels, i.e. email and phone communication. As we continue to make advances, oureach efforts will proceed in-kind. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our phase I research plan was completed as planned and led to several key developments in furthering our understanding of the unique features of our commercial insect diet for the Western Corn Rootworm (WCR), Diabrotica virgifera. As stated in our original proposal, the target impact of our developments through phase I was to optimize, refine and develop novel techniques to better the efficacy of a WCR diet that excludes the difficult to procure corn root powder, which is a critical constituent of existing medias used in the crop protection field. WCR, a soil inhabiting species, is difficult to treat with above ground spraying techniques making it difficult to adequately control. As a result, transformed corn plants are utilized in the US market that express insecticidal proteins within the host plant roots, however, there exists a constant concern of resistance development. To mitigate this concern, regular laboratory testing is required to monitor resistance and to develop new protein combinations. Costly growing operations are often needed to complete this work as there is yet to exist a viable artificial media to consistently rear WCR. Our formulation is the first to be commercially available and our efforts in this SBIR grant led directly to furthering our understanding of the formulation and determine better ways to optimize it. Below outlines our accomplishments per stated objective--proprietary data is available in the final report submitted to NIFA: 1.Develop pasteurization parameters that provide a sterilization process for the production of WCRMO-2 We were attempting to develop optimal pasteurization protocols for our novel Western Corn Rootworm (hereafter WCR) diet WCRMO-2. Frontier utilizes a specially designed 1A dairy standard pasteurization (flash sterilization) system that was commissioned and built for the processing of insect medias. Unfortunately, like many composite formulations, insect media is inherently dynamic in its physical properties meaning a successful pasteurization protocol for one formulation may be entirely inadequate for another. Many variables are at play within a media that must be well defined when designing a particular pasteurization protocol. Of particular note with WCRMO-2 is its sensitivity to high temperatures resulting in significant nutrient denaturing and observable decreases in overall efficacy when presented to WCR larvae. Frontier's complexflash sterilization system can be distilleddown to two essential control dials--1) Flowrate & 2) Temperature. As the temperature increases, we can also increase the flowrate which will result in a lower "dwell time" for the media at the high temperature.To determine this optimum treatment without an overly expansive and unwieldy experimental design we opted to employ response surface modeling. A series of experiments were performed to determine the effects of thermal (temperature) and time (flowrate) exposure on the quality of WCRMO-2 diet. After completing our study, it was determined thatexposure to high temperatures (110-141 °C), even if constrained to a small window of time (0.9-2.3 s), had significant deleterious effects on the quality of WCRMO-2 diet for feeding WCR larvae. The WCRMO-2 diet exposed to the high temperatures for the brief time exposure resulted in larval weight significantly smaller compared to the control diet, WCRMO-2 diet made at a temperature of 65 °C. As a result, it was determined that flash sterilization is not a suitable tool for preparing this media. 2.Evaluate feasibility of antibiotic removal from WCRMO-2 formulation through implementation of pasteurization techniques Currently, WCRMO-2 formulation contains two antibiotics (streptomycin and chlortetracycline) that are widely used in many other insect diets as the important antibacterial agents.WCRMO-2 diet was prepared in 96-well plates with varying rates of the two antibiotics. Life history parameters and evidence of diet contamination (bacteria)were evaluated after the larvae were reared on the WCRMO-2 diet for 10 days. We found that exclusion of the antibiotics resulted in nearly 100% bacterial contamination after 5 days post larval infestation. The increases in the antibiotics (up to 4x the standard rate) led to the significant reduction in larval growth and development. It has been determined that the exclusion of antibiotics is not feasible for this formulation. 3. Evaluate diet stability during transit and short-term storage A.Short-Term Storage WCRMO-2 diet was prepared in 96-well plates and stored at 5 different temperatures (-20, 4, 22, 25, and 33 °C) for 5 durations (1, 7, 14, 21, and 28 days). Insect artificial diet assays were conducted to evaluate the effects of the temperatures and the short-term storage on diet quality at each predetermined duration. The stability of WCRMO-2 diet was assessed via the evaluation of life history parameters (weight, molt, and survival) of WCR larvae reared on the diets.The temperatures, time storage, and their interactions had significant deleterious effects on the stability of WCRMO-2 diet for feeding WCR larvae. The WCRMO-2 diet exposed to higher temperatures > 4 °C resulted in delays in larval molting and reductions in larval weight. Larval survival was over 90% regardless of the temperatures evaluated, except at the highest temperature of 33°C where significant reductions in larval mortality were over 45%. Furthermore, the WCRMO-2 diet stored over 7 days had significantly negative impacts on larval molting and weight, whereas increases in larval mortality were observed after 14 days post storage. Taken together, with the diet ingredients in WCRMO-2 formulation, the storage temperatures less than 4 °C yielded the highest larval performance on WCRMO-2 diet, while the lengths of storage less than 7 days did not have significant impacts on the stability of WCRMO-2 diet. B. Transit Frontier made 96-well plates containing WCRMO-2 diet and placed them in 5 distinct boxes. These boxes wereshipped from Frontier (Newark, DE) to USDA-ARS laboratory (Columbia, MO) over approximately 24 hours.Each box was filled with either hot packs, cool packs, ice packs, or dry ice to mimic varying potential environmental conditions experienced during transit. During transit, the box temperatures were monitored using a data logger. The diet plates were exposed to 5 different averaged temperatures of 6.6, 9.7, 18.8, 22.5, and 25.2 °C with major temperature exposure from -2 °C to 27 °C . After receipt, all plates were visually examined andwere observed as having adequate physical properties (i.e., gel matrix intact, constituent separation, excess moisture, desiccation).After exposure to different temperatures during transit, the stability of the WCRMO-2 diets plates stored at 4 °C over 5 different time intervals of storage (1, 7, 14, 21, 28 days) was assessed by the life history parameters (survival, molting, and weight) of WCR larvae. Results indicated that the exposure to different temperatures from -2 °C to 27 °C within 24 hours during the transit followed by the storage at 4 °C had no significant impacts on the quality of WCRMO-2 diet assessed by life history parameters measured (survival, molting, and weight) 4. Evaluate compatibility of WCRMO-2 to detect differences in susceptibility to Bt protein Diet toxicity assays were performed to evaluate the compatibility of WCRMO-2 with a Bt protein (i.e., Cry3Bb1) on Cry3Bb1-resistant and susceptible strains.The WCRMO-2 diet was compatible with the Bt toxin tested (Cry3Bb1 protein). The LC50value, a lethal concentration that kills 50% of the insects tested, was> 100-fold different between susceptible and resistant strains of WCR. These results revealed that the diet toxicity assays with WCRMO-2 diet can differentiate the Cry3Bb1-resistant strain from the susceptible strain.

Publications

Progress 07/01/20 to 02/28/21

Outputs
Target Audience:The Western Corn Rootworm (WCR) is an economically significant pest of corn in the United States resulting in an estimated $1-2 Billion inloss annually. As it stands currently, the most promising method for control of this pest is through the identification and implementation of Bt proteins which are insecticidal by nature but are not harmful to humans. To preservethe efficacy of this important control measure for future generations, it is critical to understand how insects develop resistance to these proteins and how we can mitigate resistance development. The best way of accomplishing this feat is through direct comparative studies looking at the efficacy of these proteins and how/when resistance will develop. In order for a comprehensive resistance management program to be effective it is important to be able to compare data between researchers, institutions, and companies. Currently, there is not a commercially available WCR diet that allows for these studies to be directly compared. Further complicating this issue is the need to utilize low volume microtiter plates for adaptation to High-Throughput Screening (HTS) systems which presents a bottle neck in the time and effort it takes to fill plates. The broader target audience for our research efforts during Phase I is any researcher working on the Western Corn Rootworm pest complex. More specifically, we are focused on the agriculture research industry that expends significant capital into this area of research. We can identify a subset of this audience as divisions within this industry focused on, insecticide development, resistance and safety management, and/or insecticidal seed traits for WCR. Through our active work as a commercial insect diet specialist and insect pest rearing provider, we have extensive knowledge of the needs of this industry based on both the past experience of our leadership and through ongoing discussions with our customer base. It has become apparent that the solutions being investigated during our grant research may prove to be hugely impactful on the crop protection community as a whole. The interest of our audience to this product will be three-fold--1) A premium formulation that outperforms existing medias and allows for the elimination of costly to produce supplemental plant tissue; 2) A standardized media that allows for the direct comparison of critical resistance management data required by the EPA; and 3) A commercial solution to the bottleneck created by the tedious and inherently imperfect manual fills of microtiter plates. We plan to leverage the relationships we have built with our clients in this target audience to push for the adoption of this new commercially available WCR diet. We are optimistic that the adoption of this product across the industry will result in a measurable net-gain to the crop protection community and will provide much needed support to the ever-growing need for food security in the United States. Changes/Problems:Due to market volitility as a result of the COVID-19 pandemic, we opted to move the procurement of automation systems to Phase II and instead focused our efforts on qualification, development, and optimization of our premier Westrern Corn Rootworm diet formulation. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Our efforts during Phase I are to better understand the optimal conditions required of our WCRMO-2 formulation to provide an eventual commercial product (Phase II). As such, it is premature for us to complete any audience outreach until our work is complete and our premier product of aseptically filled WCR assay and rearing plates are ready for market launch. Our data thus far is significant and compelling and will result in publication in the coming months which will reach broadly across the research community. What do you plan to do during the next reporting period to accomplish the goals?In the next two months we intend to work concurrently on Aims 2 and 4. We will test our lower temperature pasteurization parameters to evaluate feasibility of removing antibiotics from the formulation. Although critical in preventing contamination in insect medias, antibiotics are generally understood to have a less than desirable effect on the health and development of insects. If possible, the removal of these antibiotics is preferred and will yield a better performing formulation. We will test formulations without antibiotics with and without our pasteurization protocols employed and will score for pre and post infestation contamination. We have long established tolerance thresholds for contamination that must be met to determine feasibility of antibiotic removal. Aim 4 consists of testing known insecticidal proteins on our new formulation. Datasets will be compared with previous tests on other formulations in existence to determine the compatibility of WCRMO-2 with these toxicity assays. This will be a relatively straightforward aims as the general protocols have been developed and we are merely testing our formulation for efficacy in these studies. All though straight forward, the efforts here will be critical to the eventual product. We must prove convincingly that our formulation can perform with existing procedures. It is not reasonable for us to expect researchers to retool theirlong-standing studies to meet our formulation needs. It is imperative that we lower the barrier of entry for product, not increase it. This means we must provide a formulation capable of performing these studies as they exist today--this is a goal we are confident we can achieve. Aim 3--evaluation of shelf-life and transit conditions will take time and is planned for completion in the early summer, regardless of our successful award for Phase II.

Impacts
What was accomplished under these goals? We structured our proposal with a significantburden on Aim 1 as our subsequent aims rely on the procedure developed here. We have made significant advancements in developing an adequate pasteurization procedure for WCRMO-2 that is equal to or better than our gold-standard bench top formulation. Previous studies of ours have indicated a deleterious effect of high temperatures on the efficacy of the media. As such we needed to qualify that observation in the pasteurization system. This initially led to several important questions, the most important of which was, how does varying times at high temperatures impact the efficacy of the media. In other words, does the media experiencing high temperatures for several minutes results in the decrease in efficacy? What if we were to reduce this time to seconds rather than minutes (a function the pasteurization system uniquely performs well)? Through a series of studies, we determined that time, or 'dwell time' as we refer to it, does not have a particularly significant impact on the efficacy. This required us to lower the overall temperature of the pasteurizer to within a range that we found to be optimal--150-175. As a result of our studies asecond proprietary solution was considered. This new proprietaryformulation advancement resulted inthe statistically best performing WCR larval diet ever evaluated by the USDA-ARS-BCIRL western corn rootworm research lab. This significant improvement is a direct result of this Phase I SBIR and will lead to improved WCR larval assays for the entire agriculture industry. In addition to bioassays, the ultimate goal of a larval media capable of continuous rearing without the addition of corn tissue supplements is now within reach.

Publications