Progress 07/01/18 to 02/28/19

Outputs
Target Audience:Product users will be large- and small-scale crop growers who are losing crops due to corn earworm feeding. Since our product must receive EPA approval, we will not be ready for market until 2024. However, we plan to reach farmers through farming organizations and through collaborative efforts with current biopesticide companies that already have customers. We have started discussions with the biopesticide company AgBiTech, the President of American Farm Investors Brian Luftman, and with a USDA Bt-resistance facility. We have introduced our technology through presentations at the MassBio BioEntrepreneur Bootcamp and Pitch Contest (Nov 2018), Kentucky state matching fund introduction of awardee's (February 2019), and the University of Kentucky Entrepreneur Showcase (March 2019). Changes/Problems:In Objective 1, we planned to inoculate caterpillars with our KS-45 nudivirus, mate them as adults to fertile mates, and collect a generation of eggs that will develop into sterile adults. Unfortunately, although the inoculation was 90% effective, only 67% of eggs developed into sterile adults. Based on previous experimentation though, we knew that injecting the KS-45 nudivirus into adult moths would generate a colony of almost entirely sterile moths. Indeed, 98% of the children became sterile adults, and the remaining 2% were infected with the virus. Viral dose is a key criteria to make the injection protocol successful, though, and we were able to define the appropriate KS-45 injectable dose. In Objective 2, we fed moths fresh KS-45 nudivirus daily for 9 days and collected the last laid eggs, hoping they would develop into sterile adults. We found that all of the children were infected with virus, but only 21% were sterile. We decided to next test all of our most successful KS-nudivirus strains (KS-3, KS-45, KS-51, and KS-52). Four colonies of moths were fed a particular virus daily for 9 days, and eggs were collected on days 3, 6, and 9 and allowed to develop into adult moths. 11% of KS-3, 19% of KS-52, 23% of KS-45, and 35% of KS-51 insects were sterile. The highest percent of sterile offspring were found in day 6 insects. These data indicate that the viral solutions were probably losing stability during storate at -20C and that the KS-51 nudivirus performed the best. Thus, in future feed experimentations, KS-51 will be the product virus. Objective 3 population reduction experimentation progressed as plan. This experiment is currently being repeated in triplicate but testing only the KS45 10:1, KS-45 1:3, and untreated colonies. What opportunities for training and professional development has the project provided?A new technician assisted in these experiments. She was trained in rearing and analyzing insects. The PI on the grant started professional development in entrepreurship. She attended a BioEntrepreneur bootcamp and pitch contest in Boston and performed well (was the runner-up in the contest). She has practiced these skills by discussing the project with other scientist, entrepreneurs, and potential investors within the community through the University of Kentucky's Innovation center. She has applied to the Boston 2019 MassConnect accelerator program. How have the results been disseminated to communities of interest?We do not plan to reach out to additional farmers until we undergo the EPA regulatory studies. We have, however, discussed the technology with biopesticide companies (ex. AgBiTech), the President and Founder of American Farm Investors Brian Luftman, and a USDA laboratory. Our most plausible exit strategy is that a major crop protection company will acquire our technology. However, these specific collaborations will provide insight into the regulatory process, marketing to biopesticide users, obtaining investors, and rearing millions of infected insects. Each of these collaborations required success from our Phase I study. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The corn earworm caterpillar eats crops and is the primary insect pest of soybean in the south and of sweet corn in the northeast. The corn earworm also attacks forty-five other crops, causing annual losses and control costs in excess of $2 billion in the U.S. Furthermore, the corn earworm has become resistant to some of the major pesticides and genetically modified plant toxins. The overall goal of this project is to develop an alternative strategy whereby corn earworm colonies are reduced by a moth-specific, sexually transmitting sterilizing virus. The KS-45 nudivirus strain is specific to the corn earworm, is unstable outside the insect body, and safe for humans and non-target insects. Our nudivirus technology can help crop growers protect their valuable crops from being eaten by corn earworms. Our technology can also be used to delay Bt toxin resistance, helping maintain the efficacy of transgenic crops. Objective 1, to optimize production of sterile moths using an inoculative approach. Our product is KS-45 nudivirus-infected sterilized moths. These moths will be released onto farms to mate, infect, and sterilize wild corn earworm moths. The goal of this objective is to develop a protocol for manufacturing high levels of sterile infected insects. The KS-45 virus is sexually transmitted between moths and passed from a mother to her eggs. Here, we infected corn earworm caterpillars and moths with the KS-45 nudivirus, bred the infected moths to fertile mates, and collected infected eggs. If successful, > 95% of eggs will develop into sterile moths. By infecting caterpillars with KS-45 nudivirus, 67% of eggs developed into sterile moths. In contrast, when infecting moths with KS-45 nudivirus, 98% of eggs developed into sterile moths, and the remaining 2% of moths were infected with the nudivirus. Calculations of time and effort for this method indicate that 1,000,000 sterile moths could be produced from injections of 1,500 females moths, which would take one person about 6 h if injecting four moths per minute. Our materials to rear one sterile corn earworm cost 8¢. These results enable us to develop a cost-effective procedure to generating large numbers of sterile moths that carry the KS-45 nudivirus. Objective 2, to evaluate oral routes of infection for large-scale production of sterile corn earworm moths. A virus delivery system based on feeding stations that infect feral adult moths would eliminate the need to rear, infect, and release live insects, thereby reducing costs and logistical issues associated with producing sterile insects. To investigate the potential of this approach, we fed moths different nudivirus strains in a sugar solution containing a viral stabilizer, and collected their eggs on days 3, 6, and 9. Our goal is that > 95% of eggs will develop into sterile moths. KS-51 performed the best of the four nudivirus strains tested. When the KS-51 nudivirus was fed to adult moths, 35% of their eggs developed into sterile adults (5% day 3, 48% day 6, and 17% day 9 insects were sterile). While this is below our goal of 95%, the percentage of sterile insects exceeded previous results (the highest percentage was previously 24%). Furthermore, we found that the virus is unstable during storage in the sucrose solution after 6 days. In our assessment, it is only possible to develop a viral cocktail product if it is stable. Ongoing studies are looking to stabilize the nudivirus. Objective 3, to test efficacy of KS-45 sterile moths generated in Objective 1. Our product is KS-45 nudivirus-infected sterilized moths. These moths will be released onto farms to mate, infect, and sterilize wild corn earworm moths, causing a significant reduction in corn earworm insects. The goals of this objective are to determine if releasing KS-45 sterilized moths could reduce a wild colony and to determine how many sterilized moths need to be released to have that reduction. In this experiment, seven Generation 0 "wild" colonies were established with 10 uninfected males and 10 uninfected female moths. In three of these colonies, KS-45 males were released into the colony. In colony 1, one sterile KS-45 male was released (10:1 uninfected to infected ratio); in colony 2, ten sterile KS-45 males were released (1:1 ratio); in colony 3, thirty sterile KS-45 males were released (1:3 ratio). A naturally-occurring nudivirus strain (the "wild-type" strain) was also released into three control colonies at the same ratios. The last colony served as an untreated control. Generation 1 eggs were collected every 2 days until egg production ceased. As adults, some generation 1 moths were allowed to mate and lay generation 2 eggs; other adults were dissected to determine if their reproductive tract was malformed and they were sterile. Our goal was that at least one KS-45 colony would have a reduction in egg production (i.e. lay less eggs and in less days than the untreated colony) and contain infected and sterile moths in each generation. The wild-type colonies had a slight reduction in egg production in Generation 1, but egg production was similar or better than the untreated colony in generations 2 and 3. The KS-45 1:1 colony did not have a significant reduction in egg production until generation 3. In the KS-45 1:3 colony, three KS-45 sterile males were released for every uninfected male. In generation 1, 67% of the insects were sterile and their egg production correspondingly was significantly reduced. Unexpectedly, in generation 2, only 27% of the insects were sterile and egg production was higher than the untreated colony. In generation 3, the sterilizing effect resumed, and egg production was significantly lower than the untreated colony. Although, the 1:3 colony showed an immediate response to a release of sterile KS-45 infected males, it was actually less effective than the 10:1 colony in reducing egg production. Thus, we do not have evidence that releasing a super-abundance of sterile moths is beneficial or necessary. In the KS-45 10:1 colony, one KS-45 sterile male was released for every 10 uninfected male. The 10:1 colony showed a 20% egg reduction in the first generation, but a significant 95% egg reduction and collapse in the population relative to the untreated control was observed in the second generation. This continued into the third generation where a 99.7% reduction in egg production was observed. This colony reached our goal criteria. To track virus infections in the 10:1 KS-45 colony, we dissected all moths to determine if their reproductive tract was malformed (indicating sterility) and harvested the abdomens from each of the fertile moths. We then isolated viral DNA, and performed PCR to determine if the fertile moths were infected with the nudivirus. The data show that over 75% of moths were infected in generation 1 while ≥ 95% of moths carried the virus in generations 2 and 3. These data indicate that KS-45 strain is an effective agent for reducing corn earworm populations in laboratory cage studies at low release levels. In summary, Phase I experiments have shown that it is possible to produce large numbers of sterile insects at minimal cost above what is required to simply rear the insect. We have also confirmed that injection of female moths and raising her sterile progeny is a better approach than infecting caterpillars. Feeding virus to adult moths will produce infections, but the instability of the virus is a significant problem that must be overcome before this approach can become feasible. And finally, virulent KS-nudivirus strains can reduce corn earworm populations by over 99% under the conditions of our experimentation. Taken together, these experiments support development of time- and cost-effective production systems for the manufacture of sterile corn earworm insects and the use of these sterile, virus-infected insects to suppress corn earworm populations.

Publications

• Type: Websites Status: Other Year Published: 2018 Citation: https://lepidext.com

Progress 07/01/18 to 02/28/19

Outputs
Target Audience:Product users will be large- and small-scale crop growers who are losing crops due to corn earworm feeding. We can reach our target audience through state extension groups that collect state-by-state corn earworm population data and could help market the product by informing users. We can also reach our target audience through agricultural companies that may want to commercialize the product, and through social media such as Facebook farming groups. Our data collected in this grant will be used to obtained an experimental use permit from the Environmental protection agency, and vetted by patent examiners to approve an international patent. Changes/Problems:Our goal for objectives 1 and 2 was to produce a generation of sterile moths using two different approaches. However only 67% and 37% of the test generation was sterile for the respective objectives. We have changed the approach in Objective 1. Instead of inoculating individual insects at 3rd instar larvae to make them sterile, we are injecting female moths and collecting her sterile offspring (Objective 1 experiment 3). Our previous data suggest any offspring laid after oviposition day 1 will be sterile. Thus, we are confident this approach will produce a generation of sterile moths. Due to the low percent of sterile offspring generated in objective 2 (37%), we did not test population suppression with a release of these insects in objective 3 as proposed. Instead, we are using the generation with 67% sterility (Objective 1 experiment 2) and will use the generation from Objective 1 experiment 3, which we believe will be 100% sterile. We have already seen good results from the experiment 2 insects, so we expect superior results using experiment 3 insects. What opportunities for training and professional development has the project provided?A technician is being trained how to rear the corn earworm and how to evaluate sterility in the adult moths. How have the results been disseminated to communities of interest?Without EPA approval, we cannot market our nudivirus as of yet. But we are beginning to use this data to discuss with potential investors and agricultural companies. What do you plan to do during the next reporting period to accomplish the goals?By the next reporting period, we will have determined how many sterile moths we can generate using breeding approach (Objective 1 experiment 3). We have counted the number of F1 eggs laid each day by 10 females injected with the KS45 virus. The hatched F1 caterpillars are currently be reared to adult moths, when they will be evaluated for sterility. After this evaluation, we will be able to know how many sterile insects this injection approach produced. This number will be used to determine the cost of procedure for generating sterile moths. By the next reporting period, we will also determine how quickly and to what level we can suppress a corn earworm colony by releasing KS45 sterile males (Objective 3). We have already seen a next generation insect suppression by releasing KS45 males at a 3:1 ratio (to uninfected males). This suppression should be amplified in the next generation. We must still evaluate the next generation insects to determine how well the virus is propagating through the colony.

Impacts
What was accomplished under these goals? The corn earworm caterpillar is a destructive pest that eats over 46 cultivated crops and cost U.S farmers over $2 billion each year. Our HzNV-2 nudivirus can sterilize moths and eliminate subsequent generations. Crop growers will be greatly benefitted by the removal of one of the most pesticide- and trangenic crop-resistance insect pests. The goal of objective 1 was to optimize production of sterile moths using an inoculative approach. We inoculated young larvae with KS45 HzNV-2 virus using a simple pin prick approach. The virus deteriorated the reproductive tract in 50% of the inoculated larvae; These larvae became sterile adults. Of these adults, we released the KS45 inoculated male moths into a population of uninfected female and male moths, and we were able to produce a next generation of moths in which 55% were sterile. This experiment was repeated. We inoculated young larvae with KS45 HzNV-2 virus using the same pin prick approach, but with fresher and more virus. The virus deteriorated the reproductive tract in 90% of the inoculated larvae. These larvae became sterile adults, and of these insects, the KS45 inoculated male moths were released into a population of uninfected female and male moths. 67% of the next generation insects were sterile, and 100% were infected with the virus. Since our goal was to acheive a next generation with 100% sterility, we are continuing with a different approach. We injected KS45 virus into female adult moths and allowed them to mate with uninfected males. Eggs were collected daily until no more eggs were laid. Each hatched larvae will be reared to adults and the percent sterility will be determined. Currently, this generation of insect is in the caterpillar life stage. The goal of objective 2 was to evaluate oral routes of infection for large scale production of sterile H. zea insects. Each mating chamber housed 20 moths, which were fed per diem sucrose solution or sucrose solution containing wild-type HzNV-2 virus, the mutated KS45 HzNV-2 virus, or the mutated KS45 HzNV-2 virus with 0.5% of a viral stabilizing agent, Blankophor. Progeny eggs were collected and reared to adult moths. In order to determine if the virus can be ingested and still deteriorate the reproductive tract of the progeny, each progeny adult was evaluated for sterility. 17% of the wild-type virus progeny, 2% of the KS45/Blankophore progeny, and no KS45 progeny were sterile. This finding indicates that feeding is currently not a plausible process for generating high numbers of sterile insects. This experiment was repeated, but uninfected moths were fed more KS45 virus with an increase in the stabilizing agent, Blankophor (to 1%). Progeny eggs were collected and reared to adult moths, which were then evaluated for sterility. We were able to increase sterility from 2% to 37%, and the gonadal moths were infected with the virus. This finding indicates that we can feed moths KS45 virus and infect every insect in the next generation. The goal of objective 3 is to test the efficacy of KS45 sterile moths (generated during the first two objectives) in collapsing a native colony. Since a low number of KS45 sterile moths were generated from objective 2, the efficacy of these sterile moths were not tested at this time. We released objective 1 KS45 sterile moths at 1:1, 1:3, and 10:1 ratios (uninfected to infected male), released WT sterile moths at the same ratios, and released uninfected moths into a 'native' colony in 37 gal aquariums. Eggs were collected every two days for 8 days, and hatched larvae were reared to adult moths. Most of these moths will be evaluated for female plug production, male sterility, carrier status, and the ability to lay eggs. This is currently ongoing. The rest of these moths were placed back into the aquarium for mating. The moths in all but one of the aquariums have laid eggs for 12 day; the KS45 1:3 aquarium stopped laying eggs after 8 day, indicating the beginnings of a colony collapse. This experiment continues for 1 or 2 more generations.

Publications