USING INSECT EXOSOMES TO INCREASE RNAI EFFICIENCY IN PEST INSECTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1016132
• Grant No.: 2018-67030-28498
• Cumulative Award Amt.: $100,000.00
• Proposal No.: 2017-09394
• Project Start Date: Aug 15, 2018
• Project End Date: Aug 14, 2021
• Grant Year: 2018
• Program Code: [A1801]- Exploratory: Exploratory Research

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
Entomology

Non Technical Summary
RNA interference (RNAi), a mechanism that uses double-stranded RNA (dsRNA) to suppress the transcript level of a target gene, has been used extensively to study the functions of specific genes and has enormous potential for controlling insect pests, particularly in cases where pests have become resistant to control by traditional chemical insecticides. Unfortunately, not all insects are uniformly susceptible to RNAi (dipterans and lepidopterans are refractory to treatment with dsRNA), and this variability has limited the widespread implementation of this strategy for insect pest control. Coleopterans often demonstrate a highly robust response to RNAi, even in cultured beetle cells, and this proposal seeks to enhance RNAi in refractory insects using a novel method which exploits this effect. We will use extracellular vesicles, called exosomes, derived from cultured beetle cells to act as vehicles to transport dsRNA into lepidopteran or dipteran target cells. In Objective 1, we will characterize the RNA and protein compositions of exosomes derived from cultured red flour beetle (RFB) and southern corn rootworm (SCR) cells using Illumina RNA and short RNA sequencing as well as LC-MS to identify critical components of exosomes with and without dsRNA treatment. In Objective 2, we will test the ability of RFB and SCR exosomes to enter and induce an RNAi response in lepidopteran and dipteran cultured cells and whole insects. This project will enhance our understanding of exosomes and has the potential to revolutionize application of RNAi techniques and make them more applicable to control a wider range of insect pests.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1150	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1150 - Toxicology;

Keywords

insect

rnai

beetle

cell culture

exosome

transcriptome

Goals / Objectives
RNA interference (RNAi) has enormous potential as a pest control strategy, but not all insects are equally susceptible to treatment with double-stranded (dsRNA) to elicit an RNAi response. Whereas beetles tend to produce a robust RNAi response, treatment of other insect groups with dsRNA can have little to no effect regardless of the route of exposure (oral or injected). Interestingly, cultured beetle cells from Southern corn rootworm (SCR;Diabrotica undecimpunctata) are extremely sensitive to treatment with dsRNA in media, resulting in >95% suppression of the target gene at very low concentrations. In addition, conditioned media harvested from SCRcells seems to be able to enhance RNAi in cultured cells from insects that are less susceptible, potentially through transport of dsRNAs or siRNAs in extracellular vesicles called exosomes. Very little information is available on the composition of insect exosomes or their role in insect physiology or intercellular communication, but in mammals, exosome components have been well described. Furthermore, exosomes have been used as vehicles to deliver specific nucleic acids or proteins in therapeutic situations. Accordingly, we hypothesize that insect exosomes can enhance RNAi efficiency by serving as efficient vehicles for delivery of dsRNA into cells and/or insects, particularly in RNAi-refractory insect species.The major goals of this project are to: 1)increase our understanding of the composition of exosomes generated by insect cells; 2) to enhance our knowledge of the roles exosomes may play in insect physiology and intercellular communication; and 3) to utilize the inherent ability of cultured SCR cells to mount an RNAi response to improve RNAi responses in insects less susceptible to RNAi.

Project Methods
This project will use a variety of experimental methods to examine the protein and nucleic acid content of SCR cell exosomes as well as to evaluate the potential of exosomes derived from SCR cells to enhance RNAi in refractory insects.Initial experiments will evaluate the homogeneity of our exosome samples by direct observation using both Nanosight particle tracking (Nanosight, Salisbury, UK) and transmission electron microscopy. Initial observations suggest that exosomes derived from cultured beetle cells are uniform in size with mean diameters of 97 and 103 nm for RFB and SCR cells, respectively, using polymer-based methods for exosome isolation. However, Nanosight analysis does not distinguish clumped EVs or protein aggregates from exosomes. Thus, we will also include analysis by transmission electron microscopy to ensure that the particles that are being measured are actually exosomes (exosomes have characteristic cup-shaped appearance under electron microscopy48). Additionally, we will compare the homogeneity of exosomes using both ultracentrifugation and polymer-based isolation methods to determine which method yields the most consistent exosome samples. Exosome isolations will also be carried out under standardized conditions (serum-free media) at standardized time points (typically 48 h) and media volumes to maximize consistency of preparations. Subsequent analysis of RFB (red flour beetle) and SCR exosomes will include identification of protein and RNA contents. Protein lysates from both RFB and SCR exosomes initially will be harvested from exosomes and run on a 1D gel to determine the complexity of the mixture. If it is not too complex, as suggested by our preliminary data from RFB cells, then whole lysates from exosome preparations will be trypsinized and subject to LC-MS/MS analysis. If more complex, we will resort to identifying proteins by cutting out major bands from the gels and submitting for analysis of LC-MS/MS. Results from both species will be searched against the Tcas5.2 genome47 to identify protein sequences corresponding to those of T. castaneum. Proteins will also be compared to results obtained in Drosophila6 and ExoCarta49, an online database for proteins, RNAs, and lipids identified in human exosomes. Proteins identified that were unexpected in exosome preparations will be further scrutinized to ensure that their inclusion was not the result of sample contamination. In addition, exosome proteins from beetle cells treated with dseGFP will be examined to identify any that are specifically associated with dsRNA treatment or RNAi responses. RNA transcriptome analysis will be performed on RNAs isolated from RFB and SCR exosomes isolated from cells treated or not with dseGFP to identify key RNAs contained in insect exosomes and to determine what species of RNA is used to carry the RNAi response to other cells. RNAs will be separated by electrophoresis into short (<40 bp) and long (>40 bp) lengths. Short and long RNAs (three per treatment for each of the SCR and RFB cell lines for a total of 12 libraries for short and 12 libraries for long RNAs) will separately be used to create strand-specific libraries and will be subject to Illumina sequencing to generate 10 million 50 nt single reads per library. We will also include transcriptome analysis (300 nt insert for 150 bp paired-end reads on an Illumina HiSeq 2000; 20 million reads resulting in 6 Gbp of sequence information and >30X genome coverage) of the SCR cell line to facilitate RNA and protein sequence identification.Initial experiments to determine the ability of beetle exosomes to enhance RNAi will parallel those in our preliminary data, wherein exosomes isolated from RFB and SCR cells will be tested for their ability to enter other cultured insect cells. Exosomes will be fluorescently labeled with SytoSelect RNA stain (Fisher Scientific; excess stain is removed via column filtration as suggested by the manufacturer's instructions) and then used to treat cultured insect cells grown on glass coverslips. Fluorescent labeling of target cells by labeled exosomes will be visualized on a Zeiss 700 confocal microscope in the Center of Excellence for Epithelial Function in Health and Disease located in the College of Veterinary Medicine at Kansas State University. Insect cell lines will be chosen according to the susceptibility of that organism or insect order to RNAi. For example, lepidopterans and dipterans typically are incapable of mounting an efficient RNAi response following feeding or injection of dsRNA4. There are a wide variety of cell lines available from lepidopteran species, and we will select both commercially available lines (Sf9) and cell lines available from USDA laboratories (see D. Stanley letter of support), including from European corn borer (ECB, Ostrinia nubilalis), tobacco budworm (Heliothis viriscens), and cotton bollworm (Helicoverpa armigera). For dipterans, we will use a Drosophila S2 cell line stably expressing a GFP reporter gene construct. In addition, we will also consider using reporter gene constructs in the other cell lines to expedite data collection as we have previously. Following our labeling experiments, we will begin assessing the ability of exosomes to suppress transcript levels in target cells. These experiments will occur in several phases to help assure success. First, trying to capitalize on the inherent ability of beetle cells to mount a systemic RNAi response (as our preliminary data suggest in Aag2 cells), we will treat SCR and RFB cells directly with dsRNA (dseGFP as control and a specific target gene, such as Snf7, a lethal target used extensively in RNAi experiments1) in media. The dsRNA sequences will depend on the species of the target cells, but the same genes will be targeted for suppression in each cell line to avoid variability caused by the susceptibility of different genes to RNAi. After 48 h, exosomes will be harvested and used to treat target cells, and transcript level will be subsequently evaluated by RT-qPCR. If unsuccessful, we will change tactics to artificially load dsRNAs into exosomes derived from untreated SCR and RFB cells by either treating the exosomes directly with long dsRNA (beetle exosomes may take up dsRNA directly from media like cultured cells) or by transfecting the exosomes using a commercially available product (ExoTransfect, Bio101) which has been developed for loading mammalian exosomes with siRNAs. Then we will perform the experiments outlined above with the artificially loaded exosomes and assay transcript suppression. These in vitro experiments will then be followed by a series of in vivo experiments that will ascertain the ability of beetle cell-derived exosomes to induce an RNAi response in whole insects. The exosome preparations (carrying dsRNAs targeting Snf7 or another acceptable lethal gene) that were most effective at suppressing transcript level in our in vitro assays will then be fed (mixed with food or fed in water) and/or injected into 3rd instar larvae of ECB or German cockroach 3rd instar nymphs. These insects are typically insensitive to oral RNAi and ECB is also refractory to injected dsRNA. Following treatment, total RNA will be isolated from the insects, and RT-qPCR will be used to assess transcript levels.

Progress 08/15/18 to 08/14/21

Outputs
Target Audience:The research conducted in this proposal will be directed at informing fellow scientists about the nature and uses of exosomes in insect development and survival as well as their use in RNAi experimentation. Further, advancements that increase RNAi efficiency in refractory insects will be of interest not only to federal and academic scientists, but also to industrial scientists looking for novel ways to apply RNAi technology to insect control. In addition, since insect control is of paramount significance to growers, the knowledge and understanding about RNAi basic mechanisms and delivery methods will be important for growers to implement new methods of insect pest control and reduce reliance on chemical pesticides. In this reporting period, the target audience reached included scientists involved in physiological, cellular, molecular, toxicological, and public health aspects of research in entomology. Changes/Problems:This two-year exploratory grant was given a one year no-cost extension as a result of the COVID pandemic and the loss of research time. The extra year has allowed us to complete many of our goals for the project. What opportunities for training and professional development has the project provided?This project has led to four presentations by the graduate student at the annual meetings of the Entomological Society of America (ESA) in 2018, 2019, and 2020 and the North Central Branch of ESA in 2019. This Exploratory grant has permitted the graduate student to attend these meetings, improve her presentation skills (she was awarded first place in the student 10-minute presentation competition for the Pesticide Biochemistry and Toxicology section), and make connections with other students and scientists in this research discipline. In addition, the graduate student has participated in student activities within the department and has taken a leadership role in the Entomology student organization (Popenoe Entomology Club). The networking and leadership development that the graduate student has received would not be possible without the awarding of this Exploratory grant. In addition to the development of the graduate student, the PD was invited to present at a symposium on mosquito control at the national annual meeting of the ESA based on his work with insect exosomes and RNAi resulting at least in part from this project. In addition, the PD was invited to present at another symposium, this one focusing on RNAi, at the 2020 annual meeting of the ESA . ThePD was also invited to present the results of the work at two international meetings focused on RNAi in China, and to meet with an array of international experts on RNAi in insects from five different countries for networking and discussion of future collaborations. How have the results been disseminated to communities of interest?The results have been and will continue to be disseminated to communities of interest through presentations at local (departmental seminar), regional (guest seminars, branch meetings of ESA), national (peer-reviewed publications, presentations at national meetings), and international (presentations at international meetings and open-access journals). See publications and presentations list from the progress and final reports. 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 project produced a wealth of new information about the cargoes that exosomes carry, their physiological roles in insects, and their ability to enhance RNAi responses in insects less susceptible to RNAi. To better understand exosome cargoes in insects (Goal 1), we sequenced small RNAs (sRNAs) and micro RNAs (miRNAs) from exosomes isolated from either untreated SCR cells or those treated with long double-stranded RNA (dsRNA) targeting GFP. Our analysis revealed a variety of miRNAs that are carried in SCR exosomes with and without dsRNA treatment. They had a bimodal size distribution with peaks at 21-22 nucleotides and 28 nucleotides in length. Many of these miRNAs were similar to those that have been identified and named in Tribolium castaneum, a model insect that is also a beetle like SCR, but there were also a number of unique miRNAs and miRNA variants that do not match the Tribolium database. There were also a diversity of mRNAs identified from exosome, many of them appearing in both treated and untreated samples, but there was also a number of mRNAs that had significantly different expression with dsRNA treatment of the parent cells. Our experiments included determining if small interfering RNAs (siRNAs) derived from long dsRNAs would also be carried inside exosomes as cargo as suggested by our preliminary data. Small RNAs matching the sequence of GFP, the sequence for the long dsRNA used to treat SCR cells, were detected in exosomes, indicating that exosomes carry systemic RNAi signals in the form of sRNAs of about 21-22 nucleotides in length. In addition, confocal experiments showed that exosomes derived from SCR cells and stained with SYBR-select RNA stain were able to enter other cultured SCR cells as well as those of the Yellow Fever mosquito (Aedes aegypti). These results, along with those below, support the idea that exosomes serve as intracellular messenger vehicles that can carry specific cargoes to target cells, and that part of this messenger service includes carrying systemic RNAi signals (Goal 2). These results are being prepared for publication as part of a Master's thesis (graduation in Fall 2021) and a peer-reviewed journal article for 2022. In addition to characterizing their cargoes and physiological functions, this project also sought to use SCR-derived exosomes to enhance RNAi responses in insects with limited RNAi responses (Goal 3). RNAi responses from both A. aegypti and Ostrinia nubilalis cultured cells (Aag2 and OnFB3, respectively) were found to be significantly less efficient that those in SCR cells, with the Aag2 cells nearly 1,000,000 fold less sensitive and OnFB3 cells showing no response at all even to 10 µg of target dsRNA per mL of media. Using exosomes from SCR cells treated with target dsRNA for Aag2 or OnFB3 cells, however, resulted in a significant increase in RNAi efficiency. Nearly 85% suppression of target transcript levels was achieved in Aag2 cells as was 30% suppression in OnFB3 cells, indicating that exosomes can induce and enhance RNAi responses in insect cells that are less sensitive to dsRNA treatment. In addition to these in vitro experiments, injection of SCR exosomes from cells treated with dsRNA specific to subunit A of the A. aegypti vacuolar ATPase, resulted in 70-80% suppression of target transcripts in adult female mosquitoes, whereas injection of an equal amount of target dsRNA had no effect. These results show that exosomes can enhance RNAi responses in insects both in vitro and in vivo, though further investigation is necessary to evaluate their effectiveness for inducing RNA through feeding.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Cooper, AMW, K Silver, J Zhang, Y Park, KY Zhu. 2019. Molecular mechanisms influencing efficiency of RNA interference in insects. Pest Management Science. 75: 18-28. doi: 10.1002/ps.5126. PMID: 29931761.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Silver, K, AMW Cooper, KY Zhu. 2021. Strategies for enhancing RNAi efficiency in insects. Pest Management Science. 77: 2645-2658.
• Type: Other Status: Accepted Year Published: 2021 Citation: Silver, K*. Unraveling systemic RNA interference responses in insects. Department of Entomology Seminar Series. Virginia Tech University. February 16, 2021. Virtual.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Silver, K*, K Featherston, S Fatehi, Y Park, KY Zhu, B Londono. Evidence for the roles of extracellular vesicles in systemic RNAi responses in southern corn rootworm. New Insights into the Development of RNA Products for Controlling Agricultural and Medically Important Insect Pests. Annual Meeting of the Entomological Society of America. November 11-25, 2020. Virtual.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Featherston, K*, KY Zhu, Y Park, K Silver. Characterizing exosomal miRNAs from a Diabrotica cell line. PBT 10 min talk. Annual Meeting of the Entomological Society of America, November 11-25, 2020. Virtual.

Progress 08/15/19 to 08/14/20

Outputs
Target Audience:The research conducted in this proposal will be directed at informing fellow scientists about the nature and uses of exosomes in insect development and survival as well as their use in RNAi experimentation. Further, advancements that increase RNAi efficiency in refractory insects will be of interest not only to federal and academic scientists, but also to industrial scientists looking for novel ways to apply RNAi technology to insect control. In addition, since insect control is of paramount significance to growers, the knowledge and understanding about RNAi basic mechanisms and delivery methods will be important for growers to implement new methods of insect pest control and reduce reliance on chemical pesticides. In this reporting period, the target audience reached included scientists involved in physiological, cellular, molecular, toxicological, and public health aspects of research in entomology. Changes/Problems:Major changes or problems encountered in this reporting year include the worldwide COVID-19 pandemic that resulted in hibernation of of the PD's laboratory activities during a university-wide period of limited activities. Progress was still made in analyzing data from sequencing experiments, but the halting of laboratory activities resulted in the PD's request for a one-year no-cost extension. The methods and goals of the Exploratory grant have not been altered in any way, just slowed significantly by the months-long hiatus for laboratory research. What opportunities for training and professional development has the project provided?This project has led to one presentation by a graduate student at the annual meeting of the Entomological Society of America (ESA). This Exploratory grant has permitted thegraduate student to attend these meetings, improve her presentation skills (she was awarded first place in the student 10-minute presentation competition for the Pesticide Biochemistry and Toxicology section), and make connections with other students and scientists in this research discipline. In addition, the graduate student has participated in student activities within the department and has taken a leadership role in the Entomology student organization (Popenoe Entomology Club). The networking and leadership development that the graduate student has received would not be possible without the awarding of this Exploratory grant. In addition to the development of the graduate student, the PD wasinvited to present at a symposium on mosquito control at the national annual meeting of the ESA based on his work with insect exosomes and RNAi resulting at least in part from this project. In addition, the PD was invited to present at another symposium, this one focusing on RNAi, at the 2020 annual meeting of the ESA that will occur virtually this year. How have the results been disseminated to communities of interest?Some of our results have been disseminated in the form of oral presentations by the graduate student working on the project and the PD (see publications/presentations under Products). These include presentations by the PD and his graduate student at the 2019 Annual meeting of ESA. In addition, the PD has also presented departmental seminars to both Entomology and Plant Pathology Departments at Kansas State University during the Fall 2019 semester. Three peer-reviewed publications (a review on strategies for enhancing RNAi in refractory iinsects, a review on insect exosomes that will serve as the graduate student's introductory chapter in her thesis, and a research article on exosome microRNAs) are all in preparation for submission late in 2020 or early 2021. What do you plan to do during the next reporting period to accomplish the goals?To address determining the protein and nucleic acid composition of exosomes derived from SCR cells (goal 1), we will complete our analysis of the microRNA sequencing data and begin analysis of both mRNA data from exosomes and the SCR cell line. This work will be supplemented by submiting protein gel bands derived from exosome lysates for identification of proteins carried within or on SCR exosomes, results from which will enhance our understanding of how exosomes are synthesized in insects and how they carry out their physiological roles (goal 2). We will also complete experiments with Helicoverpa armigera (cotton bollworm), Ostrinia nubilalis (European corn borer), and Heliothis virescens (tobacco budworm) to demonstrate the ability of SCR-derived exosomes to enhance RNAi in refractory insect species (goal 3).

Impacts
What was accomplished under these goals? During the second year of the project, we have made excellent progress in goals 1 and 2, characterizing the composition of exosomes derived from SCR cells and determining the roles of exosomes in insect physiology and intercellular communication. Analysis of sequence data has revealed a number of microRNAs that are carried by exosomes produced by the SCR cell line. In addition, following treatment of SCR cells with double-stranded RNA, we have shown that there are several unique microRNAs included in the exosomes as well as siRNAs derived from the dsRNA used to treat the cells. These results show that exosomes arecarrying RNAi signals to other cells.The newly identified micorRNAs are being named and characterized, and the data is being prepared for a Master's student's thesis and a publication. For goal 3, we have begun generating data for sensitivity of cultured Ostrinia nubilalis (European corn borer) cells to treatment with double-stranded RNA. These experiments will quickly advance to testing the effect of exosomes on enhancing RNAi in these cells. These experiments will then be following by similar experiments with cultured cells from Helicoverpa armigera (cotton bollworm)and Heliothis virescens (tobacco budworm). In addition to tests in O. nubilalis, we have also conducted experiments determining the effects of injecting exosomes into Yellow fever mosquitoes (Aedes aegypti) on RNAi in that species. Our results suggest that exosomes significantly enhance RNAi responses in this species, and these data are being prepared for a publication late in 2020 or early 2021.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Silver, K*, K Featherston, S Menghwar, S Fatehi, Y Park, KY Zhu, B Londono. Enhancing RNAi in Aedes aegypti using insight from beetles. Meeting of the Entomological Society of America. St. Louis, MO, November 17-20, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Featherston, K*, Y Park, KY Zhu, KS Silver. Characterizing exosome cargoes from a Diabrotica cell line. Meeting of the Entomological Society of America. St. Louis, MO, November 17-20, 2019.

Progress 08/15/18 to 08/14/19

Outputs
Target Audience:The research conducted in this proposal will be directed at informing fellow scientists about the nature and uses of exosomes in insect development and survival as well as their use in RNAi experimentation. Further, advancements that increase RNAi efficiency in refractory insects will be of interest not only to federal and academic scientists, but also to industrial scientists looking for novel ways to apply RNAi technology to insect control. In addition, since insect control is of paramount significance to growers, the knowledge and understanding about RNAi basic mechanisms and delivery methods will be important for growers to implement new methods of insect pest control and reduce reliance on chemical pesticides. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has led to one presentation by a graduate student at a branch meeting of the Entomological Society of America as well as a planned presentation at the annual national meeting in November. This Exploratory grant has permitted the graduate student to attend these meetings, improve her presentation skills (she was awarded second place in the student 10 minute presentation competition for the Pesticide Biochemistry and Toxicology section), and make connections with other students and scientists in this research discipline. In addition, the graduate student has participated in student activities within the department and has taken a leadership role in the Entomology student organization (Popenoe Entomology Club). The networking and leadership development that the graduate student has received would not be possible without the awarding of this Exploratory grant. In addition to the development of the graduate student, the PD was also invited to present the results of the work at two international meetings focused on RNAi in China (see publications/presentations list), and to meet with an array of international experts on RNAi in insects from five different countries for networking and discussion of future collaborations. The PD has also been invited to present at a symposium on mosquito control at the national annual meeting of the Entomological Society of America based on his work with insect exosomes and RNAi resulting at least in part from this project. How have the results been disseminated to communities of interest?Some of our results have been disseminated in the form of oral presentations by the graduate student working on the project and the PD (see publications/presentations under Products). These include an oral presentation by the graduate student at the North Central Branch Meeting of the Entomological Society of America (Featherston, K*, Y Park, KY Zhu, KS Silver. Characterizing exosomes derived from a Diabrotica cell line. PBT 10 min talk. Entomological Society of America North Central Branch Meeting. Cincinnati, OH. March 17-20, 2019) and two international presentations by the PD (Silver, K*, K Featherston, AMW Cooper, Y Park, KY Zhu. Novel strategies for enhancing RNAi in pest insects. 2nd International Conference on Insect Pest Management. Guiyang, Guizhou, China. July 26-29, 2019; andSilver, K*, K Featherston, AMW Cooper, Y Park, KY Zhu. The ins and outs of systemic RNAi in insects. RNAi Mechanisms and Applications. Shanxi University, Taiyuan, Shanxi, China. July 22-25). Further, the graduate student and the PD will both give oral presentations based on our work on insect exosomes at the annual national meeting of the Entomological Society of America in November 2019. What do you plan to do during the next reporting period to accomplish the goals?To address determining the protein and nucleic acid composition of exosomes derived from SCR cells (goal 1), we will complete our analysis of the sequencing data and submit protein gel bands for identification,which will show the normal RNA content of exosomes. By examining the content of exosomes following treatment of SCR cells with dsRNA, we will also establish the form of RNA (short or long) and hopefully any proteinsthat may be loaded into exosomes as a part of an RNAi response, providing valuable insight into how a systemic RNAi response spreads from cell to cell (goal 2). We will also complete experiments evaluating the sensitivity of insect cells from Lepidopteran insects to RNAi, their ability to take up labelled exosomes derived from SCR cells, and enhancement of RNAi responses in cultured Lepidopteran cells by SCR exosomes (goal 3). Finally, we will also use the SCR exosomes in in vivo studies with European corn borer (O. nubilalis) to demonstrate the validity of effects on RNAi responses observed in vitro.

Impacts
What was accomplished under these goals? During the first year of the project, we have made excellent progress in goal 1, characterizing the composition of exosomes derived from SCR cells. We have received sequence data on small RNAs contained in SCR exosomes and analysis of these sequences is in progrss. Further, we have also submitted samples for mRNA sequencing of RNA isolated from exosomes and areawaiting the return of the sequencingdata. We have also completed initial experiments separating proteins isolated from SCR exosomes and plan to move forward with band cutting and protein identification soon. For goal 2, we have produced similar sequencing data as in goal 1, however, small RNA and mRNA sequences are produced from exosomes isolated from SCR cells treated with double-stranded RNA (dsRNA) with complementarity to GFP. As in goal 1, analysis of small RNA sequences is in progress, we are awaiting the mRNA sequencing data, and protein identification will begin soon. For goal 3, our progress has included receiving cultured insect cells for Helicoverpa armigera (cotton bollworm), Ostrinia nubilalis (European corn borer), and Heliothis virescens (tobacco budworm). Experiments examining RNAi efficiency, uptake of labeled exosomes, and enhancing of RNAi efficiency will begin in the next two months, and procedures for each of these sets of experiments have already been developed and optimized.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Silver, K*, K Featherston, AMW Cooper, Y Park, KY Zhu. Novel strategies for enhancing RNAi in pest insects. 2nd International Conference on Insect Pest Management. Guiyang, Guizhou, China. July 26-29, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Silver, K*, K Featherston, AMW Cooper, Y Park, KY Zhu. The ins and outs of systemic RNAi in insects. RNAi Mechanisms and Applications. Shanxi University, Taiyuan, Shanxi, China. July 22-25.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Featherston, K*, Y Park, KY Zhu, KS Silver. Characterizing exosomes derived from a Diabrotica cell line. PBT 10 min talk. Entomological Society of America North Central Branch Meeting. Cincinnati, OH. March 17-20, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Silver, K*, K Featherston, S Menghwar, S Fatehi, Y Park, KY Zhu, B Londono. Enhancing RNAi in Aedes aegypti using insight from beetles. Meeting of the Entomological Society of America. St. Louis, MO, November 17-20, 2019.