Progress 04/01/23 to 03/31/24
Outputs Target Audience:The primary target audiences of this study are the researchers interested in RNA interference (RNAi), insect molecular biology, and insect pest management. Because RNAi has been shown to have its great potential for pest management, industries and pesticide regulatory agencies involved in pest management may also be interested in our findings. Changes/Problems:Our research progress has been delayed due to the process of hiring a highly qualified postdoctoral fellow for the project. We identified and offered the position to two well-trained recent Ph.D. graduates in the U.S. at two different time points, but both finally declined our offers due to their other employment opportunities. We then made an offer to an international researcher (one of the three candidates) with extensive research experience in the related areas. However, the researcher experienced a significant delay in obtaining his visa. However, we are optimistic that he will start to work within a few weeks. We will quickly catch up with the research progress during the second year of the project and plan to request a no-cost extension for one year in the third year of the project. What opportunities for training and professional development has the project provided?This project allowed for the training of one Ph.D. graduate student and two undergraduate students in the first year of the project. The graduate student made excellent progress with her Ph.D. program. In presenting her research from this project, she won the third-place award in the Ph.D. graduate student poster competition at the Entomological Society of America North Central Branch Conference (March 24-27, 2024). The graduate student was also selected by the Graduate School as one of three students to represent K-State for attending the AAAS Catalyzing Advocacy in Science and Engineering (CASE) workshop in Washington, DC (April 2024). The two undergraduate students completed their undergraduate research experience (URE) by participating in this project. Each student made a poster presentation at the 2023 Entomology Undergraduate Research Symposium at K-State (December 8, 2023). How have the results been disseminated to communities of interest?Relevant research was disseminated to scientific communities of interest through oral and poster presentations at international, national, and regional conferences. The PD was invited as a keynote speaker to present RNAi research at the Second International Molecular Plant Protection Congress in Turkey (May 15-18, 2023). Results from this project were also presented at the national meeting of the Entomological Society of America (Nov. 5-8, 2023), and at the 79th annual meeting of the Entomological Society of America North Central Branch by the PD (Mar. 24-27, 2024). Through the Undergraduate Research Experience (URE) program in the Department of Entomology at K-State, two undergraduate students completed their research, and each made a poster presentation at the 2023 Entomology Undergraduate Research Symposium at K-State (December 8, 2023). What do you plan to do during the next reporting period to accomplish the goals?Due to a delay in recruiting a highly qualified postdoctoral fellow for the project, we will be working very hard to catch up with our research progress. We will generate recombinant Dicer-2 protein from each of the two insect species (the red flour beetle, the diamondback moth), profile siRNA populations using recombinant Dicer-2 proteins, sequence and map the siRNA against long dsRNA for each insect species, design and produce insect species-specific siRNA constructs, and perform RNAi bioassay for each siRNA construct to determine their efficacy against the target genes in each insect species. Further, we will continue to optimize the synthesis of RNA microsphere (RMS) which contains the repeated hairpin RNA units and evaluate the gene silencing efficiency of RMS against the diamondback moth larvae by feeding. We will start to write manuscripts based on our results from this project.
Impacts What was accomplished under these goals?
The progress of our research in the first year was delayed due to the challenge of recruiting highly qualified research personnel including a graduate student and a postdoctoral research fellow for the project. After long searches, a highly experienced graduate student was recruited and started to work on the project in July 2023 whereas a highly qualified postdoctoral fellow was also recruited and will start his research shortly. With the help of these highly experienced researchers, we would be able to make rapid progress during the remaining funding period. Our research progresses for the first year are highlighted as follows: We have successfully established a laboratory colony of the diamondback moth using artificial diet. The egg masses were placed into Styrofoam cups for them to hatch and for the larvae to develop on the diet. After the larvae developed into a desirable stage, they were collected for laboratory studies. To maintain the diamondback moth colony in the laboratory, a certain number of the diamondback moth larvae were allowed to develop into pupae. The rearing cups with the pupae were placed in a rearing cage in a growth chamber. The adults were fed with 5% sugar solution and allowed to lay their eggs on aluminum foil treated with cabbage leaf juice. The egg strips were then used to produce the larvae. We further developed a feeding-based RNAi bioassay method using cabbage leaf discs for the diamondback moth larvae. We found that a method using 10 third-instar larvae per glass vial (19 mm in diameter and 51 mm in height) containing one cabbage leaf disc (11 mm in diameter) was most appropriate for the RNAi bioassay. We identified candidate RNAi target genes encoding chitin synthase 1 (CHS1), a key enzyme involved in the biosynthesis of new chitin for insect exoskeleton, and chitinase 10 (CHT10), an enzyme involved in the degradation of old chitin in insect midgut and exoskeleton, from both the red flour beetle and the diamondback moth. Based on the identified cDNA sequences, we designed three pairs of PCR primers for each gene and for each of the two insect species. The feasibility of using these primer sets to amplify approximately 500 bp cDNA fragments were validated by PCR. The alignments of their deduced protein sequences with those identified in other insect species using Neighbor-joining methos in Mega X revealed the identities of these sequences for the synthesis of double-stranded RNA (dsRNA). To address the challenges associated with the low RNAi efficiency in insects, particularly in lepidopteran insect pests, and with the complicated procedures to produce the conventional RNAi nanoparticles, we used a new technique known as rolling circle transcription (RCT) to generate RNA microspheres (RMS) to suppress the expression of CHS1 and CHT10 in the diamondback moth larvae by feeding. The RCT technology relies on the transcription based on a circular single-stranded DNA (ssDNA) template through the action of T7 RNA polymerase to produce long, single-stranded RNA containing multiple hairpin RNA unit repeats. The resulting RNA molecule forms a stable microsphere structure without a need for additional nanomaterials. The structures of the RMS can protect RNA from degradation during siRNA delivery and be successfully transported into the cytoplasm of the cells. Using siRNA design software, we designed several 75-base long ssDNA templates for RMS synthesis. In addition, we tried to optimize the synthesis methods to improve the RMS yield. Our initial characterization of RMS using a scanning electron microscope showed the size of approximately 180 micrometers in diameter. We also included experiments for comparisons between the long doubled-stranded RNA (dsRNA) and the RMS targeting the same genes in the diamondback moth larvae. We found that there was significant knockdown of CHT10 gene 3 days after treatment with the CHT10 dsRNA and significant knockdown of CHS1 gene 7 days after treatment with the CHS1 dsRNA. These results suggest that the timeline of suppression could vary by target gene. When the diamondback moth larvae were fed the RMS to target CHS1, we found a significantly increased larval mortality as compared with that of the control larvae fed the GFP RMS on day 3. However, we would need to adjust the sampling timepoints and bioassay methods to avoid the larval mortality in the control which might have confounded our results.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Zhang F., Zhang Y. C., Yu Z. T., Zeng B., Sun H., Xie Y. Q., Zhu K. Y., Gao C. F. 2024. The G932C mutation of chitin synthase 1 gene (CHS1) mediates buprofezin resistance as confirmed by CRISPR/Cas9-mediated knock-in approach in the brown planthopper, Nilaparvata lugens. Pestic. Biochem. Physiol. 202: 105953 (https://doi.org/10.1016/j.pestbp.2024.105953).
Zhao Y., Liu W., Zhao X., Yu Z., Guo H., Yang Y., Merzendorfer H., Zhu K. Y., Zhang J. 2024. Low-density lipoprotein receptor-related protein 2 (LRP2) is required for lipid export in the midgut of the migratory locust, Locusta migratoria. J. Integr. Agr. 23: 1618-1633 (https://doi.org/10.1016/j.jia.2023.07.027).
Zhu Y., Kong L., Wang X., Xu J., Qian X., Yang Y., Xu Z., Zhu K. Y. 2023. Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate). Pestic. Biochem. Physiol. 197: 105680 (https://doi.org/10.1016/j.pestbp.2023.105680).
Wang Y., Li H., Liu X., Gao L., Fan Y., Zhu K. Y., Zhang J. 2023. Three alternative splicing variants of Loquacious play different roles in miRNA- and siRNA-mediated RNAi pathways in Locusta migratoria. RNA Biol. 20: 323-333 (https://doi.org/10.1080/15476286.2023.2223484).
Zhang Y.-C., Gao Y., Ye W.-N., Peng Y.-X., Zhu K. Y., Gao C.-F. 2023. CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae). Pest Manag. Sci. 79: 2239-2246 (https://doi.org/10.1002/ps.7404).
Han P., Chen D., Fan J., Zhang J., Jiang S., Zhu K. Y., Zhang J. 2023. Genetically engineered Metarhizium anisopliae expressing dsRNA of Apolipophorin-D exhibits enhanced insecticidal virulence against Locusta migratoria. Entomol. Gen. 43: 167-175 (10.1127/entomologia/2023/1772).
Xiao D., Yao J., Gao X., Zhu K. Y. 2023. Clathrin-dependent endocytosis plays a critical role in larval and pupal development, and female oocyte production in the red flour beetle (Tribolium castaneum). Pest Manag. Sci. 79: 1731-1742 (https://doi.org/10.1002/ps.7348).
Zhao Y., Liu W., Zhao X., Yu Z., Guo H., Yang Y., Moussian B., Zhu K. Y., Zhang J. 2023. Lipophorin receptor is required for the accumulations of cuticular hydrocarbons and ovarian neutral lipids in Locusta migratoria. Inte. J. Biol. Macromol. 236: 123746 (https://doi.org/10.1016/j.ijbiomac.2023.123746).
Fatehi S., Aikins M., Phillips T. W., Brown S., Zhu K. Y., Scully E. D., Park Y. 2023. Characterization of Iflavirus in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae). Insects 14: 220 (https://www.mdpi.com/2075-4450/14/3/220).
Zeng B., Chen F. R., Liu Y. T., Guo D., Zhang Y. J., Feng Z. R., Wang L. X., Vontas J., Wu S. F., Zhu K. Y., Gao C. F. 2023. A chitin synthase mutation confers widespread resistance to buprofezin, a chitin synthesis inhibitor, in the brown planthopper, Nilaparvata lugens. J. Pest Sci. 96: 819-832 (https://doi.org/10.1007/s10340-022-01538-9).
- Type:
Book Chapters
Status:
Accepted
Year Published:
2024
Citation:
Wang Y., Zhang J., Song H., Shi X., Zhu K. Y., Zhang J. 2024. Chapter A: The migratory locust as a model for studying the mechanisms of RNA interference. RNAi Book (in press).
Liu W., Liu X., Zhao X., Zhu K. Y., Zhang J. 2024. Chapter B: RNA interference in the migratory locust: Functional studies of the genes in the formation and development of cuticles. RNAi Book (in press).
Zhang J., Zhu K. Y. et al., 2024. Development of Insect Cuticle and Pest Management. Science Press, Beijing, China (In Chinese). pp. 254. (ISBN: 9787030775757)
Cooper A. M. W., Silver K., Zhu K. Y. 2022. Chapter 19: RNA interference. pp. xx-xx. In: Liu D. [ed.], Handbook of Molecular Biotechnology. CRC Press, Boca Raton, FL, USA (in press).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Johnson R., Zhu K. Y., Comparing the RNAi efficiency of long double stranded RNA and RNA microspheres in suppressing gene expression in the diamondback moth, Plutella xylostella. Poster (D37) presented by RJ at the 79th Annual Meeting of the Entomological Society of America North Central Branch, Fort Collins, CO. Mar. 24-27, 2024.
Zhu K. Y., RNAi-based insect pest management: Promises, challenges, and opportunities. College of Biological Science and Technology, Taiyuan Normal University, Jinzhong City, Shanxi Province, China. Dec. 29, 2023 (INVITED).
Zhu K. Y., RNAi-based insect pest management: Promises, challenges, and opportunities. College of Plant Protection, Nanjing Agricultural University, Nanjing, China. Dec. 19, 2023 (INVITED).
Zhu K. Y., RNAi-based insect pest management: Promises, challenges, and opportunities. NeoAgro, Zhejiang Academy of Agricultural Sciences, Hangzhou, China. Dec. 15, 2023 (INVITED).
Gibson J., Johnson R., Zhu K. Y., Efficacy of RNAi delivered via artificial diet on the diamondback moth, Plutella xylostella. Poster presented by JG at the 2023 Entomology Undergraduate Research Symposium at Kansas State University, Manhattan, KS. Dec. 8, 2023.
Hulse K., Johnson R., Zhu K. Y., Feeding-based RNAi against chitin-related genes in the diamondback moth, Plutella xylostella. Poster presented by KH at the 2023 Entomology Undergraduate Research Symposium at Kansas State University, Manhattan, KS. Dec. 8, 2023.
Zhu K. Y., Implication of a potential mechanism of resistance to RNAi-based biopesticides in insects. Section Symposium, Entomology 2023: National Meeting of the Entomological Society of America, National Harbor, MD. Nov. 5-8, 2023 (INVITED).
Zhu K. Y., Zhang J., Breaking down the barriers: Strategies to enhance RNAi efficiency in insects. Presented by KYZ at the Second International Molecular Plant Protection Congress, Orhangazi-Bursa, Turkey. May 15-18, 2023 (INVITED KEYNOTE SPEECH).
Zhu K. Y., Breaking down the barriers: Strategies to enhance RNAi efficiency in insects. Insect Group Meeting, Department of Biochemistry and Biophysics, Kansas State University, Manhattan, KS. Jan. 27, 2023.
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