High-throughput transcriptome mining and in silico screening reveal saccharide feeding stimulants for Cydia pomonella larvae

Abstract

The codling moth (Cydia pomonella) is a globally significant internal-feeding pest of apple and other pome fruits, yet its cryptic larval behavior poses challenges to conventional control strategies. To identify effective feeding stimulants for manipulating larval-stage behavior, we established an integrative pipeline combining large-scale transcriptomic profiling, in silico receptor-ligand docking, and behavioral assays. Based on transcriptomes spanning 10 tissues and 21 developmental stages, we annotated 59 gustatory receptors (GRs) (CpomGRs) and 53 ionotropic receptors (CpomIRs). Candidates enriched in chemosensory tissues and larval stages were selected for structural modeling. Computational docking of 117 apple-related metabolites against selected receptors identified high-affinity interactions, particularly between sugar compounds and GRs/IRs. Behavioral feeding assays confirmed that isoquercitrin, a flavonoid glycoside, significantly enhanced larval feeding in a dose-dependent manner, with 1‰ concentration yielding effects comparable to a full sugar mixture. This study demonstrates the utility of transcriptome-guided in silico screening for discovering behaviorally active tastants and provides a foundation for developing bait-based control strategies targeting codling moth larvae. The approach outlined here offers a scalable framework for identifying chemosensory targets and feeding stimulants in other cryptic insect pests.

Keywords

chemotaxis assay / codling moth / gustatory receptor / ionotropic receptor

References

[1] Barnes, M. (1991). Codling moth occurrence, host race formation, and damage. Tortricid pests: their biology, natural enemies and control, 5, 313-327.

[2] Kadoić Balaško, M., Bažok, R., Mikac, K. M., Lemic, D., & Pajač Živković, I. (2020). Pest management challenges and control practices in codling moth: A review. Insects, 11(1), 38. https://doi.org/10.3390/insects11010038

[3] Witzgall, P., Stelinski, L., Gut, L., & Thomson, D. (2008). Codling moth management and chemical ecology. Annual Review of Entomology, 53(1), 503-522. https://doi.org/10.1146/annurev.ento.53.103106.093323

[4] Varela, L., Welter, S., Jones, V., Brunner, J., & Riedl, H. (1993). Monitoring and characterization of insecticide resistance in codling moth (Lepidoptera: Tortricidae) in four Western States. Journal of Economic Entomology, 86(1), 1-10. https://doi.org/10.1093/jee/86.1.1

[5] Gill, H. K., & Garg, H. (2014). Pesticide: Environmental impacts and management strategies. Pesticides-toxic Aspects, 8(187), 10-5772. https://doi.org/10.5772/57399

[6] Amat, C., Marion-Poll, F., Navarro-Roldán, M. A., & Gemeno, C. (2022). Gustatory function of sensilla chaetica on the labial palps and antennae of three tortricid moths (Lepidoptera: Tortricidae). Scientific Reports, 12(1), 18882. https://doi.org/10.1038/s41598-022-21825-w

[7] Charmillot, P. J., Hofer, D., & Pasquier, D. (2000). Attract and kill: A new method for control of the codling moth Cydia pomonella. Entomologia Experimentalis et Applicata, 94(2), 211-216. https://doi.org/10.1046/j.1570-7458.2000.00621.x

[8] Chapman, R. F. (2003). Contact chemoreception in feeding by phytophagous insects. Annual Review of Entomology, 48(1), 455-484. https://doi.org/10.1146/annurev.ento.48.091801.112629

[9] Acquavia, M. A., Pascale, R., Foti, L., Carlucci, G., Scrano, L., Martelli, G., Brienza, M., Coviello, D., Bianco, G., & Lelario, F. (2021). Analytical methods for extraction and identification of primary and secondary metabolites of apple (Malus domestica) fruits: A review. Separations, 8(7), 91. https://doi.org/10.3390/separations8070091

[10] Yang, Z., Lin, M., Yang, X., Zhu, C., Wu, D., & Chen, K. (2023). Mechanisms of the response of apple fruit to postharvest compression damage analyzed by integrated transcriptome and metabolome. Food Chemistry X, 20, 100972. https://doi.org/10.1016/j.fochx.2023.100972

[11] Yang, Z., Lin, M., Yang, X., Wu, D., & Chen, K. (2023). Comprehensive analysis of transcriptome and metabolome provides insights into the stress response mechanisms of apple fruit to postharvest impact damage. Food Chemistry: Molecular Sciences, 7, 100176. https://doi.org/10.1016/j.fochms.2023.100176

[12] Risticevic, S., Souza-Silva, E. A., Gionfriddo, E., DeEll, J. R., Cochran, J., Hopkins, W. S., & Pawliszyn, J. (2020). Application of in vivo solid phase microextraction (SPME) in capturing metabolome of apple (Malus ×domestica Borkh.) fruit. Scientific Reports, 10(1), 6724. https://doi.org/10.1038/s41598-020-63817-8

[13] Wang, H., Wang, S., Fan, M. M., Zhang, S. H., Sun, L. L., & Zhao, Z. Y. (2021). Metabolomic insights into the browning of the peel of bagging 'Rui Xue' apple fruit. BMC Plant Biology, 21(1), 209. https://doi.org/10.1186/s12870-021-02974-y

[14] Zhang, X., Xu, J., Xu, Z., Sun, X., Zhu, J., & Zhang, Y. (2020). Analysis of antioxidant activity and flavonoid metabolites in peel and flesh of red-fleshed apple varieties. Molecules, 25(8), 1968. https://doi.org/10.3390/molecules25081968

[15] Vittani, L., Populin, F., Stuerz, S., Buehlmann, A., Khomenko, I., Biasioli, F., Bühlmann-Schütz, S., Vrhovsek, U., Masuero, D., Zanella, A., Busatto, N., & Costa, F. (2023). Comparative transcriptome and metabolite survey reveal key pathways involved in the control of the chilling injury disorder superficial scald in two apple cultivars, 'Granny Smith' and 'Ladina. Frontiers in Plant Science, 14, 1150046. https://doi.org/10.3389/fpls.2023.1150046

[16] Lin, M. H. (2021). Integrated transcriptome and metabolome analysis of post-harvest apples response to mechanical damage. Doctoral Thesis. Zhejiang University. [In Chinese with English abstract]. https://doi.org/10.27461/d.cnki.gzjdx.2021.003030.154pp.

[17] Wu, Q. (2021). Studies on the phenolic compounds by widely targeted metabolomics and flavonoid extractionand purification of ‘hongmantang’ apple fruit. Master thesis. Shanxi Agricultural University. [In Chinese with English abstract]. https://doi.org/10.27285/d.cnki.gsxnu.2021.000683.72pp.

[18] Jia, Y., Ma, J., Li, X., Xu, Z., & Li, B. (2024). Analysis of aroma substances in apple fruits of different rootstock-scion combinations at ripening stage. South China Fruits, 53(5), 178-187. [In Chinese with English abstract]. https://doi.org/10.13938/j.issn.1007-1431.20230361

[19] Liu, Y. (2023). Gene for important differential metabolites between sweet core and non sweet core of Aksu Fuji apple. Master Thesis. Tarim University. [In Chinese with English abstract]. https://doi.org/10.27708/d.cnki.gtlmd.2023.000539.74pp.

[20] Yu, J. (2022). The apple bHLH transcription factor MdbHLH3 functions in regulating the fruit sugar and malate metabolism. Doctoral Thesis. Shandong Agricultural University. [In Chinese with English abstract]. https://doi.org/10.27277/d.cnki.gsdnu.2022.000076.123pp.

[21] Yao, J. (2024). Metabolomics reveals the effect of bagging on fruit development and quality of apple. Master Thesis. Henan Agricultural University. [In Chinese with English abstract]. https://doi.org/10.27117/d.cnki.ghenu.2024.000603.54pp.

[22] Tao, H. (2023). Effects of exogenous trehalose on leaf photosynthesis and fruit quality of “Gala” apple under water stress. Doctoral Thesis. Northwest A&F University. [In Chinese with English abstract]. https://doi.org/10.27409/d.cnki.gxbnu.2023.002549.119pp.

[23] Shen, Y. (2022). Study on microbial species and diversity on apples and metabolism of apple Penicillium expansum infection based on omics technology. Doctoral Thesis. Chinese Academy of Agricultural Sciences. [In Chinese with English abstract]. https://doi.org/10.27630/d.cnki.gznky.2022.000084.106pp.

[24] Ladd, T. L., (1988). Japanese beetle (Coleoptera: Scarabaeidae): Influence of sugars on feeding response of larvae. Journal of Economic Entomology, 81(5), 1390-1393. https://doi.org/10.1093/jee/81.5.1390

[25] Wang, X., Ji, Q., Zheng, X., Zhang, J., Wang, R., Wang, X., Peng, W., Guo, J., & Zhao, Y. (2024). Consumption of citric acid by bees promotes the gland development and enhances royal jelly quality. Life, 14(3), 340. https://doi.org/10.3390/life14030340

[26] Di, C., Ning, C., Huang, L. Q., & Wang, C. Z. (2017). Design of larval chemical attractants based on odorant response spectra of odorant receptors in the cotton bollworm. Insect Biochemistry and Molecular Biology, 84, 48-62. https://doi.org/10.1016/j.ibmb.2017.03.007

[27] Wink, M. (2018). Plant secondary metabolites modulate insect behavior-steps toward addiction? Frontiers in Physiology, 9, 364. https://doi.org/10.3389/fphys.2018.00364

[28] Elsayed, G. (2011). Plant secondary substances and insects behaviour. Archives of Phytopathology and Plant Protection, 44(16), 1534-1549. https://doi.org/10.1080/03235408.2010.507957

[29] Bernays, E. A., Gonzalez, N., Angel, J., & Bright, K. L. (1994). Food mixing by generalist grasshoppers: Plant secondary compounds structure the pattern of feeding. Journal of Insect Behavior, 8(2), 161-180. https://doi.org/10.1007/BF01988902

[30] Shrestha, B., & Lee, Y. (2023). Molecular sensors in the taste system of Drosophila. Genes & Genomics, 45(6), 693-707. https://doi.org/10.1007/s13258-023-01370-0

[31] Agnihotri, A. R., Roy, A. A., & Joshi, R. S. (2016). Gustatory receptors in Lepidoptera: Chemosensation and beyond. Insect Molecular Biology, 25(5), 519-529. https://doi.org/10.1111/imb.12246

[32] Rimal, S., & Lee, Y. (2018). The multidimensional ionotropic receptors of Drosophila melanogaster. Insect Molecular Biology, 27(1), 1-7. https://doi.org/10.1111/imb.12347

[33] Wan, F., Yin, C., Tang, R., Chen, M., Wu, Q., Huang, C., Qian, W., Rota-Stabelli, O., Yang, N., Wang, S., Wang, G., Zhang, G., Guo, J., Gu, L. A., Chen, L., Xing, L., Xi, Y., Liu, F., Lin, K., … Li, F. (2019). A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance. Nature Communications, 10(1), 4237. https://doi.org/10.1038/s41467-019-12175-9

[34] Xu, L., Tang, K. Y., Chen, X. F., Tao, Y., Jiang, H. B., & Wang, J. J. (2021). Comparative transcriptomic analysis reveals female-biased olfactory genes potentially involved in plant volatile-mediated oviposition behavior of Bactrocera dorsalis. BMC Genomics, 22, 1-11. https://doi.org/10.1186/s12864-020-07325-z

[35] Roche, D. B., & McGuffin, L. J. (2016). In silico identification and characterization of protein-ligand binding sites. Methods in Molecular Biology, 1414, 1-21. https://doi.org/10.1007/978-1-4939-3569-7_1

[36] Zhou, X., Zheng, W., Li, Y., Pearce, R., Zhang, C., Bell, E. W., Zhang, G., & Zhang, Y. (2022). I-TASSER-MTD: A deep-learning-based platform for multi-domain protein structure and function prediction. Nature Protocols, 17(10), 2326-2353. https://doi.org/10.1038/s41596-022-00728-0

[37] Renthal, R. (2024). Arthropod repellent interactions with olfactory receptors and ionotropic receptors analyzed by molecular modeling. Current Research in Insect Science, 5, 100082. https://doi.org/10.1016/j.cris.2024.100082

[38] Gomes, J. V., Singh-Bhagania, S., Cenci, M., Chacon Cordon, C., Singh, M., & Butterwick, J. A. (2024). The molecular basis of sugar detection by an insect taste receptor. Nature, 629(8010), 228-234. https://doi.org/10.1038/s41586-024-07255-w

[39] Yang, J., Guo, H., Jiang, N. J., Tang, R., Li, G. C., Huang, L. Q., van Loon, J. J. A., & Wang, C. Z. (2021). Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae. PLoS Genetics, 17(7), e1009527. https://doi.org/10.1371/journal.pgen.1009527

[40] Kim, D., Paggi, J. M., Park, C., Bennett, C., & Salzberg, S. L. (2019). Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nature Biotechnology, 37(8), 907-915. https://doi.org/10.1038/s41587-019-0201-4

[41] Bengtsson, J. M., Trona, F., Montagné, N., Anfora, G., Ignell, R., Witzgall, P., & Jacquin-Joly, E. (2012). Putative chemosensory receptors of the codling moth, Cydia pomonella, identified by antennal transcriptome analysis. PLoS One, 7(2), e31620. https://doi.org/10.1371/journal.pone.0031620

[42] Walker, W. B., Gonzalez, F., Garczynski, S. F., & Witzgall, P. (2016). The chemosensory receptors of codling moth Cydia pomonella-expression in larvae and adults. Scientific Reports, 6(1), 23518. https://doi.org/10.1038/srep23518

[43] Walker, W. B., Cattaneo, A. M., Stout, J. L., Evans, M. L., & Garczynski, S. F. (2023). Chemosensory receptor expression in the abdomen tip of the female codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae). Insects, 14(12), 948. https://doi.org/10.3390/insects14120948

[44] Chen, C., Chen, H., Zhang, Y., Thomas, H. R., Frank, M. H., He, Y., & Xia, R. (2020). TBtools: An integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13(8), 1194-1202. https://doi.org/10.1016/j.molp.2020.06.009

[45] Pertea, M., Pertea, G. M., Antonescu, C. M., Chang, T. C., Mendell, J. T., & Salzberg, S. L. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33(3), 290-295. https://doi.org/10.1038/nbt.3122

[46] Minh, B. Q., Schmidt, H. A., Chernomor, O., Schrempf, D., Woodhams, M. D., Von Haeseler, A., & Lanfear, R. (2020). IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution, 37(5), 1530-1534. https://doi.org/10.1093/molbev/msaa015

[47] Letunic, I., & Bork, P. (2021). Interactive tree of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Research, 49(W1), W293-W296. https://doi.org/10.1093/nar/gkab301

[48] Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C. C., O'Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., … Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493-500. https://doi.org/10.1038/s41586-024-07487-w

[49] Trott, O., & Olson, A. J. (2010). AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry, 31(2), 455-461. https://doi.org/10.1002/jcc.21334

[50] Li, Z., Ouyang, L., Lu, Y., Peng, Q., Qiao, X., Wu, Q., Zhang, B., Liu, B., Wan, F., & Qian, W. (2024). Antibiotics suppress the expression of antimicrobial peptides and increase sensitivity of Cydia pomonella to granulosis virus. Science of the Total Environment, 947, 174612. https://doi.org/10.1016/j.scitotenv.2024.174612

[51] Montell, C. (2009). A taste of the Drosophila gustatory receptors. Current Opinion in Neurobiology, 19(4), 345-353. https://doi.org/10.1016/j.conb.2009.07.001

[52] Zhang, Y. V., Ni, J., & Montell, C. (2013). The molecular basis for attractive salt-taste coding in Drosophila. Science, 340(6138), 1334-1338. https://doi.org/10.1126/science.1234133

[53] Sahu, D., Rathor, L. S., Dwivedi, S. D., Shah, K., Chauhan, N. S., Singh, M. R., & Singh, D. (2024). A review on molecular docking as an interpretative tool for molecular targets in disease management. Assay and Drug Development Technologies, 22(1), 40-50. https://doi.org/10.1089/adt.2023.060

[54] Zhong, Y. Z., Xie, M. H., Huang, C., Zhang, X., Cao, L., Chen, H. L., Zhang, F., Wan, F. H., Han, R. C., & Tang, R. (2022). Characterizations of botanical attractant of Halyomorpha halys and selection of relevant deorphanization candidates via computational approach. Scientific Reports, 12(1), 4170. https://doi.org/10.1038/s41598-022-07840-x

[55] Pott, D. M., Vallarino, J. G., & Osorio, S. (2020). Metabolite changes during postharvest storage: Effects on fruit quality traits. Metabolites, 10(5), 187. https://doi.org/10.3390/metabo10050187

[56] Hern, A., & Dorn, S. (2004). A female-specific attractant for the codling moth, Cydia pomonella, from apple fruit volatiles. Naturwissenschaften, 91(2), 77-80. https://doi.org/10.1007/s00114-003-0484-6

[57] Pszczolkowski, M. A. (2023). Prospects of codling moth management on apples with botanical antifeedants and repellents. Agriculture, 13(2), 311. https://doi.org/10.3390/agriculture13020311

[58] Zhou, Y., Huang, C., Fu, G., Tang, R., Yang, N., Liu, W., Qian, W., & Wan, F. (2024). Molecular and functional characterization of three general odorant-binding protein 2 genes in Cydia pomonella (Lepidoptera: Tortricidae). International Journal of Molecular Sciences, 25(3), 1746. https://doi.org/10.3390/ijms25031746

[59] Brahma, A., Frank, D. D., Pastor, P. D. H., Piekarski, P. K., Wang, W., Luo, J. D., Carroll, T. S., & Kronauer, D. J. (2023). Transcriptional and post-transcriptional control of odorant receptor choice in ants. Current Biology, 33(24), 5456-5466. https://doi.org/10.1016/j.cub.2023.11.025

[60] Huang, J., Gut, L. J., & Miller, J. R. (2013). Separating the attractant from the toxicant improves attract-and-kill of codling moth (Lepidoptera: Tortricidae). Journal of Economic Entomology, 106(5), 2144-2150. https://doi.org/10.1603/EC13152

[61] Pszczolkowski, M. A., & Brown, J. J. (2014). Enhancement of insecticides against codling moth (Lepidoptera: Tortricidae) with l-aspartate in laboratory and field experiments. Journal of Economic Entomology, 107(3), 1163-1171. https://doi.org/10.1603/EC13446

[62] Chandler, L. D. (1993). Use of feeding stimulants to enhance insect growth regulator-induced mortality of fall armyworm (Lepidoptera: Noctuidae) larvae. Florida Entomologist, 76(2), 316-326. https://doi.org/10.2307/3495731

[63] Kunte, N., McGraw, E., Bell, S., Held, D., & Avila, L. A. (2020). Prospects, challenges and current status of RNAi through insect feeding. Pest Management Science, 76(1), 26-41. https://doi.org/10.1002/ps.5588

[64] Li, F., Di, Z., Tian, J., Dewer, Y., Qu, C., Yang, S., & Luo, C. (2022). Silencing the gustatory receptor BtGR11 affects the sensing of sucrose in the whitefly Bemisia tabaci. Frontiers in Bioengineering and Biotechnology, 10, 1054943. https://doi.org/10.3389/fbioe.2022.1054943

[65] Fan, X. B., Mo, B. T., Li, G. C., Huang, L. Q., Guo, H., Gong, X. L., & Wang, C. Z. (2022). Mutagenesis of the odorant receptor co-receptor (Orco) reveals severe olfactory defects in the crop pest moth Helicoverpa armigera. BMC Biology, 20(1), 214. https://doi.org/10.1186/s12915-022-01411-2

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2026 The Author(s). New Plant Protection published by John Wiley & Sons Australia, Ltd on behalf of Institute of Plant Protection, Chinese Academy of Agricultural Sciences.