{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T00:51:56Z","timestamp":1789433516463,"version":"build-2803163510"},"reference-count":46,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,30]],"date-time":"2025-12-30T00:00:00Z","timestamp":1767052800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Duke University Science and Technology Initiative","award":["GM057440"],"award-info":[{"award-number":["GM057440"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>R-limonene has been integrated into various pest control practices as a repellent or an insecticide. However, how limonene induces aversion or mortality remains largely unknown. To elucidate the underlying mechanisms, we conducted behavioral, toxicological, and electrophysiological assays in Aedes aegypti, a primary vector of human diseases. To investigate whether limonene acts on voltage-gated sodium channels and\/or the Rdl (Resistance to dieldrin) receptor, two major targets of neuroactive insecticides, we characterized the effect of limonene on Ae. aegypti sodium and Rdl channels expressed in Xenopus oocytes. Limonene significantly potentiated GABA-induced chloride currents through Rdl in a concentration-dependent manner but had no effect on sodium channels. For repellency, limonene evoked spatial repellency in wild-type mosquitoes; however, the spatial repellency by limonene was significantly reduced in knockout mutants of Orco\u2212\/\u2212 (odorant receptor co-receptor) and TRPA1\u2212\/\u2212 (Transient Receptor Protein, subfamily A and member 1). These results indicate that limonene likely targets the Rdl receptor for insecticidal activity and limonene spatial repellency requires both Orco and TRPA1 channels. Our results reveal the involvement of multiple ion channels and receptors in the mosquito nervous system for limonene\u2019s insecticidal and\/or spatial repellency actions, highlighting limonene\u2019s potential as a multi-target neuroactive agent for pest control.<\/jats:p>","DOI":"10.3390\/ijms27010416","type":"journal-article","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T15:30:32Z","timestamp":1767195032000},"page":"416","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Involvement of Multiple Ion Channels and Receptors in Mediating the Insecticidal and Repellent Actions of Limonene"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-4752-0278","authenticated-orcid":false,"given":"Yuan","family":"Li","sequence":"first","affiliation":[{"name":"Department of Biology, Duke University, Durham, NC 27708, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilson","family":"Valbon","sequence":"additional","affiliation":[{"name":"Department of Biology, Duke University, Durham, NC 27708, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felipe","family":"Andreazza","sequence":"additional","affiliation":[{"name":"Department of Biology, Duke University, Durham, NC 27708, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9773-6350","authenticated-orcid":false,"given":"Ke","family":"Dong","sequence":"additional","affiliation":[{"name":"Department of Biology, Duke University, Durham, NC 27708, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"15288","DOI":"10.1039\/C4CC06147K","article-title":"Limonene: A Versatile Chemical of the Bioeconomy","volume":"50","author":"Ciriminna","year":"2014","journal-title":"Chem. Commun."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Eddin, L.B., Jha, N.K., Meeran, M.F.N., Kesari, K.K., Beiram, R., and Ojha, S. (2021). Neuroprotective Potential of Limonene and Limonene Containing Natural Products. Molecules, 26.","DOI":"10.3390\/molecules26154535"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.cbi.2018.02.007","article-title":"Limonene: Aroma of Innovation in Health and Disease","volume":"283","author":"Vieira","year":"2018","journal-title":"Chem.-Biol. Interact."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1603\/0022-0493-98.3.772","article-title":"Limonene, a Citrus Extract, for Control of Mealybugs and Scale Insects","volume":"98","author":"Hollingsworth","year":"2005","journal-title":"J. Econ. Entomol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1064","DOI":"10.1093\/jme\/tjz020","article-title":"Effects of Laboratory Grade Limonene and a Commercial Limonene-Based Insecticide on Haematobia Irritans Irritans (Muscidae: Diptera): Deterrence, Mortality, and Reproduction","volume":"56","author":"Showler","year":"2019","journal-title":"J. Med. Entomol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1420","DOI":"10.13005\/ojc\/350424","article-title":"The Activity of D-Limonene from Sweet Orange Peel (Citrus sinensis L.) Exctract as a Natural Insecticide Controller of Bedbugs (Cimex Cimicidae)","volume":"35","author":"Mursiti","year":"2019","journal-title":"Orient. J. Chem."},{"key":"ref_7","first-page":"93","article-title":"Volatile Organic Compounds Role in Selective Pollinator Visits to Commercial Melon Types","volume":"11","author":"Fernandes","year":"2019","journal-title":"J. Agric. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"20210161","DOI":"10.1098\/rspb.2021.0161","article-title":"A Sensory Bias Overrides Learned Preferences of Bumblebees for Honest Signals in Mimulus Guttatus","volume":"288","author":"Haber","year":"2021","journal-title":"Proc. R. Soc. B"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2801","DOI":"10.1007\/s00436-015-4586-9","article-title":"Research in Mosquito Control: Current Challenges for a Brighter Future","volume":"114","author":"Benelli","year":"2015","journal-title":"Parasitol. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"277","DOI":"10.4038\/jnsfsr.v50i0.11244","article-title":"Mosquito Control: A Review on the Past, Present and Future Strategies","volume":"50","author":"Karunaratne","year":"2022","journal-title":"J. Natl. Sci. Found. Sri Lanka"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1146\/annurev-ento-010814-020828","article-title":"Insecticide Resistance in Mosquitoes: Impact, Mechanisms, and Research Directions","volume":"60","author":"Liu","year":"2015","journal-title":"Annu. Rev. Entomol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1007\/s13744-016-0457-z","article-title":"Repellency and Larvicidal Activity of Essential Oils from Xylopia laevigata, Xylopia frutescens, Lippia pedunculosa, and Their Individual Compounds against Aedes Aegypti linnaeus","volume":"46","author":"Nascimento","year":"2017","journal-title":"Neotrop Entomol"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Sanei-Dehkordi, A., Moemenbellah-Fard, M.D., Saffari, M., Zarenezhad, E., and Osanloo, M. (2022). Nanoliposomes Containing Limonene and Limonene-Rich Essential Oils as Novel Larvicides against Malaria and Filariasis Mosquito Vectors. BMC Complement Med. Ther., 22.","DOI":"10.1186\/s12906-022-03624-y"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.chemosphere.2011.02.018","article-title":"Structure\u2013Activity Relationships of Larvicidal Monoterpenes and Derivatives against Aedes aegypti Linn","volume":"84","author":"Santos","year":"2011","journal-title":"Chemosphere"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1016\/j.carbpol.2008.02.013","article-title":"Preparation of Durable Insect Repellent Cotton Fabric: Limonene as Insecticide","volume":"74","author":"Hebeish","year":"2008","journal-title":"Carbohydr. Polym."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Nematollahi, N., Ross, P.A., Hoffmann, A.A., Kolev, S.D., and Steinemann, A. (2021). Limonene Emissions: Do Different Types Have Different Biological Effects?. Int. J. Environ. Res. Public Health, 18.","DOI":"10.3390\/ijerph181910505"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2553","DOI":"10.1038\/s41467-021-22847-0","article-title":"A Dual-Target Molecular Mechanism of Pyrethrum Repellency against Mosquitoes","volume":"12","author":"Liu","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.cub.2024.10.067","article-title":"Grapefruit-Derived Nootkatone Potentiates GABAergic Signaling and Acts as a Dual-Action Mosquito Repellent and Insecticide","volume":"35","author":"Triana","year":"2025","journal-title":"Curr. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1007\/s00441-020-03363-x","article-title":"Functional Properties of Insect Olfactory Receptors: Ionotropic Receptors and Odorant Receptors","volume":"383","author":"Wicher","year":"2021","journal-title":"Cell Tissue Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1016\/j.tig.2004.06.015","article-title":"The Odor Coding System of Drosophila","volume":"20","author":"Hallem","year":"2004","journal-title":"Trends Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"103213","DOI":"10.1016\/j.ibmb.2019.103213","article-title":"Odorant Receptors from Culex quinquefasciatus and Aedes aegypti Sensitive to Floral Compounds","volume":"113","author":"Zeng","year":"2019","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Vainer, Y., Wang, Y., Huff, R.M., Perets, D., Sar-Shalom, E., Yakir, E., Ghaninia, M., Coutinho-Abreu Gomes, I.V., Ruiz, C., and Rajamanickam, D. (2024). A Conserved Odorant Receptor Underpins Borneol-Mediated Repellency in Culicine Mosquitoes. bioRxiv.","DOI":"10.1101\/2023.08.01.548337"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1038\/nature12206","article-title":"Orco Mutant Mosquitoes Lose Strong Preference for Humans and Are Not Repelled by Volatile DEET","volume":"498","author":"DeGennaro","year":"2013","journal-title":"Nature"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1038\/nature10715","article-title":"Modulation of TRPA1 Thermal Sensitivity Enables Sensory Discrimination in Drosophila","volume":"481","author":"Kang","year":"2012","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e18425","DOI":"10.7554\/eLife.18425","article-title":"Nucleophile Sensitivity of Drosophila TRPA1 Underlies Light-Induced Feeding Deterrence","volume":"5","author":"Du","year":"2016","journal-title":"eLife"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.celrep.2011.11.002","article-title":"Thermosensory and Nonthermosensory Isoforms of Drosophila Melanogaster TRPA1 Reveal Heat-Sensor Domains of a ThermoTRP Channel","volume":"1","author":"Zhong","year":"2012","journal-title":"Cell Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1672","DOI":"10.1016\/j.cub.2010.08.016","article-title":"Drosophila TRPA1 Channel Is Required to Avoid the Naturally Occurring Insect Repellent Citronellal","volume":"20","author":"Kwon","year":"2010","journal-title":"Curr. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1988","DOI":"10.1016\/j.cub.2021.02.010","article-title":"The Irritant Receptor TRPA1 Mediates the Mosquito Repellent Effect of Catnip","volume":"31","author":"Melo","year":"2021","journal-title":"Curr. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Inocente, E.A., Shaya, M., Acosta, N., Rakotondraibe, L.H., and Piermarini, P.M. (2018). A Natural Agonist of Mosquito TRPA1 from the Medicinal Plant Cinnamosma fragrans That Is Toxic, Antifeedant, and Repellent to the Yellow Fever Mosquito Aedes aegypti. PLoS Neglected Trop. Dis., 12.","DOI":"10.1371\/journal.pntd.0006265"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e11750","DOI":"10.7554\/eLife.11750","article-title":"The Cation Channel TRPA1 Tunes Mosquito Thermotaxis to Host Temperatures","volume":"4","author":"Corfas","year":"2015","journal-title":"eLife"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3961","DOI":"10.1016\/j.cub.2019.09.070","article-title":"Polymodal Nociception in Drosophila Requires Alternative Splicing of TrpA1","volume":"29","author":"Gu","year":"2019","journal-title":"Curr. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3698","DOI":"10.1002\/ps.6282","article-title":"Behavioral and Physiological Responses of Drosophila Melanogaster and D. Suzukii to Volatiles from Plant Essential Oils","volume":"77","author":"Wang","year":"2021","journal-title":"Pest Manag. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"20200","DOI":"10.1038\/s41598-019-56639-w","article-title":"Diverse Sensitivities of TRPA1 from Different Mosquito Species to Thermal and Chemical Stimuli","volume":"9","author":"Li","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1016\/j.pestbp.2010.07.003","article-title":"Effects of Monoterpenoid Insecticides on [3H]-TBOB Binding in House Fly GABA Receptor and 36Cl\u2212 Uptake in American Cockroach Ventral Nerve Cord","volume":"98","author":"Tong","year":"2010","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.pestbp.2011.12.002","article-title":"Acetylcholinesterase Inhibition by Nootkatone and Carvacrol in Arthropods","volume":"102","author":"Anderson","year":"2012","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1093\/jee\/87.5.1172","article-title":"Insecticidal Properties of Several Monoterpenoids to the House Fly (Diptera: Muscidae), Red Flour Beetle (Coleoptera: Tenebrionidae), and Southern Corn Rootworm (Coleoptera: Chrysomelidae)","volume":"87","author":"Rice","year":"1994","journal-title":"J. Econ. Entomol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.1038\/sj.bjp.0705542","article-title":"Thymol, a Constituent of Thyme Essential Oil, Is a Positive Allosteric Modulator of Human GABAA Receptors and a Homo-Oligomeric GABA Receptor from Drosophila Melanogaster","volume":"140","author":"Priestley","year":"2003","journal-title":"Br. J. Pharmacol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.pestbp.2014.11.006","article-title":"Novel GABA Receptor Pesticide Targets","volume":"121","author":"Casida","year":"2015","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1016\/j.ibmb.2013.02.002","article-title":"Meta-Diamide Insecticides Acting on Distinct Sites of RDL GABA Receptor from Those for Conventional Noncompetitive Antagonists","volume":"43","author":"Nakao","year":"2013","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1002\/ps.3443","article-title":"The Phenolic Monoterpenoid Carvacrol Inhibits the Binding of Nicotine to the Housefly Nicotinic Acetylcholine Receptor","volume":"69","author":"Tong","year":"2013","journal-title":"Pest Manag. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1111\/mve.12377","article-title":"High-Throughput Screening Method for Evaluating Spatial Repellency and Vapour Toxicity to Mosquitoes","volume":"33","author":"Jiang","year":"2019","journal-title":"Med. Vet. Entomol."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Gross, A., and Coats, J. (2014). Can Green Chemistry Provide Effective Repellents?. Insect Repellents Handbook, CRC Press.","DOI":"10.1201\/b17407-7"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1396","DOI":"10.1021\/acsinfecdis.3c00161","article-title":"Insecticidal and Repellent Properties of Rapid-Acting Fluorine-Containing Compounds against Aedes aegypti Mosquitoes","volume":"9","author":"Zhu","year":"2023","journal-title":"ACS Infect. Dis."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"11785","DOI":"10.1073\/pnas.1305118110","article-title":"Molecular Evidence for Dual Pyrethroid-Receptor Sites on a Mosquito Sodium Channel","volume":"110","author":"Du","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"104214","DOI":"10.1016\/j.ibmb.2024.104214","article-title":"A Unique Mechanism of Transfluthrin Action Revealed by Mapping Its Binding Sites in the Mosquito Sodium Channel","volume":"175","author":"Egunjobi","year":"2024","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.neuro.2016.08.003","article-title":"Antagonist Pharmacology of Desensitizing and Non-Desensitizing Nicotinic Acetylcholine Receptors in Cockroach Neurons","volume":"56","author":"Salgado","year":"2016","journal-title":"Neurotoxicology"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/416\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T15:39:39Z","timestamp":1767195579000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/416"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,30]]},"references-count":46,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["ijms27010416"],"URL":"https:\/\/doi.org\/10.3390\/ijms27010416","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,30]]}}}