{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T09:23:16Z","timestamp":1787822596941,"version":"build-2784847793"},"reference-count":47,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Insects"],"abstract":"<jats:p>RNA interference (RNAi) is widely used in insect functional genomics and has potential for species-selective pest management, but its performance in Heteroptera varies among species, delivery routes, targets, and endpoints. This review evaluates processes that may constrain double-stranded RNA (dsRNA) delivery, processing, and phenotypic expression using a claim-level evidence hierarchy. Productive RNAi has been demonstrated in heteropteran lineages, particularly after injection, whereas oral and topical outcomes are more variable. Responses after oral, plant-mediated, and carrier-assisted delivery have also been reported in Miridae, although evidence remains concentrated in Pentatomidae. Extracellular degradation is the best-documented candidate constraint, but causal evidence linking a specific nuclease to oral RNAi remains restricted to Nezara viridula, and the in vivo relevance of haemolymph degradation is unresolved. Distant-tissue and systemic effects provide functional evidence of signal access, whereas restrictions imposed by the perimicrovillar membrane, limited cellular uptake, endosomal entrapment, and inefficiency at Dicer or RISC steps remain unresolved as limiting mechanisms. We propose an evidence-constrained multilevel framework that treats the delivery route as an experimental perturbation and transcript depletion, protein reduction, and phenotype as distinct outcomes. Mechanism-matched measurements and staged causal tests are required to identify context-specific constraints and guide optimisation.<\/jats:p>","DOI":"10.3390\/insects17090900","type":"journal-article","created":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T09:00:35Z","timestamp":1787821235000},"page":"900","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["What Shapes RNA Interference Responsiveness in Heteroptera (Insecta: Hemiptera)? An Evidence-Constrained Multilevel Framework for Experimental Design and Optimisation"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7569-6303","authenticated-orcid":false,"given":"Anna","family":"Zieli\u0144ska","sequence":"first","affiliation":[{"name":"Institute of Biology, University of Opole, Oleska 22, 45-052 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-9806-4466","authenticated-orcid":false,"given":"Julia","family":"Rojek","sequence":"additional","affiliation":[{"name":"Faculty of Natural Sciences and Technology, University of Opole, Kominka 6, 45-040 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1332-8331","authenticated-orcid":false,"given":"Jerzy A.","family":"Lis","sequence":"additional","affiliation":[{"name":"Institute of Biology, University of Opole, Oleska 22, 45-052 Opole, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1038\/35888","article-title":"Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans","volume":"391","author":"Fire","year":"1998","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1146\/annurev-ento-011019-025224","article-title":"Mechanisms, Applications, and Challenges of Insect RNA Interference","volume":"65","author":"Zhu","year":"2020","journal-title":"Annu. Rev. Entomol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1322","DOI":"10.1038\/nbt1359","article-title":"Control of Coleopteran Insect Pests through RNA Interference","volume":"25","author":"Baum","year":"2007","journal-title":"Nat. Biotechnol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Willow, J., and Veromann, E. (2021). Highly Variable Dietary RNAi Sensitivity Among Coleoptera. Front. Plant Sci., 12.","DOI":"10.3389\/fpls.2021.790816"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.cois.2014.09.012","article-title":"The Challenge of RNAi-Mediated Control of Hemipterans","volume":"6","author":"Christiaens","year":"2014","journal-title":"Curr. Opin. Insect Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2188","DOI":"10.1002\/ps.6153","article-title":"Current Scenario of RNAi-based Hemipteran Control","volume":"77","author":"Jain","year":"2021","journal-title":"Pest Manag. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"107034","DOI":"10.1016\/j.pestbp.2026.107034","article-title":"RNA Interference-Based Pesticides: Mechanism, Application, and Commercialization in Sustainable Pest Management","volume":"219","author":"Li","year":"2026","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1111\/j.1744-7917.2012.01550.x","article-title":"Advances in the Use of the RNA Interference Technique in Hemiptera","volume":"20","author":"Li","year":"2013","journal-title":"Insect Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4856","DOI":"10.1038\/s41598-020-60078-3","article-title":"First Transcriptome of the Neotropical Pest Euschistus heros (Hemiptera: Pentatomidae) with Dissection of Its siRNA Machinery","volume":"10","author":"Cagliari","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1002\/ps.6017","article-title":"RNAi-mediated Mortality in Southern Green Stinkbug Nezara viridula by Oral Delivery of dsRNA","volume":"77","author":"Sharma","year":"2021","journal-title":"Pest Manag. Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1002\/ps.5167","article-title":"Liposome Encapsulation and EDTA Formulation of dsRNA Targeting Essential Genes Increase Oral RNAi-caused Mortality in the Neotropical Stink Bug Euschistus heros","volume":"75","author":"Castellanos","year":"2019","journal-title":"Pest Manag. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Sharma, R., Taning, C.N.T., Smagghe, G., and Christiaens, O. (2021). Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezara viridula. Insects, 12.","DOI":"10.3390\/insects12020115"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3720","DOI":"10.1038\/s41598-018-22035-z","article-title":"Improving RNAi in the Brown Marmorated Stink Bug: Identification of Target Genes and Reference Genes for RT-qPCR","volume":"8","author":"Mogilicherla","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e00698","DOI":"10.1002\/adbi.202400698","article-title":"Double-Stranded DNA Reduces dsRNA Degradation in the Saliva and Significantly Enhanced RNAi-Mediated Gene Silencing in Halyomorpha halys","volume":"9","author":"Amineni","year":"2025","journal-title":"Adv. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"17059","DOI":"10.1038\/s41598-017-17134-2","article-title":"Comparative Analysis of Double-Stranded RNA Degradation and Processing in Insects","volume":"7","author":"Singh","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.jinsphys.2011.12.014","article-title":"Saliva of Lygus lineolaris Digests Double Stranded Ribonucleic Acids","volume":"58","author":"Allen","year":"2012","journal-title":"J. Insect Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1111\/1744-7917.13211","article-title":"Gene Cloning, Protein Expression, and Enzymatic Characterization of a Double-stranded RNA Degrading Enzyme in Apolygus lucorum","volume":"31","author":"Zhang","year":"2024","journal-title":"Insect Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1186\/s12951-025-03155-x","article-title":"Nano-Delivery Platform with Strong Protection and Efficient Delivery: Preparation of Self-Assembled RNA Pesticide with Dual RNAi Targets against Apolygus lucorum","volume":"23","author":"Qiao","year":"2025","journal-title":"J. Nanobiotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Liu, F., Yang, B., Zhang, A., Ding, D., and Wang, G. (2019). Plant-Mediated RNAi for Controlling Apolygus lucorum. Front. Plant Sci., 10.","DOI":"10.3389\/fpls.2019.00064"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"105481","DOI":"10.1016\/j.pestbp.2023.105481","article-title":"An Oral dsRNA Delivery System Based on Chitosan Induces G Protein-Coupled Receptor Kinase 2 Gene Silencing for Apolygus lucorum Control","volume":"194","author":"Qiao","year":"2023","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1016\/j.ibmb.2006.05.012","article-title":"RNA Interference of the Salivary Gland Nitrophorin 2 in the Triatomine Bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA Ingestion or Injection","volume":"36","author":"Araujo","year":"2006","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"105295","DOI":"10.1016\/j.pestbp.2022.105295","article-title":"Topical Delivery of dsRNA in Two Hemipteran Species: Evaluation of RNAi Specificity and Non-Target Effects","volume":"189","author":"Finetti","year":"2023","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Moriyama, M., Hosokawa, T., Tanahashi, M., Nikoh, N., and Fukatsu, T. (2016). Suppression of Bedbug\u2019s Reproduction by RNA Interference of Vitellogenin. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0153984"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"10396","DOI":"10.1038\/s41598-022-14620-0","article-title":"RNAi-Induced Knockdown of white Gene in the Southern Green Stink Bug (Nezara viridula L.)","volume":"12","author":"Souza","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.asd.2003.12.002","article-title":"Occurrence of Midgut Perimicrovillar Membranes in Paraneopteran Insect Orders with Comments on Their Function and Evolutionary Significance","volume":"33","author":"Silva","year":"2004","journal-title":"Arthropod Struct. Dev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1743","DOI":"10.1007\/s00709-016-1051-2","article-title":"Ultrastructure of the Midgut in Heteroptera (Hemiptera) with Different Feeding Habits","volume":"254","author":"Santos","year":"2017","journal-title":"Protoplasma"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1017\/S0007485315000929","article-title":"Origin, Evolution and Function of the Hemipteran Perimicrovillar Membrane with Emphasis on Reduviidae That Transmit Chagas Disease","volume":"106","author":"Zenteno","year":"2016","journal-title":"Bull. Entomol. Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"710","DOI":"10.1016\/j.jinsphys.2009.12.010","article-title":"Subcellular Fractionation of Midgut Cells of the Sunn Pest Eurygaster integriceps (Hemiptera: Scutelleridae): Enzyme Markers of Microvillar and Perimicrovillar Membranes","volume":"56","author":"Allahyari","year":"2010","journal-title":"J. Insect Physiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1002\/arch.20452","article-title":"Secretion and Formation of Perimicrovillar Membrane in the Digestive System of the Sunn Pest, Eurygaster integriceps (Hemiptera: Scutelleridae) in Response to Feeding","volume":"78","author":"Mehrabadi","year":"2011","journal-title":"Arch. Insect Biochem. Physiol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Schvartzman, C., Fresia, P., Murchio, S., Mujica, M.V., and Dalla-Rizza, M. (2022). RNAi in Piezodorus guildinii (Hemiptera: Pentatomidae): Transcriptome Assembly for the Development of Pest Control Strategies. Front. Plant Sci., 13.","DOI":"10.3389\/fpls.2022.804839"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Bansal, R., Mittapelly, P., Chen, Y., Mamidala, P., Zhao, C., and Michel, A. (2016). Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0152730"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Nishide, Y., Kageyama, D., Tanaka, Y., Yokoi, K., Jouraku, A., Futahashi, R., and Fukatsu, T. (2021). Effectiveness of Orally-Delivered Double-Stranded RNA on Gene Silencing in the Stinkbug Plautia stali. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0245081"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1093\/jisesa\/iey045","article-title":"RNA Interference of the Muscle actin Gene in Bed Bugs: Exploring Injection Versus Topical Application for dsRNA Delivery","volume":"18","author":"Basnet","year":"2018","journal-title":"J. Insect Sci."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Zhu, F., Sams, S., Moural, T., Haynes, K.F., Potter, M.F., and Palli, S.R. (2012). RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0031037"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3683","DOI":"10.1242\/dev.127.17.3683","article-title":"RNAi Analysis of Deformed, Proboscipedia and Sex Combs Reduced in the Milkweed Bug Oncopeltus fasciatus: Novel Roles for Hox Genes in the Hemipteran Head","volume":"127","author":"Hughes","year":"2000","journal-title":"Development"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1016\/j.ydbio.2005.08.010","article-title":"Hox Gene Function and Interaction in the Milkweed Bug Oncopeltus fasciatus (Hemiptera)","volume":"287","author":"Angelini","year":"2005","journal-title":"Dev. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.ydbio.2009.01.032","article-title":"Diverging Functions of Scr between Embryonic and Post-Embryonic Development in a Hemimetabolous Insect, Oncopeltus fasciatus","volume":"329","author":"Chesebro","year":"2009","journal-title":"Dev. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"107238","DOI":"10.1016\/j.pestbp.2026.107238","article-title":"The Effects of dsRNases on RNA Interference Efficiency in Insects and Future Research Directions","volume":"222","author":"Liu","year":"2026","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1038\/ncb1439","article-title":"The Endocytic Pathway Mediates Cell Entry of dsRNA to Induce RNAi Silencing","volume":"8","author":"Saleh","year":"2006","journal-title":"Nat. Cell Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"14370","DOI":"10.1074\/jbc.M513868200","article-title":"Double-Stranded RNA Is Internalized by Scavenger Receptor-Mediated Endocytosis in Drosophila S2 Cells","volume":"281","author":"Ulvila","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.ibmb.2017.09.011","article-title":"Accumulation of dsRNA in Endosomes Contributes to Inefficient RNA Interference in the Fall Armyworm, Spodoptera frugiperda","volume":"90","author":"Yoon","year":"2017","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e21678","DOI":"10.1002\/arch.21678","article-title":"Lipids Help Double-stranded RNA in Endosomal Escape and Improve RNA Interference in the Fall Armyworm, Spodoptera frugiperda","volume":"104","author":"Gurusamy","year":"2020","journal-title":"Arch. Insect Biochem. Physiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1002\/ps.5126","article-title":"Molecular Mechanisms Influencing Efficiency of RNA Interference in Insects","volume":"75","author":"Cooper","year":"2019","journal-title":"Pest Manag. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.pestbp.2018.08.005","article-title":"The Mysteries of Insect RNAi: A Focus on dsRNA Uptake and Transport","volume":"151","author":"Fishilevich","year":"2018","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Jain, R.G., Robinson, K.E., Fletcher, S.J., and Mitter, N. (2020). RNAi-Based Functional Genomics in Hemiptera. Insects, 11.","DOI":"10.3390\/insects11090557"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Joga, M.R., Zotti, M.J., Smagghe, G., and Christiaens, O. (2016). RNAi Efficiency, Systemic Properties, and Novel Delivery Methods for Pest Insect Control: What We Know So Far. Front. Physiol., 7.","DOI":"10.3389\/fphys.2016.00553"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Christiaens, O., Whyard, S., V\u00e9lez, A.M., and Smagghe, G. (2020). Double-Stranded RNA Technology to Control Insect Pests: Current Status and Challenges. Front. Plant Sci., 11.","DOI":"10.3389\/fpls.2020.00451"}],"container-title":["Insects"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4450\/17\/9\/900\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T09:09:43Z","timestamp":1787821783000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4450\/17\/9\/900"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,27]]},"references-count":47,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["insects17090900"],"URL":"https:\/\/doi.org\/10.3390\/insects17090900","relation":{},"ISSN":["2075-4450"],"issn-type":[{"value":"2075-4450","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,27]]}}}