What Shapes RNA Interference Responsiveness in Heteroptera (Insecta: Hemiptera)? An Evidence-Constrained Multilevel Framework for Experimental Design and Optimisation.

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.