Differential sensitivity of RDL1 and RDL2 GABA receptors to Broflanilide, Fluxametamide, Fluralaner, and Fipronil in Heliothis virescens and Helicoverpa zea

Authors: Du, Yuzhe; Scheibener, Shane; Allen, Kerry; Little, Nathan; Reddy, Gadi

Submitted to: Pesticide Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2026
Publication Date: 3/17/2026
Citation: Du, Y., Scheibener, S.A., Allen, K.C., Little, N., Reddy, G.V. 2026. Differential sensitivity of RDL1 and RDL2 GABA receptors to Broflanilide, Fluxametamide, Fluralaner, and Fipronil in Heliothis virescens and Helicoverpa zea. Pesticide Biochemistry and Physiology. 220(107061):. https://doi.org/10.1016/j.pestbp.2026.107061.
DOI: https://doi.org/10.1016/j.pestbp.2026.107061

Interpretive Summary: Farmers in the Mississippi Delta face major losses from the tobacco budworm and corn earworm, two pests that frequently develop resistance to insecticides used in cotton and corn production. To better understand how these insects respond to new chemistries, we studied their GABA-gated chloride channels using Xenopus laevis oocytes. We examined two receptor types, RDL1 and RDL2, from both species and found that RDL2 was about 2.5 times more sensitive to GABA than RDL1. When tested with four insecticides—broflanilide, fluxametamide, fluralaner, and fipronil—all compounds strongly blocked channel activity, with Farmers in the Mississippi Delta face major losses from the tobacco budworm and corn earworm, two pests that frequently develop resistance to insecticides used in cotton and corn production. To better understand how these insects respond to new chemistries, we studied their GABA-gated chloride channels using Xenopus laevis oocytes. We examined two receptor types, RDL1 and RDL2, from both species and found that RDL2 was about 2.5 times more sensitive to GABA than RDL1. When tested with four insecticides—broflanilide, fluxametamide, fluralaner, and fipronil—all compounds strongly blocked channel activity, with RDL1 showing greater sensitivity than RDL2. This reduced sensitivity in RDL2 may contribute to the development of insecticide resistance in field populations.These findings highlight the importance of considering receptor diversity when designing new control tools and support the development of safer, more effective insecticides. This research delivers direct benefits to farmers by improving the consistency of pest control, protecting crop yields, and reducing economic losses. It advances USDA mission areas and aligns with the Secretary of Agriculture’s priorities by promoting farm profitability, strengthening agricultural resilience, and supporting sustainable, science-based pest-management solutions that serve farmers, stakeholders, and U.S. agriculture.

Technical Abstract: Insect '-aminobutyric acid-gated chloride channels (GABACls) are major targets of non-competitive antagonist (NCA) insecticides. In this study, we cloned and functionally characterized two isoforms of RDL (resistant to dieldrin) subunits -HvRDL1 and HvRDL2 from the tobacco budworm (Heliothis virescens) and ; HzRDL1, HzRDL2 the corn earworm (Helicoverpa zea), two economically important lepidopteran pests in the Mississippi Delta. Sequence analysis revealed that HvRDL1 and HzRDL1 were identical at the amino acid level, while HvRDL2 and HzRDL2 differed by only two residues despite possessing 50–60 synonymous nucleotide substitutions. Functional characterization using two-electrode voltage clamp in Xenopus laevis oocytes showed that RDL1 and RDL2 differ in their responses to GABA agonism and NCA antagonism in both H. virescense and H. zea. All four isoforms formed functional homomeric receptors activated by GABA, with EC50 values of 30.8 µM and 28.8 µM for HvRDL1and HzRDL1, respectively, and 12.0 µM and 11.9 µM for HvRDL2 and HzRDL2, respectively, indicating that RDL2 isoforms were approximately 2.5-fold more sensitive to GABA activation thanRDL1 receptors. In insecticide assays, broflanilide, fluxametamide, fluralaner, and fipronil strongly inhibited GABA (100 µM)-induced currents. In RDL1 isoforms, these four NCAs produced rapid and robust inhibition, with IC50 values of 0.59, 0.31, 0.59, and 0.43 nM for HvRDL1, and 0.50, 0.47, 0.55, and 0.39 nM for HzRDL1, respectively. In contrast, inhibition of RDL2 isoforms occurred more slowly, with IC50 values of 5.1, 1.2, 1.5, and 0.7 nM for HvRDL2, and 5.9, 4.03, 3.06, and 1.05 nM for HzRDL2 respectively. Across all four NCAs, RDL2 consistently exhibited 2-10-fold reduced sensitivity compared to RDL1. Additionally, NCA dose-response curve exhibited steeper Hill coefficients in RDL1 than in RDL2, suggesting differences in cooperative binding. Overall, these findings provide molecular insight into the differential interactions of NCA insecticides with RDL isoforms in H. virescens and H. zea. This work advances our understanding of novel NCA modes of action and supports the development of more sustainable management strategies for lepidoptera pest control.