Ionotropic γ-aminobutyric acid (GABA) receptors are major inhibitory receptors in insect central nervous systems, with their diversity partly generated by alternative splicing. In Drosophila melanogaster, mutually exclusive alternative splicing (MEAS) of the resistant to dieldrin gene (Rdl) exon 3 (E3) produces Rdl-3a and Rdl-3b, but its regulatory mechanism remains unclear. In the present study, two intronic regulatory elements (IREs) and one exonic regulatory element (ERE) were identified in the exon 2 (E2)–exon 4 (E4) region of DmRdl precursor mRNA (pre-mRNA). The IRE1 mutation decreased exon 3b (E3b) inclusion from 54.12% to 3.95%, whereas the IRE2 mutation increased E3b inclusion from 61.70% to 97.82%. The ERE mutation significantly increased E3b inclusion both in vitro and in vivo, while the IRE mutations were lethal in vivo. The results of RNA pull-down, mass spectrometry, and RNAi demonstrated that the splicing factors, CG30122 and Rbp1-like bind to the ERE and exert opposite effects on E3b inclusion. The results of STRING network analysis supported the functional interaction between CG30122 and Rbp1-like, while molecular docking provided structural support for their potential interaction with the ERE-containing RNA sequence. This study reveals the molecular regulatory network underlying the MEAS of DmRdl E3, and and provides a foundation for investigating other MEAS exons, such as E6 and E9 in insect Rdl.

The fall armyworm (FAW) Spodoptera frugiperda is a polyphagous pest that causes significant damage to various crops and rapidly develops resistance to insecticides. Broflanilide, a novel meta-diamide insecticide, has shown effectiveness against lepidopteran pests, but the risk of resistance and associated fitness costs in FAW remain unclear. This study evaluated the development of resistance to broflanilide over nine generations of selection using the diet incorporation method at the 70% lethal concentration (LC₇₀) concentration. Following nine generations of selection, the LC₅₀ value increased from 0.134 mg/kg to 0.232 mg/kg, showing a 1.73-fold increase in resistance ratio (RR). The calculated heritability of resistance (h²) was 0.084, which suggested that resistance of FAW against broflanilide is evolving at a slow rate. Based on the projected rate of resistance progression, a 10-fold increase in LC₅₀ would take between 30.1 and 66.4 generations, assuming selection mortality rates of 90% and 50%, respectively. Fitness costs were evaluated using age-stage, two-sex life table analysis, revealing reduced fecundity and pupal weight in the broflanilide-selected (Brof-SEL) strain compared to the wild-type. The relative fitness of the Brof-SEL strain was 0.38, indicating trade-offs in biological traits. These findings suggested a low risk of rapid resistance development against broflanilide. However, effective integrated pest management strategies against FAW require the judicious use of this insecticide in combination with biological control measures, including the deployment of parasitoids and predators, to promote a more environmentally sustainable approach.