Chunqing Zhao

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 use of insect-proof nets (IPNs) are an environmentally friendly physical method for preventing insect pests from entering the greenhouse and damaging the vegetables. Bok choy Brassica rapa L. subs. chinensis Brassicaceae, a widely cultivated vegetable in China, is commonly planted in the conventional greenhouses built with plastic films (PFs), IPNs and sliding doors (SDs), and named as PF-SD. However, the effectiveness of controlling agricultural insect pests in PF-SD greenhouse is low due to improper mesh sizes of IPNs and easily-deformative SDs. In this study, the rainproof performance of IPN-ZD greenhouse was compared with that of the open field, and the effects of pest damage, temperature and relative humidity as well as the growth and metabolites of bok choy in the novel greenhouse (IPN-ZD) equipped with 29-mesh IPNs and zipper doors (ZDs) were compared with those of the PF-SD greenhouse at Nanjing, China (31.5°N, 119.0°E) in autumn. The results indicated that the IPN-ZD greenhouse was able to reduce rainfall infiltration compared to that of the open field and increased the yield by 44.9%, reduced internal temperatures, significantly reduced the leaf damage rate from 15.93% to 5.94% and metabolite contents (chlorophyll a and chlorophyll b ) in bok choy compared with those in the PF-SD greenhouse. In conclusion, the IPN-ZD greenhouse could reduce the leaf damage rate, improve the integrity, yield and quality of bok choy and is potentially beneficial to the environment and ecology compared with the PF-SD greenhouse. Significance of the study: What was already known about this subject? Insect-proof nets (IPNs) are widely used in greenhouse vegetable cultivation as a physical pest control measure. However, their effectiveness can be affected by inappropriate mesh sizes, inadequate sealing and ventilation conditions. What are the new findings? This study developed a novel greenhouse structure equipped with 29-mesh IPNs and zipper doors (IPN-ZD). Compared with the conventional PF-SD greenhouse, the IPN-ZD greenhouse significantly reduced the leaf damage rate from 15.93% to 5.94% and increased bok choy yield by 44.9% without pesticide application. It also reduced rainfall infiltration and internal temperature. What are the expected impacts on horticulture? The IPN-ZD greenhouse provides a practical physical pest-management approach for protected vegetable production, with the potential to reduce leaf damage and improve crop yield while supporting environmentally sustainable horticultural production.

Background/Objectives: Glutamate-gated chloride channels (GluCls) are essential inhibitory neurotransmitter receptors in insects and key targets of several insecticides. In Spodoptera frugiperda (FAW), mutually exclusive alternative splicing (MEAS) of exon 3 in SfGluCl produces three splice variants (SfGluCl 3A, SfGluCl 3B and SfGluCl 3C), yet the cis-regulatory elements involved in this splicing event remain unknown. This study aimed to identify candidate cis-regulatory regions associated with exon 3 MEAS and characterize their effects on exon 3 selection in a Sf9 cell-based minigene system. Methods: Semi-quantitative RT-PCR was used to assess the expression levels of three splice variants of exon 3 in SfGluCl across developmental stages. Comparative sequence analysis, combined with intron-deletion and exon-exchange assays, were performed to identify candidate cis-regulatory regions. A total of 27 constructs (23 intron-deletion and 4 exon-exchange constructs) were generated and analyzed using a minigene splicing assay in Sf9 cells. Results: Three splice variants were consistently detected across all developmental stages. In the Sf9 cell-based minigene system, two candidate cis-regulatory regions affecting SfGluCl 3B inclusion were identified: a 20 bp intronic region spanning nucleotides +13 to +32 downstream of the 5′ splice junction in the intron between exons 3B and 3C, corresponding to the region deleted in ExonB-C (Exon 3B-exon 3C)-Intdel52, and an 11 bp region within exon 3B containing six nucleotide substitutions in Ex3B (Exchanged exon 3B)-SubA2. Deleting the ExonB-C-Intdel52 or modifying the Ex3B-SubA2 significantly increased the SfGluCl 3B inclusion. Conclusions: These candidate intronic and exonic regions can influence exon 3B inclusion in the Sf9 cell-based minigene system. Although their functional effects and in vivo relevance remain to be validated in further study, our findings still provide a basis for future studies of GluCl splice variant regulation, receptor biology, and insecticide response in insects.

Fipronil (FIP) is a phenylpyrazole insecticide that acts as a noncompetitive pore blocker of insect type A γ-aminobutyric acid chloride channels (GABAARs). Although genetic and mutagenesis studies have long implicated the pore-lining TM2 helix, particularly the 2′ and 6′ positions, in FIP action, the structural basis by which this asymmetric phenylpyrazole is accommodated within the pseudo-fivefold symmetric chloride-channel pore has remained unknown. Here, we determined a 3.7 Å cryo-EM structure of FIP bound to an ELIC-based chimera containing the transmembrane domain of human GABAAR α1 subunit, providing the first structural view of phenylpyrazole recognition within the GABAA receptor pore. The structure reveals a single FIP molecule occupying the lower TM2-lined ion-conduction pathway in an elongated asymmetric pose. The phenyl group is positioned near the hydrophobic 2′ layer, whereas the trifluoromethylsulfinyl moiety extends toward the polar 6′ threonine ring. Molecular dynamics simulations support the stability of this configuration and reveal repeated formation of a hydrogen bond between the sulfoxide group and the 6′T side chain. Consistent with the structural model, substitutions at the equivalent 2′A and 6′T positions in the Drosophila homomeric RDL receptor markedly reduced FIP sensitivity, providing functional validation of the proposed binding mechanism. Structural comparison with picrotoxin- and pregnenolone sulfate-bound GABAARs further demonstrates that FIP exploits a conserved lower-TM2 inhibitory site shared by chemically diverse pore blockers. Together, these findings define the structural basis of FIP recognition and noncompetitive inhibition through direct pore occlusion, provide a mechanistic explanation for resistance-associated pore mutations, and establish a framework for the rational design of next-generation phenylpyrazole insecticides.

The rice stem borer (RSB), Chilo suppressalis, is one of the most destructive rice pests in China and chlorantraniliprole has been extensively used for its control over the past decade. However, the continuous and intensive application of chlorantraniliprole has accelerated the development of resistant RSB populations in field, thereby threatening sustainable rice production. In this study, a field resistant strain of RSB exhibited a 181.76-fold resistance level to chlorantraniliprole compared to a susceptible strain. To explore the potential involvement of ATP-binding cassette (ABC) transporters in chlorantraniliprole resistance, four candidate ABC transporter genes (CsABCC1, CsABCC3, CsABCA3 and CsABCD2) were analyzed in resistant and susceptible strains. Compared to the susceptible strain, the expressional levels of CsABCC1 and CsABCC3 were significantly upregulated by 1.58- and 1.38-fold, respectively, whereas of CsABCA3 and CsABCD2 showed non-significant differences in the resistant strain. RNA interference assays demonstrated that naked dsRNA induced only limited gene silencing, while chitosan-mediated dsRNA delivery significantly improved RNAi efficiency. Following feeding with chitosan-coated dsCsABCC1 and dsCsABCC3, the expression levels of both genes were reduced by 44.63% and 38.49%, respectively, relative to the control and the larval mortality increased following chlorantraniliprole treatment to 63.33% and 56.67%, respectively. In addition, silencing CsABCC1 caused a greater reduction in larval weight after insecticide treatment. These findings indicated that CsABCC1 and CsABCC3 are involved in chlorantraniliprole detoxification and may contribute to resistance. Overall, this study provides evidence for the functional involvement of ABC transporters in chlorantraniliprole resistance and highlights chitosan-mediated RNAi as a promising complementary approach for resistance management within integrated pest management programs.

The ionotropic γ-aminobutyric acid receptor (iGABAR) is an important insecticidal molecular target. However, the native iGABARs composition remains unknown in insect. Here, CsRdl1, truncated transcripts of CsRdl2 and CsLcch3 were obtained in the rice stem borer (RSB), Chilo suppressalis Walker. The N-terminal-truncated CsRDL2 (∆N-CsRDL2) and N-terminal-truncated CsLCCH3 (∆N-CsLCCH3) were deduced and studied in vivo, and desmethyl-broflanilide (DMBF) binding characteristics were simulated in silico. Genome and transcriptome analyses revealed truncated transcripts of CsRdl2 and CsLcch3 encoded 48 kDa of ∆N-CsRDL2 and 37 kDa of ∆N-CsLCCH3, respectively. The CsRDL1, CsRDL2 and CsLCCH3 were detected respectively from native iGABARs at molecular weights (Mws) ≥ 440 kDa in BN-PAGE. In BN/SDS-PAGE, three CsRDL1 bands (~54, ~55 and ~70 kDa), one CsRDL2 band (~48 kDa) and one CsLCCH3 band (~37 kDa) were identified in native iGABARs at Mws ≥ 669 kDa, and corresponded to CsRDL1ad, CsRDL1bd, post-translationally modified CsRDL1, ∆N-CsRDL2 and ∆N-CsLCCH3, respectively. Immunofluorescence confirmed these three subunits distributed in the same region of adult heads. Finally, DMBF displayed higher binding affinities for heteromeric iGABARs than for homomeric CsRDL1 iGABAR in silico. These findings confirm that ∆N-CsRDL2 and ∆N-CsLCCH3 in native iGABARs might support the rational design of novel insecticides.

Rice planthoppers, including Nilaparvata lugens (Stål), Laodelphax striatellus (Fallén), and Sogatella furcifera (Horváth), pose substantial threats to global rice production through direct feeding damage and transmission of viral diseases. Although chemical control remains the primary management approach, insecticide concentrations below lethal levels (also referred to as ‘sublethal doses’) produce significant biological effects that extend well beyond immediate mortality. This review synthesises current knowledge on how sublethal insecticide exposure fundamentally alters rice planthopper biology through multiple pathways, including nerve system disruption, hormonal imbalance, cellular stress, and heritable genetic changes. Sublethal exposure modifies rice planthopper behaviour by disrupting feeding activity and movement coordination, while simultaneously activating detoxification enzyme systems (cytochrome P450s, glutathione‐ S ‐transferases, and carboxylesterases) that promote insecticide resistance. Reproductive impacts are substantial, with egg production commonly reduced by more than 60%, alongside delayed egg‐laying and damaged ovarian tissues. Interestingly, low doses occasionally stimulate increased reproduction, causing temporary population increases. Developmental effects include prolonged immature stages, enhanced production of winged forms capable of long‐distance migration, and impacts that persist across multiple generations through epigenetic mechanisms. The ecological consequences including pest resurgence, secondary pest outbreak, and impact on non‐target organisms are equally important. Insecticides affect natural enemies (predators and parasitoids) more severely than pests and as a result, surviving rice planthopper populations can rebound rapidly when predation pressure decreases. Broad‐spectrum insecticide applications also enable formerly minor pests to become major problems. Furthermore, beneficial organisms such as pollinators, parasitic wasps, and aquatic predators suffer collateral damage, compromising essential ecosystem functions and creating feedback loops that progressively reduce pesticide effectiveness. Following this review, sustainable rice planthopper management requires integrated strategies combining resistant rice varieties, biological control organisms, precision application technologies, and innovative approaches such as RNA interference‐based biopesticides. Future research should prioritise quantifying impacts on beneficial insects, understanding molecular mechanisms of transgenerational effects, and developing practical decision‐support tools for farmers.

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.

DanBean, the larva of Clanis bilineata Walker (Lepidoptera: Sphingidae), is not only an agricultural pest, but also an important economic insect in China. However, predation by two-spotted ground beetle Chlaenius bioculatus (Coleoptera: Carabidae) in breeding areas substantially reduces the egg production of DanBean. The use of chemical insecticides and soil-buried traps results in the mortality of DanBean and its pupae, respectively. Therefore, we aimed to identify highly efficient attractants and to design a novel, non-buried trap for the eco-friendly control of C. bioculatus . Five attractants with varying compositions were evaluated to determine the most effective blend-attractants. The traditional trap (Barber pot) was used as a control. Attractant No. 5 (brown sugar: liquor: white vinegar: starch = 1: 1: 1: 1) demonstrated the highest attraction ability for C. bioculatus . A novel trap, referred to as the “conical-shape trap”, was developed with a funnel-shaped design using 3D printing technology and featured a mesh barrier to reduce entry resistance for C. bioculatus . The trapping efficiency of the novel trap was lower than the traditional trap, however, the attractant (No. 5) and conical-shape traps are suitable for field application in breeding areas of DanBean. In conclusion, we offer an effective and environmentally sustainable method for controlling C. bioculatus and supporting DanBean production.