The dynamics of N6-methyladenine RNA modification in resistant and susceptible rice varieties responding to rice stem borer damage.

N6-methyladenosine (m6A) is the most prevalent modification in cellular RNA which orchestrates diverse physiological and pathological processes during stress response. However, the differential m6A modifications that cope with herbivore stress in resistant and susceptible crop varieties remain unclear. Here, we found that rice stem borer (RSB) larvae grew better on indica rice (e.g., MH63, IR64, Nanjing 11) than on japonica rice varieties (e.g., Nipponbare, Zhonghua 11, Xiushui 11). Then, transcriptome-wide m6A profiling of representative resistant (Nipponbare) and susceptible (MH63) rice varieties were performed using a nanopore direct RNA sequencing approach, to reveal variety-specific m6A modifications against RSB. Upon RSB infestation, m6A methylation occurred in actively expressed genes in Nipponbare and MH63, but the number of methylation sites decreased across rice chromosomes. Integrative analysis showed that m6A methylation levels were closely associated with transcriptional regulation. Genes involved in herbivorous resistance related to mitogen-activated protein kinase, jasmonic acid (JA), and terpenoid biosynthesis pathways, as well as JA-mediated trypsin protease inhibitors, were heavily methylated by m6A, and their expression was more pronounced in RSB-infested Nipponbare than in RSB-infested MH63, which may have contributed to RSB resistance in Nipponbare. Therefore, dynamics of m6A modifications act as the main regulatory strategy for expression of genes involved in plant-insect interactions, which is attributed to differential responses of resistant and susceptible rice varieties to RSB infestation. These findings could contribute to developing molecular breeding strategies for controlling herbivorous pests.

Effects of eugenol on physicochemical properties of sturgeon skin collagen-chitosan composite membrane.

In this study, a series of collagen-chitosan-eugenol (CO-CS-Eu) flow-casting composite films were prepared using collagen from sturgeon skin, chitosan, and eugenol. The physicochemical properties, mechanical properties, microstructure, as well as antioxidant and antimicrobial activities of the composite membranes were investigated by various characterization techniques. The findings revealed that the inclusion of eugenol augmented the thickness of the film, darkened its color, reduced the transparency, and enhanced the ultraviolet light-blocking capabilities, with the physicochemical properties of the CO-CS-0.25%Eu film being notably favorable. Eugenol generates increasingly intricate matrices that disperse within the system, thereby modifying the optical properties of the material. Furthermore, the tensile strength of the film decreased from 70.97 to 20.32 MPa, indicating that eugenol enhances the fluidity and ductility of the film. Added eugenol also exhibited structural impact by loosening the film cross-section and decreasing its density. The Fourier transform infrared spectroscopy results revealed the occurrence of several intermolecular interactions among collagen, chitosan, and eugenol. Moreover, the incorporation of eugenol bolstered the antioxidant and antimicrobial capabilities of the composite film. This is primarily attributed to the abundant phenolic/hydroxyl groups present in eugenol, which can react with free radicals by forming phenoxy groups and neutralizing hydroxyl groups. Consequently, inclusion of eugenol substantially enhances the freshness retention performance of the composite film. PRACTICAL APPLICATION: ● The CO-CS-Eu film utilizes collagen from sturgeon skin, improving the use of sturgeon resources.● Different concentrations of eugenol altered its synergistic effect with chitosan.● The CO-CS-Eu film is composed of natural products with safe and edible properties.

Identification and characterization of cold-responsive aquaporins from the larvae of a crambid pest Agriphila aeneociliella (Eversmann) (Lepidoptera: Crambidae).

As small ectotherms, insects need to cope with the challenges of winter cold by regulating the water content through water transport. Aquaporins (AQPs) are key players to enhance the cold resistance by mediating essential homeostatic processes in many animals but remain poorly characterized in insects. Agriphila aeneociliella is a newly discovered winter wheat pest in China, and its early-stage larvae have strong tolerance to low temperature stress. Six AQP genes were identified, which belong to five AQP subfamilies (RPIP, Eglp, AQP12L, PRIP, DRIP). All of them contained six hydrophobic transmembrane helices (TMHs) and two relatively conservative Asparagine-Proline-Alanine motifs. The three-dimensional homology modeling showed that the six TMHs folded into an hourglass-like shape, and the imperceptible replace of four ar/R residues in contraction region had critical effects on changing the pore size of channels. Moreover, the transcript levels of AaAQP 1, 3, and 6 increased significantly with the treatment time below 0 °C. Combined with the results of pore radius variation, it is suggested that AaAQP1 and AaAQP3 may be considered to be the key anti-hypothermia proteins in A. aeneociliella by regulating rapid cell dehydration and allowing the influx of extracellular cold resistance molecules, thus avoiding death in winter.

G3'MTMD3 in the insect GABA receptor subunit, RDL, confers resistance to broflanilide and fluralaner.

Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). In this study, we used in silico analysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3') in the third transmembrane domain (TMD3) with methionine (G3'M TMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer, Chilo suppressalis (CsRDL) in oocytes of the African clawed frog, Xenopus laevis, where the G3'MTMD3 mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3'MTMD3 was introduced into the Rdl gene of the fruit fly, Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3'MTMD3 did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3'MTMD3 were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3'MTMD3. Moreover, the M3'GTMD3 mutation in the mouse Mus musculus α1ß2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincing in vitro and in vivo evidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, our findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.

Shisa reduces the sensitivity of homomeric RDL channel to GABA in the two-spotted spider mite, Tetranychus urticae Koch.

The γ-aminobutyric acid receptors (GABARs) mediate fast inhibitory transmission in central nervous system of insects and are important targets of insecticides. An auxiliary subunit, Shisa7, was identified in mammals as a single-passing transmembrane protein. However, the homology gene(s) of Shisa in invertebrates has not been reported to date. In the present study, a homolog Shisa gene was identified from the two-spotted spider mite, Tetranychus urticae Koch. Its open reading frame had 927 base pairs and encoded 308 amino acid residues, which has a typical Shisa domain at 13th-181st amino acid residues. According to the phylogenetic tree, the invertebrate Shisa was categorized apart with those of vertebrate, and TuShisa showed closest relationship with the Shisa9 of velvet mite, Dinothrombium tinctorium (L.). In the electrophysiological assay with two-electrode voltage clamp, the GABA-activated TuRDL channel was functionally formed in the Africa clawed frog Xenopus laevis (Daudin) oocytes (EC50 = 53.34 µM). No GABA-activated current could be observed in TuShisa-expressed oocytes, whereas TuShisa could reduce the sensitivity of TuRDL/TuShisa (mass ratio of 1: 4) channel to GABA. The homology structural models of TuRDL and TuShisa were built by the SWISS-MODEL server, their interaction was predicted using Z-DOCK and three predicted hydrogen bonds and interface residues were analysed by PyMOL. Meanwhile, the key interface residues of TuShisa affected the stability of complex were calculated by Discovery Studio 2019. In conclusion, the TuShisa, as the first reported invertebrate Shisa, was explored and functionally examined as the GABARs auxiliary subunit. Our findings provide a basis for research of invertebrate Shisa.

The acute toxicity, mechanism, bioconcentration and elimination of fluxametamide on zebrafish (Danio rerio).

Fluxametamide is a completely novel and the first isoxazoline insecticide used to control agricultural pests and has high insecticidal properties. To expand its usage in the paddy field, its potential toxicological effects on fish are necessary to make clear. In this study, the acute toxicity, bioconcentration and elimination of fluxametamide to zebrafish Danio rerio, and the action mode of it on the heteromeric Drα1ß2Sγ2 and Drα1ß2S GABA receptor was respectively determined by HPLC and two-electrode voltage clamp technique. Fluxametamide exhibited high toxicity to D. rerio, whereas slightly inhibited the GABA-stimulated current of Drα1ß2Sγ2 or Drα1ß2S. It showed high bioconcentration level in D. rerio at 0.0314 mg L-1 and 0.157 mg L-1, with bioconcentration factors at steady state of 1491.55 and 2875.28, respectively. The concentration of fluxametamide in D. rerio rapidly decreased from 47.84 ± 0.12 to 9.77 ± 1.13 mg kg-1 in 0.0314 mg L-1 or from 393.19 ± 0.46 to 46.93 ± 2.88 mg kg-1 in 0.157 mg L-1 within 10 days, and steadily kept at a low level after 18 days. In conclusion, fluxametamide has highly acute toxicity to D. rerio, and might induce high bioconcentration in a short time. As we know, this is the first report to provide a theoretical basis for evaluating the potential risk of fluxametamide on fish, and guidance for the application of fluxametamide.

Synthesis and activity-detection of photoswitchable ligands with fipronil to insect.

BACKGROUND: Ionotropic γ-aminobutyric acid (GABA) receptor (GABAR) in an insect is the major inhibitory receptor and is one of the most important targets for insecticides. Due to the high spatiotemporal resolution of GABAR, the photopharmacological ligands acting on it in vertebrates but not insect have been developed. RESULTS: In this study, two types of photochromic ligands (PCLs) including DTFIPs (DTFIP1 and DTFIP2) and ABFIPs (p-, m-, and o-ABFIP) were synthesized by incorporating photoswitch azobenzene or dithienylethene into fipronil (FIP), which is the antagonist of insect GABAR. Their photomodulation was measured by mosquito larval behavior, and their potential action mechanism was explored by the two-electrode voltage clamp (TEVC) technique in vitro. DTFIP1 and m-ABFIP exhibited the most significant difference of insecticidal activity by about 90- and 5-fold to mosquito larvae between non-irradiated and irradiated formation, respectively, and allowed for optical control of mosquito swimming activity. TEVC assay results indicated that m-ABFIP and DTFIP1 enable optical control over the homomeric LsRDL-type GABAR, which is achieved by regulating the chloride channel of resistance to dieldrin (RDL)-type GABAR by photoisomerization. CONCLUSION: Our results suggested that PCLs synthesized from fipronil provide an alternative and precise tool for studying insect ionotropic GABARs and GABA-dependent behavior. © 2022 Society of Chemical Industry.

Diagnostic Value of LH Peak Value of the GnRH Stimulation Test for Girls with Precocious Puberty and Its Correlation with Body Mass Index.

Objective: To analyze the diagnostic value of luteinizing hormone (LH) peak value of the gonadotropin-releasing hormone (GnRH) stimulation test for girls with precocious puberty and its correlation with body mass index (BMI). Methods: A total of 230 girls with precocious puberty who came to our hospital for testing from June 2019 to June 2021 were selected and divided into a true group (n = 130) and sham group (n = 100) according to the results of the GnRH stimulation test. According to the BMI, the true group was further divided into a normal group (48 cases), overweight group (43 cases), and obese group (39 cases). The GnRH stimulation test was performed on all subjects, and the basal value and peak value of LH and the basal value and peak value of follicle-stimulating hormone (FSH) were recorded. The general data and serological indexes of the true group and the sham group were compared. Indicators of the GnRH stimulation test, breast stage, bone age, BMI, uterine volume, ovarian volume, and serological indicators (leptin, sex hormone-binding protein (SHBG), and adiponectin (APN)) were compared among the normal group, the overweight group, and the obese group. Results: There were no significant differences in age and breast stage between the true group and the sham group (P > 0.05). There were statistically significant differences in bone age, BMI, uterine volume, and ovarian volume between the two groups (P < 0.05). The LH base value, LH peak value, FSH base value, and FSH peak value in the true group were higher than those in the sham group, and the differences were statistically significant (P < 0.05). ROC curve analysis showed that the AUC of LH peak value in diagnosing girls with precocious puberty was 0.973, which was higher than 0.895, 0.875, and 0.912 of LH base value, FSH base value, and FSH peak value, respectively. There were statistically significant differences in LH base value, LH peak value, FSH base value, breast development stage, bone age, BMI, SHBG, leptin, and APN among the normal group, overweight group, and obese group (P < 0.05), but there were no significant differences in FSH peak value, uterine volume, and ovarian volume among the three groups (P > 0.05). There was a negative correlation between BMI, LH peak value, and FSH base value (P < 0.05), but there was no significant correlation between BMI and FSH peak value (P > 0.05). Conclusion: The LH peak value of the GnRH stimulation test has high diagnostic value for girls with precocious puberty, and BMI is negatively correlated with the LH peak value of CPP children.

5-(4-Pyridinyl)-3-isothiazolols as Competitive Antagonists of Insect GABA Receptors: Design, Synthesis, and a New Mechanism Leading to Insecticidal Effects.

Ionotropic γ-aminobutyric acid (GABA) receptors (iGABARs) are validated targets of drugs and insecticides. Our previous studies showed that the competitive antagonists of insect iGABARs exhibit insecticidal activities and that the 3-isothiazolol scaffold is used as a lead for developing novel iGABAR antagonists. Here, we designed a novel series of 4-aryl-5-(4-pyridinyl)-3-isothiazolol (4-API) analogs that have various aromatic substituents at the 4-position. Two-electrode voltage clamp experiments showed that all synthesized 4-APIs exhibited antagonistic activity against Musca domestica and Spodoptera litura iGABARs (RDL) expressed in oocytes of Xenopus laevis at 100 µM. Of the 4-APIs, the 4-(1,1'-biphenylyl) analog was the most potent antagonist with IC50s of 7.1 and 9.9 µM against M. domestica and S. litura RDL receptors, respectively. This analog also showed a certain insecticidal activity against S. litura larvae, with >75% mortality at 100 µg/g diet. Molecular docking studies with a M. domestica iGABAR model indicated that the π-π stacking interactions formed between the pyridinyl ring and Y252 and between the 4-substituted aromatic group and Y107 might be important for antagonism by the 4-(1,1'-biphenylyl) analog. Our studies provide important information for designing novel iGABAR antagonists and suggest that the 4-APIs acting on iGABARs are promising insecticide leads for further studies.

Optical Regulation of GABA Receptor by Visible Light via Azobenzene-Phenylpyrazole.

Photopharmacology provides superior temporal and spatial resolution for the study of biological functions of γ-aminobutyric acid receptors (GABARs), and photopharmacology based on GABARs has significantly advanced the research of neurons and cells. Herein, we reported the azobenzene-phenylpyrazole (ABPs) for controlling the function of GABARs by light. The insecticidal activity of ABPs against Aedes albopictus larvae was different before and after light illumination. ABP3 (1 µM) could induce depolarization of dorsal unpaired median neurons and enable the real-time photoregulation of mosquito larval behavior. An electrophysiological experiment indicated that ABP3 had different inhibitory effects on GABARs before and after illumination. ABPs realized optical control of GABARs utilizing visible light, providing valuable supplements to the existing GABAR photocontrol tools.

Novel insecticidal 1,6-dihydro-6-iminopyridazine derivatives as competitive antagonists of insect RDL GABA receptors.

BACKGROUND: The ionotropic γ-aminobutyric acid (GABA) receptor (iGABAR) is an important target for insecticides and parasiticides. Our previous studies showed that competitive antagonists (CAs) of insect iGABARs have the potential to be used for developing novel insecticides and that the structural modification of gabazine (a representative CA of mammalian iGABARs) could lead to the identification of novel CAs of insect iGABARs. RESULTS: In the present study, a novel series of 1,3-di- and 1,3,5-trisubstituted 1,6-dihydro-6-iminopyridazines (DIPs) was designed using a versatile strategy and synthesized using facile methods. Electrophysiological studies showed that several target DIPs (30 µM) exhibited excellent antagonistic activities against common cutworm and housefly iGABARs consisting of RDL subunits. The IC50 values of 3-(4-methoxyphenyl), 3-(4-trifluoromethoxyphenyl), 3-(4-biphenylylphenyl), 3-(2-naphthyl), 3-(3,4-methylenedioxyphenyl), and 3,5-(4-methoxyphenyl) analogs ranged from 2.2 to 24.8 µM. Additionally, several 1,3-disubstituted DIPs, especially 3-(4-trifluoromethoxyphenyl) and 3-(3,4-methylenedioxyphenyl) analogs, exhibited moderate insecticidal activity against common cutworm larvae, with >60% mortality at a concentration of 100 mg kg-1 . Molecular docking studies showed that the oxygen atom on the three-substituted aromatic ring could form a hydrogen bond with Arg254, which may enhance the activity of these DIPs against housefly iGABARs. CONCLUSION: This systematic study indicated that the presence of a carboxyl side chain shorter by one methylene than that of gabazine at the 1-position of the pyridazine ring is effective for maintaining the stable binding of these DIPs in insect iGABARs. Our study provides important information for the design of novel insect iGABAR CAs. © 2022 Society of Chemical Industry.

Neuroligin 3 from common cutworm enhances the GABA-induced current of recombinant SlRDL1 channel.

BACKGROUND: Neuroligin (NLG) protein is a nerve cell adhesion molecule and plays a key role in the precision apposition of presynaptic domains on inhibitory and excitatory synapses. Existing studies mainly focused on the function of NLG3 against the excitatory channel. However, the interaction between insect NLG3 and ionotropic GABA receptor, which is the main inhibitory channel, remains unclear. In this study, the Nlg3 of common cutworm (CCW), Spodoptera litura Fabricius, one important agricultural Lepidopteron, is selected to explore its function in the inhibitory channel. RESULTS: The SlNlg3 was obtained and the SlNLG3 contains the characteristic features including transmembrane domain, PDZ-binding motif and type-B carboxylesterases signature 2 motif. The SlNlg3 messenger RNA (mRNA) was most abundant in midgut, and exhibited multiple expression patterns in different developmental stages and tissues or body parts. Compared with the single injection of SlRDL1, the median effective concentration value of GABA in activating currents was smaller in Xenopus laevis oocytes co-injected with SlRDL1 and SlNlg3. In addition, SlNlg3 could enhance the GABA-induced current of homomeric SlRDL1 channel from -391.86 ± 15.41 to -2152.51 ± 30.09 nA. DsSlNlg3 depressed the expression level of SlNlg3 mRNA more than 64.29% at 6 h. After exposure to median lethal dose of fluralaner, the mortality of CCW injected with dsSlNlg3 was significantly decreased by 13.34% and 30.00% at 24 and 48 h, respectively, compared to injection of dsEGFP. CONCLUSION: NLG3 should have physiological function on ionotropic GABA receptor in vitro, which provided a favorable foundation for further research on the physiological function of Nlg gene in Lepidopteron. © 2021 Society of Chemical Industry.

Novel meta-diamide insecticide, broflanilide, suppresses the population of common cutworm Spodoptera litura through its lethal and sublethal effects.

BACKGROUND: Broflanilide has been registered in China for the control of Lepidoptera and Coleoptera pests, and is widely used to control the target pests at lethal and sublethal levels. The lethal and sublethal effects of broflanilide on the common cutworm (CCW) Spodoptera litura Fabricius, a representative Lepidopteran pest in agricultural crops, were examined to explore its ecological influence on pests. RESULTS: In F0 , broflanilide had little influence on the hatchability of eggs, but significantly reduced the neonate survival rate. The lethal activity of broflanilide towards third-instar larvae and adults was 0.13 mg kg-1 (LD50 ) and 3.59 mg L-1 (LC50 ) respectively at 48 h. After being treated with a sublethal dose (LD10 and LD30 ) of broflanilide, the duration of third- to sixth-instar larvae and the mean fecundity of reproductive females were significantly increased, but pupation rate, weight of pupae and life-cycle rate were significantly decreased. In F1 , the duration of F1 larvae and the doubling time were prolonged, whereas the rates of pupation and the life cycle were decreased by 14.92% and 18.00%, respectively. The intrinsic rate of increase, finite rate of increase and net reproductive rate in the sublethal group were lower than in the control group. The relative fitness of F1 treated by LD10 and LD30 was 0.81 and 0.66, respectively. CONSLUSION: Broflanilide not only has highly lethal activity, but also suppresses the population growth and progeny of CCW, as a critical factor for guidelines of its usage in the field.

Lateral and Longitudinal Driving Behavior Prediction Based on Improved Deep Belief Network.

Accurately predicting driving behavior can help to avoid potential improper maneuvers of human drivers, thus guaranteeing safe driving for intelligent vehicles. In this paper, we propose a novel deep belief network (DBN), called MSR-DBN, by integrating a multi-target sigmoid regression (MSR) layer with DBN to predict the front wheel angle and speed of the ego vehicle. Precisely, the MSR-DBN consists of two sub-networks: one is for the front wheel angle, and the other one is for speed. This MSR-DBN model allows ones to optimize lateral and longitudinal behavior predictions through a systematic testing method. In addition, we consider the historical states of the ego vehicle and surrounding vehicles and the driver's operations as inputs to predict driving behaviors in a real-world environment. Comparison of the prediction results of MSR-DBN with a general DBN model, back propagation (BP) neural network, support vector regression (SVR), and radical basis function (RBF) neural network, demonstrates that the proposed MSR-DBN outperforms the others in terms of accuracy and robustness.

Influence of three insecticides targeting GABA receptor on fall armyworm Spodoptera frugiperda: Analyses from individual, biochemical and molecular levels.

The fall armyworm (FAW) Spodoptera frugiperda (Lepidoptera: Noctuidae) is a severe agricultural pest, which has invaded into China in 2019 and caused heavy damage to maize. The γ-aminobutyric acid receptor (GABAR)-targeted insecticides including broflanilide, fluralaner and fipronil exhibit high toxicity towards lepidopteran pests. However, whether they could be used for control of FAW and their possible mode of action in FAW remain unclear. In this study, broflanilide, fluralaner and fipronil exhibited high oral toxicity in FAW larvae with median lethal dose (LD50) values of 0.677, 0.711, and 23.577 mg kg-1 (active ingredient/ artificial food), respectively. In the electrophysiological assay, fluralaner and fipronil could strongly inhibit GABA-induced currents of homomeric FAW resistance to dieldrin 1 (RDL1) receptor with median inhibitory concentration (IC50) values of 5.018 nM (95% confidence interval (CI) 2.864-8.789) and 8.595 nM (95% CI 5.105-14.47), respectively, whereas broflanilide could not. In addition, the cytochrome P450 (P450), glutathione-S-transferase (GST) and carboxylesterase (CarE) activities were positively response to broflanilide, P450 and GST to fluralaner, and GST and CarE to fipronil, respectively, compared with those of control. In conclusion, we firstly reported a notable insecticidal activity of three representative GABAR-targeted insecticides to FAW in vivo, and in vitro using electrophysiological assay. The GST is the primary detoxification enzyme for three tested insecticides. Our results would guide the rotational use of GABAR-targeted insecticides in field.

Transcript level is a key factor affecting RNAi efficiency.

Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.

Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA.

RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.

Novel alternative splicing of GABA receptor RDL exon 9 from Laodelphax striatellus modulates agonist potency.

The resistance to dieldrin gene (RDL) encodes the primary subunit of the insect ionotropic γ-aminobutyric acid (GABA) receptor (GABAR), which is the target of phenylpyrazole and isoxazoline insecticides. The splice variants in exons 3 and 6 of RDL, which have been widely explored in many insects, modulate the agonist potency of the homomeric RDL GABAR and potentially play an important role in the development of insects. In the present study, four splice variants of exon 9 were identified in RDL of the small brown planthopper, Laodelphax striatellus (LsRDL), resulting in LsRDL-9a, LsRDL-9a', LsRDL-9b, and LsRDL-9c. LsRDL-9a has one more amino acid (E, glutamic acid) compared with LsRDL-9a', and LsRDL-9b lacked two amino acids and had seven different amino acids compared with LsRDL-9c. Two-electrode voltage-clamp recording on LsRDLs expressed in Xenopus oocytes showed that alternative splicing of exon 9 has significant impact on LsRDL sensitivity to the agonists GABA and ß-alanine, whereas no significant difference was observed in the potencies of the non-competitive antagonists (NCAs) ethiprole and fluralaner on the splice variants. Our results suggest that alternative splicing of RDL exon 9 broadens functional capabilities of the GABAR in L. striatellus by influencing the action of GABA.

Exploring the Interaction Mechanism of Desmethyl-broflanilide in Insect GABA Receptors and Screening Potential Antagonists by In Silico Simulations.

Broflanilide, a novel insecticide, is classified as a negative allosteric modulator (NAM) of insect γ-aminobutyric acid (GABA) receptors (GABARs) as desmethyl-broflanilide (DMBF) allosterically inhibits the GABA-induced responses. The G277M mutation of the Drosophila melanogaster GABAR subunit has been reported to abolish the inhibitory activity of DMBF. The binding mode of DMBF in insect GABARs needs to be clarified to understand the underlying mechanism of this mutation and to develop novel, efficient NAMs of insect GABARs. Here, we found that a hydrogen bond formed between DMBF and G277 of the D. melanogaster GABAR model might be the key interaction for the antagonism of DMBF by in silico simulations. The volume increase induced by the G277M mutation blocks the entrance of the binding pocket, making it difficult for DMBF to enter the binding pocket and thereby decreasing its activity. The following virtual screening and bioassay results identified a novel NAM candidate of insect GABARs. Overall, we reported a possible binding mode of DMBF in insect GABARs and proposed the insensitivity mechanism of the G277M mutant GABAR to DMBF using molecular simulations. The identified NAM candidates might provide more alternatives or potentials for the design of GABAR-targeting insecticides.

Identification of a double-stranded RNA-degrading nuclease influencing both ingestion and injection RNA interference efficiency in the red flour beetle Tribolium castaneum.

RNA interference (RNAi) efficiency dramatically varies among different insects and among administration methods. Numerous studies have revealed that a poor RNAi response is usually associated with a high double-stranded RNA (dsRNA)-degrading activity. Using the red flour beetle Tribolium castaneum, we conducted genome-wide identification of genes encoding dsRNA-degrading nucleases of the DNA/RNA non-specific endonuclease superfamily. To achieve a robust RNAi response in T. castaneum, four dsRNase genes were identified in the genome that seemed to be the potential factors reducing RNAi efficacy. Analysis of biochemical properties revealed that optimal conditions for the dsRNA-degrading activity were alkaline (pH 8.0) in the absence of Mg2+ at 37 °C. The dsRNA-degrading activity was predominantly present in the gut, and via heterologous expression and RNAi experimentation, gut-specific TcdsRNase1 was confirmed as the major nuclease performing dsRNA degradation. After a knockdown of the TcdsRNase1 nuclease activity, RNAi efficiency improved from 38.6% to 58.9% and from 20.9% to 53.9% for injection and ingestion of dsRNA, respectively. Our results contribute to a comprehensive understanding of the mechanisms influencing dsRNA stability and even RNAi efficiency in T. castaneum and point to a good method for improving RNAi efficiency through downregulation of the relevant nuclease activity.