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.

VmSom1 is Essential for Growth, Development, Maintenance of Cell Wall Integrity and Virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Infestation of Rice Striped Stem Borer (Chilo suppressalis) Larvae Induces Emission of Volatile Organic Compounds in Rice and Repels Female Adult Oviposition.

Plants regulate the biosynthesis and emission of metabolic compounds to manage herbivorous stresses. In this study, as a destructive pest, the pre-infestation of rice striped stem borer (SSB, Chilo suppressalis) larvae on rice (Oryza sativa) reduced the subsequent SSB female adult oviposition preference. Widely targeted volatilomics and transcriptome sequencing were used to identify released volatile metabolic profiles and differentially expressed genes in SSB-infested and uninfested rice plants. SSB infestation significantly altered the accumulation of 71 volatile organic compounds (VOCs), including 13 terpenoids. A total of 7897 significantly differentially expressed genes were identified, and genes involved in the terpenoid and phenylpropanoid metabolic pathways were highly enriched. Correlation analysis revealed that DEGs in terpenoid metabolism-related pathways were likely involved in the regulation of VOC biosynthesis in SSB-infested rice plants. Furthermore, two terpenoids, (-)-carvone and cedrol, were selected to analyse the behaviour of SSB and predators. Y-tube olfactometer tests demonstrated that both (-)-carvone and cedrol could repel SSB adults at higher concentrations; (-)-carvone could simultaneously attract the natural enemies of SSB, Cotesia chilonis and Trichogramma japonicum, and cedrol could only attract T. japonicum at lower concentrations. These findings provide a better understanding of the response of rice plants to SSB and contribute to the development of new strategies to control herbivorous pests.

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.

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.

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.

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.

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.

Identification of transcriptome and fluralaner responsive genes in the common cutworm Spodoptera litura Fabricius, based on RNA-seq.

BACKGROUND: Fluralaner is a novel isoxazoline insecticide with a unique action site on the γ-aminobutyric acid receptor (GABAR), shows excellent activity on agricultural pests including the common cutworm Spodoptera litura, and significantly influences the development and fecundity of S. litura at either lethal or sublethal doses. Herein, Illumina HiSeq Xten (IHX) platform was used to explore the transcriptome of S. litura and to identify genes responding to fluralaner exposure. RESULTS: A total of 16,572 genes, including 451 newly identified genes, were observed in the S. litura transcriptome and annotated according to the COG, GO, KEGG and NR databases. These genes included 156 detoxification enzyme genes [107 cytochrome P450 enzymes (P450s), 30 glutathione S-transferases (GSTs) and 19 carboxylesterases (CarEs)] and 24 insecticide-targeted genes [5 ionotropic GABARs, 1 glutamate-gated chloride channel (GluCl), 2 voltage-gated sodium channels (VGSCs), 13 nicotinic acetylcholine receptors (nAChRs), 2 acetylcholinesterases (AChEs) and 1 ryanodine receptor (RyR)]. There were 3275 and 2491 differentially expressed genes (DEGs) in S. litura treated with LC30 or LC50 concentrations of fluralaner, respectively. Among the DEGs, 20 related to detoxification [16 P450s, 1 GST and 3 CarEs] and 5 were growth-related genes (1 chitin and 4 juvenile hormone synthesis genes). For 26 randomly selected DEGs, real-time quantitative PCR (RT-qPCR) results showed that the relative expression levels of genes encoding several P450s, GSTs, heat shock protein (HSP) 68, vacuolar protein sorting-associated protein 13 (VPSAP13), sodium-coupled monocarboxylate transporter 1 (SCMT1), pupal cuticle protein (PCP), protein takeout (PT) and low density lipoprotein receptor adapter protein 1-B (LDLRAP1-B) were significantly up-regulated. Conversely, genes encoding esterase, sulfotransferase 1C4, proton-coupled folate transporter, chitinase 10, gelsolin-related protein of 125 kDa (GRP), fibroin heavy chain (FHC), fatty acid synthase and some P450s were significantly down-regulated in response to fluralaner. CONCLUSIONS: The transcriptome in this study provides more effective resources for the further study of S. litura whilst the DEGs identified sheds further light on the molecular response to fluralaner.

Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

RNA interference (RNAi) efficiency varies among insects. RNAi is highly efficient and systemic in coleopteran insects but quite variable and inefficient in lepidopteran insects. Degradation of double-stranded RNA (dsRNA) by double-stranded ribonucleases (dsRNases) is thought to contribute to the variability in RNAi efficiency observed among insects. One or two dsRNases involved in dsRNA digestion have been identified in a few insects. To understand the contribution of dsRNases to reduced RNAi efficiency in lepidopteran insects, we searched the transcriptome of Spodoptera litura and identified six genes coding for DNA/RNA non-specific endonucleases. Phylogenetic analysis revealed the evolutionary expansion of dsRNase genes in insects. The mRNA levels of three midgut-specific dsRNases increased during the larval stage, and the highest dsRNA-degrading activity was detected in third-instar larvae. Proteins produced via the expression of three midgut-specific dsRNases, and the widely expressed dsRNase3, in a baculovirus system showed dsRNase activity for four out of five dsRNases tested. In addition, the increase in dsRNA-degrading activity and upregulation of dsRNase1 and 2 in larvae fed on cabbage leaves suggests that the diet of S. litura can influence dsRNase expression, dsRNA stability, and thus probably RNAi efficiency. This is the first report that multiple dsRNases function together in an RNAi-recalcitrant insect. The data included in this paper suggest that multiple dsRNases coded by the S. litura genome might contribute to the lower and variable RNAi efficiency reported in this and other lepidopteran insects.

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis, biological activity, and molecular docking studies.

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC50 values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC50 values of 2.0 and 0.9 µM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100 µg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.

Identification of the ionotropic GABA receptor-like subunits from the striped stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae).

Ionotropic γ-aminobutyric acid (GABA) receptors (GABARs) mediate rapid inhibitory neurotransmission in both vertebrates and invertebrates, and are important molecular targets of insecticides. However, components of insect GABARs remain elusive. In addition to CsRDL1 and CsRDL2, the complementary DNAs (cDNAs) of another two GABA receptor-like subunits, CsLCCH3 and Cs8916, were identified from the rice striped stem borer, Chilo suppressalis Walker in the present study. Both CsLCCH3 and Cs8916 subunits shared common structural features, such as a highly-conserved Cys-loop structure, six distinct regions involved in ligand binding (loops A-F), and four transmembrane domains (TM 1-4). Transcript analysis demonstrated that the relative mRNA expression levels of both CsLCCH3 and Cs8916 subunits were the highest in the ventral nerve cord. Regarding developmental stage, transcript levels of both subunits were highest in eggs. Injections of double-stranded RNAs (dsRNAs), including dsRDL1, dsRDL2, dsLCCH3, or ds8916, significantly reduced mRNA abundance after 24 and 48 h. However, no observable effects on the development of C. suppressalis were observed. Injection of dsRDL1 or dsRDL2 did significantly reduce the mortality of C. suppressalis treated with fluralaner. Our results indicated that CsRDLs mediated the susceptibility of C. suppressalis to fluralaner, whereas CsLCCH3 and CsL8916 did not. The current investigation enhances our knowledge of Lepidopteran GABARs and offers a molecular basis for the development of novel insecticides to control C. suppressalis.

Catalyst-free assembly of giant tris(heteroaryl)methanes: synthesis of novel pharmacophoric triads and model sterically crowded tris(heteroaryl/aryl)methyl cation salts.

A series of giant tris(heteroaryl)methanes are easily assembled by one-pot three-component synthesis by simple reflux in ethanol without catalyst or additives. Diversely substituted indoles (Ar1) react with quinoline aldehydes, quinolone aldehydes, chromone aldehydes, and fluorene aldehydes (Ar2CHO) and coumarins (Ar3) in 1:1:1 ratio to form the corresponding tris(heteroaryl)methanes (Ar1Ar2Ar3)CH along with (Ar1Ar1Ar2)CH triads. A series of new 2:1 triads were also synthesized by coupling substituted indoles with Ar2CHO. The coupling reactions could also be carried out in water (at circa 80 °C) but with chemoselectivity favoring (Ar1Ar1Ar2)CH over (Ar1Ar2Ar3)CH. The molecular structure of a representative (Ar1Ar2Ar3)CH triad was confirmed by X-ray analysis. Model tris(heteroaryl/aryl)methylium salts were generated by reaction with DDQ/HPF6 and studied by NMR and by DFT and GIAO-DFT.

Toxicity and sublethal effects of fluralaner on Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

The increasing occurrence of resistance to chemical insecticides in insect pest populations is a serious threat to the integrity of current pest management strategies, and exploring new alternative chemistries is one important way to overcome this obstacle. Fluralaner, as a novel isoxazoline insecticide, has broad spectrum activity against a variety of insect pests, but little data is available about its effect on Lepidopterans. The effects of fluralaner on Spodoptera litura Fabricius, a widespread and polyphagous pest, were evaluated in the present study. Our results showed younger larvae were more susceptible to fluralaner treatment, but feeding and topical applications were similarly effective in 3rd instar larvae. Synergism assays indicated that piperonyl butoxide (PBO) could increase the toxicity of fluralaner to S. litura to a certain degree and P450 may be involved in the detoxification of fluralaner in vivo. Sublethal developmental effects included reduced larval body weight, decreased pupation and emergence, and notched wings in adults, accompanied by changes in the transcript levels of chitinase 5 (CHT5) and juvenile hormone acid methyltransferase (Jhamt), genes vital for insect development. Above results manifested that fluralaner is highly toxic to S. litura larvae via either topical or oral application and provide an indication of how this insecticide is metabolized in vivo. Further, our results provided a foundation for further development of fluralaner as a new tool in insect pest management.

GABAA receptor target of tetramethylenedisulfotetramine.

Use of the highly toxic and easily prepared rodenticide tetramethylenedisulfotetramine (TETS) was banned after thousands of accidental or intentional human poisonings, but it is of continued concern as a chemical threat agent. TETS is a noncompetitive blocker of the GABA type A receptor (GABAAR), but its molecular interaction has not been directly established for lack of a suitable radioligand to localize the binding site. We synthesized [(14)C]TETS (14 mCi/mmol, radiochemical purity >99%) by reacting sulfamide with H(14)CHO and s-trioxane then completion of the sequential cyclization with excess HCHO. The outstanding radiocarbon sensitivity of accelerator mass spectrometry (AMS) allowed the use of [(14)C]TETS in neuroreceptor binding studies with rat brain membranes in comparison with the standard GABAAR radioligand 4'-ethynyl-4-n-[(3)H]propylbicycloorthobenzoate ([(3)H]EBOB) (46 Ci/mmol), illustrating the use of AMS for characterizing the binding sites of high-affinity (14)C radioligands. Fourteen noncompetitive antagonists of widely diverse chemotypes assayed at 1 or 10 µM inhibited [(14)C]TETS and [(3)H]EBOB binding to a similar extent (r(2) = 0.71). Molecular dynamics simulations of these 14 toxicants in the pore region of the α1ß2γ2 GABAAR predict unique and significant polar interactions for TETS with α1T1' and γ2S2', which are not observed for EBOB or the GABAergic insecticides. Several GABAAR modulators similarly inhibited [(14)C]TETS and [(3)H]EBOB binding, including midazolam, flurazepam, avermectin Ba1, baclofen, isoguvacine, and propofol, at 1 or 10 µM, providing an in vitro system for recognizing candidate antidotes.

Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae).

Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system.

Biodegradation of nicosulfuron by a novel Alcaligenes faecalis strain ZWS11.

A bacterial strain ZWS11 was isolated from sulfonylurea herbicide-contaminated farmland soil and identified as a potential nicosulfuron-degrading bacterium. Based on morphological and physicochemical characterization of the bacterium and phylogenetic analysis of the 16S rRNA sequence, strain ZWS11 was identified as Alcaligenes faecalis. The effects of the initial concentration of nicosulfuron, inoculation volume, and medium pH on degradation of nicosulfuron were investigated. Strain ZWS11 could degrade 80.56% of the initial nicosulfuron supplemented at 500.0mg/L under the conditions of pH7.0, 180r/min and 30°C after incubation for 6days. Strain ZWS11 was also capable of degrading rimsulfuron, tribenuron-methyl and thifensulfuron-methyl. Four metabolites from biodegradation of nicosulfuron were identified, which were 2-aminosulfonyl-N, N-dimethylnicotinamide (M1), 4, 6-dihydroxypyrimidine (M2), 2-amino-4, 6-dimethoxypyrimidine (M3) and 2-(1-(4,6-dimethoxy-pyrimidin-2-yl)-ureido)-N,N-dimethyl-nicotinamide (M4). Among the metabolites detected, M2 was reported for the first time. Possible biodegradation pathways of nicosulfuron by strain ZWS11 were proposed. The degradation proceeded mainly via cleavage of the sulfonylurea bridge, O-dealkylation, and contraction of the sulfonylurea bridge by elimination of a sulfur dioxide group. The results provide valuable information for degradation of nicosulfuron in contaminated environments.

Comparative Insecticidal Activity and Mechanism of Isocycloseram versus Other GABAergic Insecticides against the Fall Armyworm.

The fall armyworm (FAW) is a serious agricultural pest and has developed resistance to multiple insecticides. It is necessary to introduce novel insecticide(s) for controlling FAW. Isocycloseram is a completely novel isoxazoline insecticide. However, its activity and mode of action against FAW have not been reported. In this study, isocycloseram exhibited a higher insecticidal activity (LC50 = 0.26 mg/kg) than fipronil (LC50 = 7.72 mg/kg) against FAW. The median inhibitory concentration (IC50) of isocycloseram (IC50 = 8.52 nM) was almost equal to that of the desmethyl-broflanilide (IC50 = 7.32 nM) to the SfrRDL1 receptor. The IC50 of isocycloseram to the SfrRDL2 receptor was 11.13 nM, which was obviously less than that of desmethyl-broflanilide, dieldrin, fipronil, fluxametamide. Compared with the SfrRDL2 receptor, the SfrRDL1 receptor exhibited higher sensitivity to GABAergic insecticides. The recombinant SfrGluCl receptor was successfully stimulated by l-glutamate; however, the currents were low and weakly inhibited by isocycloseram at 10 µM. In conclusion, our results provided the theoretical basis for usage of GABAergic insecticides for controlling FAW.

Control efficacy and joint toxicity of broflanilide mixed with commercial insecticides to an underground pest, the black cutworm in highland barley.

BACKGROUND: The highland barley, Hordeum vulgare L., is a staple food crop with superior nutritional functions in Xizang, China. It is often damaged by the black cutworm, Agrotis ipsilon (Hufnagel), which is an underground pest and difficult to effectively manage. To introduce a novel insecticide with unique mode of action, broflanilide (BFL) and its binary mixtures with chlorantraniliprole (CAP), fluxametamide, ß-cypermethrin or imidacloprid were screened out as seed treatment to control black cutworm in highland barley in the present study. RESULTS: In the laboratory bioassays, BFL had outstanding insecticidal activity to black cutworm with a median lethal dose (LD50) of 0.07 mg kg-1. The mixture of BFL × CAP at the concentration ratio of 7:40 exhibited the highest synergistic effect with a co-toxicity coefficient of 280.48. In the greenhouse pot experiments, BFL and BFL × CAP seed treatments at 8 g a.i. kg-1 seed could effectively control black cutworm, with a low percentage of injured seedlings <20% and high control efficacies of 93.33-100% during a period of 3-12 days after seed emergence. Moreover, BFL and BFL × CAP seed treatments could promote the seed germination and seedling growth of highland barley at the tested temperatures of 15, 20 and 25 °C. CONCLUSION: Our results indicated that BFL and BFL × CAP were effective and promising insecticides as seed treatment to control black cutworm in highland barley. © 2024 Society of Chemical Industry.

Transcriptome-wide N6-methyladenosine profiling reveals growth-defense trade-offs in the response of rice to brown planthopper (Nilaparvata lugens) infestation.

BACKGROUND: N6-Methyladenosine (m6A) is a common messenger RNA (mRNA) modification that affects various physiological processes in stress responses. However, the role of m6A modifications in plants responses to herbivore stress remains unclear. RESULTS: Here, we found that an infestation of brown planthopper (Nilaparvata lugens) female adults enhanced the resistance of rice to N. lugens. The m6A methylome analysis of N. lugens-infested and uninfested rice samples was performed to explore the interaction between rice and N. lugens. The m6A methylation mainly occurred in genes that were actively expressed in rice following N. lugens infestation, while an analysis of the whole-genomic mRNA distribution of m6A showed that N. lugens infestation caused an overall decrease in the number of m6A methylation sites across the chromosomes. The m6A methylation of genes involved in the m6A modification machinery and several defense-related phytohormones (jasmonic acid and salicylic acid) pathways was increased in N. lugens-infested rice compared to that in uninfested rice. In contrast, m6A modification levels of growth-related phytohormone (auxin and gibberellin) biosynthesis-related genes were significantly attenuated during N. lugens infestation, accompanied by the down-regulated expression of these transcripts, indicating that rice growth was restricted during N. lugens attack to rapidly optimize resource allocation for plant defense. Integrative analysis of the differential patterns of m6A methylation and the corresponding transcripts showed a positive correlation between m6A methylation and transcriptional regulation. CONCLUSION: The m6A modification is an important strategy for regulating the expression of genes involved in rice defense and growth during rice-N. lugens interactions. These findings provide new ideas for formulating strategies to control herbivorous pests. © 2024 Society of Chemical Industry.