Metabolism-Based Nontarget-Site Mechanism Is the Main Cause of a Four-Way Resistance in Shortawn Foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

The Cys-2088-Arg mutation in the ACCase gene and enhanced metabolism confer cyhalofop-butyl resistance in Chinese sprangletop (Leptochloa chinensis).

Acetyl-CoA carboxylase (ACCase)-inhibiting herbicides are among the most commonly used herbicides to control grassy weeds, especially Leptochloa chinensis, in rice fields across China. Herein, we collected a suspected resistant (R) population of L. chinensis (HFLJ16) from Lujiang county in Anhui Province. Whole plant dose response tests showed that, compared with the susceptible (S) population, the R population showed high resistance to cyhalofop-butyl (22-fold) and displayed cross-resistance to metamifop (9.7-fold), fenoxaprop-P-ethyl (18.7-fold), quizalofop-P-ethyl (7.6-fold), clodinafop-propargyl (12-fold) and clethodim (8.4-fold). We detected an amino acid substitution (Cys-2088-Arg) in the ACCase of resistant L. chinensis. However, ACCase gene expression levels were not significantly different (P > 0.05) between R plants and S plants, without or with cyhalofop-butyl treatment. Furthermore, pretreatment with piperonyl butoxide (PBO, a cytochrome P450 monooxygenase (CYP450) inhibitor) or 4-chloro-7-nitrobenzoxadiazole (NBD-Cl, a glutathione-S-transferase (GST) inhibitor), inhibited the resistance of the R population to cyhalofop-butyl significantly (by approximately 60% and 26%, respectively). Liquid chromatography tandem mass spectrometry analysis showed that R plants metabolized cyhalofop-butyl and cyhalofop acid (its metabolite) significantly faster than S plants. Three CYP450 genes, one GST gene, and two ABC transporter genes were induced by cyhalofop-butyl and were overexpressed in the R population. Overall, GST-associated detoxification, CYP450 enhancement, and target-site gene mutation are responsible for the resistance of L. chinensis to cyhalofop-butyl.

Functional analysis of three odorant receptors in Plutella xylostella response to repellent activity of 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Plutella xylostella is an important pest showing resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Previous study showed that 2,3-dimethyl-6-(1-hydroxy)-pyrazine has significant repellent activity to P. xylostella adult which mainly effect to the olfactory system, however the molecular targets and mechanism are still unclear. Based on the RNA-Seq and RT-qPCR data, eight ORs (Odorant receptor) in P. xylostella were selected as candidate targets response to repellent activity of 2,3-dimethyl-6-(1-hydroxy)-pyrazine. Here, most of the ORs in P. xylostella were clustered into three branches, which showed similar functions such as recognition, feeding, and oviposition. PxylOR29, PxylOR31, and PxylOR46 were identified as the potential molecular targets based on the results of repellent activity and EAG response tests to the adults which have been injected with dsRNA, respectively. Additionally, the three ORs were higher expressed in antenna of P. xylostella, followed by those in the head segment. Furthermore, it was found that the bindings between these three ORs and 2,3-dimethyl-6-(1-hydroxy)-pyrazine mainly depend on the hydrophobic effect of active cavities, and the binding to PxylOR31 was more stabler and easier with an energy of -16.34 kcal/mol, together with the π-π T-shaped interaction at PHE195 site. These findings pave the way for the complete understanding of pyrazine repellent mechanisms.

Sublethal effect and detoxifying metabolism of metaflumizone and indoxacarb on the fall armyworm, Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera, Noctuidae), is a highly polyphagous invasive pest that damages various crops. Pesticide control is the most common and effective strategy to control FAW. In this study, we evaluated the toxicity of metaflumizone and indoxacarb against third-instar FAW larvae using the insecticide-incorporated artificial diet method under laboratory conditions. Both metaflumizone and indoxacarb exhibited substantial toxicity against FAW, with LC50 values of 2.43 and 14.66 mg/L at 72 h, respectively. The sublethal effects of metaflumizone and indoxacarb on parental and F1 generation FAW were investigated by exposing third-instar larvae to LC10 and LC30 concentrations of these insecticides. Sublethal exposure to these two insecticides significantly shortened adult longevity, extended pupal developmental times and led to reduced pupal weight, pupation rates, and adult fecundity in the treated parental generation and F1 generation at LC10 or LC30 concentrations, in comparison to the control group. The larval developmental times were shortened in the parental generation but prolonged in the F1 generation, after being treated with sublethal concentrations of metaflumizone. Furthermore, larvae exposed to LC10 or LC30 concentrations of indoxacarb exhibited elevated activity levels of cytochrome P450 monooxygenase and glutathione S-transferase, which coincides with the observed synergistic effect of piperonyl butoxide and diethyl maleate. In conclusion, the high toxicity and negative impact of metaflumizone and indoxacarb on FAW provided significant implications for the rational utilization of insecticides against this pest.

Inhalation bioaccessibility of imidacloprid in particulate matter: Implications for risk assessment during spraying.

Adverse health outcomes due to the inhalation of pesticide residues in atmospheric particulate matter (PM) are gaining global attention. Quantitative health risk assessments of pesticide inhalation exposure highlight the need to understand the bioaccessibility of pesticide residues. Herein, the inhalation bioaccessibility of imidacloprid in PM was determined using three commonly used in vitro lung modeling methods (Artificial Lysosomal Fluid, Gamble Solution, and Simulated Lung Fluid). To validate its feasibility and effectiveness, we evaluated the bioavailability of imidacloprid using a mouse nasal instillation assay. The in vitro inhalation bioaccessibility of imidacloprid was extracted using Gamble Solution with a solid-liquid ratio of 1/1000, an oscillation rate of 150 r/min, and an extraction time of 24 h, showed a strong linear correlation with its in vivo liver-based bioavailability (R2 =0.8928). Moreover, the margin of exposure was incorporated into the inhalation exposure risk assessment, considering both formulations and nozzles. The inhalation unit exposure of imidacloprid for residents was 0.95-4.09 ng/m3. The margin of exposure for imidacloprid was determined to be acceptable when considering inhalation bioaccessibility. Taken together, these results indicate that the inhalation bioaccessibility of pesticides should be incorporated into assessments of human health risks posed by PM particles.

Identification and functional characterization of two antenna-specifc odorant-binding proteins in Plutella xylostella response to 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Plutella xylostella is an important cruciferous crop pest with a serious resistance to multiple insecticides, a novel natural compound, 2,3-dimethyl-6-(1-hydroxy)-pyrazine were isolated, that showed significant repellent activity against P. xylostella with olfactory system as a potential target. Eight odorant-binding proteins (OBPs) were determined as candidate target genes using RT-qPCR (Quantitative reverse transcription PCR), most of them were clustered with OBPs from Spodoptera frugiperda. Fluorescence competitive binding assays showed that PxylPBP2 (Pheromone binding protein) and PxylOBP3 had Ki values of 7.13 ± 0.41 µM and 9.56 ± 0.35 µM, indicating a high binding affinity to the pyrazine. Moreover, the binding style between these two OBPs and the pyrazine was determined as a hydrophobic interaction by using molecular docking. The binding between PxylPBP2 and the pyrazine was found to be more stable, and the carbon atoms of C-2 and C-3 in this pyrazine showed potential optimization characteristics. Both PxylPBP2 and PxylOBP3 were highly expressed in the antennae of both sexes. These results can be used to design and develop novel green pesticides with the pyrazine as the active or lead compound to reduce the utilization of chemical pesticides and postpone development of resistance.

Ecological Toxicity of Cyantraniliprole against Procambarus clarkii: Histopathology, Oxidative Stress, and Intestinal Microbiota.

Cyantraniliprole is a novel insecticide recently introduced for rice pest control that may cause potential threats to the red swamp crayfish (Procambarus clarkii) in rice-crayfish coculture systems. In this study, we investigated the acute toxicity of cyantraniliprole against P. clarkii with a LC50 value of 149.77 mg/L (96 h), first. Some abnormal behaviors of P. clarkii treated with 125 mg/L cyantraniliprole, including incunabular hyperexcitability, imbalance, inactivity, and increased excretion were observed. Moreover, it was observed that exposure to 5 mg/L cyantraniliprole for 14 days resulted in histopathological alterations in abdominal muscle, gills, hepatopancreas, and intestines. Furthermore, exposure to 0.05 and 5 mg/L cyantraniliprole induced increased activities of several oxidative stress-related enzymes, which was verified by the upregulation of related genes. Additionally, dysregulation of the intestinal microbiota was determined via 16S rRNA sequencing. These results will provide the basis for the utilization of cyantraniliprole in the fields of rice-crayfish integrated system.

Fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon resistance status in Beckmannia syzigachne from wheat fields across Anhui Province, China.

Severe infestations of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) in wheat fields throughout Anhui Province, China, pose a significant threat to local agricultural production. This study aims to evaluate the susceptibility of 37 B. syzigachne populations collected from diverse wheat fields in Anhui Province to three commonly used herbicides: fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon. Single-dose testing revealed that out of the 37 populations, 31, 26, and 11 populations had either evolved or were evolving resistance to fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon, respectively. Among them, 25 populations displayed concurrent resistance to both fenoxaprop-P-ethyl and mesosulfuron-ethyl, while eight exhibited resistance to all three tested herbicides. Whole-plant bioassays confirmed that approximately 84% of the fenoxaprop-P-ethyl-resistant populations manifested high-level resistance (resistance index (RI) ≥10); 62% of the mesosulfuron-ethyl-resistant populations and 82% of the isoproturon-resistant populations exhibited low- to moderate-level resistance (2 ≤ RI <10). Three distinct target-site mutations were identified in 27% of fenoxaprop-P-ethyl-resistant populations, with no known resistance mutations detected in the remaining herbicide-resistant populations. The inhibition of cytochrome P450s (P450s) and/or glutathione S-transferases (GSTs) substantially increased susceptibility in the majority of resistant populations lacking mutations at the herbicide target site. In conclusion, resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl was widespread in B. syzigachne within Anhui Province's wheat fields, while resistance to isoproturon was rapidly evolving due to its escalating usage. Target-site mutations were present in approximately one-third of fenoxaprop-P-ethyl-resistant populations, and alternative mechanisms involving P450s and/or GSTs could explain the resistance observed in most of the remaining populations.

Quaternized chitosan-based organic-inorganic nanohybrid nanoparticles loaded with prothioconazole for efficient management of fungal diseases with minimal environmental impact.

Poor foliar deposition and retention of pesticides results in serious pesticide residues and environmental pollution. Organic-inorganic hybridized nanoparticles (OIHN), combining the advantages of organic and inorganic materials, can be used as carriers to load pesticides for efficient and safe application. Herein, a novel multifunctional OIHN composed of mesoporous silica nanoparticles (MSNs) and cationic chitosan quaternary ammonium salt (HACC) was constructed and used as a delivery system for prothioconazole (PTC). The resultant PTC@MSNs-HACC exhibited a remarkable loading capacity of 39.07 wt% and demonstrated enhanced PTC release (31.47 %) under alkaline conditions. The UV-shielding properties of MSNs efficiently shielded PTC from photodegradation, increasing its photostability by over threefold. The strong positive charge of HACC conferred excellent adhesion of PTC@MSNs-HACC to fungal cell membranes, leading to high deposition on wheat leaves with improved rain-wash resistance (increased by 30 %). Consequently, PTC@MSNs-HACC (EC50: 12.48 mg/L) exhibited superior wheat scab control compared to PTC emulsifiable concentrate (EC50: 28.49 mg/L). Additionally, PTC@MSNs-HACC displayed excellent uptake and transport in plants, ensuring plant safety and reducing toxicity to zebrafish by >1-fold. The potential application of the developed PTC@MSNs-HACC in agricultural production holds significant promise and is anticipated to find widespread use in the future.

Residue Behavior of Chiral Fungicide Prothioconazole and Its Major Chiral Metabolite in Flour Product Processing.

This study systematically investigates the stereoselective metabolism and residue behavior of chiral pesticide prothioconazole enantiomers during the steaming, baking, and frying of steamed buns, bread, and deep-fried dough sticks. The results show that steaming, baking, and frying can significantly promote the degradation of the prothioconazole enantiomers. In low- and high-concentration treatments, the degradation rates of prothioconazole enantiomers were over 96.0% and 45.4%, respectively, and the residual concentration of prothioconazole-desthio enantiomers was less than 32.7 µg/kg (excluding fried processing). During the processing of steamed buns, bread, and deep-fried dough sticks, the enantiomer fraction (EF) value of the prothioconazole enantiomer was close to 0.5, and the stereoselectivity was not significant. During the processing of steamed buns (low concentration), bread (low and high concentrations), and deep-fried dough sticks (low concentration), the stereoselectivity of prothioconazole-desthio was significant, and preferential enantiomer degradation occurred. Following the analysis of 120 flour product samples, the residual risk.

In vitro inhalation bioaccessibility and health risk assessment of difenoconazole in the atmosphere.

BACKGROUND: Assessment of the risk of pesticide inhalation in populations around farmland is necessary because inhalation is one of the ways in which pesticides can risk human health. This study aimed to identify the inhalation risk of difenoconazole on humans by using dose-response and exposure assessments. RESULTS: In the field simulation application, respiratory exposure in populations around farmland ranged from 71 to 430 ng/m3 . Using response surface methodology, the maximum bioaccessibility of difenoconazole in three simulated lung fluids was 35.33% in Gamble's solution (GS), 34.12% in artificial lysosomal fluid (ALF), and 42.06% in simulated interstitial lung fluid (SLF). Taking the proliferation activity of the A549 cell model as the endpoint, the benchmark dose limit and benchmark dose of difenoconazole on A549 cells were 16.36 and 5.60 mg/kg, respectively. The margin of exposure to difenoconazole in GS, ALF and SLF were, respectively, 8.66 × 105 to 5.28 × 106 , 8.97 × 105 to 5.47 × 106 and 7.28 × 105 to 4.44 × 106 . CONCLUSION: The risk assessment results indicate that under all circumstances, applying difenoconazole is safe for populations around farmland. However, a fan-shaped nozzle, suspension concentrate and greater inhalation height increase the risk of inhalation. © 2023 Society of Chemical Industry.

Transcript Level and Sequence Matching Are Key Determinants of Off-Target Effects in RNAi.

Double-stranded RNA (dsRNA) pesticides, those based on RNA interference (RNAi) technology utilizing dsRNA, have shown potential for pest control. However, the off-target effects of dsRNA pose limitations to the widespread application of RNAi and raise concerns regarding potential side effects on other beneficial organisms. The precise impact and underlying factors of these off-target effects are still not well understood. Here, we found that the transcript level and sequence matching jointly regulate off-target effects of dsRNA. The much lower expressed target genes were knocked down to a lesser extent than genes with higher expression levels, and the critical sequence identity of off-target effects is approximately 80%. Moreover, off-target effects could be triggered by a contiguous matching sequence length exceeding 15 nt as well as nearly perfectly matching sequences with one or two base mismatches exceeding 19 nt. Increasing the dosage of dsRNA leads to more severe off-target effects. However, the length of mismatched dsRNA, the choice of different RNAi targets, and the location of target sites within the same gene do not affect the severity of off-target effects. These parameters can be used to guide the design of possibly selective sequences for RNAi, optimize the specificity and efficiency of dsRNA, and facilitate practical applications of RNAi for pest control.

Pyrrolizidine Alkaloids in Tea (Camellia sinensis L.) from Weeds through Weed-Soil-Tea Transfer and Risk Assessment of Tea Intake.

Pyrrolizidine alkaloids (PAs) have been detected in tea and can threaten human health. However, the specific source of PAs in tea is still unclear. Here, 88 dried tea products collected from six major tea-producing areas in Anhui Province, China, were analyzed. The detection frequency was 76%. The content of total PAs in dried tea was between 1.1 and 90.5 µg/kg, which was all below the MRL recommended by the European Union (150 µg/kg). In the Shexian tea garden, PAs in the weeds and weed rhizospheric soil around tea plants and the fresh tea leaves were analyzed. Intermedine (Im), intermedine-N-oxide (ImNO), and jacobine-N-oxide (JbNO) were transferred through the weed-to-soil-to-tea route into the fresh tea leaves; only Im and ImNO were detected in dried tea samples. Potential risk of the total PAs in the tea infusion was assessed according to the margin of exposure method, and it might be a low concern for public health.

Knockdown of the glutamate-gated chloride channel gene decreases emamectin benzoate susceptibility in the fall armyworm, Spodoptera frugiperda.

Emamectin benzoate (EB), a derivative of avermectin, is the primary insecticide used to control the fall armyworm (FAW) in China. However, the specific molecular targets of EB against FAW remain unclear. In this study, we cloned the glutamate-gated chloride channel (GluCl) gene, which is known to be a primary molecular target for avermectin. We first investigated the transcript levels of SfGluCl in FAW and found that the expression level of SfGluCl in the head and nerve cord was significantly higher than that in other tissues. Furthermore, we found that the expression level of SfGluCl was significantly higher in eggs than that in other developmental stages, including larvae, pupae, and adults. Additionally, we identified three variable splice forms of SfGluCl in exons 3 and 9 and found that their splice frequencies remained unaffected by treatment with the LC50 of EB. RNAi mediated knockdown of SfGluCl showed a significant reduction of 42% and 65% after 48 and 72 h of dsRNA feeding, respectively. Importantly, knockdown of SfGluCl sifgnificantly reduced LC50 and LC90 EB treatment induced mortality of FAW larvae by 15% and 44%, respectively, compared to the control group feeding by dsEGFP. In contrast, there were no significant changes in the mortality of FAW larvae treated with the control insecticides chlorantraniliprole and spinetoram. Finally, molecular docking simulations revealed that EB bound to the large amino-terminal extracellular domain of SfGluCl by forming five hydrogen bonds, two alkyl hydrophobic interactions and one salt bridge. These findings strongly suggest that GluCl may serve as one of the molecular targets of EB in FAW, shedding light on the mode of action of this important insecticide.

Risk assessment of honeybee larvae exposure to pyrethroid insecticides in beebread and honey.

Honeybee is an essential pollinator to crops, evaluation to the risk assessment of honeybee larvae exposure to pesticides residue in the bee bread and honey is an important strategy to protect the bee colony due to the mixture of these two matrices is main food for 3-day-old honeybee larvae. In this study, a continuous survey to the residue of five pyrethroid insecticides in bee bread and honey between 2018 and 2020 from 17 major cultivation provinces which can be determined as Northeast, Northwest, Eastern, Central, Southwest, and Southern of China, there was at least one type II pyrethroid insecticide was detected in 54.7 % of the bee bread samples and 43.4 % of the honey. Then, we assayed the acute toxicity of type II pyrethroid insecticides based on the detection results, the LD50 value was 0.2201 µg/larva (beta-cyhalothrin), 0.4507 µg/larva (bifenthrin), 2.0840 µg/larva (fenvalerate), 0.0530 µg/larva (deltamethrin), and 0.1640 µg/larva (beta-cypermethrin), respectively. Finally, the hazard quotient was calculated as larval oral ranged from 0.046 × 10-3 to 2.128 × 10-3. Together, these empirical findings provide further insight into the accurate contamination of honey bee colonies caused by chemical pesticides, which can be used as a valuable guidance for the beekeeping industry and pesticide regulation.

Cytochrome P450s-Involved Enhanced Metabolism Contributes to the High Level of Nicosulfuron Resistance in Digitaria sanguinalis from China.

Large crabgrass (Digitaria sanguinalis (L.) Scop.) is one of the major malignant grass weeds in Chinese maize (Zea mays L.) fields, and it has recently developed resistance to the acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron. This study focused on a suspected nicosulfuron-resistant (R) population (LJ-01) of D. sanguinalis, collected from Lujiang County in Anhui Province, China, to explore the resistance level and potential resistance mechanism. Whole-plant dose-response testing confirmed that the LJ-01 population evolved a high level of resistance to nicosulfuron (11.5-fold) compared to the susceptible (S) population, DY-02. The ALS gene sequencing and relative expression assay of the R plants indicated that target gene mutation and overexpression were not responsible for the resistance phenotype. However, pretreatment with malathion, a known cytochrome P450 monooxygenase (P450) inhibitor, alleviated the resistance of the R population to nicosulfuron by approximately 36%. High-performance liquid chromatography (HPLC) analysis revealed that the R plants metabolized nicosulfuron faster than the S plants. Moreover, cross-resistance testing suggested that the R population exhibited low levels of resistance to thifensulfuron-methyl and pyrazosulfuron-ethyl, but it remained susceptible to rimsulfuron. Multiple resistance patterns showed that the R population evolved low resistance to the photosystem inhibitors bromoxynil octanoate and atrazine and sensitivity to the acetyl-CoA carboxylase (ACCase) inhibitor cyhalofop-butyl and the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors tembotrione, mesotrione, and topramezone. This study reports, for the first time, the simultaneous resistance to ALS and different photosystem inhibitors in D. sanguinalis. The nicosulfuron resistance observed in the R population could primarily be attributed to an enhanced metabolism involving P450 enzymes.

Expression profile and function analysis of MsCSP17 and MsCSP18 in the larval development of Mythimna separata.

Chemosensory proteins (CSPs) were necessary for insect sensory system to perform important processes such as feeding, mating, spawning, and avoiding natural enemies. However, their functions in non-olfactory organs have been poorly studied. To clarify the function of CSPs in the development of Mythimna separata (Walker) larvae, two CSP genes, MsCSP17 and MsCSP18, were identified from larval integument transcriptome dataset. Both of MsCSP17 and MsCSP18 contained four conserved cysteine sites (C × (6)-C × (18)-C × (2)-C), with a signal peptide at the N-terminal. RT-qPCR analysis showed that MsCSP17 and MsCSP18 have different expression patterns among different developmental stages and tissues. MsCSP17 was highly expressed in 1st-4th instar larvae, and MsCSP18 had high expression in adults. Both genes were expressed highly in larval head, thorax, integument and mandible. Moreover, both of MsCSP17 and MsCSP18 were lowly expressed in larval integuments when larvae molted for 6 h and 9 h from 3rd to 4th instar, but highly at the beginning and end phase during molting. After injection of dsMsCSP17 and dsMsCSP18, the expression levels of two genes decreased significantly, with the body weight of larvae decreased, the mortality increased, and the eclosion rate decreased. It was suggested that MsCSP17 and MsCSP18 contributed to the development of M. separata larvae.

Identification of odorant receptors of Tribolium confusum in response to limonene repellent activity.

Tribolium confusum is an important storage pest showing significant resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Present study attempts to explore the molecular mechanism about the repellent activity of limonene. When treatment concentration of limonene was 200.00 µg/cm2, the repellent level remained at grade V after 24 hours. Our study showed that limonene could be distinguished by T. confusum antenna with a maximal electroantennography test value of 0.90 mV. Simultaneously, 88 upregulated and 98 downregulated genes were sequenced in limonene-repellent T. confusum, and RT-qPCR analysis showed that four down-regulated and one up-regulated OR genes play an important role in the response to limonene. The repellent rate was decreased by 22.13% mediated with a knockdown of dsTconOR93, while the EAG value of the female and male adults was reduced to 0.26 mV (49.06%) and 0.20 mV (54.05%), respectively. In conclusion, limonene had a strong repellent activity against T. confusum and TconOR93 gene was determined to be a major effector in perception of limonene. This study provides a basis for the development of limonene as a novel botanical pesticide for the control to storage pests, which will reduce the utilization of chemical pesticides and postpone the development of resistance.

Exposure to sublethal concentrations of thiacloprid insecticide modulated the expression of microRNAs in honeybees (Apis mellifera L.).

This study aimed to investigate the sublethal effects of thiacloprid on microRNA (miRNA) expression in honeybees (Apis mellifera L.) and the role of a specific miRNA, ame-miR-283-5p, in thiacloprid resistance. The high-throughput small RNA-sequencing was used to analyze global miRNA expression profile changes in honeybees orally exposed to sublethal concentrations of thiacloprid (20 mg/L and 4 mg/L) for 72 h. Thiacloprid at 20 mg/L had no observed adverse effects. In addition, bees were fed with miRNA mimics or inhibitors to increase or decrease ame-miR-283-5p expression, and its effects on P450 gene expression (CYP9Q2 and CYP9Q3) were examined. Thiacloprid susceptibility was also detected. The results showed that treatment with thiacloprid at 20 mg/L and 4 mg/L induced 11 and five differentially expressed miRNAs (DEMs), respectively. Bioinformatic analysis suggested that the DEMs are mainly involved in the regulation of growth and development, metabolism, nerve conduction, and behavior. ame-miR-283-5p was downregulated by both concentrations, which was validated using quantitative real-time reverse transcription PCR analysis. Enhancing ame-miR-283-5p expression significantly inhibited CYP9Q2 mRNA and protein expression, and increased thiacloprid toxicity, while reducing ame-miR-283-5p expression significantly promoted CYP9Q2 expression, and decreased thiacloprid susceptibility. Our study revealed a novel role of miRNAs in insecticide resistance in honeybees.

Identification and characterization of odorant receptors in Plutella xylostella antenna response to 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Diamondback moth (Plutella xylostella), a worldwide migratory pest that is developing strong resistance to various chemical insecticides. It has been determined that four natural pyrazines isolated from Allium tuberosum showed significant repellent activity to P. xylostella, but the molecular target still unknown. In the present study, a novel synthetic route for 2,3-dimethyl-6-(1-hydroxy)-pyrazine which has the most significant repellent activity with a purity of 90.60% was established. Simultaneously, the bioassay result declared that the repellent grade was IV at a dosage of 0.01 mg which was the same as to the published data. Transcriptomics analysis detected 1643 upregulated and 3837 downregulated genes in P. xylostella antennae following this pyrazine exposure. Then, 2142 differentially expressed genes were annotated using Gene Ontology and 2757 genes were annotated by Kyoto Encyclopedia of Genes and Genomes. Moreover, this procedure identified 84 odour perception-related genes, 58 odorant receptor (OR) genes including 57 conventional ORs and the odorant receptor co-receptor (Orco, atypical odorant receptor) gene, and 26 odorant-binding protein (OBP) genes. Based on quantitative real time PCR (RT-qPCR) and differential expression results, 9 OR genes including the Orco were cloned and characterised. In summary, this study provides important basis for the utilization of pyrazines as the main active ingredients or lead compounds to developing new botanical pesticides, which will reduce application of chemical pesticides and postpone the development of resistance.