Identification and Functional Characterization of Carboxylesterase Genes Involved in Spirodiclofen Resistance in Panonychus citri (McGregor).

Overexpression of carboxyl/cholinesterase (CCE) genes has been reported to be associated with many cases of pesticide resistance in arthropods. However, it has been rarely documented that CCE genes participate in spirodiclofen resistance in Panonychus citri. In previous research, we found that spirodiclofen resistance is related to increased P450 and CCE enzyme activities in P. citri. In this study, we identified two CCE genes, PcCCE3 and PcCCE5, which were significantly upregulated in spirodiclofen-resistant strain and after exposure to spirodiclofen. RNA interference of PcCCE3 and PcCCE5 increased the spirodiclofen susceptibility in P. citri. In vitro metabolism indicated that PcCCE3 and PcCCE5 could interact with spirodiclofen, but metabolites were detected only in the PcCCE3 treatment. Our results indicated that PcCCE3 participates in spirodiclofen resistance through direct metabolism, and PcCCE5 may be involved in the spirodiclofen resistance by passive binding and sequestration, which provides new insights into spirodiclofen resistance in P. citri.

Cytochrome P450 CYP81A104 in Eleusine indica confers resistance to multiherbicide with different modes of action.

BACKGROUND: Developing herbicide-resistant (HR) crop cultivars is an efficient way to control weeds and minimize crop yield losses. However, widespread and long-term herbicide application has led to the evolution of resistant weeds. Here, we established a resistant (R) E. indica population, collected from imidazolinone-resistant rice cultivar fields. RESULTS: The R population evolved 4.5-fold resistance to imazamox. Acetolactate synthase (ALS) gene sequencing and ALS activity assays excluded the effect of target-site resistance in this population. P450 inhibitor malathion pretreatment significantly reversed resistance to imazamox. RNA sequencing showed that a P450 gene CYP81A104 was expressed higher in R versus susceptible (S) plants. Arabidopsis overexpressing CYP81A104 showed resistance to ALS inhibitors (imazamox, tribenuron-methyl, penoxsulam and flucarbazone-sodium), PSII inhibitor (bentazone), hydroxyphenyl pyruvate dioxygenase inhibitor (mesotrione) and auxin mimics (MCPA), which was generally consistent with the results presented in the R population. CONCLUSION: This study confirmed that the CYP81A104 gene endowed resistance to multiherbicides with different modes-of-action. Our findings provide an insight into the molecular characteristics of resistance and contribute to formulating an appropriate strategy for weed management in HR crops. © 2024 Society of Chemical Industry.

Metabolism-Based Herbicide Resistance to Mesosulfuron-methyl and Identification of Candidate Genes in Bromus japonicus.

The evolved resistance of Bromus japonicus Houtt. to ALS-inhibiting herbicides is well established. Previous studies have primarily focused on target-site resistance; however, non-target-site resistance has not been well characterized. This investigation demonstrated that ALS gene sequencing did not detect any previously known resistance mutations in a mesosulfuron-methyl-resistant (MR) population, and notably, treatment with the P450 monooxygenase (P450) inhibitor malathion markedly heightened susceptibility to mesosulfuron-methyl. Utilizing UPLC-MS/MS analysis confirmed elevated mesosulfuron-methyl metabolism in MR plants. The integration of Isoform Sequencing (Iso-Seq) and RNA Sequencing (RNA-Seq) facilitated the identification of candidate genes associated with non-target sites in a subpopulation with two generations of herbicide selection. Through qRT-PCR analysis, 21 differentially expressed genes were characterized, and among these, 10 genes (comprising three P450s, two glutathione S-transferases, one glycosyltransferase, two ATP-binding cassette transporters, one oxidase, and one hydrolase) exhibited constitutive upregulation in resistant plants. Our findings substantiated that increased herbicide metabolism is a driving force behind mesosulfuron-methyl resistance in this B. japonicus population.

A natural glutathione S-transferase gene GSTU23 confers metabolic resistance to metamifop in Echinochloa crus-galli.

Herbicides are essential for farmers to control weed. However, prolonged use of herbicides has caused the development of herbicide resistance in weeds. Here, the resistant Echinochloa crus-galli (RL5) was obtained by continuous treatment with metamifop for five generations in paddy fields. RL5 plants showed a 13.7-fold higher resistance to metamifop compared to susceptible E. crus-galli (SL5) plants. Pre-treatment with GST inhibitor (NBD-Cl) significantly increased the susceptibility of RL5 plants to metamifop. Faster metamifop metabolism and higher GST activity in RL5 plants than in SL5 plants were also confirmed, highlighting the role of GST in metabolic resistance. RNA-Seq analysis identified EcGSTU23 as a candidate gene, and this gene was up-regulated in RL5 and field-resistant E. crus-galli plants. Furthermore, the EcGSTU23 gene was overexpressed in the transgenic EcGSTU23-Maize, and the EcGSTU23-Maize showed resistance to metamifop. In vitro metabolic studies also revealed that the purified EcGSTU23 displayed catalytic activity in glutathione (GSH) conjugation, and metamifop was rapidly metabolized in the co-incubation system containing EcGSTU23 protein. These results provide direct experimental evidence of EcGSTU23's involvement in the metabolic resistance of E. crus-galli to metamifop. Understanding the resistance mechanism can help in devising effective strategies to combat herbicide resistance and breeding of genetically modified herbicide resistant crops.

Target gene overexpression and enhanced metabolism confer resistance to nicosulfuron in Eriochloa villosa (Thunb.).

Eriochloa villosa (Thunb.) Kunth is a troublesome weed widely distributed in maize (Zea mays L.) fields in Northeast China. Many populations of E. villosa have evolved resistance to nicosulfuron herbicides, which inhibit acetolactate synthase (ALS). The objectives of this research were to confirm that E. villosa is resistant to nicosulfuron and to investigate the basis of nicosulfuron resistance. Whole-plant dose-response studies revealed that the R population had not developed a high level of cross-resistance and exhibited greater resistant (25.62-fold) to nicosulfuron than that of the S population and had not yet developed a high level of cross-resistance. An in vitro ALS activity assay demonstrated that the I50 of nicosulfuron was 6.87-fold greater in the R population than the S population. However, based on ALS gene sequencing, the target ALS gene in the R population did not contain mutations. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that ALS gene expression between the R and S populations was significantly different after nicosulfuron application, but no differences were observed in the gene copy number. After the cytochrome P450 inhibitor malathion or the GST inhibitor NBD-Cl was applied, the resistant E. villosa population exhibited increased sensitivity to nicosulfuron. Based on the activities of GSTs and P450s, the activities of the R population were greater than those of the S population after nicosulfuron application. This is the first report that the resistance of E. villosa to ALS inhibitors results from increased target gene expression and increased metabolism. These findings provide a theoretical foundation for the effective control of herbicide-resistant E. villosa.

Identification and transcriptional response of ATP-binding cassette transporters to beta-cypermethrin in the poultry red mite, Dermanyssus gallinae.

Dermanyssus gallinae, a worldwide pest in birds, has developed varying degrees of resistance to insecticides. The ATP-binding cassette (ABC) transporters are essential for the removal of xenobiotics from arthropods. However, our knowledge about ABC transporter proteins in D. gallinae is limited. Forty ABC transporters were identified in the transcriptome and genome of D. gallinae. The resistant population displayed an augmented metabolic rate for beta-cypermethrin compared to the susceptible group, with a remarkable increase in the content of ABC transporters. Verapamil was found able to increase the toxicity of beta-cypermethrin in the resistant population. Results from qRT-PCR analysis showed that eleven ABC transcripts were more highly expressed in the resistant population than the susceptible group at all stages of development, and beta-cypermethrin was observed to be able to induce the expression of DgABCA5, DgABCB4, DgABCD3, DgABCE1 and DgABCG5 in D. gallinae. RNAi-mediated knockdown of the five genes was observed to increase the susceptibility of resistant mites to beta-cypermethrin. These results suggest that ABC transporters, DgABCA5, DgABCB4, DgABCD3, DgABCE1 and DgABCG5 genes, may be related to beta-cypermethrin resistance in D. gallinae. This research will serve as a foundation for further studies on mechanism of insecticide resistance, which could be beneficial for controlling D. gallinae.

Monitoring detoxification enzyme levels and resistance of Tetranychus urticae against some METI-group chemicals in Türkiye cotton fields.

Acaricides used against Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) in cotton fields cause control failure over time. To determine the resistance status of T. urticae populations to tebufenpyrad and bifenazate, different populations collected from Aydin (AYD), Adana (ADA), Sanliurfa (SAN), and Diyarbakir (DIY) provinces of Türkiye, between 2019 and 2020, were subjected to diagnostic dose bioassays. Firstly, the spider mites were eliminated with a discriminating dose. Afterwards, LC50 and LC90 of the remaining populations were determined and the ten highest resistant populations were selected. The highest phenotypic resistance to bifenazate was observed in AYD4 and DIY2 (LC50 57.14 mg L- 1 with 85.01-fold and LC50 30.15 mg L- 1with 44.86-fold, respectively), while the lowest phenotypic resistance was found in SAN6 (LC50 1.5 mg L- 1; 2.28-fold). Considering the phenotypic resistance to tebufenpyrad, the highest resistance was found in AYD4 population (LC50 96.81 mg L- 1; 12.92-fold), while the lowest - in DIY28 population (LC50 21.23 mg L- 1; 2.83-fold). In pharmacokinetic studies, the ADA16 population was compared with the sensitive German Susceptible Strain population and it was determined that carboxylesterase activity was statistically higher (1.46 ± 0.04 nmol/min/mg protein enzyme activation 2.70-fold). The highest activation of glutathione S-transferase was detected in ADA16 (1.49 ± 0.01 nmol/min/mg protein; 2.32-fold). No mutations were found in PSST (METI 1), the point mutation site for tebufenpyrad, and Cytb (METI 3), the point mutation site for bifenazate. In terms of phenotypic resistance, bifenazate was found to be moderately resistant in two populations (85.01 and 44.86-fold), while tebufenpyrad was moderately resistant in one population (12.92-fold). This study showed that both acaricides are still effective against T. urticae populations.

Impact of the exposure of sublethal dose of mosquito coil on the development of insecticide resistance in Aedes aegypti (L.) (Diptera: Culicidae).

Mosquito coil is commonly used in many African households for protection against mosquito bites. The coil usually has semi-volatile pyrethroids as an active ingredient, which usually diffuse across open space, and the cloud either kills mosquitoes that are exposed, or mosquitoes can be exposed to sublethal doses of the insecticides. This study was conducted to assess the impact of sublethal doses of mosquito coil on the development of insecticide resistance in Aedes aegypti, a major vector for dengue fever and several other arboviral diseases. A laboratory colony of Ae. aegypti was exposed to sublethal doses of a meperfluthrin-based mosquito coil in a Peet-Grady chamber once per generation for 16 generations. The susceptibility of the exposed colony to a diagnostic dose of the mosquito coil as well as to three other insecticides was determined. Three different kdr mutations and five enzyme activities were evaluated in both the exposed and control colonies. After 16 generations of sublethal exposure to mosquito coils, the full diagnostic dose of the coil caused 68% mortality to the exposed colony compared to 100% mortality in the control colony. Mortality caused by deltamethrin (0.05%) was also significantly lower in the exposed colony. The frequency of 1016I kdr mutation as well as MFO and alpha esterase activities were higher in the exposed colony compared to the control colony. This study provides evidence of the development of pyrethroid resistance in an Ae. aegypti population due to sublethal exposure to mosquito coil for 16 generations. Given the large-scale use of mosquito coils in many African households, its role as a pyrethroid resistance selection source should be taken into consideration when designing resistance management strategies.

Genetic surveillance of insecticide resistance in African Anopheles populations to inform malaria vector control.

Insecticide resistance in malaria vector populations poses a major threat to malaria control, which relies largely on insecticidal interventions. Contemporary vector-control strategies focus on combatting resistance using multiple insecticides with differing modes of action within the mosquito. However, diverse genetic resistance mechanisms are present in vector populations, and continue to evolve. Knowledge of the spatial distribution of these genetic mechanisms, and how they impact the efficacy of different insecticidal products, is critical to inform intervention deployment decisions. We developed a catalogue of genetic-resistance mechanisms in African malaria vectors that could guide molecular surveillance. We highlight situations where intervention deployment has led to resistance evolution and spread, and identify challenges in understanding and mitigating the epidemiological impacts of resistance.

Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin.

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

The impact of agrochemical pollutant mixtures on the selection of insecticide resistance in the malaria vector Anopheles gambiae: insights from experimental evolution and transcriptomics.

BACKGROUND: There are several indications that pesticides used in agriculture contribute to the emergence and spread of resistance of mosquitoes to vector control insecticides. However, the impact of such an indirect selection pressure has rarely been quantified and the molecular mechanisms involved are still poorly characterized. In this context, experimental selection with different agrochemical mixtures was conducted in Anopheles gambiae. The multi-generational impact of agrochemicals on insecticide resistance was evaluated by phenotypic and molecular approaches. METHODS: Mosquito larvae were selected for 30 generations with three different agrochemical mixtures containing (i) insecticides, (ii) non-insecticides compounds, and (iii) both insecticide and non-insecticide compounds. Every five generations, the resistance of adults to deltamethrin and bendiocarb was monitored using bioassays. The frequencies of the kdr (L995F) and ace1 (G119S) target-site mutations were monitored every 10 generations. RNAseq was performed on all lines at generation 30 in order to identify gene transcription level variations and polymorphisms associated with each selection regime. RESULTS: Larval selection with agrochemical mixtures did not affect bendiocarb resistance and did not select for ace1 mutation. Contrastingly, an increased deltamethrin resistance was observed in the three selected lines. Such increased resistance was not majorly associated with the presence of kdr L995F mutation in selected lines. RNA-seq identified 63 candidate resistance genes over-transcribed in at least one selected line. These include genes coding for detoxification enzymes or cuticular proteins previously associated with insecticide resistance, and other genes potentially associated with chemical stress response. Combining an allele frequency filtering with a Bayesian FST-based genome scan allowed to identify genes under selection across multiple genomic loci, supporting a multigenic adaptive response to agrochemical mixtures. CONCLUSION: This study supports the role of agrochemical contaminants as a significant larval selection pressure favouring insecticide resistance in malaria vectors. Such selection pressures likely impact kdr mutations and detoxification enzymes, but also more generalist mechanisms such as cuticle resistance, which could potentially lead to cross-tolerance to unrelated insecticide compounds. Such indirect effect of global landscape pollution on mosquito resistance to public health insecticides deserves further attention since it can affect the nature and dynamics of resistance alleles circulating in malaria vectors and impact the efficacy of control vector strategies.

RNA and protein biomarkers for detecting enhanced metabolic resistance to herbicides mesosulfuron-methyl and fenoxaprop-ethyl in black-grass (Alopecurus myosuroides).

BACKGROUND: The evolution of non-target site resistance (NTSR) to herbicides leads to a significant reduction in herbicide control of agricultural weed species. Detecting NTSR in weed populations prior to herbicide treatment would provide valuable information for effective weed control. While not all NTSR mechanisms have been fully identified, enhanced metabolic resistance (EMR) is one of the better studied, conferring tolerance through increased herbicide detoxification. Confirming EMR towards specific herbicides conventionally involves detecting metabolites of the active herbicide molecule in planta, but this approach is time-consuming and requires access to well-equipped laboratories. RESULTS: In this study, we explored the potential of using molecular biomarkers to detect EMR before herbicide treatment in black-grass (Alopecurus myosuroides). We tested the reliability of selected biomarkers to predict EMR and survival after herbicide treatments in both reference and 27 field-derived black-grass populations collected from sites across the UK. The combined analysis of the constitutive expression of biomarkers and metabolism studies confirmed three proteins, namely, AmGSTF1, AmGSTU2 and AmOPR1, as differential biomarkers of EMR toward the herbicides fenoxaprop-ethyl and mesosulfuron in black-grass. CONCLUSION: Our findings demonstrate that there is potential to use molecular biomarkers to detect EMR toward specific herbicides in black-grass without reference to metabolism analysis. However, biomarker development must include testing at both transcript and protein levels in order to be reliable indicators of resistance. This work is a first step towards more robust resistance biomarker development, which could be expanded into other herbicide chemistries for on-farm testing and monitoring EMR in uncharacterised black-grass populations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Trp-574-Leu mutation and metabolic resistance by cytochrome P450 gene conferred high resistance to ALS-inhibiting herbicides in Descurainia sophia.

Descurainia sophia (flixweed) is a troublesome weed in winter wheat fields in North China. Resistant D. sophia populations with different acetolactate synthetase (ALS) mutations have been reported in recent years. In addition, metabolic resistance to ALS-inhibiting herbicides has also been identified. In this study, we collected and purified two resistant D. sophia populations (R1 and R2), which were collected from winter wheat fields where tribenuron-methyl provided no control of D. sophia at 30 g a.i. ha-1. Whole plant bioassay and ALS activity assay results showed the R1 and R2 populations had evolved high-level resistance to tribenuron-methyl and florasulam and cross-resistance to imazethapyr and pyrithiobac­sodium. The two ALS genes were cloned from the leaves of R1 and R2 populations, ALS1 (2004 bp) and ALS2 (1998 bp). A mutation of Trp 574 to Leu in ALS1 was present in both R1 and R2. ALS1 and ALS2 were cloned from R1 and R2 populations respectively and transferred into Arabidopsis thaliana. Homozygous T3 transgenic seedlings with ALS1 of R1 or R2 were resistant to ALS-inhibiting herbicides and the resistant levels were the same. Transgenic seedlings with ALS2 from R1 or R2 were susceptible to ALS-inhibiting herbicides. Treatment with cytochrome P450 inhibitor malathion decreased the resistant levels to tribenuron-methyl in R1 and R2. RNA-Seq was used to identify target cytochrome P450 genes possibly involved in resistance to ALS-inhibiting herbicides. There were five up-regulated differentially expressed cytochrome P450 genes: CYP72A15, CYP83B1, CYP81D8, CYP72A13 and CYP71A12. Among of them, CYP72A15 had the highest expression level in R1 and R2 populations. The R1 and R2 populations of D. sophia have evolved resistance to ALS-inhibiting herbicides due to Trp 574 Leu mutation in ALS1 and possibly other mechanisms. The resistant function of CYP72A15 needs further research.

Target-site and non-target-site based resistance to clodinafop-propargyl in wild oats (Avena fatua L.).

Wild oat (Avena fatua L.) is a common and problematic weed in wheat fields in China. In recent years, farmers found it increasingly difficult to control A. fatua using acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. The purpose of this study was to identify the molecular basis of clodinafop-propargyl resistance in A. fatua. In comparison to the S1496 population, whole dose response studies revealed that the R1623 and R1625 populations were 71.71- and 67.76-fold resistant to clodinafop-propargyl, respectively. The two resistant A. fatua populations displayed high resistance to fenoxaprop-p-ethyl (APP) and low resistance to clethodim (CHD) and pinoxaden (PPZ), but they were still sensitive to the ALS inhibitors mesosulfuron-methyl and pyroxsulam. An Ile-2041-Asn mutation was identified in both resistant individual plants. The copy number and relative expression of the ACCase gene in the resistant population were not significantly different from those in the S1496 population. Under the application of 2160 g ai ha -1 of clodinafop-propargyl, the fresh weight of the R1623 population was reduced to 74.9%; however, pretreatment with the application of the cytochrome P450 inhibitor malathion and the GST inhibitor NBD-Cl reduced the fresh weight to 50.91% and 47.16%, respectively, which proved the presence of metabolic resistance. This is the first report of an Ile-2041-Asn mutation and probable metabolic resistance in A. fatua, resulting in resistance to clodinafop-propargyl.

Enhanced metabolic resistance mechanism endows resistance to metamifop in Echinochloa crus-galli (L.) P. Beauv.

Barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.), one of the worst weeds in paddy fields in China, has been frequently reported evolving resistance to acetyl-CoA carboxylase (ACCase) inhibiting herbicides. However, in the previous research, more attention was paid to target-site resistance (TSR) mechanisms, the non-target-site resistance (NTSR) mechanisms have not been well-established. In this study, the potential mechanism of resistance in a metamifop-resistant E. crus-galli collected from Kunshan city, Jiangsu Province, China was investigated. Dose-response assays showed that the phenotypic resistant population (JS-R) has evolved 4.3-fold resistance to metamifop compared with the phenotypic susceptible population (YN-S). The ACCase CT gene sequencing and relative ACCase gene expression levels studies showed that no mutations were detected in the ACCase CT gene in both YN-S and JS-R, and there was no significant difference in the relative ACCase gene expression between YN-S and JS-R. After the pre-processing of glutathione-S-transferase (GSTs) inhibitor NBD-Cl, the resistance level of JS-R to metamifop was reversed 18.73%. Furthermore, the GSTs activity of JS-R plants was significantly enhanced compared to that of YN-S plants. UPLC-MS/MS revealed that JS-R plants had faster metabolic rates to metamifop than YN-S plants. Meanwhile, the JS-R popultion exhibited resistant to cyhalofop-butyl and penoxsulam. In summary, this study presented a novel discovery regarding the global emergence of metabolic resistance to metamifop in E. crus-galli. The low-level resistance observed in the JS-R population was not found to be related to TSR but rather appeared to be primarily associated with the overexpression of genes in the GSTs metabolic enzyme superfamily.

Target-Site and Metabolic Resistance Mechanisms to Penoxsulam in Late Watergrass (Echinochloa phyllopogon) in China.

Echinochloa phyllopogon, a malignant weed in Northeast China's paddy fields, is currently presenting escalating resistance concerns. Our study centered on the HJHL-715 E. phyllopogon population, which showed heightened resistance to penoxsulam, through a whole-plant bioassay. Pretreatment with a P450 inhibitor malathion significantly increased penoxsulam sensitivity in resistant plants. In order to determine the resistance mechanism of the resistant population, we purified the resistant population from individual plants and isolated target-site resistance (TSR) and nontarget-site resistance (NTSR) materials. Pro-197-Thr and Trp-574-Leu mutations in acetolactate synthase (ALS) 1 and ALS2 of the resistant population drove reduced sensitivity of penoxsulam to the target-site ALS, the primary resistance mechanisms. To fully understand the NTSR mechanism, NTSR materials were investigated by using RNA-sequencing (RNA-seq) combined with a reference genome. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis further supported the enhanced penoxsulam metabolism in NTSR materials. Gene expression data and quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation confirmed 29 overexpressed genes under penoxsulam treatment, with 16 genes concurrently upregulated with quinclorac and metamifop treatment. Overall, our study confirmed coexisting TSR and NTSR mechanisms in E. phyllopogon's resistance to ALS inhibitors.

The effect of voluntary exercise on light cycle stress-induced metabolic resistance.

PURPOSE: Disruption of circadian genes affects metabolic homeostasis. Regular exercise programs prevent metabolic dysfunction and alter circadian gene expression In this study, we investigated whether exercise affects light stress-induced circadian rhythm derangement and metabolic resistance. METHODS: A circadian rhythm derangement mouse model was designed by extending the light exposure by two hours (14 L/10 D) for three weeks. Nine-weekold male mice were single-caged and divided into four groups: sedentary groups with or without light stress, and voluntary wheel-trained groups with or without light stress. In addition, differentiated myotubes were cultured in the presence of dexamethasone with or without 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). The comprehensive laboratory animal monitoring system was used to analyze the metabolic changes in mice. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the mRNA expression levels of circadian genes in animal and cell culture models. RESULTS: Three weeks of light stress reduced the running distance and increased the weight of mice. In addition, VO2 consumption and heat production were increased during the night cycle under non-stress conditions but not under stress conditions. PCR analysis revealed that exercise and stress altered the expression levels of circadian genes in the hypothalamus and quadriceps muscles. mRNA expression levels of period circadian regulator 1 were downregulated in the quadriceps muscles of the stressed sedentary group compared to that in muscles of the non-stressed sedentary group. Furthermore, differentiated myotube cells cultured in the presence of dexamethasone, with or without AICAR, showed distinct oscillation patterns at various time points. CONCLUSION: Our study demonstrates that exercise partially prevents metabolic disruption by regulating the circadian gene expression in skeletal muscles.

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.

Deltamethrin and transfluthrin select for distinct transcriptomic responses in the malaria vector Anopheles gambiae.

BACKGROUND: The widespread use of pyrethroid insecticides in Africa has led to the development of strong resistance in Anopheles mosquitoes. Introducing new active ingredients can contribute to overcome this phenomenon and ensure the effectiveness of vector control strategies. Transfluthrin is a polyfluorinated pyrethroid whose structural conformation was thought to prevent its metabolism by cytochrome P450 monooxygenases in malaria vectors, thus representing a potential alternative for managing P450-mediated resistance occurring in the field. In this study, a controlled selection was used to compare the dynamics of resistance between transfluthrin and the widely used pyrethroid deltamethrin in the mosquito Anopheles gambiae. Then, the associated molecular mechanisms were investigated using target-site mutation genotyping and RNA-seq. METHODS: A field-derived line of An. gambiae carrying resistance alleles at low frequencies was used as starting material for a controlled selection experiment. Adult females were selected across 33 generations with deltamethrin or transfluthrin, resulting in three distinct lines: the Delta-R line (selected with deltamethrin), the Transflu-R line (selected with transfluthrin) and the Tiassale-S line (maintained without selection). Deltamethrin and transfluthrin resistance levels were monitored in each selected line throughout the selection process, as well as the frequency of the L1014F kdr mutation. At generation 17, cross-resistance to other public health insecticides was investigated and transcriptomes were sequenced to compare gene transcription variations and polymorphisms associated with adaptation to each insecticide. RESULTS: A rapid increase in resistance to deltamethrin and transfluthrin was observed throughout the selection process in each selected line in association with an increased frequency of the L1014F kdr mutation. Transcriptomic data support a broader response to transfluthrin selection as compared to deltamethrin selection. For instance, multiple detoxification enzymes and cuticle proteins were specifically over-transcribed in the Transflu-R line including the known pyrethroid metabolizers CYP6M2, CYP9K1 and CYP6AA1 together with other genes previously associated with resistance in An. gambiae. CONCLUSION: This study confirms that recurrent exposure of adult mosquitoes to pyrethroids in a public health context can rapidly select for various resistance mechanisms. In particular, it indicates that in addition to target site mutations, the polyfluorinated pyrethroid transfluthrin can select for a broad metabolic response, which includes some P450s previously associated to resistance to classical pyrethroids. This unexpected finding highlights the need for an in-depth study on the adaptive response of mosquitoes to newly introduced active ingredients in order to effectively guide and support decision-making programmes in malaria control.

The dynamics of deltamethrin resistance evolution in Aedes albopictus has an impact on fitness and dengue virus type-2 vectorial capacity.

BACKGROUND: Worldwide invasion and expansion of Aedes albopictus, an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness. RESULTS: A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534F/S) in the voltage gated sodium channel (vgsc) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534S/S) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity. CONCLUSIONS: This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus. These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.