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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.

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Potatoes hold the distinction of being the largest non-cereal food crop globally. The application of insecticides has been the most common technology for pest control. The repeated use of synthetic insecticides of the same chemical class and frequent applications have resulted in the emergence of insecticide resistance. Two closely related pests that feed on potato crops are the potato tuber moth, Phthorimaea operculella, and the tomato leafminer, Phthorimaea absoluta (syn. Tuta absoluta). Previous studies indicated the existence of insecticide resistance to various classes of insecticides including organophosphates, carbamates, and pyrethroids in field populations of P. operculella and P. absoluta. However, the exact mechanisms of insecticide resistance in P. operculella and to a lesser extent P. absoluta remain still poorly understood. Detecting resistance genotypes is crucial for the prediction and management of insecticide resistance. In this study, we identified multiple genetic mutations related to insecticide resistance in two species of Phthorimaea. An unexpected genetic divergence on target-site mutations was observed between P. operculella and P. absoluta. Three mutations (A201S, L231V, and F290V) in Ace1 (acetylcholinesterase), four mutations (M918T, L925M, T928I, and L1014F) in VGSC (voltage-gated sodium channel), and one mutation (A301S) in RDL (GABA-gated chloride channel) have been detected with varying frequencies in Chinese P. absoluta field populations. In contrast, P. operculella field populations showed three mutations (F158Y, A201S, and L231V) in Ace1, one mutation (L1014F) in VGSC at a lower frequency, and no mutation in RDL. These findings suggest that pyrethroids, organophosphates, and carbamates are likely to be ineffective in controlling P. absoluta, but not P. operculella. These findings contributed to a deeper understanding of the presence of target-site mutations conferring resistance to commonly used (and cheap) classes of insecticides in two closely related potato pests. It is recommended to consider the resistance status of both pests for the implementation of resistance management strategies in potatoes.

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With the continuous development of advanced packaging technology in heterogeneous semiconductor integration, the delamination failure problem in a dynamic service environment has gradually become a key factor limiting the reliability of packaging devices. In this paper, the delamination failure mechanism of polymer-based packaging devices is clarified by summarizing the relevant literature and the latest research solutions are proposed. The results show that, at the microscopic scale, thermal stress and moisture damage are still the two main mechanisms of two-phase interface failure of encapsulation devices. Additionally, the application of emerging technologies such as RDL structure modification and self-healing polymers can significantly improve the thermal stress state of encapsulation devices and enhance their moisture resistance, which can improve the anti-delamination reliability of polymer-based encapsulation devices. In addition, this paper provides theoretical support for subsequent research and optimization of polymer-based packages by summarizing the microscopic failure mechanism of delamination at the two-phase interface and introducing the latest solutions.

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Photosensitive polyimides (PSPIs) have been widely developed in microelectronics, which is due to their excellent thermal properties and reasonable dielectric properties and can be directly patterned to simplify the processing steps. In this study, 3 µm~7 µm thick PSPI films were deposited on different substrates, including Si, 50 nm SiN, 50 nm SiO2, 100 nm Cu, and 100 nm Al, for the optimization of the process of integration with Cu films. In situ temperature-dependent resistance measurements were conducted by using a four-point probe system to study the changes in resistance of the 70 nm thick Cu films on different dielectrics with thick diffusion films of 30 nm Mn, Co, and W films in a N2 ambient. The lowest possible change in thickness due to annealing at the higher temperature ranges of 325 °C to 375 °C is displayed, which suggests the high stability of PSPI. The PSPI films show good adhesion with each Cu diffusion barrier up to 350 °C, and we believe that this will be helpful for new packaging applications, such as a 3D IC with a Cu interconnect.

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We investigated insecticide resistance profiles of field populations of the German cockroach, Blattella germanica (L.), collected from central regions of Thailand. Seven strains (PW, RB, MTH, MTS, TL, AY, and SP) were evaluated with diagnostic doses (DD; 3 × LD95 generated from a susceptible strain) of deltamethrin, fipronil, and imidacloprid using topical assays and compared with a susceptible strain (DMSC). Results showed fipronil (2-27% mortality), deltamethrin (16-58% mortality), and imidacloprid (15-75% mortality) resistance in the field strains. Synergism studies with piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) in combination with the DD of insecticides significantly increased (P < 0.05) mortality of the test insects of the field strains suggesting the involvement of P450 monooxygenase and esterase pathways of detoxification. Gel bait evaluations demonstrated that all field-collected strains were resistant to Maxforce Forte (0.05% fipronil), Maxforce Fusion (2.15% imidacloprid), and Advion Cockroach Gel Bait (0.6% indoxacarb) with mean survival times ranging from 1.87-8.27, 1.77-11.72, and 1.19-3.56 days, respectively. Molecular detection revealed that the Rdl mutation was completely homozygous in all field-collected strains except in the PW strain. Field-collected strains were screened for 3 voltage-gated sodium channel (VGSC) mutations associated with pyrethroid resistance. The L993F mutation was present in 5 strains, but no C764R and E434K mutations were detected.

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The circuitry underlying the detection of visual motion in Drosophila melanogaster is one of the best studied networks in neuroscience. Lately, electron microscopy reconstructions, algorithmic models, and functional studies have proposed a common motif for the cellular circuitry of an elementary motion detector based on both supralinear enhancement for preferred direction and sublinear suppression for null-direction motion. In T5 cells, however, all columnar input neurons (Tm1, Tm2, Tm4, and Tm9) are excitatory. So, how is null-direction suppression realized there? Using two-photon calcium imaging in combination with thermogenetics, optogenetics, apoptotics, and pharmacology, we discovered that it is via CT1, the GABAergic large-field amacrine cell, where the different processes have previously been shown to act in an electrically isolated way. Within each column, CT1 receives excitatory input from Tm9 and Tm1 and provides the sign-inverted, now inhibitory input signal onto T5. Ablating CT1 or knocking down GABA-receptor subunit Rdl significantly broadened the directional tuning of T5 cells. It thus appears that the signal of Tm1 and Tm9 is used both as an excitatory input for preferred direction enhancement and, through a sign inversion within the Tm1/Tm9-CT1 microcircuit, as an inhibitory input for null-direction suppression.

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BACKGROUND: Abamectin is one of the main insecticides used for the control of Plutella xylostella, a destructive pest of cruciferous crops. Target-site mutation plays an important role in insecticide resistance. A point mutation (D472N) has been reported in the Rdl1 γ-aminobutyric acid receptor (GABAR) in P. xylostella, but its roles in insecticide resistance remain unknown. RESULTS: In this study, the D472N mutation of the Rdl1 GABAR was detected in several field populations of P. xylostella and showed a positive correlation with abamectin resistance. A knock-in homozygous mutation strain (D472N-KI) of P. xylostella was successfully constructed using CRISPR/Cas9 coupled with homology-directed repair, and the bioassay results demonstrated that compared with the susceptible strain, the D472N-KI strain had 11.1- and 3.7-fold increased resistance to abamectin and endosulfan, respectively. There was no difference in resistance to fipronil, broflanilide or isocycloseram, which also target the GABAR. In addition, the total fecundity of the D472N-KI strain was significantly reduced by 50.0%. CONCLUSION: Our results suggest that the homozygous D472N mutation in Rdl1 confers a low level of resistance to abamectin in P. xylostella but causes significant fecundity disadvantages, which may delay the development of resistance to some extent. These results lay a foundation for further understanding the mechanisms of abamectin resistance in insect pests. © 2022 Society of Chemical Industry.

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Nanotwinned copper is a potential microelectronic interconnection material due to its superior strength and conductivity, however, its filling ability is urgently needed to improve before its application in the field of advanced packaging. The effect of additive (sodium thiazolinyl dithiopropane sulphonate, SH110) addition on the surface roughness, microstructure, mechanical properties and filling capacity of nanotwinned copper films was investigated. The surface roughness and grain size were firstly reduced then increased with the increasing concentrations of SH110, reaching the minimum value at 10 ppm. It was noticed that copper films with 10 ppm SH110 also possessed superior tensile strength and elongation, which were measured as 481 MPa and 3.68% on average of 12µm thick samples by dynamic thermo-mechanical analyzer. Further, their uniformity and flatness of redistributed layers (RDLs) were controlled as 2% and 1.9%, which were significantly improved compared to the samples without SH110 (7.6% and 4.7%). As demonstrated by linear sweep voltammetry analysis and galvanostatic measurement, the SH110 could cooperate well with gelatin and serve as a combination of accelerator and leveler, resulting in the improvement of filling capacity for nanotwinned copper RDLs.

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Populations of Blattella germanica (L.) (German cockroach) have been documented worldwide to be resistant to a wide variety of insecticides with multiple modes of action. The phenylpyrazole insecticide fipronil has been used extensively to control German cockroach populations, exclusively in baits, yet the highest reported fipronil resistance is 38-fold in a single population. We evaluated five populations of German cockroaches, collected in 2018-2019 in apartments in North Carolina and assayed in 2019, to determine the status of fipronil resistance in the state. Resistance ratios in field-collected strains ranged from 22.4 to 37.2, indicating little change in fipronil resistance over the past 20 yr. In contrast, resistance to pyrethroids continues to escalate. We also assessed the roles of detoxification enzymes in fipronil resistance with four synergists previously shown to diminish metabolic resistance to various insecticides in German cockroaches-piperonyl butoxide, S,S,S-tributyl phosphorotrithioate, diethyl maleate, and triphenyl phosphate. These enzymes appear to play a variable role in fipronil resistance. We also sequenced a fragment of the Rdl (resistant to dieldrin) gene that encodes a subunit of the GABA receptor. Our findings showed that all field-collected strains are homozygous for a mutation that substitutes serine for an alanine (A302S) in Rdl, and confers low resistance to fipronil. Understanding why cockroaches rapidly evolve high levels of resistance to some insecticides and not others, despite intensive selection pressure, will contribute to more efficacious pest management.

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Nootkatone, a sesquiterpenoid isolated from Alaskan yellow cedar (Cupressus nootkatensis), is known to possess insect repellent and acaricidal properties and has recently been registered for commercial use by the Environmental Protection Agency. Previous studies failed to elucidate the mechanism of action of nootkatone, but we found a molecular overlay of picrotoxinin and nootkatone indicated a high degree of structural and electrostatic similarity. We therefore tested the hypothesis that nootkatone was a GABA-gated chloride channel antagonist, similar to picrotoxinin. The KD50 and LD50 of nootkatone on the insecticide-susceptible strain of Drosophila melanogaster (CSOR) showed resistance ratios of 8 and 11, respectively, compared to the cyclodiene-resistant strain of RDL1675, indicating significant cross-resistance. Nootkatone reversed GABA-mediated block of the larval CSOR central nervous system; nerve firing of 78 ± 17% of baseline in the CSOR strain was significantly different from 24 ± 11% of baseline firing in the RDL1675 strain (p = 0.035). This finding indicated that the resistance was expressed within the nervous system. Patch clamp recordings on D. melanogaster central neurons mirrored extracellular recordings where nootkatone inhibited GABA-stimulated currents by 44 ± 9% at 100 µM, whereas chloride current was inhibited 4.5-fold less at 100 µM in RDL1675. Taken together, these data suggest nootkatone toxicity in D. melanogaster is mediated through GABA receptor antagonism.

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Vector control interventions including long-lasting insecticidal nets and indoor residual spraying are important for malaria control and elimination. And effectiveness of these interventions depends entirely on the high level of susceptibility of malaria vectors to insecticides. However, the insecticide resistance in majority of mosquito vector species across African countries is a serious threat to the success of vector control efforts with the extensive use of insecticides, while no data on insecticide resistance was reported from Sierra Leone in the past decade. In the present study, the polymerase chain reaction was applied for the identification of species of 757 dry adult female Anopheles gambiae mosquitoes reared from larvae collected from four districts in Sierra Leone during May and June 2018. And the mutations of kdr, rdl, ace-1 genes in An. gambiae were detected using SNaPshot and sequencing. As a result, one sample from Western Area Rural district belonged to Anopheles melas, and 748 An. gambiae were identified. Furthermore, the rdl mutations, kdr west mutations and ace-1 mutation were found. The overall frequency was 35.7%, 0.3%, 97.6% and 4.5% in A296G rdl, A296S rdl, kdrW and ace-1, respectively. The frequencies of A296G rdl mutation (P < 0.001), kdrW mutation (P = 0.001) and ace-1 mutation (P < 0.001) were unevenly distributed in four districts, respectively, while no statistical significance was found in A296S rdl mutation (P = 0.868). In addition, multiple resistance patterns were also found. In conclusion, multiple mutations involved in insecticide resistance in An. gambiae populations in Sierra Leone were detected in the kdrW, A296G rdl and ace-1 alleles in the present study. It is necessary to monitor vector susceptibility levels to insecticides used in this country, and update the insecticide resistance monitoring and management strategy.

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The resistance of Rhipicephalus microplus to pyrethroids is widely dispersed worldwide and has been associated with several nucleotide substitutions in its target site, the para-sodium ion channel (Na-channel) gene. The resistance of the tick to fipronil has been increasing in South America, and mutations in the GABA-gated chloride channel (GABA-Cl) have been described in fipronil-resistant tick strains. We developed a multiplex allele-specific PCR (mAS-PCR) to screen for single-nucleotide polymorphisms (SNPs) associated with the resistance to pyrethroids (knockdown resistance or kdr) and fipronil (resistance to dieldrin or rdl) in susceptible tick populations from Uruguay (n = 11) and the Rio Grande do Sul state in Southern Brazil (n = 15). Toxicological in vitro assays with larvae and adults were used to confirm the resistance to cypermethrin, flumethrin, and fipronil. Three SNPs in the Na-channel gene were investigated (C190A, G215 T, and T2134A), and the mAS-PCR included the detection of an SNP (G858 T) coding a non-synonymous mutation in the GABA-Cl gene. C190A was present in all pyrethroid-resistant populations from Uruguay and Brazil, most frequently homozygous. The SNPs G215 T and T2134A were not found. Of the seventeen fipronil-resistant populations, fourteen presented at least one mutant GABA-Cl gene allele, more frequently in heterozygosis. Other mechanisms apart from target site insensitivity may be involved in fipronil resistance since in some resistant populations, the SNP G858 T was not detected. Sixteen (61,5%) of the populations presented individuals with simultaneous mutations in the Na-channel and GABA-Cl genes. This could be a significant problem for the future control of R. microplus. This study shows the wide dispersion of a pyrethroid resistance-associated SNP in high frequency in the region. Fipronil resistance mutations are also dispersed across the region and increasing.

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BACKGROUND: Immigrants are disproportionally impacted by HIV infection in Europe and in Spain. Immigrants are also identified as a vulnerable population during economic crises. Various socioeconomic barriers hinder HIV-positive immigrants from accessing healthcare services in the host country. As a result of the 2008 financial crisis, Spain has implemented multiple austerity measures, one of which was the enactments of Royal Decree Law (RDL) 16/2012 and Royal Decree (RD) 1192/2012 which abolished universal healthcare coverage. In this context, this study examined: 1) Participants' mixed experiences in accessing health care after the enactment of 2012 RDL and RD, and 2) Distress felt by the participants and their experiences as HIV-positive immigrants living in Spain. METHODS: Participants were recruited through a nongovernmental organization (NGO) during routine visits at the center. A total of 12 participants were interviewed to reach data saturation. Participants were HIV-positive immigrants living in Spain for 1 or more years, allowing for substantial experience with navigating the healthcare system. Thematic analysis was performed to identify common themes in participants' experiences living as HIV-positive individuals in Spain and in accessing healthcare. RESULTS: Four primary themes were identified. The primary systemic barrier to accessing health care encountered by participants was the inability to fulfill the requirement of having proof of registration in an Autonomous Community for the required time period, thus not being able to apply for a public health insurance card and utilize free care services. Participants identified a positive impact of third party (NGO, social worker, friend/family member) guidance on their experience of applying for a public health insurance card. Participants expressed experiencing emotional or physical (eg, side effects of medication) distress in adapting to life as HIV-positive individuals. Participants also identified experiencing discrimination while living as HIV-positive immigrants in Spain. CONCLUSIONS: HIV-positive immigrants are underserved in Spain. They encounter systemic barriers while accessing healthcare services, and experience fear and/or discrimination. The study underscores the role of NGOs in helping HIV-positive immigrants navigate the healthcare system. More research is needed on comprehensive approaches to address healthcare needs of HIV-positive immigrants in Spain.

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The phenylpyrazole insecticide fipronil blocks resistance to dieldrin (RDL) γ-aminobutyric acid (GABA) receptors in insects, thereby impairing inhibitory neurotransmission. Some insect species, such as the diamondback moth (Plutella xylostella), possess more than one Rdl gene. The involvement of multiple Rdls in fipronil toxicity and resistance remains largely unknown. In this study, we investigated the roles of two Rdl genes, PxRdl1 and PxRdl2, in P. xylostella fipronil action. In Xenopus oocytes, PxRDL2 receptors were 40 times less sensitive to fipronil than PxRDL1. PxRDL2 receptors were also less sensitive to GABA compared with PxRDL1. Knockout of the fipronil-sensitive PxRdl1 reduced the fipronil potency 10-fold, whereas knockout of the fipronil-resistant PxRdl2 enhanced the fipronil potency 4.4-fold. Furthermore, in two fipronil-resistant diamondback moth field populations, PxRdl2 expression was elevated 3.7- and 4.1-fold compared with a susceptible strain, whereas PxRdl1 expression was comparable among the resistant and susceptible strains. Collectively, our results indicate antagonistic effects of PxRDL1 and PxRDL2 on fipronil action in vivo and suggest that enhanced expression of fipronil-resistant PxRdl2 is potentially a new mechanism of fipronil resistance in insects.

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BACKGROUND: Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] show high insecticide activity by acting as antagonists to the insect resistance to dieldrin (RDL) γ-aminobutyric acid (GABA) receptors. In contrast, low-level antagonist activities of meta-diamides have been demonstrated against the human GABA type A receptor (GABAA R) α1ß2γ2S, mammalian GABAA R α1ß3γ2S, and the human glycine receptor (GlyR) α1ß. Glycine residue 336 in the membrane-spanning region M3 of the Drosophila RDL GABA receptor is essential for its high sensitivity to meta-diamide 7, [3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide]. RESULTS: We examined the effects of an equivalent mutation (M288G) in spontaneously opened human GABAA R ß3 homomers using membrane potential assay. Picrotoxin and fipronil blocked spontaneously opened human GABAA Rs ß3 and ß3-M286G in a concentration-dependent manner. In contrast, meta-diamide 7 did not block spontaneously opened GABAA R ß3 homomers, although meta-diamide 7 blocked spontaneously opened GABAA R ß3-M286G homomers. In addition, inhibitory potency of meta-diamide 7 for GABA-induced membrane potential change in cells expressing GABAA R α1ß3-M286G was much higher than that in cells expressing GABAA R α1ß3. In the same way, the equivalent mutation (A288G) in GlyR α1 increased the inhibitory potency of meta-diamide 7 for GlyRs α1 and α1ß. CONCLUSION: Studies substituting an equivalent mutation (M288G) in spontaneously opening human GABAA R ß3 homomers and human GABAA Rs α1ß3 heteromers suggest that M286 in human GABAA R ß3 is important for the low sensitivity to meta-diamide 7. In this study, we summarize the mechanisms underlying the selectivity of meta-diamides between insect RDL and human GABA and glycine receptors. © 2020 Society of Chemical Industry.

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BACKGROUND: Insect Rdl GABA receptor is an important insecticide target. To design a novel insecticide, studies on the structures of homologous pentameric ligand-gated ion channels provide information about important amino acids that are necessary for the function of insect Rdl GABA receptors. RESULTS: L9'A, T12'A, T13'A, T13'S, M15'S, and M15'N mutations in the Drosophila Rdl GABA receptor subunit caused the protein to spontaneously adopt the open state conformation. In contrast, the S16'A, S16'T, S17'A, and S17'H mutant homomers showed the same levels of agonist and antagonist sensitivity as the wild-type receptor. The G336M mutation in the Drosophila Rdl GABA receptor abolished the agonist activities of ivermectin and milbemectin, but the F339M mutation did not. Additionally, the F339M mutation caused spontaneous opening of the receptor. In the Drosophila Rdl model, the hydrophobic girdle plays an important role in stabilization of the closed state. Mutations which decrease hydrophobic interactions resulted in spontaneous opening, supporting the importance of the hydrophobic girdle for keeping the channel closed. Through a mutational study of transmembrane 3 (TM3) cytoplasmic domain and Rdl GABA receptor modeling, hydrophobic interactions between TM3 and TM4 and intersubunit interaction were demonstrated to be important for channel gating. Alternatively, the intrasubunit interaction between TM2 and TM3 domains were less important for channel gating in case of Drosophila Rdl GABA receptor. CONCLUSION: This study demonstrates important amino acids critical to the function of the Drosophila Rdl GABA receptor based on the mutational studies and Drosophila Rdl GABA receptor modeling approach. © 2020 Society of Chemical Industry.

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BACKGROUND: The cys-loop ligand-gated ion channels, including the glutamate-gated chloride channel (GluCl) and GABA-gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength. RESULTS: The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker-assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50-fold. Next, we used CRISPR-Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin. CONCLUSION: Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. © 2020 Society of Chemical Industry.

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

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The resistance to dieldrin gene (Rdl) encodes a subunit of the insect γ-amino butyric acid (GABA) receptor, and the encoded Rdl subunit is a major target site for cyclodiene and phenylpyrazole insecticides. Since the substitution of a single amino acid (Ala to Ser/Gly at position 302) of the Drosophila melanogaster Rdl gene was first identified to confer high level resistance to dieldrin, mutations at the equivalent positions have been reported to confer resistance to dieldrin and/or fipronil in a wide range of different insects. In the cotton bollworm Helicoverpa armigera, there are two Rdl homologs (HaRdl-1 and HaRdl-2) in close proximity on the Z chromosome, which as wild-type sequences, encode alanine and serine respectively at amino acid position 302. In the present study, we used the CRISPR/Cas9 gene editing approach to knock out HaRdl-1 and HaRdl-2 and establish two homozygous knockout strains (ΔRdl-1 and ΔRdl-2). The ΔRdl-1 strain showed low levels of resistance (8.0- to 9.3-fold) to three cyclodiene insecticides (endosulfan, aldrin and dieldrin) compared with the background SCD strain. In contrast, toxicity of the three cyclodiene insecticides to the ΔRdl-2 strain increased significantly (3.6- to 6.3-fold) when compared with the SCD strain. Genetic analysis indicated the obtained resistance to endosulfan and dieldrin in the ΔRdl-1 strain was sex-linked, which is consistent with the fact that HaRdl-1 locus is located on the Z chromosome. The above results demonstrate that both HaRdl-1 and HaRdl-2 are important determinants for the susceptibility of H. armigera SCD strain to the three cyclodiene insecticides, but have opposite effects. It was also found that HaRdl-1 and HaRdl-2 are involved, to some extent, in mediating sensitivity of H. armigera to avermectin and fipronil respectively. We speculate that the HaRdl-1 and HaRdl-2 subunits have different pharmacological properties, which contribute to the differential sensitivities of H. armigera to the tested cyclodienes and other insecticides.

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It is known that a single mutation exerts moderate resistance to pesticide, while double mutations (DM) cause severe resistance problem through synergistic effect, and even result in failure application of pesticides. However, little is known about how double mutations would synergistically cause much high resistance level. In this work, computational studies were performed on the interaction of fipronil with N. lugens RDL-GABA receptors, to see how single and double mutations changed receptor structure properties and then conferred distinct resistance levels. The A2'S mutation displayed relative weak influence on receptor structure properties. The R0'Q mutation, which has not been detected in the absence of A2'S, however could deeply alter the electrostatic potential around the inner pore region and significantly narrow the bottom region around -2'Pro. For the DM system, the synergistic effect of two mutations lead to similar pore diameters to the WT system, except for the slightly reduced middle part. Docking study and binding free energy calculation revealed that fipronil displayed binding potencies in the order of WT > A2'S > R0'Q > DM systems, coinciding well with the reported fipronil sensitivity trends and resistance levels.

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