Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae)

Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field‐collected population of Spodoptera exigua. The field‐collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH‐S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH‐S strain by crossing WF with WH‐S, followed by three rounds of backcrossing with WH‐S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH‐S strain. Compared with WH‐S, the near‐isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.     

CRISPR/Cas9 mediated G4946E substitution in the ryanodine receptor of Spodoptera exigua confers high levels of resistance to diamide insecticides

Diamide insecticides selectively activate insect ryanodine receptors (RyRs), inducing uncontrolled release of calcium ions, and causing muscle contraction, paralysis and eventually death. The RyR^G4946E substitution associated with diamide resistance has been identified in three lepidopteran pests, Plutella xylostella, Tuta absoluta and Chilo suppressalis. Recently, the T. absoluta RyR^G4946V mutation was knocked into the model insect Drosophila melanogaster by CRISPR/Cas9 mediated genome editing and provided in vivo functional confirmation for its role in diamide resistance. In the present study, we successfully introduced the RyR^G4946E mutation with CRISPR/Cas9 technology into a lepidopteran pest of global importance, Spodoptera exigua. The genome-edited strain (named 4946E) homozygous for the SeRyR^G4946E mutation exhibited 223-, 336- and >1000-fold resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively when compared to the wild type strain (WHS) of S. exigua. Reciprocal crossing experiments revealed that the target-site resistance in strain 4946E underlies an autosomal and almost recessive mode of inheritance for anthranilic diamides, whereas it was completely recessive for flubendiamide. Our results not only provided in vivo functional validation of the RyR^G4946E mutation in conferring high levels of resistance to diamide insecticides for the first time in a controlled genetic background of a lepidopteran pest, but also revealed slight differences on the level of resistance between anthranilic diamides (chlorantraniliprole and cyantraniliprole) and flubendiamide conferred by the SeRyR^G4946E mutation.     

Geographic spread,genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth,Plutella xylostella

Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [³H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4–S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where diamide insecticides largely failed to control diamondback moth populations. Thus, our study highlights the global importance of the G4946E RyR target-site mutation, which as a mechanism on its own, confers high-level resistance to diamide insecticides in diamondback moth.     

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer,Tuta absoluta (Lepidoptera: Gelechiidae)

Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.     

Resistance to diamide insecticides in diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor

Diamide insecticides such as chlorantraniliprole and flubendiamide are a new class of insecticide that selectively target insect ryanodine receptors (RyR), a distinct class of homo-tetrameric calcium release channels which play a pivotal role in calcium homeostasis in numerous cell types. Resistance to these insecticides has recently been reported in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a global lepidopteran pest of cruciferous crops. In the present study a region of the gene encoding the proposed diamide binding site of the RyR from P. xylostella collected from the Philippines and Thailand and found to be over 200-fold resistant to both chlorantraniliprole and flubendiamide compared to susceptible strains, were amplified by RT-PCR and sequenced. Comparison of the sequence with those from several susceptible reference strains revealed non-synonymous mutations in each of the resistant strains that in both cases lead to a glycine to glutamic acid substitution (G4946E) in the protein. The independent evolution of the same amino acid substitution within a highly conserved region of the proposed diamide binding site in two geographically separated resistant strains of P. xylostella strongly suggests a causal association with diamide resistance. Furthermore we designed a pyrosequencing-based diagnostic assay for resistance monitoring purposes that can be used to detect the G4946E mutation in field-collected samples of diamondback moth. The implications of the reported findings for resistance management strategies are discussed.     

Functional and genetic characteristics of Chlorantraniliprole resistance in the diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth (Plutella xylostella) is a globally distributed and important economic pest, and it has developed resistance to all conventional insecticide classes used in the field. Chlorantraniliprole is a new chemical class of insecticide that acts as a conformation‐sensitive activator of the insect ryanodine receptor (RyR). In the present study, a field strain (16.3‐fold resistance to chlorantraniliprole) was collected in Korea and lab‐selected with chlorantraniliprole for more than one year. The resulting strain presented 2,157‐fold resistance to chlorantraniliprole. A point mutation (G4946E) in the RyR gene was observed at a high frequency in the resistant strain. Enzyme assays indicated that glutathione S‐transferase (GST) and P450 activity in the resistant strain were 2.4‐ and 1.96‐times higher than that of the susceptible strain, respectively. The expression of the RyR, GST (sigma, omega, and zeta) and CYP321E1 gene was higher in the resistant strain than in the susceptible strain. The F₁ progeny resulting from reciprocal crosses did not reveal maternal effects or a diamide‐susceptible phenotype, which suggests an autosomal nearly recessive mode of inheritance. In addition, we surveyed the susceptibility to 13 insecticides (3 diamides, 2 synthetic pyrethroids, 2 spinosyns, 1 organophosphate, 1 oxadiazine, 1 avermectin, and 3 others) in the chlorantraniliprole‐resistant strain. The resistant strain exhibited high cross‐resistance to flubendiamide (5,910 fold) and showed no cross‐resistance to spinetoram, spinosad, indoxacarb, and metaflumizone. These results can serve as an important basis for guiding the use of insecticides in the field.     

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47–0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.     

Rapid development of resistance to diamide insecticides in the smaller tea tortrix,Adoxophyes honmai (Lepidoptera: Tortricidae), in the tea fields of Shizuoka Prefecture,Japan

We investigated the susceptibility of the smaller tea tortrix, Adoxophyes honmai Yasuda, to diamide insecticides in the Shimada-Yui tea fields in Shizuoka Prefecture, Japan, from 2006 to 2011. By 2011, the insects had developed significant resistance even to concentrations far above the registration concentrations of two diamides, flubendiamide and chlorantraniliprole. The lethal concentration 50 (LC₅₀) values of flubendiamide showed a rapid annual increase from 16.2 ppm in 2007 to 161 ppm in August 2011, exceeding the registration concentration of 100 ppm in 2010 and 2011. The LC₅₀ values of chlorantraniliprole increased sharply from 25.3 ppm in 2010 to 98.8 ppm in August 2011, exceeding the registration concentration of 50 ppm. The LC₅₀ values for flubendiamide and chlorantraniliprole at 10 days after treatment in insects collected in August 2011 were 105-fold and 77.2-fold higher, respectively, than those in a susceptible strain.     

Detection of ryanodine receptor target-site mutations in diamide insecticide-resistant Spodoptera frugiperda in China

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a widely distributed pest of corn. Since it invaded China in 2018, it has caused serious damage to local corn production. Chlorantraniliprole, an anthranilic diamide insecticide, has been widely used to control lepidopteran pests. Tetrachloropyramid is a new allosteric modulator insecticide developed based on chlorantraniliprole, so it has a similar mechanism and insecticidal effect. In this study, we investigated resistance levels to chlorantraniliprole and tetrachloropyramid in S. frugiperda from 13 populations in China. Among the populations tested, the relative highest resistance to chlorantraniliprole occurred in the Guangzhou population, and the most susceptible to chlorantraniliprole was found in the Wuhan population. The lethal dosage LD₅₀ value of the Guangzhou population against chlorantraniliprole was 27.8-fold higher than that of the Wuhan population. Minimal differences were observed among S. frugiperda populations in terms of sensitivity to tetrachloropyramid. Heterozygous mutations at the I4734 site of the ryanodine receptor (RyR) were found, while no mutations were found in the G4891 site. The mutations were detected in only two of the 786 individuals analyzed, one from the Qinzhou population and other from the Anshun population (frequency below 2% in both cases). There were no significant differences in the expression levels of RyR between Guangzhou and Wuhan populations. In summary, our results indicate that: (i) S. frugiperda has low resistance levels to diamide insecticides in China; and (ii) the differences in relative resistance among the 13 populations analyzed are not caused by the mutations in RyR or the expression of RyR.     

Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides

Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors.     

新型二酰胺类杀虫剂对鱼尼丁受体作用的分子机理

最近发现了一类新型二酰胺类杀虫剂——氟虫酰胺和氯虫酰胺,其作用靶标是鱼尼丁受体 (ryanodine receptors, RyRs)。本文对RyR的结构与功能、电压门控钙离子通道和鱼尼丁受体钙离子释放通道对细胞质钙离子内环境稳定的调节以及二酰胺类杀虫剂对RyRs作用的分子机理进行综述。二酰胺类杀虫剂使昆虫RyR通道处于持续的开放状态,引发钙离子从肌质网腔内大量释放,破坏了细胞质钙离子内环境的稳定,从而产生不同的药物学特性。这些变化都是由一个不同于鱼尼丁在RyR上的结合部位介导的。该类杀虫剂的作用对昆虫RyR s是高度专一的,结果产生选择毒性。由于二酰胺类杀虫剂的结构独特,作用方式新颖,对鳞翅目害虫效果好、杀虫谱广,对各种益虫和天敌安全,并对现用的杀虫剂无交互抗性,故它们非常适合于抗性治理和IPM。     

Discovery of cyantraniliprole,a potent and selective anthranilic diamide ryanodine receptor activator with cross-spectrum insecticidal activity

Anthranilic diamides are an exceptionally active class of insect control chemistry that selectively activates insect ryanodine receptors causing mortality from uncontrolled release of calcium ion stores in muscle cells. Work in this area led to the successful commercialization of chlorantraniliprole for control of Lepidoptera and other insect pests at very low application rates. In search of lower log P analogs with improved plant systemic properties, exploration of cyano-substituted anthranilic diamides culminated in the discovery of a second product candidate, cyantraniliprole, having excellent activity against a wide range of pests from multiple insect orders. Here we report on the chemistry, biology and structure–activity trends for a series of cyanoanthranilic diamides from which cyantraniliprole was selected for commercial development.     

湖北四个地区草地贪夜蛾田间种群的杀虫剂敏感性及靶标突变检测

【目的】测定湖北草地贪夜蛾田间种群对4种杀虫剂的敏感性,并对杀虫剂靶标Ace-1,VGSC和RyR的基因型和突变频率进行检测,以明确湖北草地贪夜蛾田间种群药剂敏感性现状,进而指导田间科学用药。【方法】采集湖北黄冈、武穴、咸宁和荆州4个地区玉米田中的草地贪夜蛾幼虫,采用浸叶法测定其对氯虫苯甲酰胺、乙基多杀菌素、茚虫威和甲维盐4种杀虫剂的敏感性;提取4个种群80头个体的单头总RNA合成cDNA,利用特异性引物进行PCR扩增,获得目的基因片段,根据序列比对和测序峰图分析,确定Ace-1,VGSC和RyR的基因型和靶标突变位点的突变频率。【结果】生测结果表明,湖北这4个田间种群的草地贪夜蛾幼虫对氯虫苯甲酰胺、乙基多杀菌素、茚虫威和甲维盐均比较敏感,敏感性从高到低依次为:甲维盐乙基多杀菌素氯虫苯甲酰胺茚虫威。分子检测结果表明,湖北草地贪夜蛾这4个田间种群Ace-1基因在A201S, G227A和F290V位点均存在抗性杂合突变,且在F290V位点检测到抗性纯合突变,而VGSC和RyR两个基因均没有检测到靶标位点突变。【结论】湖北草地贪夜蛾4个田间种群对氯虫苯甲酰胺、乙基多杀菌素、茚虫威和甲维盐这4种杀虫剂均较敏感,但这4个草地贪夜蛾田间种群均携带有对有机磷和氨基甲酸酯类杀虫剂的抗性基因,存在潜在的抗性风险,田间防治建议少用或不用该类杀虫剂,同时需要进一步加强抗性监测工作。     

双酰胺类杀虫剂对棉铃虫和松毛虫赤眼蜂的选择毒力

为了指导棉铃虫防控中化学杀虫剂和天敌昆虫协调使用,本文采用浸叶法和玻管药膜法测定了4种双酰胺类杀虫剂对棉铃虫3龄幼虫和松毛虫赤眼蜂成蜂的毒力.结果表明,供试双酰胺类杀虫剂中氯虫苯甲酰胺、氟苯虫酰胺和氟氰虫酰胺对棉铃虫幼虫表现出了较高的毒力水平(LC50分别为0.2882、0.3894、0.8609 mg/L),稍低于对照药剂甲氨基阿维菌素苯甲酸盐(简称:甲维盐)(LC50为0.06493 mg/L);而溴氰虫酰胺对棉铃虫的毒力(LC50为3.7638 mg/L)明显低于甲维盐.从对天敌昆虫安全性来看,氯虫苯甲酰胺、氟苯虫酰胺和氟氰虫酰胺对松毛虫赤眼蜂的毒性(LC50分别为1.0060、0.9933、1.1541 mg/L)明显低于甲维盐(其LC50为0.1646 mg/L),表现出了较高的安全性;但是,溴氰虫酰胺对松毛虫赤眼蜂毒性较高(LC50为0.4952 mg/L),安全性较差.本研究将为棉铃虫防控中药剂与天敌协调使用提供依据.     

FastD: Fast detection of insecticide target‐site mutations and overexpressed detoxification genes in insect populations from RNA‐Seq data

Target‐site mutations and detoxification gene overexpression are two major mechanisms conferring insecticide resistance. Molecular assays applied to detect these resistance genetic markers are time‐consuming and with high false‐positive rates. RNA‐Seq data contains information on the variations within expressed genomic regions and expression of detoxification genes. However, there is no corresponding method to detect resistance markers at present. Here, we collected 66 reported resistance mutations of four insecticide targets (AChE, VGSC, RyR, and nAChR) from 82 insect species. Next, we obtained 403 sequences of the four target genes and 12,665 sequences of three kinds of detoxification genes including P450s, GSTs, and CCEs. Then, we developed a Perl program, FastD, to detect target‐site mutations and overexpressed detoxification genes from RNA‐Seq data and constructed a web server for FastD (http://www.insect-genome.com/fastd). The estimation of FastD on simulated RNA‐Seq data showed high sensitivity and specificity. We applied FastD to detect resistant markers in 15 populations of six insects, Plutella xylostella, Aphis gossypii, Anopheles arabiensis, Musca domestica, Leptinotarsa decemlineata and Apis mellifera. Results showed that 11 RyR mutations in P. xylostella, one nAChR mutation in A. gossypii, one VGSC mutation in A. arabiensis and five VGSC mutations in M. domestica were found to be with frequency difference >40% between resistant and susceptible populations including previously confirmed mutations G4946E in RyR, R81T in nAChR and L1014F in VGSC. And 49 detoxification genes were found to be overexpressed in resistant populations compared with susceptible populations including previously confirmed detoxification genes CYP6BG1, CYP6CY22, CYP6CY13, CYP6P3, CYP6M2, CYP6P4 and CYP4G16. The candidate target‐site mutations and detoxification genes were worth further validation. Resistance estimates according to confirmed markers were consistent with population phenotypes, confirming the reliability of this program in predicting population resistance at omics‐level.     

Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active- and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6(54/84) variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystal-structure analysis of the V6(54/84) variant bound to ritonavir and indinavir shows structural changes in the 80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggest possible changes in the dynamics of flap opening and closing.     

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms—multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates—were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.     

Identification of Genotypic Changes in Human Immunodeficiency Virus Protease That Correlate with Reduced Susceptibility to the Protease Inhibitor Lopinavir among Viral Isolates from Protease Inhibitor-Experienced Patients

The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with reduced susceptibility. Mutations at positions 82, 54, 10, 63, 71, and 84 were most closely associated with relatively modest (4- and 10-fold) changes in phenotype, while the K20M/R and F53L mutations, in conjunction with multiple other mutations, were associated with >20- and >40-fold-reduced susceptibility, respectively. The median 50% inhibitory concentrations (IC(50)) of lopinavir against isolates with 0 to 3, 4 or 5, 6 or 7, and 8 to 10 of the above 11 mutations were 0.8-, 2.7-, 13.5-, and 44.0-fold higher, respectively, than the IC(50) against wild-type HIV. On average, the IC(50) of lopinavir increased by 1.74-fold per mutation in isolates containing three or more mutations. Each of the 16 viruses that displayed a >20-fold change in susceptibility contained mutations at residues 10, 54, 63, and 82 and/or 84, along with a median of three mutations at residues 20, 24, 46, 53, 71, and 90. The number of protease mutations from the 11 identified in these analyses (the lopinavir mutation score) may be useful for the interpretation of HIV genotypic resistance testing with respect to lopinavir-ritonavir (Kaletra) regimens and may provide insight into the genetic barrier to resistance to lopinavir-ritonavir in both antiretroviral therapy-naive and protease inhibitor-experienced patients.     

A novel point mutation of acetylcholinesterase in a trichlorfon-resistant strain of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Acetylcholinesterase (AChE) is the target enzyme of organophosphorus and carbamate insecticides. We applied trichlorfon to select resistant strains of Bactrocera dorsalis Hendel in the laboratory. Two trichlorfon-resistant strains, the Tri-R₁ strain with 18.23-fold resistance and the Tri-R₂ strain with 69.5-fold resistance, were obtained. Three known mutations, I159V, G433S and Q588R were identified in AChE of two resistant strains, and a novel mutation, G365A, was identified in the more resistant Tri-R₂ strain. The modeled 3-D-structure of AChE showed that G365A and G433S are closely adjacent in the gorge above the catalytic site S235. Mutations of G365A and G433S resulted in a steric hindrance by stronger Van der Waals force between two sites. Such a minor structural change might block insecticides from squeezing through the gorge to reach the active site, but not the natural substrate. Compared with the susceptible strain, the AChE activity of the Tri-R₁ strain and the Tri-R₂ strain was 0.87- and 0.67-fold, the K _m value of the Tri-R₁ strain and the Tri-R₂ strain was 0.11- and 0.10-fold, the V _max value of two resistant strains was 0.26- and 0.15-fold, whereas, the I ₅₀ to trichlorfon significantly increased by 9.07- and 13.19-fold. These results suggested that the novel point mutation G365A of AChE might be involved in increasing resistance to trichlorfon in the resistant strain of oriental fruit fly.