二斑叶螨不同抗性品系最佳内参基因的筛选及CYP392E亚家族基因的表达分析

【目的】 筛选二斑叶螨Tetranychus urticae敏感品系（SS）、 抗阿维菌素品系(Av-R)和抗螺虫乙酯品系（Sp-R）中合适的内参基因, 研究二斑叶螨不同抗性品系CYP392E亚家族基因表达水平的变化。【方法】采用实时荧光定量PCR （quantitative real time PCR, qRT-PCR）技术, 对二斑叶螨α-tubulin, β-actin, ELFn, GAPDH, 5.8S rRNA基因和SDHA共6个看家基因的表达稳定性进行分析以期筛选出合适的内参基因, 并在此基础上进一步分析二斑叶螨不同品系P450酶系CYP392E亚家族基因的表达量差异。【结果】在二斑叶螨SS, Av-R和Sp-R品系中稳定性最高的内参基因为ELFn。以ELFn为内参基因, CYP392E7基因相对表达量在二斑叶螨Av-R品系中显著高于SS品系(P<0.05), 为后者的2.18倍, 而在Sp-R品系和SS品系中差异不显著; 其余基因的相对表达量在2个抗性品系中均没有增加, Av-R品系的CYP392E4, CYP392E9和CYP392E10基因以及Sp-R品系的CYP392E1和CYP392E9基因相对表达量甚至显著下调, 分别为SS品系的48%, 74%, 65%, 63%和73%。【结论】在二斑叶螨SS, Av-R和Sp-R品系中ELFn为理想的内参基因, Av-R品系CYP392E4, CYP392E7, CYP392E9和CYP392E10基因相对表达量的显著变化可能与二斑叶螨对阿维菌素的抗性形成有关, Sp-R品系CYP392E1和CYP392E9基因也可能与二斑叶螨对螺虫乙酯的抗性形成有一定联系。     

Lethal and sublethal effects of spiromesifen,spirotetramat and spirodiclofen on Tetranychus urticae Koch (Acari: Tetranychidae)

Lethal and sublethal effects of spirotetramat, spiromesifen and spirodiclofen were assessed on Tetranychus urticae Koch. Leaf disc bioassay was used in all experiments. In this study, toxicity of these compounds was tested on T. urticae eggs and adults. Ovicidal activity was observed in all of the compounds tested, but only spirodiclofen was considerably effective against adult mites. Up to 24?h-old adult females were placed on leaf discs treated with sublethal concentrations (LC₁₀ or LC₂₅). Twenty-four hours after exposure to treated discs, 20 females showing no visible symptoms of poisoning were transferred to untreated leaf discs. The leaf discs were changed daily for up to five days. The number of eggs laid during this period was recorded. The survival rate, total number of laid eggs per female and egg hatching rate were calculated. All above-mentioned parameters were slightly lower in treated females compared with controls.     

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (k_cat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (k_cat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.     

Can insecticide mixtures be considered to surmount neonicotinoid resistance in Bemisia tabaci?

Cotton whitefly, Bemisia tabaci is an important polyphagous pest worldwide. It is exposed to various chemical insecticides throughout the year, resulting in the rapid development of insecticide resistance. Mixtures of insecticides with distinct modes of action could enhance the toxicity of chemicals more effectively than sequences or rotations in resistant pest populations. Bioassays were conducted to study the efficacy of mixtures of neonicotinoid and ketoenol insecticides at different ratios against a laboratory susceptible (Lab-WB) and a neonicotinoid resistant (TMX-SEL) strain of B. tabaci Asia I. The results showed that mixtures of imidacloprid, acetamiprid, thiamethoxam or dinotefuran with spiromesifen at 1:1, 1:10 and 1:20 ratios and of imidacloprid, thiamethoxam or dinotefuran with spirotetramat at 1:1 ratio significantly increased (p < 0.05) toxicity to neonicotinoids in TMX-SEL strain. The combination indices of each tested neonicotinoids + ketoenols at 1:1 ratio and of acetamiprid + spiromesifen, and imidacloprid or dinotefuran + spirotetramat at 1:10 ratio for TMX-SEL strain were significantly below 1, suggesting synergistic interactions. The inhibitors PBO and DEF largely overcame resistance to the tested neonicotinoids, while none of the synergists significantly restored the susceptibility of B. tabaci to ketoenols. Increased activities of P450 monooxygenase and esterase were observed in TMX-SEL strain with an elevated 2.76 and 1.32-fold, respectively. Mixtures of neonicotinoids with spiromesifen or spirotetramat at a 1:1 ratio could be used to restore the neonicotinoid susceptibility in B. tabaci.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): I. Properties of the Herbicide Target Enzymes Acetyl-Coenzyme A Carboxylase and Acetolactate Synthase

Lolium rigidum biotype SR4/84 is resistant to the herbicides diclofop-methyl and chlorsulfuron when grown in the field, in pots, and in hydroponics. Similar extractable activities and affinities for acetyl-coenzyme A of carboxylase (ACCase), an enzyme inhibited by diclofop-methyl, were found for susceptible and resistant L. rigidum. ACCase activity from both biotypes was inhibited by diclofop-methyl, diclofop acid, haloxyfop acid, fluazifop acid, sethoxydim, and tralkoxydim but not by chlorsulfuron or trifluralin. Exposure of plants to diclofop-methyl did not induce any changes in either the extractable activities or the herbicide inhibition kinetics of ACCase. It is concluded that, in contrast to diclofop resistance in L. multiflorum and diclofop tolerance in many dicots, the basis of resistance to diclofop-methyl and to other aryloxyphenoxypropionate and cyclohexanedione herbicides in L. rigidum is not due to the altered inhibition characteristics or expression of the enzyme ACCase. The extractable activities and substrate affinity of acetolactate synthase (ALS), an enzyme inhibited by chlorsulfuron, from susceptible and resistant biotypes of L. rigidum were similar. ALS from susceptible and resistant plants was equally inhibited by chlorsulfuron. Prior exposure of plants to 100 millimolar chlorsulfuron did not affect the inhibition kinetics. It is concluded that resistance to chlorsulfuron is not caused by alterations in either the expression or inhibition characteristics of ALS.     

Expression of Xenobiotic Metabolizing Cytochrome P450 Genes in a Spinosad-Resistant Musca domestica L. Strain

Background

Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.

Results

Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.

Conclusion

CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.     

Resistant Acetyl-CoA Carboxylase is a Mechanism of Herbicide Resistance in a Biotype of Avena sterilis ssp. ludoviciana

A biotype of Avena sterilis ssp. ludoviciana is highly resistantto a range of herbicides which inhibit a key enzyme in fattyacid synthesis, acetyl-CoA carboxylase (ACCase). Possible mechanismsof herbicide resistance were investigated in this biotype. Acetyl-CoAcarboxylase from the resistant biotype is less sensitive toinhibition by herbicides to which resistance is expressed. I₅₀values for herbicide inhibition of ACCase were 52 to 6 timesgreater in the resistant biotype than in the susceptible biotype.This was the only major difference found between the resistantand susceptible biotypes. The amount of ACCase in the meristemsof the resistant and susceptible is similar during ontogenyand no difference was found in distribution of ACCase betweenthe two biotypes. Uptake, translocation and metabolism of [¹⁴C]diclofop-methylwere not different between the two biotypes. In vivo, ACCaseactivity in the meristems of the susceptible biotype was greatlyinhibited by herbicide application whereas only 25% inhibitionoccurred in the resistant biotype. Depolarisation of plasmamembrane potential by 50 µM diclofop acid was observedin both biotypes and neither biotype showed recovery of themembrane potential following removal of the herbicide. Hence,a modified form of ACCase appears to be the major determinantof resistance in this resistant wild oat biotype. (Received February 10, 1994; Accepted March 11, 1994)     

二斑叶螨多重抗性品系最优内参基因的筛选及CYP392A亚家族基因的表达分析

【目的】筛选出二斑叶螨Tetranychus urticae Koch抗甲氰菊酯、阿维菌素及螺螨酯混剂的实时定量PCR最优内参基因。【方法】选取5.8S rRNA, α-tubulin, TBP, β-actin, ELFn, RPL13a, GAPDH和SDHA 8个候选内参基因,以GeNorm, BestKeeper和Normfinder 3个软件分析这8个基因在二斑叶螨多重抗性品系中的表达稳定性, 并以筛选的内参基因分析二斑叶螨P450酶系CYP392A亚家族基因的表达水平。【结果】经GeNorm, BestKeeper和Normfinder 3个软件综合评价确定ELFn基因为二斑叶螨敏感品系(susceptible strain, SS)和多重抗性品系(multi-pesticide resistant strain, Mp-R)各发育阶段的最优内参基因。以ELFn为内参基因对二斑叶螨CYP392A亚家族16个基因表达量进行分析,结果表明：经多重抗性选育40代后,Mp-R品系卵期CYP392A1表达量显著上调;CYP392A16基因在各发育阶段表达量极显著高于SS品系相应发育阶段;其他基因表达量在敏感品系和抗性品系之间差异不显著。【结论】筛选出了SS和Mp R品系中各发育阶段最佳内参基因为EFLn;Mp-R品系CYP392A亚家族16个基因的表达量在幼螨和若螨阶段低于卵与成螨阶段,其中CYP392A16基因在二斑叶螨多重抗性的形成中起主要作用。该结果为二斑叶螨多重抗性研究奠定了一定基础。     

Resistance to acaricides in Italian strains of <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: toxicological and enzymatic assays

Problems with Tetranychus urticae are frequently reported in protected crops in Italy, particularly in roses where many introduced acaricides show a progressive loss of effectiveness. We have conducted bioassays to assess the response of some Italian strains of T. urticae to a number of acaricides. These include compounds that were widespread and frequently used in the past, but also some recently registered compounds. We investigated two T. urticae strains collected from rose growers where control failures were reported (SAN and PSE), together with a strain collected from unsprayed vegetables (BOSA). Adult females of the rose strains (SAN and PSE) were resistant to tebufenpyrad (Resistant Ratio—RR, RR₅₀ = 48.4 and 163.6) and fenpyroximate (RR₅₀ = 74.1 and 25.9) when compared to the susceptible BOSA strain. Lethal concentrations for these products were higher than the registered field rate. The PSE strain proved to be highly resistant to abamectin (RR₅₀ = 1,294.1). Variation in bifenazate susceptibility was detected amongst strains, but LC₉₀ values of SAN and PSE were still in the range of the registered field rate. In egg bioassays, the SAN and PSE strains exhibited high resistance levels to clofentezine (RR₅₀ = 66,473 and 170,714), hexythiazox (RR₅₀ = 70,244 and 159,493) and flufenoxuron (RR₅₀ = 61.9 and 117.9). But the recently introduced ovi/larvicides etoxazole and spirodiclofen exhibited high activity on all strains. The activity of detoxifying enzymes such as esterases, glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (MFOs) was determined in these strains as a preliminary attempt to identify potential resistance mechanisms. Enzymatic assays showed that the rose strains exhibited 2.66 and 1.95-fold increased MFOs activity compared to the susceptible strain. Assays for GSTs revealed that only the SAN strain exhibited a significantly higher activity. In contrast, only the PSE strain showed a significant higher hydrolysis of 1-naphthyl acetate.     

Establishment of an acaricide-susceptible Tetranychus urticae strain and its species confirmation based on morphological and molecular characters

Acquisition of a reference Tetranychus strain that is completely susceptible to acaricides and retains an identical genetic background to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish a strain completely susceptible to various acaricides, we collected Tetranychus mite populations from several regions in South Korea, including both remote and heavily cultivated regions. We tested their suitability as a susceptible reference strain by determining baseline susceptibility to six acaricides and by determining species identity as Tetranychus urticae. The UD strain, originally collected from a remote island region, was found to be most susceptible to all five major acaricides tested and was confirmed to as T. urticae on the basis of both morphological and molecular evidence. Moreover, molecular phylogenetic analysis revealed that the UD strain is an ancestral strain of other prevalently collected green-type strains. Taken together, we propose that the UD strain can be used as a susceptible reference strain for T. urticae resistance studies as it provides baseline susceptibility to acaricides and possesses a common genetic background with most other acaricide-resistant strains.     

Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): insights into resistance evolution in a hexaploid species

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids.     

Milbemectin resistance in <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae): selection,stability and cross-resistance to abamectin

Studies on artificial laboratory selections with milbemectin, stability of milbemectin resistance and possible cross-resistance with abamectin were carried out with Tetranychus urticae Koch to provide basic information for a milbemectin resistance management program. Selections for resistance and susceptibility to milbemectin were performed in a population of T. urticae, collected from a commercial chrysanthemum field in the State of São Paulo, Brazil. After six selections for resistance and five selections for susceptibility, susceptible (S) and resistant (R) strains of T. urticae to milbemectin were obtained. The resistance ratio (R/S) at the LC₅₀ reached 409-fold value. The stability of milbemectin resistance was also studied under laboratory conditions, using a population with initial frequency of 75% of resistant mites. The frequencies of milbemectin resistance were evaluated monthly for a period of 7 months. In order to observe possible correlation between milbemectin and abamectin resistance, the frequencies of abamectin resistance were also evaluated for that population, during the same period. The frequency of milbemectin resistance decreased from 75 to 14.5%, while the percentage of abamectin resistant mites decreased from 57 to 9.1%, in 7 months. The frequencies of milbemectin and abamectin resistance were also evaluated in 25 field populations of T. urticae, collected from several crops in the State of São Paulo. The frequencies of milbemectin resistance varied from 4.1 to 89.5%, and of abamectin, from 7.0 to 90.5%. A positive and significant correlation was observed between the frequencies of milbemectin and abamectin resistance, indicating positive cross-resistance between these acaricides. The results indicate that abamectin should be avoided for managing milbemectin resistance in T. urticae. This is the first report on milbemectin resistance in T. urticae in Brazil.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Accord insertion in the 5′ flanking region of CYP6G1 confers nicotine resistance in Drosophila melanogaster

What has driven the sweep of the Accord retrotransposon insertion allele of CYP6G1 in the natural populations of Drosophila melanogaster is unknown. Previous studies on the DDT selection hypothesis produced conflicting data. To reexamine the DDT selection hypothesis and search for alternative explanations, we conducted a series of correlation and genetic linkage experiments with eight D. melanogaster natural populations collected from California (CM1, CM2, CM3, and CM7) and Africa (AM2, AM3, AM4, AM7). Diagnostic PCR showed that CM1, CM2, CM7, and AM3 have the Accord insertion in the CYP6G1 locus, whereas the other four strains do not. RT-PCR analysis exhibits a 100% correlation between Accord insertion and CYP6G1 overexpression. However, among the four strains with Accord-mediated CYP6G1 overexpression only CM1 and CM7 are resistant to DDT, and the other two strains (CM2 and AM3), like the four Accord-free strains, are susceptible to DDT. By contrast, all the four strains with Accord-mediated CYP6G1 overexpression are resistant to nicotine, a plant allelochemical. Genetic crosses between DDT resistant and susceptible Accord-insertion strains, as well as crosses between Accord-insertion and Accord-free strains demonstrated that Accord insertion and CYP6G1 overexpression are genetically linked to nicotine resistance rather than DDT resistance. These results suggest that naturally-occurring allelochemicals such as nicotine are the initial driving force for the worldwide prevalence of the Accord insertion allele of CYP6G1 in D. melanogaster natural populations.     

Ambulatory dispersal of the susceptible and propargite-resistant strains of Tetranychus urticae and its influence on pesticide resistance dynamics

The distribution of resistant individuals is determined by the amount of movement between populations. The differential rate of dispersal of a susceptible and a pesticide-resistant strain could influence the resistance dynamics under field conditions. The dispersal rate and dispersal efficiency of the susceptible and propargite-resistant strains of Tetranychus urticae were measured in separate-release and mixed-release experiments. The diffusion coefficient (D) of both strains did not differ significantly (P > 0.344) and an estimate of the asymptotic rate of advance (2 √ rD) (for one generation) was estimated at 0.1047 and 0.0930 cm per degree day for the susceptible and propargite-resistant strains, respectively. The dispersal efficiency of the two strains differed significantly (P < 0.005) as more susceptible mites than propargite-resistant mites crossed into specified zones more quickly after 290 and 366 degree days. Significantly (P < 0.05) higher number of susceptible adults, immatures and eggs were found in the outer most zone of an arena as compared to that of the propargite-resistant mites. The bioassay of the two strains showed a similar pattern of the spread of the adult females across the specified zones in the mixed-release experiment. The relatively lower dispersive tendency of the propargite-resistant T. urticae and the smaller proportion of adult females exhibiting that behaviour increase the chances of developing resistant ‘hotspots’ in field specially after an acaricide application.     

Molecular mechanisms conferring asymmetrical cross-resistance between tebufenozide and abamectin in Plutella xylostella

Based on the confirmation of asymmetrical cross-resistance between abamectin and tebufenozide in Plutella xylostella, the present work proved that the cytochrome P450 monooxygenase plays a decisive role in cross-resistance, and the expression of various cytochrome P450 (CYP450) genes in different strains was surveyed to elucidate the molecular basis of the underlying mechanisms. Enzyme analysis showed the activity of cytochrome P450 monooxygenase was notable enhanced in the strains resistant to both tebufenozide (3.07-fold) and abamectin (3.37-fold), suggesting that the enhancement of cytochrome P450 monooxygenase is the main detoxification mechanism responsible for the cross-resistance. CYP4M7 (64.58-fold) and CYP6K1 (41.97-fold) had extremely high expression levels in the Teb-R strain, selected using tebufenozide, which was highly resistant to tebufenozide (RR 185.5) and moderately cross-resistant to abamectin (RR 41.0). When this strain was subjected to further selection using abamectin, the resultant Aba-R strain showed a higher expression of CYP6K1 (60.32-fold). However, the expression of CYP4M7 was reduced (10.62-fold). Correspondingly, the Aba-R strain became more resistant to abamectin (RR 593.8) and less resistant to tebufenozide (RR 28.0). Therefore, we concluded that the over expression of CYP4M7 was the main cause for tebufenozide resistance, and that CYP6K1 mainly conferred abamectin resistance. The asymmetrical cross-resistance occurred because tebufenozide selection not only enhanced the expression of CYP4M7, but also that of CYP6K1. This is the first report on the molecular mechanism of asymmetrical cross-resistance between insecticides.     

Molecular characterization of antibiotic resistant Escherichia coli isolates recovered from food samples and outpatient Clinics,KSA

Multidrug-resistant Escherichia coli is one of the most important public health concern worldwide that can be transferred through the food of animal origin to human being causing serious infection. The genetic responsibility of such resistant genes (Plasmids, integrons, and transposons) can be easily transmitted from the resistant strain to another. Therefore, the main objectives of the study is the molecular characterization of the resistant Escherichia coli isolates recovered from food samples and human isolates collected from outpatient clinics, KSA especially the resistance strains against aminoglycoside resistance genes which are responsible for the resistance against gentamicin and the resistance caused β-lactamases genes. Examination of food samples revealed 120 Escherichia coli isolates (22.22%) (30 strains O26: K60, 28 strains O128: K67, 20 strains O111: K58, 18 strains O126: K58, 10 strains O55: K59, 9 strains O86: K61 and 5 strains O157: H7). All the strains were highly resistance to penicillin, amoxicillin-clavulanic and erythromycin with a percentage of 100%, while the resistance to gentamicin, ampicillin, oxytetracycline, chloramphenicol, norfloxacin, trimethoprim, and nalidixic acid were 83%, 75%, 65.3%, 55.8%, 36.5%, 30.7% and 26.9% respectively. On the other hand, 59.6% of tested strains were sensitive to ciprofloxacin. Positive amplification of 896?bp fragments specific for aacC2 genes were observed by PCR designated for the detection of the aminoglycoside resistance genes. Meanwhile, multiplex PCR designed to detect the ampicillin and amoxicillin-clavulanic acid resistant E. coli isolates revealed positive amplification of 516?bp fragments specific for BlaTEM gene with all the resistant strains to ampicillin and amoxicillin-clavulanic acid. Moreover, positive amplification of 392?bp fragments specific for BlaSHV resistant gene were observed with (60.52%) of E. coli isolate. While all the tested strains were negative for amplification of BlaOXA_1.     

二点叶螨对甲氰菊酯、氧乐果和四螨嗪抗药性的选育、衰退和恢复

以兰州吐鲁沟公园的二点叶螨Tetranychus urticae 为敏感品系,分别用氧乐果、甲氰菊酯、四螨嗪喷雾处理15次,其抗性水平分别为38.5、479.8和67.3倍。将抗性品系分别与敏感品系进行杂交和回交的结果表明：抗氧乐果品系的显性系数DRS（R♀×S♂）为0.4700、D_SR（S♀×R♂）为0.4749;抗甲氰菊酯品系的DRS（R♀×S♂）为0.5155, DSR为0.5237;抗四螨嗪品系的DRS为0.3134, DSR为0.2466。表明二点叶螨对这3种药剂的抗性均是由单个不完全显性基因所控制。在连续10个月不接触药剂的情况下,3个抗性品系的抗药性都有下降,抗氧乐果品系的抗性下降最快,只有敏感品系的抗性倍数的3.6倍;抗甲氰菊酯种群的抗性下降较慢,为敏感品系的95.9倍。再经甲氰菊酯、氧乐果、四螨嗪分别连续15次喷雾处理后,3个抗性种群的抗性水平又再度回升,抗甲氰菊酯品系回升较快,抗性为敏感品系的523.5倍,抗四螨嗪品系次之,抗氧乐果的品系抗性恢复最慢。