Determination of acaricide resistance allele frequencies in field populations of Tetranychus urticae using quantitative sequencing

Resistances to monocrotophos, fenpropathrin and abamectin in Tetranychus urticae are primarily conferred by reduced sensitivities of respective target sites [i.e., acetylcholinesterase (TuAChE), voltage-sensitive sodium channel (TuVSSC) and glutamate-gated chloride channel (TuGluCl)], which are due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, a quantitative sequencing (QS) protocol was developed for the determination of the resistance-associated mutation frequencies in T. urticae. Standard prediction equations revealed high correlation coefficients (r² = 0.993–0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower and higher detection limits for the four resistance mutations were 3.7–13.1% (7.8 ± 3.3%) and 89.4–97.3% (93.3 ± 3.2%), respectively, suggesting that QS can be employed as a preliminary monitoring tool for the detection of resistance allele frequencies, which ranged approximately 7.8–93.3% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 26 T. urticae populations. The two TuAChE mutations responsible for monocrotophos resistance were almost saturated in most field populations. The TuVSSC L1022V mutation tentatively associated with fenpropathrin resistance was also found in 9 field populations. However, the TuGluCl G323D mutation conferring abamectin resistance was found only in one field population, suggesting that abamectin resistance is not yet widespread. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of T. urticae.     

Monitoring of carbamate and organophosphate resistance levels in Nilaparvata lugens based on bioassay and quantitative sequencing

The resistance levels to carbamate (CB) and organophosphate (OP) insecticides were determined by topical application in 14 field strains of Nilaparvata lugens. The resistance levels of N. lugens to CB and OP were 1.3–47.5-fold and 1.4–14.4-fold higher than a susceptible strain, respectively. A quantitative sequencing (QS) protocol was established to determine the allele frequencies of four acetylcholinesterase point mutations putatively associated with CB and OP resistance. The allele frequencies of the four mutations (G119A, F/Y330S, F331H and I332L) in field strains' ranges are ca. 0.0–51.7%, 0.0–88.9%, 5.1–56.0% and 6.7–57.3%, respectively. The F331H and I332L were tightly linked to each other, suggesting that these mutations may occur simultaneously. In correlation analysis, G119A was not well correlated with actual resistance levels (r² = < 0.232), whereas F331H and I332L showed a better correlation with the resistance levels of benzofuranyl methylcarbamates (r² = 0.595). This finding indicates that F331H and I332L mutation frequencies may be used as molecular markers for detecting carbamate resistance in N. lugens. A QS protocol detecting the F331H and I332L mutation frequencies could therefore be employed as a supportive tool for the rapid monitoring of CB insecticide resistance levels in N. lugens.     

北京和河北地区小菜蛾的抗药性动态

【目的】了解北京和河北地区小菜蛾Plutella xylostella(L.)对主要防治药剂的抗药性现状,为小菜蛾的有效防治提供参考。【方法】2011—2015年,采用浸叶法监测了小菜蛾对9种药剂的抗药性。【结果】小菜蛾对氯虫苯甲酰胺、多杀菌素和丁醚脲均为敏感水平,对Bt制剂、虫螨腈和茚虫威个别年份出现中等抗性水平,但总体上为敏感状态,对阿维菌素和氟啶脲保持中等抗性水平,对高效氯氰菊酯为中等至高水平抗性。【结论】在北京和河北地区高效氯氰菊酯不适用于小菜蛾的防治,少用或暂停使用阿维菌素,其它药剂可交替或轮换使用。     

寄主胁迫下小菜蛾种群的RAPD分析

采用RAPD-PCR技术,研究了取食6种不同十字花科寄主植物第4、9、15代小菜蛾Plutella xylostella(L.)种群的DNA多态性。通过17条引物的重复PCR扩增实验,利用NTsys软件对电泳条带统计分析,构建了遗传距离矩阵和聚类分析图谱。结果表明:取食芥蓝的小菜蛾种群与其它5种寄主种群遗传距离较远(0.6523～0.8246),独立聚为一枝;取食萝卜的小菜蛾种群遗传距离与甘蓝上种群较近(0.3443),聚为一枝,并且与白菜、芥菜和菜心上小菜蛾种群遗传距离较近,逐次聚为一枝,芥蓝、萝卜及甘蓝寄主种群随代次的增多种群分化出现稳定规律性。各寄主种群间遗传距离平均值和范围、多态性条带占总条带数目的比例随代次增多而增大,说明种群分化程度随代次增多而加深。     

小菜蛾系统调查及抗药性监测方法

小菜蛾Plutella xylostella(L.)是十字花科作物重要害虫。其种群消长受地理条件、气候变化和耕作方式等多种因素的影响。小菜蛾也是抗药性最强的害虫,几乎对所有杀虫剂产生了抗药性。针对小菜蛾的生活习性和抗药性,统一规范了田间种群系统调查和抗药性监测方法,为其种群动态及抗药性监测提供技术支撑。     

Biochemical basis of organophosphate and carbamate resistance in Asian citrus psyllid

The Asian citrus psyllid, Diaphorina citri Kuwayama, is a worldwide pest of citrus, which vectors the putative causal pathogen of huanglongbing. Current management practices warrant continuous monitoring of field populations for insecticide resistance. Baseline activities of acetylcholinesterase (AChE), general esterase, and glutathione S-transferase as well as sensitivity of AChE to selected organophosphate and carbamate insecticides were established for a susceptible laboratory strain (Lab) and compared with several field populations of D. citri from Florida. The specific activity of AChE in various D. citri populations ranged from 0.77 to 1.29 microM min(-1) mg of protein(-1); the Lab strain was characterized by the highest activity. Although reduced AChE sensitivity was observed in the Lab strain compared with field populations, overlap of 95% confidence intervals of I50 values (concentration required for 50% AChE activity inhibition) suggests no significant difference in AChE sensitivity among all populations tested for a given insecticide. There was no significant evidence of target site insensitivity in field populations that were exposed to the selected organophosphate and carbamate insecticides tested. The specific activity of general esterase and glutathione S-transferase was lowest in the Lab strain and was generally comparable to that of the field populations evaluated. The current data provide a mode-of-action specific baseline for future monitoring of resistance to organophosphate and carbamate insecticides in populations of D. citri.     

Temperature‐sensitive fitness cost of insecticide resistance in Chinese populations of the diamondback moth Plutella xylostella

Alleles conferring a higher adaptive value in one environment may have a detrimental impact on fitness in another environment. Alleles conferring resistance to pesticides and drugs provide textbook examples of this trade‐off as, in addition to conferring resistance to these molecules, they frequently decrease fitness in pesticide/drug‐free environments. We show here that resistance to chlorpyrifos, an organophosphate (OP), in Chinese populations of the diamondback moth, Plutella xylostella, is conferred by two mutations of ace1 – the gene encoding the acetylcholinesterase enzyme targeted by OPs – affecting the amino acid sequence of the corresponding protein. These mutations were always linked, consistent with the segregation of a single resistance allele, ace1R, carrying both mutations, in the populations studied. We monitored the frequency of ace1R (by genotyping more than 20 000 adults) and the level of resistance (through bioassays on more than 50 000 individuals) over several generations. We found that the ace1R resistance allele was costly in the absence of insecticide and that this cost was likely recessive. This fitness costs involved a decrease in fecundity: females from resistant strains laid 20% fewer eggs, on average, than females from susceptible strains. Finally, we found that the fitness costs associated with the ace1R allele were greater at high temperatures. At least two life history traits were involved: longevity and fecundity. The relative longevity of resistant individuals was affected only at high temperatures and the relative fecundity of resistant females – which was already affected at temperatures optimal for development – decreased further at high temperatures. The implications of these findings for resistance management are discussed.     

Mechanisms of organophosphate and carbamate resistance in Culex quinquefasciatus from Saudi Arabia

Broad spectrum organophosphate resistance in Culex quinquefasciatus Say from Saudi Arabia is inherited as a semi-dominant characteristic. The resistance has a metabolic basis and confers cross-resistance against the carbamate propoxur. Organophosphate-selected strains contain two elevated esterases with the same electrophoretic mobilities as those in resistant Cx quinquefasciatus from Sri Lanka and a range of African locations. Alteration in the sensitivity of acetylcholinesterase to insecticide inhibition does not play a major role in resistance. There was a significant increase in the amount of Cytochrome P450 in Cx quinquefasciatus lines selected with the pyrethroid permethrin or with the organophosphate pirimiphos-methyl, but no change in lines selected with five other organophosphates or propoxur, compared to the parental strain, which suggests that oxidases are involved in the P450 mediated resistance to both permethrin and pirimiphos-methyl.     

Inheritance of resistance and cross resistance pattern in indoxacarb-resistant diamondback moth Plutella xylostella L.

Leaf-dip assay of Plutella xylostella against indoxacarb showed that the concentration that produced 50% mortality (LC₅₀) of indoxacarb ranged from 20.1 to 11.9 ppm, with highest in Nasik and lowest levels in Coimbatore strains. In selection studies, the LC₅₀ of indoxacarb was 18.5 ppm at generation 1 (G1), which increased to 31.3-fold (167.8 ppm) resistance after ten exposed generations (G10) as compared to unexposed. The LC₅₀ of quinalphos was 74.4 ppm, which increased to 10.0-fold (631.5 ppm) resistance after G10. The LC₅₀ of cypermethrin resistant strain resulted in an 11.5-fold increase in resistance after G10. In P. xylostella , heritability (h²) after ten generations of selection was estimated at 0.4. The number of generations required for tenfold increase in LC₅₀ (1/R) were 6.7. The response to indoxacarb selection in P. xylostella was 0.2 and the selection differential was estimated as 0.4. The phenotypic standard deviation was 0.2. Reciprocal crosses between indoxacarb-resistant and susceptible strains showed that the inheritance of indoxacarb resistance was autosomal. The degree of heritability (D_LC) (0.4, 0.4) indicated incomplete recessive inheritance of indoxacarb resistance.     

Quantitative determination of allele frequency in pooled DNA by using sequencing method

Quantitative determination of the allele frequency of single-nucleotide polymorphism (SNP) in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present such a simple, accurate, and inexpensive method for quantitative determining the allele frequency in pooled DNA samples. Three steps of DNA pooling, PCR amplification and sequencing are involved in this assay. Although direct determination of the allele frequency from the two allele-specific fluorescence intensities is possible, correction for differential response of alleles is important. We explored the effect of differential response of alleles on test statistics and provide a solution to this problem based on heterozygous fluorescence intensities. We demonstrate the accuracy and reliability of this assay on pooled DNA samples with pre-determined allele frequencies from 7.1% to 53.9%. The accuracy of allele frequency measurements is high, with a correlation coefficient of r2 = 0.997 between measured and known frequencies. We believe that by providing a means for SNP genotyping up to hundreds of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies.     

Detection and selection of organophosphate and carbamate resistance in Culex quinquefasciatus from Saudi Arabia

Resistance to a number of organophosphorus insecticides and the carbamate propoxur has been detected in a field population of Culex quinquefasciatus Say from Jeddah, Saudi Arabia. Cross resistance studies indicate that the same mechanism(s) may produce resistance to the full range of these compounds.     

Sex pheromone production and response in Korean populations of the diamondback moth, Plutella xylostella

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae), is a worldwide pest of cruciferous crops. We examined the female pheromone production and male response to various pheromone blends in two Korean populations. Gas chromatography (GC) and GC‐mass spectrometry (MS) analyses of pheromone gland extracts revealed that females produce (Z)‐11‐hexadecenal (Z11‐16:Ald), (Z)‐11‐hexadecenyl acetate (Z11‐16:OAc), and (Z)‐11‐hexadecen‐1‐ol (Z11‐16:OH) in a ratio of 8:100:18. However, (Z)‐9‐tetradecenyl acetate (Z9‐14:OAc), a previously reported component of the sex attractant of a Canadian P. xylostella population was not detected in gland extracts of the Korean one. Field tests showed that Z11‐16:Ald and Z11‐16:OAc are essential for attraction of male moths, and the highest attraction is obtained with a 10:90 blend mimicking the blend found in gland extracts. Addition of 1 or 10% of Z11‐16:OH to the 10:90 blend of Z11‐16:Ald and Z11‐16:OAc significantly increased attraction. However, attraction was strongly antagonized by the addition of as little as 0.1% of Z9‐14:OAc to the most attractive ternary blend. The ternary blend of Z11‐16:Ald, Z11‐16:OAc, and Z11‐16:OH at a ratio of 10:90:1 was more effective at catching P. xylostella males than the Japanese three‐component blend or the Canadian four‐component blend in Korea. These results suggest that there is geographical variation in the pheromone systems of this species.     

SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries

High-density single-nucleotide polymorphism (SNP) arrays have revolutionized the ability of genome-wide association studies to detect genomic regions harboring sequence variants that affect complex traits. Extensive numbers of validated SNPs with known allele frequencies are essential to construct genotyping assays with broad utility. We describe an economical, efficient, single-step method for SNP discovery, validation and characterization that uses deep sequencing of reduced representation libraries (RRLs) from specified target populations. Using nearly 50 million sequences generated on an Illumina Genome Analyzer from DNA of 66 cattle representing three populations, we identified 62,042 putative SNPs and predicted their allele frequencies. Genotype data for these 66 individuals validated 92% of 23,357 selected genome-wide SNPs, with a genotypic and sequence allele frequency correlation of r = 0.67. This approach for simultaneous de novo discovery of high-quality SNPs and population characterization of allele frequencies may be applied to any species with at least a partially sequenced genome.     

小菜蛾对杀虫双和杀螟丹抗性的现实遗传力

利用室内选育119代的小菜蛾Plutella xylostella抗性品系,分析了小菜蛾对杀虫双和杀螟丹抗药性的现实遗传力,并对遗传力和杀虫剂的杀死率对抗性发展速率的影响进行了预测,结果表明,室内继代药剂选择119代后,小菜蛾对杀虫双和杀螟丹抗性的遗传力很低（h^2=0.03)。因此,小菜蛾对这两种药剂产生更高水平抗性的潜力不大,在选择的前半段（F30）,杀虫双和杀螟丹的抗性遗传力（分别为0.14t 0.11)高于选择后半段（F80)的遗传力（均为0.05),选择前、后半段的选择差数没有明显差别。即使在室内选择条件下,假如斜率=2.0(δp=0.5),h^2=0.05,群体中70%个体致死时要获得10倍的抗性,约需34代,田间条件下,由于等位基因频率的变化、环境变异等因素的影响,抗性增长10倍则需要更长的时间,因此田间条件下小菜蛾在短期内不易对杀虫双和杀螟丹产生高水平抗性。     

小菜蛾对阿维菌素抗性基因的AFLP连锁图谱的构建

世界性害虫小菜蛾Plutella xylostella (L.) 田间种群已对阿维菌素药剂产生了高抗药性。本研究以小菜蛾对阿维菌素的抗性品系、敏感品系和回交群体组建作图家系,再利用AFLP技术构建小菜蛾对阿维菌素抗性基因的连锁图谱。用10组AFLP引物组合对未经药剂处理的小菜蛾成虫DNA选择性扩增,共获得1 044条可区分的DNA条带,271条带具有多态性,χ²检验表明只有123个位点符合1∶1 （P=0.05）,其中的112个构成28个连锁群,其总长度为1 222.7 cM; 用10组AFLP引物对经药剂处理后存活的小菜蛾成虫DNA扩增,所得的DNA条带中有54个条带符合分离比1∶1,经Mapmaker 3.0对多态性位点分析,只有E1M4-15、E1M1-4和E1M4-2标记位点位于同一连锁群,3个AFLP标记与抗性基因的遗传距离分别为0 cM,8.3 cM和13.1 cM。比较分析得出,抗性基因所在的连锁群是第5连锁群。结果显示AFLP连锁图谱对小菜蛾对阿维菌素的抗性监测具有很好的应用潜力。     

Monitoring and characterization of diamondback moth (Lepidoptera: Plutellidae) resistance to spinosad

Fourteen populations of the diamondback moth, Plutella xylostella (L.), were collected from fields of crucifer vegetables in the United States, Mexico, and Thailand in 1999 and 2000 for susceptibility tests with spinosad. Most populations were susceptible to spinosad and similar to earlier baseline values, but populations from Thailand and Hawaii showed high levels of tolerance. A statewide survey in Hawaii in 2000 and 2001 indicated resistance problems on several islands. One colony collected in October 2000 from Pearl City, HI, was subjected to further selection pressure, using spinosad in the laboratory, and then was used as the resistant strain (Pearl-Sel) for other tests. Spray tests using the recommended field rates of spinosad on potted broccoli plants in the greenhouse confirmed that field control failures due to resistance were possible in the areas of these collections. Analysis of probit lines from F1 reciprocal crosses between the Pearl-Sel and S strain indicated that resistance to spinosad was inherited autosomally and was incompletely recessive. A direct test of monogenic inheritance based on the F1 x Pearl-Sel backcrosses suggested that resistance to spinosad was probably controlled by one locus. The synergists S,S,S-tributyl phosphorotrithioate and piperonyl butoxide did not enhance the toxicity of spinosad to the resistant colony, indicating metabolic mediated detoxification was probably not responsible for the spinosad resistance. Two field colonies in Hawaii that were resistant to spinosad were not cross-resistant to emamectin benzoate or indoxacarb. Resistance developed in Hawaii due to the continuous cultivation of crucifers in which as many as 50 applications of spinosad per year may have been made to a common population of P. xylostella in sequential plantings, although each grower might have used the labeled restrictions for resistance management. Resistance management strategies will need to address such cropping and pest management practices.     

高原春油菜田小菜蛾种群动态和抗药性的监测

【目的】为了准确掌握典型春油菜种植区小菜蛾Plutella xylostella(L.)种群变化动态和抗药性现状。【方法】诱捕法调查了青海高原小菜蛾成虫发生动态、室内用浸渍法测定了小菜蛾田间种群的抗性倍数,并进行了田间药效试验。【结果】青海省小菜蛾一般一年发生3代,但2 500 m以上的地区第3代成虫数量较第1代、第2代明显下降。在我省高原春油菜区,每日20:00至次日晨4:00是小菜蛾成虫发生主要的时间段。小菜蛾在青海省不能越冬。湟中点小菜蛾对溴虫腈产生低水平抗性;对多杀菌素、丁醚脲产生中等抗性水平;对Bt、高效氯氰菊酯、茚虫威产生高水平的抗性;对阿维菌素、啶虫隆、氯虫苯甲酰胺产生极高水平抗性。互助点小菜蛾对溴虫腈、丁醚脲产生低水平抗性;对多杀菌素、啶虫隆产生中等抗性水平;对Bt、氯虫苯甲酰胺、茚虫威产生高水平抗性;对阿维菌素产生极高水平抗性。小菜蛾的抗性监测结果与田间药效结果基本一致,溴虫腈的抗性倍数最低,田间防治效果好于其他参试药剂。【结论】青海省小菜蛾年发生代数较少,且不能越冬。春油菜田小菜蛾已对大部分农药产生了抗药性。     

Investigation of resistance mechanisms to fipronil in diamondback moth (Lepidoptera: Plutellidae)

The SZ-F strain of Plutella xylostella (L.) (Lepidoptera: Plutellidae) was derived from a field-collected strain (SZ) by 20 generations of continuous selection with fipronil. The selection resulted in 300-fold increase in resistance to fipronil, and 3.5- and 6.5-fold cross-resistance to dieldrin and endosulfan, respectively, in the SZ-F strain compared with the unselected SZ strain. Analysis of detoxification enzyme activities and bioassay with synergists indicated that metabolic mechanisms are not important to fipronil resistance of the SZ-F strain and that the fipronil resistance is most likely attributed to target site insensitivity. The genomic DNA fragments flanking the second membrane-spanning region of Rdl gamma-aminobutyric acid (GABA) receptor gene from P. xylostella, PxRdl, were cloned and sequenced. A single allele of the PxRdl gene (encoding A302 or allele PxRdl-Ala) was found in both the Roth (susceptible) and SZ strains. In addition to the wild-type allele PxRdl-Ala, the resistant SZ-F strain carried a mutant PxRdl allele with the conserved amino acid replacement A302(GGC)--> S302(TCC) (allele PxRdl-Ser). The mutant PxRdl-Ser allele frequency in the SZ-F strain was 30%. After treatment of 20 mg/l fipronil on the SZ-F strain, the PxRdl-Ser allele frequency in the survivors increased to 57%. High frequency of the PxRdl-Ala allele remaining in the resistant SZ-F strain suggested that the A302S mutation in the PxRdl gene is partially associated with fipronil resistance and that other mutation(s) in the PxRdl gene or other Rdl-like subunit(s) may contribute to fipronil resistance.