二斑叶螨对甲氰菊酯的抗性选育及解毒酶活力变化

为了明确二斑叶螨Tetranychus urticae Koch对甲氰菊酯产生抗性的机理,在室内模拟田间药剂的选择压力, 用甲氰菊酯对二斑叶螨敏感品系（S）进行逐代汰选,选育至38代时, 获得了抗性倍数( resistance ratio, RR)为247.35的抗甲氰菊酯品系（Fe-R）。对S和Fe-R解毒酶活性的分析表明,Fe-R₃₈体内羧酸酯酶（carboxylesterase, CarE）、酸性磷酸酯酶(acid phosphatase, ACP)、 碱性磷酸酯酶(alkaline phosphatase, ALP)、谷胱甘肽-S-转移酶(glutathione s-transferase, GSTs)和多功能氧化酶(mixed function oxidase, MFO)较S体内相应酶的活力显著升高（P< 0.05）,其相对比值（R/S）分别为1.822,13.941,3.789,4.262和17.386。此外,筛选至第9,19,25,32代时,除Fe-R₂₅和Fe-R₃₂的MFO活性与S相比有显著性差异（P< 0.05）外,其余解毒酶（CarE,ACP,ALP,GSTs）的活性与S相比均无显著性差异(P>0.05)。筛选至第38代时, 5种解毒酶的活力与S相比均差异显著(P<0.05)。结果说明二斑叶螨Fe-R随着筛选代数的增加（第25代后）,MFO活性的上升可能是二斑叶螨对甲氰菊酯产生抗性的主要原因。     

Effect of clofentezine-hexythiazox resistance on life-table attributes of Tetranychus urticae Koch (Acari: Tetranychidae)

Life-table parameters of clofentezine-hexythiazox susceptible, resistant and back-crossed hybrid strains of Tetranychus urticae were studied to aid resistance management. Mean egg production (±s.e.) for the QRPH strain was 102.99 (13.452) and significantly more (P<0.05) than for strains S 67.18 (8.370) and S^QRPH 50.98 (6.180) which were not significantly different from each other. We consider the elevated level of egg production evident in the QRPH strain to be evidence of coadaption. The relative fitness of the resistant strain would make resistance management difficult.This work contributes in part for the fulfilment of the requirements of the degree of PhD at the University of Sydney.     

Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari: Tetranychidae).

Resistance of the twospotted spider mite, Tetranychus urticae Koch, to acaricides acting as mitochondrial electron transport inhibitors (METIs) is an increasing problem. Because of their high levels of cross-resistance to all commercially available METI-acaricides, a Japanese strain (AKITA) and an English strain (UK-99) of T. urticae were investigated in detail. Larvae of both strains, AKITA and UK-99, showed 1,100- and 480-fold resistance against pyridaben, 870- and 45-fold resistance against fenpyroximate, and 33- and 44-fold resistance against tebufenpyrad, respectively, in a foliar spray application bioassay compared with the susceptible strain GSS. These resistance factors remained stable even when maintained in the laboratory without further selection. Furthermore, strain AKITA showed cross-resistance to dicofol. The METI resistant strains AKITA and UK-99 showed 2.4- and 1.7-fold enhanced O-ethoxycoumarin O-deethylation (cytochrome P450) activity. Increased oxidative metabolism of the METI-acaricides in the resistant strains could be partially suppressed in vivo by the monooxygenase-inhibitor piperonyl butoxide. Reciprocal crosses of homozygous, diploid females and hemizygous, haploid males of strains GSS (susceptible) and AKITA (resistant) revealed that resistance to pyridaben and fenpyroximate was inherited incompletely dominant with slight differences between maternal and paternal inheritance. This is the first attempt to mechanistically describe METI-acaricide resistance in T. urticae. The implications for resistance management strategies are discussed.     

Multiple Resistance and Biochemical Mechanisms of Dicofol Resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) resistant to dicofol was selected with dicofol successively for 20 generations to produce the DR-20 strain. Resistance and multiple resistance levels of the DR-20 strain to 15 acaricides were determined using a spray bioassay. The DR-20 strain was extremely resistant to dicofol [resistance ratio (RR), 465]. The strain showed extremely strong resistance to acrinathrin (RR, 373) and benzoximate (RR, 197) and strong resistance to bromopropylate (RR, 136), fenbutatin oxide (RR, 65), fenpropathrin (RR, 70), fenpyroximate (RR, 68), and pyridaben (RR, 63). A RR of 11–29 was observed with abamectin, fenazaquin, milbemectin, propagite, and tebufenpyrad. The DR-20 strain exhibited low levels of resistance (RR<3) to azocyclotin and chlorfenapyr. In comparative assays with detoxifying enzymes, the DR-20 strain showed 4.7-fold higher activity in p-nitroanisole-O-demethylation and 1.6-fold higher activities in both α- and β-naphthyl acetate hydrolysis. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide, iprobenfos, triphenyl phosphate, and 4, 4-dichloro-α-methyl benzhydrol had little or no synergistic activity in the susceptible and DR-20 strains. These results suggest that employment of certain acaricides with little multiple resistance will be useful for the management of dicofol resistance in the field.     

Acaricidal and stimulatory effects of insecticides on Tetranychus urticae Koch (Acari: Tetranychidae) in cotton

Possible stimulatory or acaricidal effects of thiodicarb, dimethoate and endosulfan on the two-spotted spider mite, Tetranychus urticae Koch, were tested in a glasshouse. Population development of T . urticae was monitored, with the expectation that any stimulatory or acaricidal effects would be reflected in different population growth relative to untreated plants. Acaricidal activity of the three insecticides was also investigated using a standard bioassay technique. Population growth on thiodicarb- or dimethoate-treated plants was no different to that on untreated plants but was slower on plants treated with endosulfan. Bioassays showed that endosulfan was moderately acaricidal, that T . urticae was highly resistant to dimethoate and that thiodicarb was of very low acaricidal activity. No evidence of stimulatory effects of dimethoate or thiodicarb was found. The data suggest that stimulation is an unlikely explanation for outbreaks of T . urticae in cotton and that endosulfan sprayed for control of Helicoverpa spp. may delay the development of mite outbreaks.     

二斑叶螨抗螺螨酯品系GST基因的克隆与表达分析

【目的】揭示二斑叶螨Tetranychus urticae对螺螨酯的分子抗性机理。【方法】利用RT-PCR克隆了二斑叶螨的谷胱甘肽-S-转移酶（glutathione S-transferase, GST)基因 cDNA 全长序列, 采用生物信息学软件分析了克隆基因的编码蛋白特性; 利用实时荧光定量PCR 方法分析GST基因在二斑叶螨的螺螨酯抗性与敏感品系中的表达差异。【结果】克隆获得的谷胱甘肽-S-转移酶2个基因分别被命名为TuGSTd1和TuGSTd2 (GenBank登录号分别为： KC445659和KC445660)。序列分析发现, TuGSTd1的开放阅读框长度为648 bp, 编码215个氨基酸, 分子量约为24.47 kDa, 理论等电点为5.49; TuGSTd2的开放阅读框为648 bp, 编码215个氨基酸, 分子量约为24.57 kDa, 理论等电点为6.33。系统发育分析表明这两个基因与桔全爪螨Panonychus citri Delta家族的GST基因的氨基酸序列一致性为93%。实时荧光定量PCR 结果表明, TuGSTd1和TuGSTd2在二斑叶螨抗螺螨酯品系中的相对表达量分别为敏感品系的5.60和3.75 倍。【结论】 GST基因在二斑叶螨抗螺螨酯品系中的相对表达量均显著高于敏感品系, 据此推测GST基因的过量表达可能与其对螺螨酯的抗性形成有关。     

Life table parameters of an indigenous strain of Neoseiulus californicus McGregor (Acari: Phytoseiidae) when fed Tetranychus urticae Koch (Acari: Tetranychidae)

The life table of the indigenous Neoseiulus californicus was studied at different temperatures and 65 ± 5% relative humidity under conditions of 16 h light : 8 h dark (LD 16:8). The total developmental period from egg to adult varied from 3.0 to 14.0 days at 15 to 35°C. Survival to adulthood ranges from 86.21 to 93.94%, with the highest rate at 25°C. The lower threshold temperature from egg to adult stages of females and males was 10.84 and 10.72°C, respectively, and the thermal constant was 57.14 degree‐days (DD) for females and 56.18 DD for males. Total number of eggs laid by each female was the highest (70.38 eggs) at 25°C, whereas average daily fecundity was the highest (3.69 eggs/female/day) at 30°C. The net reproductive rate was the highest (48.49) at 25°C and lowest (26.18) at 30°C. Mean generation time decreased from 19.04 to 11.47 days with increasing temperature from 20 to 30°C. Both intrinsic rate of natural increase (0.284) and finite rate of increase (1.32) were maximum at 30°C. Adult longevity was the highest (42.75 days for females and 32.60 days for males) at 20°C and lowest (22.70 days for females and 15.30 days for males) at 30°C. Sex ratio was female biased and was the highest (78.08) at 25°C and lowest (70.24) at 30°C. Developmental data of five constant temperatures, temperature thresholds and thermal requirements may be used to predict the occurrence, number of generations and population dynamics of N. californicus as an important biocontrol agent of Tetranychus urticae.     

Sublethal effects of spirodiclofen on the demographic parameters of Tetranychus urticae Koch (Acari: Tetranychidae)

Abstract

The two-spotted spider mite, Tetranychus urticae Koch is one of the most common pests of agricultural crops. The use of sublethal doses can reduce the need of pesticides and prevent pests’ resistance, which is an important step in integrated pest management and safely controlling this pest. The sublethal effects (LC₅, LC₁₅ and LC₃₅) of spirodiclofen on demographic parameters of T. urticae based on the age-stage, two-sex life table theory were investigated under laboratory conditions [25?±?2?°C, 60?±?5% RH and 16:8 (L:D) h]. The longevity, total life span, oviposition period and total fecundity reduced after exposure to LC₁₅ and LC₃₅. Oviposition period ranged from 5.64 to 10.92?days. The gross reproduction rate (GRR), net reproductive rate (R₀) and the finite rate of increase (λ) of the mites treated with LC₁₅ and LC₃₅ were inferior to the results when treated with LC_5, and control. The values of intrinsic rate of increase (r) was 0.2209?day^?1 in the control and this was reduced to 0.1714?day^?1 in LC₃₅. Based on these results, considering the negative impact of spirodiclofen on the population growth of T. urticae, this acaricide can be helpful to achieve an efficient control for T. urticae in frame of integrated pest management.     

Genetic distinctness between two forms of Tetranychus urticae Koch (Acari: Tetranychidae) detected by electrophoresis

The genetic relatedness between the green and red forms of the two-spotted spider mite, Tetranychus urticae Koch, was analysed using an allozyme marker, MDH, detected by polyacrylamide gel electrophoresis. Cross experiments showed that each of the two forms had form-specific alleles for MDH. Furthermore, there was no overlapping in the alleles for MDH between the field populations of the two forms, suggesting that no gene flow would occur between them in the field.     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

Nucleo-cytoplasmic interactions causing partial female sterility in the spider mite Tetranychus urticae Koch (Acari: Tetranychidae)

A case of non-reciprocal hybrid infertility between strains of the spider mite Tetranychus urticae Koch is described. Evidence is presented that the infertility results from an interaction between a chromosomal and a cytoplasmic factor. The presence or absence of the cytoplasmic factor seems to be controlled also by chromosomal genes.     

Field evaluation of Beauveria bassiana efficacy for the control of Tetranychus urticae Koch (Acari: Tetranychidae)

Trials with eggplants were conducted to evaluate field efficacy of Beauveria bassiana (Bb) applications against Tetranychus urticae, during spring of 2006 and 2007. In the 2006‐Trial, a rate of ca. 0.7 × 10¹² conidia/ha was sprayed onto plants at intervals. In the 2007‐Trial, the rates of ca. 0.8 × 10¹² and 1.6 × 10¹² conidia/ha were sprayed at 1‐ or 2‐week intervals. Based on population densities of mites and leaf damage assessed throughout the trial, the Bb sprays with 1‐ or 2‐week interval showed better control than those of 3 or 4 weeks. In both trials there was no statistical difference between 1‐week and 2‐week spray interval for all parameters measured. Furthermore, the higher rate performed significantly better than the lower rate, regardless of the intervals. No interaction was found between the spraying interval and the applied rate. Ultimately, to suppress the population density of T. urticae and prevent the eggplant damage in spring, the higher rate applied at 2‐week interval was effective.     

二斑叶螨体内感染的Wolbachia的wsp基因序列测定与分析

应用Wolbachia的wsp基因特异引物, 通过PCR扩增法检测了二斑叶螨Tetranychus urticae Koch 12个地理种群（辽宁兴城、河北昌黎、湖南长沙、河南郑州、江苏徐州、甘肃兰州、云南昆明、山东泰安、新疆石河子、宁夏银川、陕西眉县和上海）感染Wolbachia的状况。结果表明,12个地理种群全部感染了Wolbachia。克隆分离得到了它们的wsp基因序列,在GenBank的登录号依此为： AY585714、 AY585712、AY785376、AY785375、AY585713、AY785374、AY785371、AY712954、AY712955、AY785372、AY785377和AY785373。对序列进行系统发育分析,发现它们全部与B大组Ori组的Wolbachia株系十分相近或完全相同。总体来看,它们的聚类关系基本上呈平行交叉分布,在不同种群之间没有构成明显的地理分布聚集性。     

Gene flow among Tetranychus urticae (Acari: Tetranychidae) populations in Greece

Polymorphism of four enzymatic loci has been examined in 27 populations of Tetranychus urticae in relation to their geographical distribution and to two ecological parameters: open field vs. greenhouse habitats, and species of the colonized host plant. Genetic differentiation was significantly correlated to geographical distance in both types of habitat. Mite density and distribution of infested plants appear to be important factors for the population structure of T. urticae . In open field, T. urticae specimens from citrus trees were genetically more similar to other 'citrus' samples collected in different localities than they were from mites collected in the same locality on other plant species.     

Local variation in diapause characteristics of Tetranychus urticae Koch (Acarina: Tetranychidae)

Summary Diapause characteristics of nine local populations of Tetranychus urticae Koch (Acarina: Tetranychidae) occurring on rose in central Japan were investigated. The percentage of females that entered diapause at 18° C and 9L: 15D photoperiod ranged between 0 and 77%. In addition, both photoperiodic and temperature responses varied widely among these populations. We suggest that temperature response might be a better trait than photoperiodic response for selection among local populations. Females in diapause survived –24° C better than those that were not. However, no difference in cold hardiness among these populations was found. The mite responded rapidly to artificial selection for low and high diapause percentage. The response was asymmetrical, being easier in the direction of low diapause percentage. Reciprocal and back crosses using selected low and high diapause percentage cultures showed that the genetic control of diapause was complicated. Neither the lowdiapause nor the high-diapause trait dominated over the other. However, the low-diapause trait seemed to be stronger. The results of this study suggest that variation in diapause response is maintained by (1) adaptation to local environments and (2) the complexity of the genetics of diapause. Such variation may provide the genetic raw material for natural selection and is a prerequisite for the extension of ecological range.     

Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece

We investigated the mechanisms conferring resistance to methyl-parathion (44-fold) and to methomyl (8-fold) in Tetranychus urticae from Greece by studying the effect of synergists on the resistance and the kinetic characteristics of various enzymes in a resistant strain (RLAB) and a susceptible reference strain (SAMB). It is shown that S,S,S-tributyl phosphorotrithioate, a synergist that inhibits esterases and glutathione S-transferases, and piperonyl butoxide, a synergist that inhibits cytochrome P450 mediated monooxygenases, did not affect the level of methyl-parathion or methomyl resistance in RLAB and that resistance ratios to both insecticides did not change significantly in the presence of either synergist. Isoelectric focusing of esterase allozymes on single mites revealed no differences in staining intensity and glutathione S-transferase activity was not significantly different in the two strains. The activity of two cytochrome P450 monooxygenase groups was compared. No significant difference of 7-ethoxyresorufin-O-diethylase activity was observed between strains that were two-fold higher in RLAB than in SAMB. The kinetic characteristics of acetylcholinesterase, the target enzyme of organophosphates and carbamates, revealed that acetylcholinesterase in RLAB was less sensitive to inhibition by paraoxon and methomyl in comparison with SAMB. I(50), the inhibitor concentration inducing 50% decrease of acetylcholinesterase activity was greater (119- and 50-fold with paraoxon and methomyl, respectively) and the bimolecular constant k(i) was lower (39- and 47-fold with paraoxon and methomyl, respectively) in RLAB compared to SAMB.     

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.     

Evaluation of Vigna angularis (Willd.) Ohwi and Ohashi accessions for resistance to Tetranychus urticae Koch (Acari:Tetranychidae)

Under laboratory conditions 88 azuki bean and three rice bean accessions were evaluated with a leaf-disc technique to select for those which expressed an antibiotic response to the TSSM ovipositional rate. Nine of the most promising accessions determined in the screening and Erimo, a commercial cultivar used as a control, were re-evaluated to determine the ovipositional rate and fecundity. Statistically significant differences between accessions were found: some of the accessions exhibited a reduced TSSM ovipositional rate and fecundity when compared with the susceptible control. The accessions ADZU279 and ADZU514 from Japan and ADZU577 from Ivory Coast demonstrated strong inhibition to TSSM fecundity. These three accessions may possess inherent potential in a breeding programme designed to enhance resistance to T. urticae.