Expression of Ace-paralogous acetylcholinesterase of Culex tritaeniorhynchus with an amino acid substitution conferring insecticide insensitivity in baculovirus-insect cell system

In Ace paralogous acetylcholinesterase (AP-AChE) of Culex tritaeniorhynchus, an amino acid substitution, Phe455Trp, is accompanied by the insecticide insensitivity. To confirm the responsibility of the substitution to the insensitivity, AP-AChE cDNA with and without a Phe455Trp substitution and Ace orthologous AChE (AO-AChE) cDNA were expressed in a baculovirus-insect cell system and the biochemical properties of AChEs (AP-CxTI, AP-CxTS, and AO-CxT, respectively) determined. AP-CxTI which has the same level of affinity to ACh, the natural substrate, showed a drastic decline in affinity to the artificial substrates composed of a longer moiety. The sensitivity of AP-CxTI to inhibitors was extremely reduced when compared with AP-CxTS. The insensitivity to tested organophosphates was greater than to monomethyl carbamates. AO-AChE showed similar substrate specificity and a slightly higher sensitivity to inhibitors when compared with AP-CxTS. Taking the position of Phe455Trp in the acyl pocket of the active site into account, the dimensions of the acyl pocket appear to became smaller by the substitution and insensitive to inhibitors.     

Amino acid substitutions conferring insecticide insensitivity in Ace-paralogous acetylcholinesterase

Since insecticide insensitivity of acetylcholinesterase (AChE) was, found about 40 years ago, a cause of the resistance to organophosphates in the spider mite, more than 30 insect and Acarus species have added to the instance. Based on the 3-dimensional analysis of Torpedo AChE structure and sequencing of Drosophila AChE gene (Ace), amino acid substitutions conferring the insensitivity have been found in Drosophila melanogaster. However, no amino acid substitution responsible for the AChE insensitivity had been found in insects and Acari except Brachicera flies until the second type of AChE paralogous to Ace was discovered in Schizaphis graminus and Anopheles gambiae. Sequencing of Ace-paralogous AChE cDNAs has been followed in insect species of various orders. Now, various amino acid substitutions are found and correspond to different biochemical properties of insensitive AChEs in relation to the function of substituted amino acids in the 3-dimensional structure. Existence of two AChE genes raises questions about differentiation of the two genes, site of gene expression, and function of each enzyme.     

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene (Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC₅₀) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance (r² = 0.712) compared to the F439W mutation (r² = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced (r² = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.     

Spider mite control and resistance management: does a genome help?

The complete genome of the two‐spotted spider mite, Tetranychus urticae, has been reported. This is the first sequenced genome of a highly polyphagous and resistant agricultural pest. The question as to what the genome offers the community working on spider mite control is addressed. Copyright © 2012 Society of Chemical Industry     

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates

BACKROUND: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos‐methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos‐methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR‐RLFP assay developed in this study. Copyright © 2009 Society of Chemical Industry     

用杂交方法鉴定我国三种常见叶螨

本文报道通过杂交试验,连代隔离饲养观察和阳具端锤的形状区别将北京花卉上发现的绿色种叶螨、红色种叶螨和菜豆上发现的红色种叶螨作了鉴定。绿色种与两红色种之间均存在着生殖隔离,杂交产生不育后代或不能交配,因此它们是各自独立的种。绿色叶螨应是二斑叶螨(Tetranychus urticae),连代隔离饲养证明雌成螨始终体呈绿色(滞育型除外),其遗传性是稳定的,这可以作为二斑叶螨和其近似种相区别的直观特征。从阳具端锤的形状差别可以区分两种红色螨分别是硃砂叶螨(T.cinnabarinus)(采自花卉)和截形叶螨(T.truncatus)(采自菜豆)。据此,作者对国内目前通行的有关棉叶螨(T.urticae)和红叶螨(T.cinnabarinus)的学名和中文名称的混乱问题,进行了讨论。认为棉叶螨不是T.urticae,红叶螨不一定全是T.cinnabarinus。     

Spiromesifen toxicity to the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis> and selectivity to the predator <Emphasis Type="Italic">Neoseiulus californicus</Emphasis>

Tetranychus urticae Koch (Acari: Tetranychidae) is a major pest of several agricultural crops and Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) is an important natural enemy of this pest mite. The objective of the study was to evaluate the effect of spiromesifen on the development and reproduction of T. urticae, and to assess the influence of spiromesifen and other acaricides on the population growth rates of the spider mite and its predator, N. californicus. Toxicity tests with spiromesifen at different life stages of T. urticae indicated that eggs less than 72 h old were more sensitive than other development stages. The oviposition rate of T. urticae was significantly affected by spiromesifen. Experiments on the effects of spiromesifen on the growth rates of T. urticae and N. californicus indicated that spiromesifen is innocuous to the predaceous mite but highly toxic to the spider mite, leading to population suppression in 10 days. Fenpropathrin, acephate and neem oil were not harmful to N. californicus, but were not so efficient as spiromesifen in controlling T. urticae, and had significantly less influence on the population growth rates of the spider mite. Among these three products, only neem oil caused significant reduction in the growth rate of T. urticae. Chlorfenapyr, abamectin, milbemectin and diafenthiuron significantly affected the population growth rates of T. urticae and N. californicus. Spiromesifen was the most promising acaricide for managing the two-spotted spider mite, when used in combination with N. californicus.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

棉叶螨的抗药性现状与治理策略

棉叶螨也称为棉红蜘蛛,属蛛形纲叶螨科,其种类繁多,分布范围广,世代周期短,是为害棉花的一类重要害螨。目前,用于防治棉叶螨的化学药剂主要是神经毒剂及呼吸抑制剂2大类,且棉叶螨对多数药剂产生了不同程度的抗性,以二斑叶螨Tetranychus urticae为首的植食性害螨已成为世界上抗药性最严重的节肢动物之一。美国路易斯安那州棉田二斑叶螨种群对阿维菌素产生了1 415倍抗性,而国内棉花上棉叶螨主要对有机磷类药剂产生了较强抗性,最高为467倍。棉叶螨产生抗药性的机制主要涉及靶标突变及解毒代谢增强,其中靶标突变主要涉及乙酰胆碱酯酶、电压门控钠离子通道和谷氨酸门控氯离子通道等;细胞色素P450单加氧酶、羧酸酯酶和谷胱甘肽S-转移酶等一种或多种解毒酶共同参与害螨对化学药剂的解毒代谢。该文主要从棉叶螨的种类及分布、用于防治棉叶螨的化学药剂、棉叶螨的抗药性现状、抗药性机制解析和抗药性治理策略5个方面进行阐述,提出因地制宜的抗药性治理策略,旨在为棉叶螨的田间防治提供指导。     

Chlorfenapyr resistance in the spider mite Tetranychus urticae: stability, cross-resistance and monitoring of resistance

The two-spotted spider mite, Tetranychus urticae Koch, is a key pest of many agricultural crops. Studies of stability of resistance, cross-resistance relationships and monitoring of chlorfenapyr resistance were carried out with T. urticae to provide basic information necessary to define effective acaricide resistance management strategies for this pest. Chlorfenapyr resistance was shown to be stable in the absence of selection pressure under laboratory conditions. The activities of seven different acaricides against chlorfenapyr-resistant and -susceptible strains of T. urticae were evaluated. The results indicated possible positive cross-resistance between chlorfenapyr and the acaricides abamectin, propargite and etoxazole. No cross-resistance was detected for the acaricides milbemectin, fenpyroximate and diafenthiuron. A possible negatively correlated cross-resistance was observed between chlorfenapyr and spiromesifen. The evaluation of 21 T. urticae populations from several crops in the States of São Paulo, Mato Grosso, Goiás, and Bahia, in Brazil, indicated that the susceptibility of mites to chlorfenapyr was variable, with percentages of resistant mites ranging from 0.0 to 86.5%. The highest resistance frequencies were observed in ornamental plants in the State of São Paulo. Some populations from cotton and papaya also presented high frequencies of chlorfenapyr resistance. This is the first report on chlorfenapyr resistance in T. urticae on cotton and papaya in Brazil. Strategies for the management of acaricide resistance are discussed.     

Synergistic effect of Beauveria bassiana and spirodiclofen on the two-spotted spider mite (Tetranychus urticae)

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important pests in agriculture. Due to its short life cycle and high reproductive rate, this pest has become resistant to most acaricides. Therefore, finding alternative control strategies for this damaging pest is necessary. Application of microbiological agents with minimum concentrations of acaricides is a very suitable tactic in integrated pest management. In the present study, lethal effects of a chemical acaricide – spirodiclofen, and an Iranian strain of the entomopathogenic fungus Beauveria bassiana, were evaluated on different life stages of the two-spotted spider mite on two host plants, bean and cucumber. In the next step, compatibility of acaricide with the fungus was studied regarding conidial germination and vegetative growth of the fungus in the presence of different concentrations of the acaricide. Finally, the acaricide at rates of LC₂₅ with the fungus at LC₅₀ were sprayed on adult female mites. The results showed that immature stages of T. urticae were more susceptible to the acaricide whereas the adults were more susceptible to the fungus. Also, mites reared on cucumber were significantly more susceptible to the acaricide and fungus than those reared on bean. Spirodiclofen and the fungus had a synergistic effect; percentage of mortality for fungus infection was 48.6% and 53.5% on bean and cucumber, respectively, while it was 80.6% on bean and 84.5% on cucumber when the mite was sprayed with both the fungus and the acaricide.     

盐胁迫下交替呼吸对二斑叶螨侵染叶片及其间接防御的影响

为探索在遭受盐胁迫时二斑叶螨Tetranychus urticae Koch侵染叶片的交替呼吸途径的生理学功能,在实验室条件下研究了NaCl以及水杨基氧肟酸对二斑叶螨侵染叶片的间接防御反应,以及对过氧化氢含量和细胞死亡水平的影响。200 mmol/L NaCl胁迫没有明显影响二斑叶螨侵染叶片对智利小植绥螨Phytoseiulus persimilis的吸引作用,也没有提高二斑叶螨侵染叶片中过氧化氢含量和细胞死亡的水平,但显著提高了侵染叶片中交替呼吸途径的水平。200 mmol/L NaCl和1mmol/L水杨基氧肟酸的复合处理则显著降低了二斑叶螨侵染叶片对捕食螨的吸引作用,并提高了过氧化氢含量和细胞死亡的水平。研究表明,当二斑叶螨侵染叶片遭受盐胁迫时,交替呼吸途径不仅有助于维持植物的间接防御反应,而且缓解了植物体内的氧化压力和细胞死亡。     

Daily consumption and predation rate of different<Emphasis Type="Italic">Stethorus punctillum</Emphasis> instars feeding on<Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Laboratory experiments were conducted to evaluate the prey stage preference and the daily consumption of each stage of the coccinellid predatorStethorus punctillum Weise (Coleoptera: Coccinellidae) feeding on the two-spotted spider miteTetranychus urticae (Koch) (Acari: Tetranychidae). Groups of different life stages of the prey were offered (eggs, larvae, nymphs and adults). The prey preference varied with the stage ofS. punctillum. First larval instars had no significant preference among theT. urticae stages offered. Second larval instars consumed significantly more spider mite larvae in comparison with nymphs. In contrast, third larval instars indicated a strong preference for mite eggs. Significantly fewerT. urticae larvae were consumed by the fourth larval instars ofS. punctillum, in comparison with the three other mite stages. Finally, adult predators consumed significantly more mite eggs than the other stages offered. This preferential trend was similar for all adults tested, whether during the pre-oviposition or the oviposition period. http://www.phytoparasitica.org posting Feb. 17, 2004.     

Fitness costs associated with milbemectin resistance in the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite Tetranychus urticae Koch is one of the most important pests of a wide range of crops worldwide. Its control is still largely based on the use of acaricides. However, due to its short life-cycle, high fecundity and arrhenotokous reproduction, it is able to develop resistance to these compounds very rapidly. Preliminary studies for milbemectin resistance in T. urticae showed that, under laboratory conditions, such resistance was unstable in the absence of a selection pressure. The aim of this study was to evaluate the possible fitness costs associated with milbemectin resistance in T. urticae. Comparison of biological traits between resistant and susceptible strains indicated the occurrence of fitness costs associated with milbemectin resistance. The resistant strain showed a longer juvenile development period for females, lower fecundity and a higher proportion of males among the progeny. The figures for net reproductive rate (R ₀), intrinsic rate of natural increase (r _m) and finite rate of increase (λ) were higher in the susceptible strain. The instability of milbemectin resistance can be exploited in resistance management programmes.     

Synthesis of some new ether derivatives of hydroquinone and their ovicidal activity against the two-spotted spider mite (Tetranychus urticae Koch)

A new class of compounds derived from hydroquinone ethers showed high and predominantly ovicidal activity against the two-spotted spider mite, Tetranychus urticae Koch. The sequence of compounds synthesised and the factors which appeared to have contributed most to the discovery of these promising acaricides are presented.     

Biology and seasonal abundance of the two-spotted spider mite, Tetranychus urticae, on orchids and rose

Biology of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) was studied on five orchids and one rose species under laboratory conditions. The duration of developmental stages of this mite was the shortest on rose (7.17 ± 0.20 days) followed by Cymbidium (10.49 ± 1.50 days), Oncidium (11.60 ± 1.32 days), Dendrobium (12.67 ± 0.21 days), Phalaenopsis (14.47 ± 1.21 days) and Coelogyne (17.10 ± 1.29 days). The number of eggs/female laid was maximum on rose, whereas the minimum was on Phalaenopsis under the most suitable temperature (26 ± 2°C) and relative humidity (65 ± 5%) conditions. The five orchid farms (three conventionally sprayed and two unsprayed) in the northeastern region of India were surveyed during 2009 and 2010 for mite incidence. Twenty-three species and 11 hybrids of orchids were encountered as the most preferred host, 12 species as moderately preferred, and 28 species and one hybrid were found to be the least preferred orchid host for T. urticae. The mite’s incidence was recorded on orchids all year round, indicative of overlapping generations; however, two distinct population peaks were recorded during April and May. Subsequently its population declined at the start of the rainy season and remained at extremely low levels in the winter. Maximum and minimum temperatures had a significant positive correlation with mite incidence; daylight had a non-significant positive correlation; and rainfall and relative humidity had non-significant negative correlations with mite incidence. The results are discussed in relation to the most susceptible stage on orchids and suitable management practices for the two-spotted spider mite in India.     

大环内酯类新化合物天维菌素杀螨活性及田间防治效果

为明确新化合物天维菌素的杀螨活性,采用叶碟喷雾法测定了在室内天维菌素对朱砂叶螨Tetranychus cinnabarinus、柑橘红蜘蛛Panonychus citri及二斑叶螨Tetranychus urticae的毒力,并研究了其制剂对螨类害虫的田间防治效果。结果表明,天维菌素对朱砂叶螨若螨和雌成螨均具有较高的毒力,其LC50分别为0.011、0.11 mg(a.i.)/L,杀螨活性优于弥拜霉素、阿维菌素,但杀卵活性较差。田间试验中2%天维菌素微乳剂具有良好的速效性且具有一定的持效期,在有效成分8~40 mg/kg剂量下,对朱砂叶螨和二斑叶螨药剂处理14 d的防治效果分别为83.22%~92.77%和85.49%~92.64%,有效控制期在14 d以上;在有效成分75 mg/kg剂量下,对柑橘红蜘蛛防治效果最好,处理1、3、7、14 d的防治效果分别达96.53%、97.72%、98.05%和91.42%。表明天维菌素具有作为杀螨剂的潜力。     

Acaricide toxicity and resistance in larvae of different strains of Tetranychus urticae and Panonychus ulmi (Acari: Tetranychidae)

The toxicities of eight structurally different acaricidal compounds to six‐legged larvae (first motile stage) of three laboratory strains of the two‐spotted spider mite, Tetranychus urticae, and the European red mite, Panonychus ulmi, were evaluated following spray application. The larvae of five field‐derived strains of T urticae originating from France, Italy, Brazil, California and Florida were also tested for their susceptibilities to discriminating concentrations of several acaricides resulting in 95% mortality when applied to the organophosphate‐resistant laboratory reference strain WI. The spray bioassay used was robust and gave repeatable results with a wide range of acaricidal compounds, irrespective of their mode of action (ovo‐larvicides or primarily acting on motile life stages). Compounds tested were abamectin, azocyclotin, chlorpyrifos, clofentezine, deltamethrin, fenpyroximate, hexythiazox and pyridaben. Larvae of one of the laboratory strains of T urticae, AK, originally collected in Japan in 1996 and maintained without further selection pressure, exhibited 2000‐ and >4000‐fold resistance to the mitochondrial electron transport inhibitors pyridaben and fenpyroximate, respectively. Another strain of T urticae, AU, obtained from Australia and maintained in the laboratory under selection with hexythiazox and clofentezine since 1987 showed >770‐ and >1000‐fold resistance to clofentezine and hexythiazox, respectively. The same resistance pattern was observed against larvae of a laboratory strain of P ulmi, CE, also selected with hexythiazox. Larvae of one of the field‐derived strains of T urticae, BR, showed a lower susceptibility to a number of compounds, whilst the others were susceptible to all compounds except the organophosphates. © 2001 Society of Chemical Industry     

Effects of pesticides on mite predators (Acari: Phytoseiidae) and colonization of apple trees byTetranychus urticae

A 2-year survey of mite populations and pesticide use was carried out in Nova Scotia, Canada, in apple orchards where the two-spotted spider mite (Tetranychus urticae Koch) was the dominant phytophagous mite. Fungicides were the only class of pesticides that affected cumulative number ofT. urticae-days per leaf in tree canopies and colonization success — the ratio ofT. urticae-days to number of immigratingT. urticae caught in sticky bands on tree trunks. In 2000, increased numbers ofT. urticae-days in the tree canopy were associated with more frequent applications of ethylene bis-dithiocarbamate (EBDC) fungicides and with higher fungicide toxicity scores, which indicate cumulative level of suppression of the phytoseiid predatorTyphlodromus pyri Scheuten by all fungicide applications. Higher rates of colonization success were also associated with higher toxicity scores. EBDC’s applied in 2000 promotedT. urticae immigration as indicated by their counts on sticky bands. In 2000 and 2001, number ofT. pyri-days in the tree canopies was decreased by more frequent EBDC applications and by higher fungicide toxicity scores. Promotion ofT. urticae in tree canopies by EBDC’s was attributed to their toxicity toT. pyri. BothT. pyri and another phytoseiid,Amblyseius fallacis (Garman), were found in ground cover vegetation. Hence, increased immigration from the ground cover attributed to the toxicity of EBDC’s toT. pyri and, especially, toA. fallacis, which is a specialist predator of spider mites and an effective natural enemy ofT. urticae. http//www.phytoparasitica.org posting Sept. 13, 2006. Formerly, Atlantic Food and Horticulture Research Centre, now retired.     

Synthesis and acaricidal activity of phenylpiperazine derivatives

We synthesized 33 new phenylpiperazine derivatives and assessed their acaricidal activity. These derivatives were synthesized through sequential reactions consisting of the sulfonylation of 2-substituted 4-methylaniline with chlorosulfonic acid, reduction with red phosphorus and iodine, alkylation by alkyl halide, cyclization with bis(2-chloroethyl)amine hydrochloride, and N-substitution reaction of phenylpiperazines with various reagents. All phenylpiperazines synthesized were evaluated for acaricidal activity and their structure–activity relationships discussed, it was found that 4-substituted 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfanyl)phenyl]piperazine derivatives exhibited good acaricidal activity. Among them, 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfanyl)phenyl]-4-(trifluoromethylsulfonyl) piperazine showed the highest level of activity against Tetranychus urticae and provided a high level of activity against Tetranychus kanzawai and Panonychus citri. In addition, studies on the effect at various stages of T. urticae exhibited that this compound showed good activity against both adults and eggs.