Resistance Selection Against Abamectin in Tetranychus cinnabarinus(Boisduval) and Changes in Its Detoxification Enzyme Activity

The susceptible strain of Tetranychus cinnabarinus (Boisduval) was collected from the cassava germplasm resource garden of Guangxi South Subtropical Agricultural Science Research Institute and raised for 15 generations,during which resistance selection against avermectin was performed through the indoor spraying of avermectin,thereby obtaining an avermectin-resistance strain (Ab-R) with a resistance ratio of 3.25.After the indoor resistance selection,the determination of detoxification enzyme activity in the Ab-R and SS strains showed that the specific activity of carboxylesterases (CarE),glutathione-s-transferases (GSTs) and multi-functional oxidase (MFO) in Ab-R15 was 1.27,1.69 and 1.92 times of that in SS,respectively.In addition,the specific activity of MFO in Ab-R5,Ab-R10 and Ab-R15 was significantly different from that in SS;there was no significant difference in the specific activity of CarE compared with SS when screened to the F10 generation;and when screened to the F5 generation,there was no significant difference in the specific activity of GSTs compared with SS.The results showed that the significant increase in MFO activity was an important cause of resistance to avermectin in T.cinnabarinus,and CarE and GSTs were also involved in the formation of avermectin-resistant strains.

Identification of the key chitinase genes in Tetranychus cinnabarinus (Boisduval) based on the expression and sequence characteristic analysis

Chitin is an important content in the exoskeletons of arthropods, and its hydrolyzation is catalyzed by chitinases during the process of molting, thus, the chitinases are considered as ideal target to interfere the growth of arthropods. This study intends to clarify the characteristic of the chitinases during the development of Tetranychus cinnabarinus, and screen out important genes as potential control targets. The results showed that the total enzyme concentration of chitinases was significantly higher in larva, the first and second nymph than that in egg and adult. Base on the transcriptome data, six unigenes encoding chitinases were identified and their expression patterns in different developmental stages were detected. The expressions of TcCHIT1 and TcCHITIO showed high abundance during the molting process and their expression change during the developmental stages was consistent with the enzyme concentration. The full-length of these two genes were further cloned, and the structural characteristics of their proteins were analyzed by constructing the three-dimensional structure model. The results provide basic information to understand the characteristic of chitinases in T. cinnabarinus and might be considered as target for control.

Biochemical Evidences for Scopoletin Inhibits Ca^(2+)-ATPase Activity in the Carmine Spider Mite,Tetranychus cinnabarinus(Boisduval)

[Objective] This study aimed to investigate the acaricidal effect of scopoletin, and provide the biochemical evidences of scopoletin influences Ca2 +-ATPase activity and gene expressions in the Carmine Spider Mite, Tetranychus cinnabarinus.[Method] The acaricidal effects of scopoletin were investigated by slip-dip method.Exposeed to different concentrations of scopoletin(0.16-2.5 mg/ml), Ca2+-ATPase activity in vivo and protein contents were investigated. For assessing the in vitro effect, Ca2+-ATPase enzyme(200 μl) prepared from normal mites were incubated with different concentrations of scopoletin reagents. [Result] Scopoletin exhibited significant inhibitory effect on Ca2+-ATPase activity both in vivo and in vitro, and resulted in increased protein contents; kinetic analysis showed that the catalytic capability of Ca2+-ATPase was significantly reduced by scopoletin. [Conclusion] Scopoletin exhibits a significant inhibitory effect on Ca2 +- ATPase, and its acaricidal effect against T. cinnabarinus might be due to the direct inhibition of Ca2+-ATPase.

Joint Acaricidal Action of Diafenthiuron and Fenbutatin-oxide Against Tetranychus cinnabarinus(Boisduval)(Acari: Tetranychidae)

[Objective]This study was conducted to obtain a new high-efficiency acaricide mixture.[Method]The synergism acaricidal of diafenthiuron and fenbutatin-oxide against carmine spider mite was investigated,and the optimal ratio of both acaricides were determined by means of co-toxicity factors and co-toxicity coefficients(CTC).[Result]At 24 h post-treatment,the LC50values of diafenthiuron and fenbutatin-oxide against T.cinnabarinus female adults were 154.67 and 93.26 mg/L,respectively,and the synergistic mass ratios of diafenthiuron and fenbutatin-oxide ranged from 1:0.06 to 1:0.87,and the optimal diafenthiuron-to-fenbutatin-oxide ratio was 1:0.39 with the LC50and CTC of 67.87 mg/L and 188.93(CTC significantly above 100 strongly indicated synergism),respectively,and the toxicity relative to diafenthiuron and fenbutatin-oxide was 2.28 and 1.37 times,respectively.[Conclusion]The joint acaricidal action of diafenthiuron and fenbutatin-oxide strongly indicates synergism.These findings proved scientific theoretical basis for mixing diafenthiuron and fenbutatin-oxide to control spider mites and developing a new synergisic acaricide.

桑树(Morus alba)朱砂叶螨(Tetranychus cinnabarinus)实验种群的温度效应

采用生命表分析、生存分析、“王-兰-丁”模型及线性模型等分析方法，对15-28℃温区的5个恒温处理朱砂叶螨（Tetranychus cinnabarinus）实验种群进行了系统研究。结果表明，此温区内，种群在生殖、发育和生存3方面，有明显的温度效应：内禀增长力、周限增长力、净增殖力和平均日产卵量、世代平均周期、及种群倍增时间的倒数呈线性增长；而平均寿命、最大死亡年龄，随温度升高而递减；性比和实际产卵天数对温度不敏感。种群在生殖、发育和生存三者之间，采取了较为“折衷”的策略：8℃为发育临界点；13℃左右为生殖和种群增长临界温度；22℃左右为生殖和种群增长最适温度；30℃左右为发育最适温度。

中草药提取物对朱砂叶螨(Tetranychus cinnabarinus)生物活性的研究

在室内通过玻片浸渍法测定了8种供试中草药的3种提取物对朱砂叶螨成螨的触杀活性。结果表明:当提取物浓度达到2mg/ml时,旋覆花石油醚提取物,蓖麻子甲醇提取物,石楠叶石油醚提取物的杀螨活性较高。根据杀螨活性初筛结果,选取这3种触杀活性较高的中草药提取物进行毒力活性测定结果为:旋覆花石油醚提取物的LC50值为(1.1843±0.0868)mg/ml、蓖麻子甲醇提取物的LC50值为(1.2111±0.1616)mg/ml、石楠叶石油醚提取物的LC50值为(1.3731±0.0898)mg/ml。由此,这3种中草药的提取物均具有较好的杀螨生物活性。

普通钝绥螨(Amblyseius vulgaris)对朱砂叶螨(Tetranychus cinnabarinus)捕食作用的研究

研究了普通钝绥螨(Amblyseius vulgaris)对朱砂叶螨(Tetranychus cinnabarinus)不同螨态的选择捕食作用、功能反应和数量反应。结果表明,普通钝绥螨喜欢捕食朱砂叶螨的幼螨,对猎物不同螨态的功能反应均表现为Holling Ⅱ型曲线,但其捕食效率因猎物螨态和环境温度不同而有差异。猎物螨的密度对普通钝绥螨的繁殖有明显影响。

华北地区31种植物粗提物对朱砂叶螨Tetranychus cinnabarinus的触杀活性

通过华北地区31种植物粗提物对朱砂叶螨(Tetranychus cinnabarinus)成螨触杀活性的测定,发现粗提物浓度为1 mg/mL时,柠檬薄荷(Monarda didyma)、胡椒薄荷(Mentha piperita)、木本香薷(Elsholtzia staunt-oni)、猪牙皂(Gleditsia sinensis)、乌头(Aconitum carmichaeli)、鸦胆子(Brucea javanica)、龙葵(Solanum nig-rum)的石油醚粗提物,紫穗槐(Amorpha fruticosa)、艾蒿(Artemesia annua)、银杏叶(Ginkgo biloba)、老鹳草(Geranium pseudosibiricum)的氯仿粗提物,百部(Radix Stemonae)的甲醇粗提物均有明显的杀螨活性,成螨校正死亡率均在80%以上。其中致死率最高的为木本香薷的石油醚粗提物,达到100%;鬼针草(Bidens parviflo-ra)的氯仿粗提物对螨的致死率最低,为2.01%。对成螨触杀死亡率进行Duncan’s检验,发现所试植物中部分植物的杀螨活性在5%水平上差异显著,唇形科(Labiatae)植物的石油醚粗提物触杀效果明显。大部分供试植物的甲醇粗提物对害螨的触杀作用相对较差。用唇形科3种薄荷石油醚粗提物对若螨进行进一步的毒力测定,结果表明毒性最强的为柠檬薄荷粗提物,其LC50值为0.429 4 mg/mL。

朱砂叶螨Tetranychus cinnalarinus(Boisduval)蛋白质含量测定中的两种方法比较

本试验以朱砂叶螨为材料，对动物源蛋白质含量测定中的Folin酚法和考马斯亮蓝G－250法进行了比较，结果表明：Folin酚法和考马斯亮蓝法间并无显著性差异，但前者对蛋白质的敏感度高，且操作简单，建议在朱砂叶螨蛋白质测定中使用。

棉花苗期棉叶螨(Tetranychus cinnabarinus)种群密度估计方法

通过对23块棉田大样本调查数据的分析,提出了基于螨株率(Pi)、红叶率(Pj)估计棉叶螨种群密度的数学模型。并利用 Kuno(1969) ,Green(1970) ,Willson(1983) 模型建立了棉叶螨序贯抽样式.

Studies on Acaricidal Bioactivities of Artemisia annua L.Extracts Against Tetranychus cinnabarinus Bois.(Acari:Tetranychidae)

The aim of this study was to determine the best extraction technique, the most suitable solvent, the optimal plant parts, and the acaricidal activities of Artemisia annua L. The petroleum ether （30-60℃）, petroleum ether （60-90℃）, ethanol, acetone, and water parallel and sequenced extracts were obtained from the leaves, stems and roots of different period of A. annua L. in April, May, June, July and September respectively. And then the acaricidal bioactivities against Tetranychus cinnabarinus of all extracts were determined by the slide-capillary method in the laboratory. The results indicated that the acaricidal bioactivities elevated as the development of A. annua plant at the concentration of 5 mg mL-L The general tendency exhibited the sequence of July 〉 June 〉 May 〉 April, but September decreased comparing to July. However, the most effective extracts in five months were all acetone parallel extract of A. annua leaf, and the corrected mortalities treated after 48 h ranged from 74 to 100%. The median lethal concentrations （LC50） against T. cinnabarinus of acetone parallel extracts ofA. annua leaves in September, July, June, May and April were 0.5986, 0.4341, 0.8376, 0.9443 and 1.3817 mg mL^-1, respectively, treated after 48 h. The 13 groups were isolated from acetone extracts ofA. annua leaves in July by column chromatography, both the 1 lth and 12th groups exhibited strong bioactivities. The median lethal concentrations of the 1 lth and 12th groups against T. cinnabarinus were 0.3683 and 0.1586 mg mL^-1, respectively. The acetone parallel extract ofA. annua leaf in July was the most toxic to T. cinnabarinus and the corrected mortality was 100% after 48 h. The acetone parallel extract of the 1 lth and 12th groupswere the most active components, acted as the emphases in further study.

Resistance Selection of Tetranychus cinnabarinus to Three Acaricides and Its Management Strategy

The effects of retarding resistance evolution of mixing and sequencing acaricides were studied based on the resistance selection of three acaricides to Tetranychus cinnabarinus in the laboratory. Tetrany-chus cinnabarinus was continuously selected with fenpropathrin, abamectin and pyridaban for 28, 30 and 31 generations, respectively, and resistance factor increased to 65.6, 7.2 and 1.2 time, accordingly. Results of cross-resistance determination suggested that there were no cross-resistance existed among the three acaricides. Analysis of resistance selection and estimation of realized heritability (h2) of resistance to three single and two mixed acaricides showed that a mixture of fenpropathrin+abamectin would slow resistance development compared with a sequence of fenpropathrin followed by abamectin while the mixture of pyridaben+abamectin and the sequence of pyridaben followed by abamectin would effectively retard resistance evolution.

Resistance Selection of Tetranychus cinnabarinus to Fenpropathrin and Genetic Analysis

The resistant inheritance mode of Tetranychus cinnabarinus to fenpropathrin were studied based on the resistance selection in laboratory. After 40 generations selection, T. cinnabarinus developed 68.5-fold resistance to fenpropathrin. The methods of cross and back-cross between resistant （R） and sensitive （S） strains were used for exploring the inheritance mode of the resistance of this mite to fenpropathrin. The log （Lc）-p equation of F1 got from obverse （SR） and reverse （RS） crosses was intermediate between S and R and inclined to S, the dominant indices DSR and DRS were -0.83 and -0.29, respectively, which indicated that the resistance is controlled by the incompletely recessive gene; the 95% confidence limit of this two dominant indices do not superpose showing DSR and DRS have significant difference and the maternal or external karyon effect may be existed in resistance inheritance; the back-cross results of heterozygote F1 with its parents suggested that the separation of progenies F2 （SR♀× S ♂ and RS ♀ × R ♂） was consistent with Mendel＇s single gene inheritance model.

The influence of Tetranychus cinnabarinus-induced plant defense responses on Aphis gossypii development

Carmine spider mites （Tetranychus cinnabarinus） and cotton aphids （Aphis gossypii） are both serious pests of cotton, and cause reductions in yields of this key agricultural crop. In order to gain insights into how plant defense responses induced by one herbivore species affect the behavior and performance of another, we examined how infestation with T. cinnabarinus influences the development of A. gossypfi using cotton as a model. In this study, we measured the activities of several important biochemical markers and secondary metabolites in the leaves of cotton seedlings responding to infestation by T. cinnabarinus. Furthermore, the influences of T. cinnabarinus infestation on the development ofA. gossypfi in cotton were also examined. Our data showed that the activities of several key defense enzymes, including phenylalanine ammonia-lyase （PAL）, peroxidase （POD）, lipoxygenase （LOX）, and polyphenol oxidase （PPO）, were substantially increased in cotton seedlings responding to spider mite infestation. Further, the contents of gossypol and condensed tannins, key defensive compounds, were significantly enhanced in leaves of cotton seedlings following T. cinnabarinus infestation. Moreover, the T. cinnabarinus-induced production of defense enzymes and secondary metabolites was correlated with infestation density. The developmental periods of A. gossypii on cotton seedling leaves infested with T. cinnabarinus at densities of 10 and 15 individuals cm-2 were 1.16 and 1.18 times that of control, respectively. Meanwhile, the mean relative growth rates of A. gossypfi on cotton leaves infested with T. cinnabarinus at densities of 8, 10 and 15 individuals cm-2 were significantly reduced. Therefore, these data suggested that the developmental periods of A. gossypfi were significantly lengthened and the mean relative growth rates were markedly reduced when cotton aphids were reared on plants infested with high densities of spider mites. This research sheds light on the role that inducible defense responses played in plant-mediated interspecific interactions between T. cinnabarinus and A. gossypfi.

The dynamic changes of genes revealed that persistently overexpressed genes drive the evolution of cyflumetofen resistance in Tetranychus cinnabarinus

Changes in gene expression are associated with the evolution of pesticide resis-tance in arthropods.In this study,transcriptome sequencing was performed in 3 different resistance levels(low,L;medium,M;and high,H)of cyflumetofen-resistant strain(YN-CyR).A total of 1685 genes,including 97 detoxification enzyme genes,were upregulated in all 3 stages,of which 192 genes,including 11 detoxification enzyme genes,showed a continuous increase in expression level with resistance development(L to H).RNA in-terference experiments showed that overexpression of 7 genes(CYP392A1,TcGSTd05,CCE06,CYP389A1,TcGSTz01,CCE59,and CYP389C2)is involved in the development of cyflumetofen resistance in Tetranychus cinnabarinus.The recombinant CYP392A1 can effectively metabolize cyflumetofen,while CCE06 can bind and sequester cyflumetofen in vitro.We compared 2 methods for rapid screening of resistance molecular markers,in-cluding short-term induction and 1-time high-dose selection.Two detoxification enzyme genes were upregulated in the field susceptible strain(YN-S)by induction with 20%lethal concentration(LC2o)of cyflumetofen.However,16 detoxification enzyme genes were up-regulated by 1-time selection with LCso of cyflumetofen.Interestingly,the 16 genes were overexpressed in all 3 resistance stages.These results indicated that 1685 genes that were upregulated at the L stage constituted the basis of cyflumetofen resistance,of which 192 genes in which upregulation continued to increase were the main driving force for the de-velopment of resistance.Moreover,the 1-time high-dose selection is an efficient way to rapidly obtain the resistance-related genes that can aid in the development of resistance markers and resistance management in mites.

不同木薯品种对朱砂叶螨的抗性及生理基础研究

【目的】探究不同木薯品种对朱砂叶螨的抗性及其生理基础,为抗朱砂叶螨木薯品种的选育及应用推广提供参考。【方法】试验在木薯朱砂叶螨高发区进行,以6个木薯品种(桂热4号、华南205、桂垦26、华南124、南植199和西选09)为试验材料,试验期间朱砂叶螨自然危害。在朱砂叶螨危害高峰期过后,对不同木薯品种进行螨害情况调查和鉴定,计算叶片受害率、叶片脱落率和螨害指数;同时采集螨害叶片与健康叶片测定可溶性糖、可溶性蛋白、总酚、黄酮和单宁含量及过氧化物酶(POD)、过氧化氢酶(CAT)活性,比较分析不同木薯品种螨害情况与上述指标变化的关系。【结果】不同木薯品种对朱砂叶螨的抗性有明显差异,螨害指数为61.86%~91.34%,6个品种螨害指数排序为南植199>华南205>桂热4号(S)>华南124>桂垦26>西选09;南植199和华南205对朱砂叶满的抗性表现为高感,桂热4号、华南124和桂垦26号表现为感,西选09表现为中抗。西选09受螨害最轻,其叶片可溶性糖含量最低,总酚、黄酮和单宁含量及POD、CAT活性最高;南植199受螨害最重,其叶片可溶性糖含量最高,总酚、黄酮和单宁含量及POD、CAT活性最低,2个品种上述指示的差异均达极显著水平(P<0.01)。【结论】不同木薯品种对朱砂叶螨的抗性不同;叶片可溶性糖、总酚、黄酮和单宁含量及POD、CAT活性与木薯抗朱砂叶螨密切相关,可作为木薯抗朱砂叶螨评价的生理指标。

朱砂叶螨β-N-乙酰己糖胺酶基因TecHEX3的克隆及在蜕皮发育中的作用

【目的】本研究旨在探明朱砂叶螨Tetranychus cinnabarinus几丁质降解通路中的关键酶β-N-乙酰己糖胺酶基因(TecHEX3)在螨蜕皮发育中的功能,为开发新型安全的防治害螨生物农药奠定基础。【方法】利用PCR克隆朱砂叶螨TecHEX3,并进行生物信息学分析。利用RT-qPCR检测TecHEX3在朱砂叶螨不同发育阶段(卵、幼螨、若螨和成螨)以及饲喂蜕皮激素20-羟基蜕皮酮(20-hydroxyecdysone,20E)的若螨中的表达量。通过饲喂法进行朱砂叶螨雌成螨和若螨TecHEX3的RNAi,分析沉默效率,统计并观察朱砂叶螨若螨的死亡率和致死表型。【结果】成功克隆了朱砂叶螨TecHEX3(GenBank登录号:OR413561),其编码蛋白具有典型的20糖苷水解酶家族结构特征;系统发育树表明TecHEX3与二斑叶螨T.urticae TuHEX3的亲缘性最高且均属于β-N-乙酰己糖胺酶Ⅳ型。TecHEX3在朱砂叶螨不同发育阶段都有表达,在成螨期表达量最高;饲喂500 ng/μL的20E对TecHEX3的表达有最好的诱导效果。RNAi结果表明饲喂ds TecHEX3时朱砂叶螨若螨TecHEX3表达量较对照组(饲喂ds EGFP)显著下调81%;沉默TecHEX3导致朱砂叶螨蜕皮困难或蜕皮后形态异常,致死率达40.58%。【结论】TecHEX3参与朱砂叶螨几丁质降解过程,并在朱砂叶螨的蜕皮发育过程中发挥重要作用。

新型含丁烯酸内酯基三氟乙基硫醚(亚砜)类化合物的设计、合成与杀螨活性

为寻找高活性杀螨化合物,以sulfiflumin为先导化合物,保留4-甲基-2-氟-5-(2,2,2-三氟乙硫基)苯胺活性基团,通过其与氯乙酰乙酸乙酯成环合成三氟乙基硫醚丁烯酸内酯的核心骨架C1,再对核心骨架进行氧化、氯代或溴代,以及将内酯环改造为硫内酯环及二硫代酮酯环得到C2~C87个化合物,采用1H NMR、^(13)C NMR和HRMS手段对其结构进行了表征。生物活性测试数据表明,目标化合物在100 mg/L质量浓度下具有一定的杀螨活性,尤其是对朱砂叶螨Tetranychus cinnabarinus卵孵化的抑制活性较高(校正孵化抑制率67%~93%);此外,化合物C3对二斑叶螨Tetranychus urticae成虫的的校正死亡率达100%。该研究可为丁烯酸内酯基三氟乙基硫醚(亚砜)衍生物和氟吡呋喃酮衍生物的设计和改造提供研究思路,对探究该类化合物的生物活性具有参考意义。

5种药剂对朱砂叶螨杀螨活性的比较及增效组合筛选

为了筛选对朱砂叶螨具有较高杀螨活性的药剂,通过浸叶法测定和比较了螺虫乙酯等5种药剂对朱砂叶螨的毒力并两两组合筛选防治效果较好的增效混剂。结果表明:以常用杀螨剂哒螨灵作为标准杀虫剂,螺虫乙酯等4种杀虫剂对朱砂叶螨的24 h和48 h毒力均高于哒螨灵,其中乙螨唑的毒力最高,其次为多杀菌素,第3和第4分别是螺虫乙酯和联苯肼酯;在不同组合增效复配剂的筛选试验中,螺虫乙酯与乙螨唑的两种配比(3.2∶1和1∶1.25)混剂的共毒系数分别为359.33和419.98,哒螨灵与联苯肼酯配比5.6∶1的混剂的共毒系数为221.98,多杀菌素与乙螨唑配比4.8∶1的共毒系数为550.90,增效作用最明显;相比于单剂,4种混配组合均有明显的增效作用,具有一定的开发应用前景。

Molecular characterization of two novel esterase genes from carmine spider mite, Tetranychus cinnabarinus （Acarina： Tetranychidae）

Two novel esterase complementary DNAs were identified and cloned from the insecticide-susceptible strain of Tetranychus cinnabarinus （Boisduval） （Acarina： Tetrany- chidae）, which were designated as TCE1 and TCE2, respectively. The cDNA of TCE1 gene contained an open reading frame （ORF） of 1701 bp encoding 567 amino acids, and a predicted molecular weight of 62.75 kDa, the cDNA of TCE2 contained an ORF of 1680 bp encoding 560 amino acids, and a predicted molecular weight of 63.14 kDa. TCE1 and TCE2 were submitted to GenBank, accession number EU 130461 and EU 130462. The well-conserved sequence motif, GXSXG, used as a signature pattern in the esterase fam- ily are present in both TCE1 and TCE2 （GQSAG in TCE1, whereas GESAG in TCE2）, indicating that these two genes are predicted to be esterases. Comparison of the deduced amino acid sequence with the published mite esterase sequence coming from Boophilus microplus showed that TCE1 shares 33.98% identity and TCE2 shares 33.46% identity. TCE1 and TCE2 share 46.4% identity. Quantitative real-time polymerase chain reaction revealed that expression level of the TCE2 gene was relatively higher than that of the TCE1 in all instars examined except the protonymph, and the expression level of these two esterase genes in adults of T. cinnabarinus was significantly higher than that in any other instars, respectively. T. cinnabarinus is an important agricultural mite pest and esterases are important in the metabolisms of insects and mites; the genomic information obtained in this study will contribute to esterase molecular biological study on mite pest species.