舞毒蛾(Lymantria dispar)酚氧化酶的酶学特征及有机溶剂对其活性的影响

经35%饱和度硫酸铵分级沉淀,将舞毒蛾(Lymantria dispar)酚氧化酶部分纯化。测定该酶最适pH值为6.5,pH在6.5～7.5范围内酶保持稳定的活力。最适温度为35℃,当温度低于25℃时,酶具有稳定的活力。以L-多巴、邻苯二酚和焦性没食子酸为底物时,测定酚氧化酶对底物的专一性,结果表明其Km值分别为3.19、10.74和19.26 mmol.L-1。本实验还研究了有机溶剂对酶活力的影响,结果表明甲醇、乙醇、丙酮和二甲苯对舞毒蛾酚氧化酶都有持续的抑制作用,其IC50分别为0.450、.430、.41和0.13 mmol.L-1。

Control of Lymantria dispar L. by biological agents

The experiment on control of Lymantria dispar L by using different kinds ofbiological measures, including nuclear polyhedrosis virus (NPV) of Lymantria dispar L., BtMP-342,sex-attractant as well as botanical insecticide, was carried out in the forest regions of InnerMongolia in 2003. Two concentrations (2.632x10^6 PIB ? ml^(-1) and 2.632x10^7 PIB ?ml^(-1)) of Lymantria dispar L. NPV were sprayed on the 2rd-instar-larvae of L. dispar and 70% and77.8% control effect were obtained respectively. BtMP0-342 was applied to the 3rd- and 4th-instarlarvae and the control effect was around 80%. The sex-attractant provided by Canada Pacific ForestryResearch Center also showed a good result in trapping L. dispar adults. The self-produced botanicalinsecticide, which was extracted from a kind of poisonous plant distributed in Daxing'an Mountains,China, exhibited a good control result in controlling the larvae of L. dispar, and 82% mortalitywas observed when spraying primary liquid of the botanical insecticide on the 3rd-5th-instar-larvaein lab.

舞毒蛾Methuselah-like分子模拟及靶向小分子抑制剂研究

[目的]旨在通过虚拟筛选技术筛选Methuselah-like(Ldmthl1)蛋白潜在的小分子抑制剂,并通过分子动力学模拟、MM-PBSA及生物测定,探究潜在化合物对Ldmthl1蛋白的抑制能力,为针对舞毒蛾Ldmthl1研发新型靶向杀虫剂奠定理论基础。[方法]以舞毒蛾为研究对象,构建Ldmthl1同源模型,虚拟筛选Ldmthl1受体小分子抑制剂,并采用分子动力学和MM-PBSA计算结合自由能分析6种潜在小分子抑制剂与Ldmthl1受体的结合强度;通过生物测定分析6种潜在小分子抑制剂的毒力,同时将6种潜在小分子抑制剂与溴氰菊酯进行联合使用,探究6种潜在化合物的增效作用。[结果]Ldmthl1含7个跨膜结构,符合G蛋白偶联受体结构特点,评估后符合蛋白模型评估标准;分子对接获得20个候选化合物,根据结合方式及结合能确定6种小分子化合物为潜在的抑制剂;分子动力学模拟显示,6种潜在小分子抑制剂通过氢键和疏水作用力与Ldmthl1受体稳定结合;MM-PBSA计算结合自由能发现6种潜在小分子抑制剂与Ldmthl1受体结合紧密;致死浓度LC30溴氰菊酯与6种抑制剂按照1:1、1:2和2:1的比例联合使用饲喂舞毒蛾3龄幼虫,6种抑制剂与溴氰菊酯具有协同增效作用,且当混合比例为1:2时,增效作用最显著,但溴氰菊酯浓度过高时,导致抑制剂无增效作用。[结论]虚拟筛选得到6种可与Ldmthl1受体稳定结合且具有杀虫活性潜在小分子抑制剂,与溴氰菊酯联用后呈现增效作用,本研究可为研发Ldmthl1靶向新型杀虫剂奠定理论基础。

Influences of the Populus deltoids seedlings treated with exogenous methyl jasmonate on the growth and development of Lymantria dispar larvae

Lyantria dispar larvae were fed with the leaves of Populus deltoids seedlings exposed to methyl jasmonate （MeJA） for 24 h. The growth and development of the larvae were investigated, and phenolics contents in treated leaves including pyrocatechol, caffeic acid, coumarin, fernlic acid and benzoic acid were also surveyed by high-pressure liquid chromatography （HPLC）. Results indicated that approximate digestibility, efficiency of conversion of ingested food, efficiency of conversion of digested food, and weight of the larvae were inhibited obviously, especially from the sixth day, which may result from the increase of total phenolics contents in treated leaves. This result provides strong supports for MeJA acting as the airborne signal molecule between woody plants.

Molecular characterization and functional analysis of USP-1 by RNA interference in the Asian gypsy moth Lymantria dispar

Ecdysteroids play an important role in regulating diverse physiological processes in arthropods,such as molting,metamorphosis,reproduction and diapause.Ecdysteroids mediate the response by binding to a heterodimeric complex of two nuclear receptors:the ecdysone receptor and the ultraspiracle(USP).To investigate the role of USP in development of the Asian gypsy moth(Lymantria dispar),a USP cDNA was obtained from the transcriptome of L.dispar and verified by PCR.In-depth profiling of transcript levels of L.dispar USP-1(LdUSP-1)at different developmental stages and over time in thirdinstar larvae and different tissues isolated during the thirdinstar stage of L.dispar was then carried out.Transcript levels of LdUSP-1 were relatively high before 72 h in the third-instar larvae after ecdysis and in the adult male.The function of LdUSP-1 in molting was analyzed by knockdown of LdUSP-1 in third instar larvae using RNA interference.Silencing of LdUSP-1 significantly downregulated the transcript level of E75,an ecdysone-inducible gene,and of Sad,a Halloween gene.In addition,the duration of the third-instar stage was slightly shortened and larval mortality increased after the LdUSP-1 knockdown.

Life circle and bionomics of Lymantria dispar L.

The life circle and bionomics ofLymantria dispar was studied from the May to the July of 1997 in Tulihe Forestry Enterprise of Inner Mongolia. The results showed thatL. dispar has one generation in a year, larvae overwinters in eggs, and last 50 days to grow up. From the completing of larvae period to the appearing of adults lasts 11 days. The study made a basis for the period forecasting of L.dispar in Inner Mongolia.

The sex peptide receptor in the Asian gypsy moth,Lymantria dispar,is involved in development and stress resistance

The G protein-coupled receptor(GPCR)regulates downstream genes by binding to a heterotrimeric G protein.However,the function of sex peptide receptor(SPR)in lepidopteran species is mostly unknown.Understanding the physiological functions of SPR in insects is essential for exploring new insecticidal targets.In the present study,the functions of an SPR in Lymantria dispar(Asian gypsy moth;LdSPR)were investigated.The expression of LdSPR was the highest in the 6 th instar larval stage,and there was a large difference in expression between male and female adults.After LdSPR gene silencing,L.dispar larvae showed increased sensitivity to high temperature,starvation,and oxidative stress,indicating that LdSPR enhances stress resistance.These results enrich our knowledge of the function of the insect SPRs,which will lead to a better understanding of other insect GPCR family members and the identification of new targets for the development of environmentally friendly pesticides.

Molecular cloning, characterization, and antioxidant function of catalase in Lymantria dispar asiatic (Lepidoptera: Lymantriidae) under avermectin stress

The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa,theoretical pI of 5.00,aliphatic index of 29.92,grand average of hydropathicity of 0.764,and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis,LdCAT was most similar to CAT from Spodoptera litura and S.exigua.Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested,verifying the ubiquity of LdCAT in L.disapr.Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90,and in the cuticula,rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration.The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L.disapr defensing against pesticide stress and may be a good target for controlling this pest.

Synergic effect of sex pheromone(7R,8S)-cis-7,8-epoxy-2-methyloctadec-17-ene on(+)-disparlure of the Asian gypsy moth,Lymantria dispar, in Korea

field attraction test of (7R,8S)-cis-7,8-epoxy-2- methyloctadec-17-ene (= 7R,8S-epo-2me-17ene-18Hy), a trace component of the sex pheromone gland of the Asian gypsy moth,(Lymantria dispar), traps that were baited with the trace component attracted more L. dispar than traps baited with (7S,8R)-cis-7,8-epoxy-2-methyloctadec-17-ene (= 7S,8R-epo-2me-17ene-18Hy) and unbaited traps. However, traps baited with 7R,8S-epo-2me-17ene-18Hy were less attractive than traps baited with (+)-disparlure [=(7R,8S)-cis-7,8-epoxy-2-methyloctadecane], the sex pheromone of L. dispar. Combination tests with (?)-disparlure, 7R,8S-epo-2me-17ene-18Hy, and 7S,8R-epo-2me- 17ene-18Hy revealed that 7R,8S-epo-2me-17ene-18Hy acted synergistically with (?)-disparlure.

Population Dynamic and Biological Characteristics of the Gypsy Moth Lymantria dispar （Lepidoptera： Lymantriidae） in West-Siberian Geographic

Gypsy moth, Lymantria dispar （L.） is one of the most widespread defoliators of deciduous and larch forests in Kazakhstan. Preferred host plants, flying capacity of females and neonate caterpillars, preferred places for egg laying, major factors of mortality and other characteristics vary considerably between different populations of the pest. According to several year researches on gypsy moth, in Kazakhstan, the main biological characteristics of this pest in the regions were investigated. Results of research showed that gypsy moth exists in central and northern of theses regions. From biological and geographical aspects, gypsy moth which there is in Kazakhstan is close to the same species from Siberia western regions. The pest lays on the different heights of trees trunk. It was found that the number of eggs in each egg mass and egg masses laid by the pest are different. The lack of egg eaters and the over- reproduction of the pest at the natural conditions have resulted in the development of bacterial and viral diseases in the populations of this pest from the regions of Kazakhstan to Siberia west.

Primary Host Tree Species of the Gypsy Moth Lymantria dispar （Lepidoptera： Lymantriidae）in Hyrcanian Forests

Invasions by exotic insects and diseases are one of the most important threats to the stability and productivity of forest ecosystems around the world. Lymantria dispar （Lepidoptera： Lymantriidae） in Iran was observed for the first time in 1937 by Jalal Afshar in oak forests of the Guilan state region. Since its introduction, gypsy moth has spread to the northern, western and south-western regions of Iran. The largest outbreaks of gypsy moth occurred in the Talesh Oak forests of the Hyrcanian zone in 1975. The gypsy moth is a highly polyphagous folivore species that feeds on over 300 species of woody plants. Among its most preferred hosts are oaks and aspens. Some trees are resistant to the gypsy moth including honey locust, black locust, silver maple, green ash, dogwood, sycamore, horse chestnut, firs, and tulip trees. Defoliation during severe outbreaks alters the appearance of forest habitats and leads to tree mortality and changes in stand composition. This study was conducted during 2008 and 2009 in an effort to determine the primary host tree species that the gypsy moth infests in the Hyrcanian forests of lran. For that purpose all egg masses in the area were counted and conclusively significant differences in defoliation levels among tree species were found. The primary host tree species of gypsy moth in Iran was Persian ironweed, Parrotia persica.

舞毒蛾LdOA1基因克隆分析及对3种杀虫剂胁迫的响应

G蛋白偶联受体(GPCRs)是生物体内重要膜受体,通过激活三聚体G蛋白参与各种细胞内信号转导,在药物研发中具有重要的作用。从舞毒蛾无参照全转录本文库中鉴定获得GPCR家族中眼白化I型全长基因(命名为LdOA1),该基因全长453 bp,编码150个氨基酸,分子质量为17.54 kDa,理论等电点(pI)为9.76。进化树分析表明LdOA1蛋白与帝王蝶的亲缘关系较近。实时荧光定量PCR分析表明舞毒蛾3龄幼虫在溴氰菊酯、甲萘威和氧化乐果胁迫下LdOA1的表达均表现为显著抑制。

两种丛枝菌根真菌定殖青山杨后对舞毒蛾的抗性研究

【目的】研究丛枝菌根真菌(AMF)对青山杨抗虫性的影响以及舞毒蛾对AMF定殖青山杨的适应能力,为AMF定殖青山杨后抗虫性的研究提供科学依据。【方法】分别以对照(CK,无AMF定殖)、根内根孢囊霉(GI)或摩西管柄囊霉(GM)定殖的青山杨叶片饲喂舞毒蛾幼虫,统计3、4、5龄幼虫的体重、体长、头壳宽和食物利用率,测定4、5龄幼虫解毒酶酸性磷酸酯酶(ACP)和碱性磷酸酯酶(AKP)的活性。【结果】GI组3、4龄幼虫的体重显著高于对照组(P<0.05),5龄与对照组差异不显著,GM组5龄幼虫的体长、体重、头壳宽、食物利用率均显著高于对照组。GI组3龄幼虫取食量显著高于对照组(P<0.05),4、5龄显著低于对照组,GM组5龄显著高于对照组,3、4龄与对照组差异不显著。GI组ACP显著低于对照组(P<0.05),AKP在4龄显著高于对照组,5龄AKP活性显著低于对照组。GM组两种解毒酶活性均显著高于对照组。【结论】GI定殖的青山杨能促进舞毒蛾低龄幼虫的生长,对舞毒蛾老熟幼虫的生长影响不显著。GM定殖青山杨叶片促进了舞毒蛾幼虫的生长发育,提高了食物的利用效率,激发了幼虫体内磷酸酯酶活性,表明舞毒蛾对GM定植的青山杨有更好的适应性。AMF对树木及植食性昆虫的影响具有种类特异性,GI定殖对青山杨的抗虫性的影响呈中性,GM定殖对青山杨的抗舞毒蛾产生负效应。

取食不同植物对舞毒蛾幼虫生长发育及生化酶的影响

以垂柳、白桦、雪松、水杉、华山松、元宝枫饲喂舞毒蛾幼虫,研究了不同食料植物对幼虫生长发育和6种酶活性及其基因表达量的影响。结果表明:取食雪松的2龄幼虫的存活率最高为55.32%,发育历期最短为5.41 d;取食垂柳和白桦的存活率分别为41.34%和35.71%,发育历期分别为6.40 d和6.66 d;取食元宝枫、华山松、水杉的幼虫存活率显著低于取食其他3种植物的,且不能完成整个生命史。幼虫取食不同植物后,体内的6种酶活性差异显著,幼虫取食垂柳后的CarE、AChE、POD和CAT活性显著高于取食雪松的,取食雪松个体的GSTs和SOD活性显著高于取食垂柳的个体。GST、SOD活性与幼虫的存活率和发育历期呈正相关;CarE、AChE、POD、CAT活性与幼虫的体质量、头宽和体长呈正相关。取食垂柳个体的LdAChE、LdCAT的相对表达量极显著高于取食雪松的个体,LdPOD的相对表达量显著高于取食雪松的个体,取食雪松个体的LdS-OD表达量极显著高于取食垂柳的个体。舞毒蛾幼虫能够随着取食植物种类的不同调整其体内的生化酶活性及酶基因的表达水平,以满足其生长发育的需要。

Difference in volatiles of poplar induced by various damages

Three treatments including mechanical damage, Lymantria dispar attacking and daubing oral secretions of the in-sects on mechanically damaged cut were conducted on Populus simonii譖opulus pyramibalis c.v. in order to find the genuine reason leading to effective resistance response of tree to insects attacking. The release situation of the induced volatiles of the plant was analyzed by TCT-GC/MS at 24 hours after damages. The results indicated that some of the volatiles such as (Z)-3-hexenyl acetate, decanal, 3-hexenyl isovalerate, nonanal, ocimene, and 2-cyanobutane can be induced by both insects attack-ing and mechanical damage, while 2,6-dimethyl-1,3,5,7-octatetraene, 2-methyl-6-methylene-1,7-octadien-3-one, caryophyllene, Isovaleronitrile, diethyl-methyl-benzamide, and dicapryl phthalate were only induced by insects attacking. Such difference in volatiles was attributed to that there existed active components in oral sections of the larvae of Lymantria dispar

TAT-Cloan-DH-EGFP融合蛋白的原核表达及其在舞毒蛾幼虫体内的跨膜转导

【目的】为了探究滞育激素经昆虫取食的生物效应,需设计一种能够加速跨膜运输的融合蛋白,克服经昆虫消化道的降解作用。【方法】本研究以分月扇舟蛾Clostera anastomosis滞育激素基因Cloan-DH和TAT穿膜肽编码序列为基础,构建TAT-Cloan-DH融合基因;然后以增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)为标签,构建在p ET-22b载体中。在0.2 mmol/L IPTG诱导下,在大肠杆菌Escherichia coli BL21 Rosetta(DE3)中表达出TAT-CloanDH-EGFP融合蛋白。再分别将表达有TAT-Cloan-DH-EGFP和EGFP融合蛋白的大肠杆菌BL21菌体拌入人工饲料,经舞毒蛾Lymantria dispar幼虫持续取食0,8,24,48,72和90 h后,随机挑选试虫进行组织固定并制作石蜡切片,选取中肠所在体段切片进行组织荧光分析。【结果】37℃0.2 mmol/L IPTG诱导条件下,表达的TAT-Cloan-DH-EGFP融合蛋白的分子量为61 k D,主要以包涵体形式表达,融合蛋白约占细胞总蛋白含量的18.1%;随着试虫取食含TAT-Cloan-DHEGFP融合蛋白饲料的增加,虫体组织的绿色荧光强度逐渐增加,取食72 h后,组织荧光强度达到最高。【结论】实验结果说明融合蛋白TAT-Cloan-DH-EGFP可以通过消化道横向跨膜运输,到达虫体其他组织,且该过程与蛋白的摄入量成比例。

融合肽TAT-Helicokinin I对舞毒蛾生物活性研究

以美洲棉铃虫Helicoverpa zea利尿激肽Helicokinin I和穿膜肽TAT为基础合成融合肽TAT-Helicokinin I,并测试3种肽对舞毒蛾Lymantria dispar幼虫的注射活性;探究融合肽TAT-Helicokinin I和利尿激肽Helicokinin I经取食途径进入虫体后对舞毒蛾幼虫生长的影响。结果表明:TAT-Helicokinin I和Helicokinin I对舞毒蛾幼虫均表现出注射毒性,且TAT-Helicokinin I比Helicokinin I表现出更高的生物活性;60,600 pmol/头的剂量对舞毒蛾幼虫均表现出显著致死性,6 pmol/头的剂量对舞毒蛾幼虫生长无影响;Helicokinin I饲喂舞毒蛾幼虫未显现出毒性,而饲喂TAT-Helicokinin I对试虫生长表现出显著抑制作用。该研究证实TAT与Helicokinin I融合后能够降低昆虫消化道酶对利尿激肽Helicokinin I的降解。

绿原酸提高舞毒蛾核型多角体病毒(LdNPV)的致病力

【目的】明确绿原酸（chlorogenic acid,CA）对舞毒蛾核型多角体病毒Lymantria dispar necleopolyhydrovirus（Ld NPV）致病力的影响,为舞毒蛾的防治提供参考依据。【方法】采用食料给毒法进行生物测定,测定舞毒蛾2龄幼虫对单独Ld NPV及添加绿原酸的Ld NPV（CA＋Ld NPV）的剂量及致死时间响应。【结果】CA＋Ld NPV与单独Ld NPV对舞毒蛾2龄幼虫的剂量及时间响应间有显著差异,二者对舞毒蛾2龄幼虫的致死中浓度（LC50）分别为161.8 OBs/μL（95%的置信区间为105.6～235.3 OBs/μL）和264.4 OBs/μL（95%的置信区间为178.6～384.0 OBs/μL）,前者对舞毒蛾的致病力较后者强。CA＋Ld NPV对舞毒蛾2龄幼虫的致死中时间（LT_（50））较Ld NPV的短,当Ld NPV浓度为590 OBs/μL,CA＋Ld NPV及Ld NPV对舞毒蛾2龄幼虫的LT_（50）分别为9.9 d和12.3 d;当Ld NPV浓度为5 900 OBs/μL时,则LT_（50）分别为6.9 d和8.0 d。绿原酸降低了Ld NPV对舞毒蛾幼虫的致死中浓度,缩短了致死中时间。【结论】绿原酸可提高Ld NPV对舞毒蛾2龄幼虫的致病力,其机理有待进一步研究。

Bt和LdNPV混用对舞毒蛾幼虫毒杀效果的研究

测定了苏云金杆菌Bacillus thuringiensis(Bt)和舞毒蛾核型多角体病毒Lymantria dispar nu-clear polyhedrosis virus(LdNPV)混用对舞毒蛾Lymantria dispar的毒杀效果,室内联合作用时Bt稀释16 000倍对LdNPV仍具有增效作用,LdNPV浓度2.5×107,2.5×106,2.5×105OBs/mL均对Bt有增效作用;LdNPV浓度2.5×104,2.5×103OBs/mL对Bt则分别表现为相加和拮抗作用。林间套笼试验结果表明,Bt与LdNPV混用死亡高峰期出现在第8天,死亡率为11.57%,混合处理14d的累计死亡率为69.65%,均高于单剂。灌木喷洒试验结果表明,Bt和LdNPV混用的死亡高峰期有2个,分别为第5天和第16天,而Bt和LdNPV单剂的高峰分别出现在第6天和第18天。平均虫口密度降低率以混合处理最高,为38.67%。本试验证明Bt与LdNPV混用对舞毒蛾具有增效作用,并能弥补单剂缺陷。

转舞毒蛾LdCYP6AN15v1基因果蝇品系对氯虫苯甲酰胺胁迫响应

【目的】舞毒蛾是林业重要害虫,细胞色素P450是昆虫体内广泛分布的参与外源化合物代谢关键酶系,探讨P450家族基因CYP6AN15v1对杀虫剂代谢解毒功能,为舞毒蛾有效治理提供依据。【方法】通过RT-PCR法获得Ld CYP6AN15v1基因c DNA全长,采用传统酶切连接的方法构建转CYP6AN15v1基因果蝇载体,通过转基因技术获得表达Ld CYP6AN15v1果蝇品系(命名为att P40>CYP6AN15v1)。采用分光光度计法研究低剂量氯虫苯甲酰胺(7.17mg·L^(-1))处理对转基因和非转基因果蝇品系细胞色素P450活性的影响,并采用qRT-PCR法测定其对CYP6AN15v1基因表达的影响。【结果】从舞毒蛾无参照转录本文库中克隆获得CYP6AN15v1全长基因,编码512个氨基酸,蛋白分子质量为59.02 k Da;系统进化树分析表明CYP6AN15v1与甜菜夜蛾和棉铃虫关系较近。以DNA和c DNA为模板,att P40>CYP6AN15v1果蝇品系均检测到1 539 bp目的基因,表明Ld CYP6AN15v1基因成功整合到果蝇基因组。与非转基因att P40果蝇品系相比,转基因att P40>CYP6AN15v1果蝇品系对氯虫苯甲酰胺的敏感性显著降低,致死中浓度LC50为非转基因果蝇的2.92倍;低剂量(7.17 mg·L^(-1))氯虫苯甲酰胺胁迫下,舞毒蛾细胞色素P450酶活性和CYP6AN15v1基因的诱导作用呈现时间效应,att P40>CYP6AN15v1果蝇品系P450活性为非转基因果蝇的1.09~1.93倍,主要表现为诱导效应;att P40>CYP6AN15v1果蝇品系的CYP6AN15v1基因mRNA表达量呈诱导激活,其表达量为非转基因的44.54~137.80倍。【结论】利用转基因技术成功构建了转Ld CYP6AN15v1果蝇品系att P40>CYP6AN15v1;氯虫苯甲酰胺可能通过诱导Ld CYP6AN15v1基因mRNA的上调表达而增强黑腹果蝇P450酶活性,从而参与对氯虫苯甲酰胺的解毒作用。