Characteristics of Cellulases from Anoplophora glabripennis Motsch (Coleoptera: Cerambycidae)

The property of major cellulases from the guts of Anoplophora glabripennis larvae have been characterized. The optimal temperatures of both β 1,4 glucosidase (β glucosidase) and endo β 1,4 endoglucanase (endoglucanase, Cx) are 40℃. The β glucosidase was optimally active at pH 4\^8, while the optimal activity of the endoglucanase occurred at pH 4 4 5 6 The endoglucanase was active with a wide range of pH and temperature, the levels of activity from 25℃ to 50℃ were more than 80%, and the activity remained 60% between pH 3 2 and pH 7 2. The endoglucanase exhibited higher thermal stability than β glucosidase. Both enzymes lose their activities by heat treatment at 60℃. Two isozymes of endoglucanase were detected in sodium carboxymethylcellulose polyacrymide gels (CMC gel) by chemical colorization, and purified by elution from the gel slices. The molecular weights of the two isozymes were estimated as 26kD and 39kD respectively. Moreover molecular characteristics of the two isozymes are currently underway.

Phylogenetic Analysis of the 16S rDNA of a Strain Isolated from Diseased Larva of Anoplophora glabripennis (Motsch.)

[ Objective] Study on the phylogenetic analysis of the 16S rDNA and insecticidal characteristics of strain BH-1 isolated from diseased larva of Anoplophora glabripennis （Motseh.） [ Method ] The strain was identified by routine method and inoculated onto healthy Anoplophora glabripennis （Motseh.） for observing insecticidal effect, further 16S DNA was amplified by the specific primers for sequencing and homology analysis. [ Result] The mortality of second instar ofAnoplophora glabripennis（ Motseh. ） reached 72.7% 8 d after 10^10cfu/ml BH-1 was inoculated. The homology of 16S DNA sequences between BH-1 and Serratia marcescens accessed in GenBank reached 99.5%. Combined with the results of routine identification, BH-1 was identified as S. marcescens. [Conclusion] BH-1 could be used for biological control ofAnoplophora glabripennis （Motsch.）.

茉莉酸甲酯喷施和光肩星天牛(Anoplophora glabripennis(Motschulsky)咬食后五角枫释放的挥发物

茉莉酸甲酯喷施和光肩星天牛(Anoplophora glabripennis(Motschulsky)咬食后五角枫(Acer mono Maxim.)植株均诱导产生了乙酸丁酯、2-壬醇、乙酸己酯、3-甲基-2-丁醇、1-辛烯、里那醇、3-蒈烯、1-丁醇、(Z)-2-己烯醛9种正常植株中没有检测到的挥发物。两种处理均诱导或促进了萜烯类、醇类和醛类挥发物的大量释放,且乙酸-3-己烯酯、乙酸乙酯、己醇、反-2-己烯醇、乙基己醇、1-辛烯、石竹烯、法尼烯等挥发物的时序变化趋势也非常相似。因而推测光肩星天牛咬食五角枫后在植物体内诱导产生了茉莉酸信号传导途径。

Electrophysiological and Behavioral Responses of Adult Anoplophom glabripennis (Motschulsky) to Volatile Components of Host-Plant

Electrophysiological and behavioral responses of adult A. glabripennis (Motsch.) to volatiles from ashleaf maple (Acer negundo L.) were investigated to identify semiochemicals involved in host location. Measurable electroantennogram (BAG) responses were elicited to all compounds tested, the most effective antennal stimulants were trans-2-hexen-1-al,decyl aldehyde and trans-2-hexenl-ol.These profiles were similar between males and females. In Y-tube olfactometer bioassays, above three compounds with certain concentration, trans-2-hexen-l-al (1%), trans-2-hexen-l-ol (1%) and decyl aldehyde (10%), were significantly attractive to the adults in laboratory. The results show that either BAG or olfactory responding to a particular volatile compound are markedly influenced by the concentration.

Cellulase in Anoplophora glabripennis adults emerging from different host tree species

In order to investigate different kinds of cellulase in insect pests, we selected male and female adults of Anoplophora glabripennis （Motschulsky） emerging from four different host species （Populus alba var. pyramidalis, P. nigra var. thevestina （Dode） Bean., P. simonii × P. pyramidliscr cv. Opera 8277 Hsu. and Salix matsudana f. lobato-glandulosa） as our research material. The enzyme activitives of three kinds of cellulase in the intestines of the adult insects were measured. The Cx-cellulase isozymes were detected with a CMC-incorporated polyacrylamide gel. The results show that： there are no statistically significant differences between the enzyme activities of males and females emerging from the different host species. The order of magnitude in activity is： Cx-cellulase 〉 β-glycosidase 〉 C1-cellulase. When the adults emerge from the same host species, there are no statistically significant differences between male and female enzyme activities of β-glycosidase and C1-cellulase, but the enzyme activity of Cx-cellulase of males is clearly higher than that of females. The patterns and migration of Cx-cellulase isozymes of males and females emerging from differ-ent hosts trees are clearly not different, and neither are they different when emerging from the same hosts.

Combining WV-2 images and tree physiological factors to detect damage stages of Populus gansuensis by Asian longhorned beetle (Anoplophora glabripennis) at the tree level

Background:Anoplophora glabripennis(Motschulsky),commonly known as Asian longhorned beetle(ALB),is a wood-boring insect that can cause lethal infestation to multiple borer leaf trees.In Gansu Province,northwest China,ALB has caused a large number of deaths of a local tree species Populus gansuensis.The damaged area belongs to Gobi desert where every single tree is artificially planted and is extremely difficult to cultivate.Therefore,the monitoring of the ALB infestation at the individual tree level in the landscape is necessary.Moreover,the determination of an abnormal phenotype that can be obtained directly from remote-sensing images to predict the damage degree can greatly reduce the cost of field investigation and management.Methods:Multispectral WorldView-2(WV-2)images and 5 tree physiological factors were collected as experimental materials.One-way ANOVA of the tree’s physiological factors helped in determining the phenotype to predict damage degrees.The original bands of WV-2 and derived vegetation indices were used as reference data to construct the dataset of a prediction model.Variance inflation factor and stepwise regression analyses were used to eliminate collinearity and redundancy.Finally,three machine learning algorithms,i.e.,Random Forest(RF),Support Vector Machine(SVM),Classification And Regression Tree(CART),were applied and compared to find the best classifier for predicting the damage stage of individual P.gansuensis.Results:The confusion matrix of RF achieved the highest overall classification accuracy(86.2%)and the highest Kappa index value(0.804),indicating the potential of using WV-2 imaging to accurately detect damage stages of individual trees.In addition,the canopy color was found to be positively correlated with P.gansuensis’damage stages.Conclusions:A novel method was developed by combining WV-2 and tree physiological index for semi-automatic classification of three damage stages of P.gansuensis infested with ALB.The canopy color was determined as an abnormal phenotype that could be directly assessed using remote-sensing images at the tree level to predict the damage degree.These tools are highly applicable for driving quick and effective measures to reduce damage to pure poplar forests in Gansu Province,China.

Oviposition preference of Anoplophora glabripennis emerging from five host tree species under field conditions

The Asian longhorned beetle （ALB）, Anoplophora glabripennis （Motschulsky） is a polyphagous woodborer of hardwood trees. In order to well understand the oviposition preference of A. glabripennis emerging from different larval host tree species, we selected five common host tree species in the field and evaluated its oviposition preferences. The five host tree species are Acer negundo （AN）, Salix matsudana f. lobato-glandulosa （SM）, Populus nigra L. var. thevestina （Dode） Bean （PN）, P. simonii × P. pyramidalis cv. Opera 8277 （PS） and P. alba var. pyramidalis （PA）. Results show that adult beetles emerging from these five tree species almost have the same oviposition phenomenon. All adult beetles, except those emerging from PS, chewed the highest potential sites and laid most eggs on AN trees. Adult beetles emerging from AN, PN, PS and PA did not chew any sites nor laid any eggs on PA trees. These results showed that adult A. glabripennis emerging from the five host tree species did not show any clear oviposition preference for their larval host tree species in the field but they did present the most obvious preference for AN and the lowest preference for PA.

Cellulase in Anoplophora glabripennis adults fed on original host tree species and non-original host trees

Cellulase activities of Anoplophora glabripennis （Motsch.） adults from two host plants （Populus simonii x p pyramidliscr cv. Opera Hsu. and Salix matsudana Koidz） fed on three different host tree species （Acer negundo Linn., S. matsudana Koidz and P simonii x P pyramidliscr cv. Opera Hsu.） were investigated. Enzyme activities of endoglucanase and β-glucosidase in the intestines of the insects were measured. The results show that there are no statistically significant differences in the enzyme activities of endoglucanase between male and female insects from the two host plants fed on three host trees, neither inβ-glucosidase. No statistically significant differences in the enzyme activities of endoglucanase and β-glucosidase were found between males and females from the two plants fed on three host tree species, except that the endoglucanase activity in males from the opera poplar fed on Hankow willow is lower than that fed on ash-leaf maple and opera poplar.

Oviposition Site Selection of Anoplophora glabripennis in Sugar Maple (Acer saccharum) and Its Gnawing Nidus Oviposition Habits

[Objective] The paper was to explore the oviposition site selection of Anoplophora glabripennis in sugar maple and its gnawing nidus oviposition habits. [Method] Through field and indoor rearing observation, the oviposition site selection of A. glabripennis in sugar maple was studied, and the gnawing nidus oviposition habits were analyzed. [Result] The height of oviposition site of A. glabripennis in sugar maple, the number observation holes （gnawing nidus oviposition points） and bark thickness in oviposition site showed linear correlation with diameter of branch at breast height. The gnawing nidus oviposition habit of A. glabripennis in sugar maple was mainly divided into 4 stages： gnawing nidus site selection, gnawing nidus, oviposition and nidus sealing, the duration from gnawing nidus to nidus sealing was（38.29±16.47）min. [Conclusion] The study provided reference for comprehensive prevention of A. glabripennis in greening tree species sugar maple, as well as further study on reproductive behavior and regulation mechanism of A. glabripennis.

光肩星天牛种群间及其近缘种遗传关系的RAPD研究(英文)

利用RAPD技术对采自中国和美国的星天牛属Anoplophora 5个种及 8个光肩星天牛Anoplophoraglabripennis(Motschulsky)地理种群共 13个样品进行了遗传相似性分析。选用了Operon公司生产的引物H系列 2 0个 ,L系列 2 0个 ,Q系列11个共 5 1个引物 ,最后从 4 0个引物中筛选出 2 6个具有多态性的引物作为第一组用于星天牛属种间和光肩星天牛种群间分析 ,从 31个引物中筛选出 19个具有多态性的引物作为第二组单独用于光肩星天牛种群分析。根据第一组引物实验获得的RAPD聚类图及遗传距离表明 ,各个地理种群的光肩星天牛和黄斑星天牛A .nobilis都聚在一起 ,形成一个大的分枝 ,而四川星天牛A .freyi、楝星天牛A .horsfieldi和星天牛A .chinensis均在此分枝之外。来自美国纽约和芝加哥的光肩星天牛种群聚于中国光肩星天牛种群之外的另一个独立的分枝上。分布在我国宁夏、内蒙古和河北的光肩星天牛以及宁夏黄斑星天牛和山东、陕西的光肩星天牛分别聚在一起 ,而甘肃的光肩星天牛与甘肃的黄斑星天牛则聚于另一枝 ,且它们之间的遗传距离很近 ,仅为 0 132 4 ,说明这两者之间有着极其相近的亲缘关系 ,由此推断光肩星天牛和黄斑星天牛的差异很小 ,遗传关系难以区分 ,进一步证实了它们很可能是同一个种下的两个不同的型。

罹病光肩星天牛幼虫分离菌株的16S rDNA系统发育分析

[目的]研究从罹病光肩星天牛[Anoplophora glabripennis(Motsch.)]幼虫刻槽中分离的菌株BH-1的16S rDNA系统发育及该菌株杀虫特性。[方法]常规细菌鉴定,将BH-1接种健康天牛后观察杀虫效果并通过设计特异引物扩增其16S rDNA序列,进行测序及同源性分析。[结果]用1010cfu/ml BH-1菌液接种2龄天牛幼虫8 d后致死率达72.7%。BH-1与GenBank收录的Serratia marcescens的16S rDNA序列的同源性达99.5%,同时结合细菌的部分常规鉴定结果,鉴定出BH-1菌株为粘质沙雷氏菌(S.marcescens)。[结论]BH-1的发现为天牛的生物防治提供了参考。

利用RAPD研究星天牛地理种群间亲缘关系

利用随机扩增的多态性DNA(RAPD)方法对分布在我国甘肃、宁夏、内蒙、河北、西安、青岛、福建、江苏等16个地区,美国纽约、芝加哥、新泽西3个地区和韩国2个地区的光肩星天牛及其近缘种星天牛共23个地理种群进行了研究。实验筛选出17个多态性引物产生了219个多态性位点,RAPD聚类分析表明,美国和中国的光肩星天牛种群之间存在显著差异,遗传关系较远。

光肩星天牛及其近缘种mtDNA序列和基因特点

通过对光肩星天牛及其近缘种mtDNA的 7个基因片段的测序 ,分析了光肩星天牛mtDNA的特点。结果表明 ,所测的光肩星天牛mtDNA 7个基因序列与其近缘种差异达 11.3%以上 ,光肩星天牛种群及个体间碱基差异小于 3% ,大部分基因基本没有差异 ,光肩星天牛mtDNA的这一特点说明这些基因在种内较为保守 ,可用于该虫的鉴定和系统遗传分析 ;光肩星天牛与黄斑星天牛在这些基因上差异很小 ,有可能为同一个种。在COⅠ ,Cytb等基因测序中 ,发现部分个体出现差异大的序列 。

光肩星天牛气味结合蛋白AglaOBP1的克隆、表达及结合特性

利用RT-PCR技术克隆了光肩星天牛Anoplophora glabripennis Motschulsky气味结合蛋白(odorant binding proteins,OBPs)基因AglaOBP1(Gen Bank登录号:KX660670),AglaOBP1的开放阅读框长435 bp,编码144个氨基酸,其中N端有21个氨基酸组成的信号肽序列,成熟蛋白序列具有4个保守的半胱氨酸残基,AglaOPB1属于Minus-C OBP亚家族基因。AglaOBP1主要在成虫触角中表达,且雌虫触角中的表达量显著高于雄虫触角。重组蛋白AglaOBP1与34种气味配体的竞争结合实验表明,AglaOBP1具有广泛的结合谱,能与五角枫挥发物中的醇类、醛类、萜烯类和酮类物质结合,其中与顺-3-己烯-1-醇、顺-2-己烯-1-醇、1-己醇、反-2-己烯醛、反-2-癸烯醛、β-石竹烯、(+)-桧烯、α-蒎烯、1-(2,3-二甲基苯基)-乙酮的结合能力较强,结合常数分别为5.88、8.33、12.29、12.55、11.90、7.47、9.07、10.29和13.12μM。此外,配体的官能团、碳链长度、空间构型也影响AglaOBP1对气味分子的结合。AglaOBP1基因在成虫触角中高丰度表达,其重组蛋白能够与五角枫挥发物的多种组分结合,表明AglaOBP1在成虫寄主定位选择中起重要作用。

光肩星天牛气味结合蛋白AglaOBP12的基因克隆、表达及配体结合特征

【目的】克隆和鉴定光肩星天牛Anoplophora glabripennis气味结合蛋白(odorant binding proteins,OBPs)基因,明确其表达特点及与寄主植物挥发物的结合特性,有助于阐明光肩星天牛嗅觉识别的分子机制。【方法】根据光肩星天牛雌成虫触角转录组数据,利用RT-PCR克隆OBP12基因,并进行生物信息学分析。通过实时定量PCR(qRT-PCR)测定OBP12在光肩星天牛成虫触角、头(移除触角)、胸、腹、足、翅中的转录水平。利用原核表达系统和Ni离子亲和层析技术表达和纯化OBP12重组蛋白,荧光竞争结合实验测定重组蛋白与39种气味配体的结合能力。【结果】获得光肩星天牛气味结合蛋白基因AglaOBP12(GenBank登录号:KX890109)的完整编码序列,其开放阅读框长414 bp,编码137个氨基酸,N末端具有18个氨基酸组成的信号肽序列,蛋白序列具有6个保守的半胱氨酸残基,Agla OPB12属于Classical OBPs亚家族基因。qRT-PCR测定结果表明,AglaOBP12主要在成虫触角中表达,在其他组织微量表达。在待测的39种寄主植物挥发物中,重组蛋白AglaOBP12仅与19种化合物具有结合活性,表明AglaOBP12对寄主植物挥发物具有明显的选择结合特性。重组蛋白AglaOBP12与十二烷醇、十四烷醇、法尼醇、十二醛、乙酸-顺-3-己烯酯和β-石竹烯的结合能力较强,结合常数分别为1.96,0.96,1.03,0.82,0.77和0.74μmol/L。【结论】明确了AglaOBP12的核苷酸和氨基酸序列组成,重组AglaOBP12蛋白与主链有12个碳原子的醇类、醛类和萜烯类挥发物有特异性的结合活性。根据AglaOBP12基因的表达特点和重组蛋白的结合特性,推测AglaOBP12在光肩星天牛成虫定位补充营养寄主植物中发挥重要作用。

光肩星天牛mRNA差异显示体系的建立与触角差异表达基因的筛选

【目的】建立一套适合光肩星天牛的mRNA差异显示(DDRT-PCR)体系,并对光肩星天牛触角差异表达基因进行初步研究。【方法】以光肩星天牛雌、雄个体的触角和去触角的身体部分为材料,对影响mRNA差异显示体系的主要因子进行优化,并研究了其差异表达基因。【结果】建立了适合光肩星天牛mRNA差异显示的反应体系,并筛选得到最适的PCR退火温度。使用此体系分离得到28条触角差异表达基因片段,其中9个片段的翻译产物分别与觅食相关蛋白、MYB转录因子、β-乙酰葡萄糖胺转移酶、化学感受蛋白11、表皮蛋白peritrophins1-I、甘油激酶、抗菌肽Alo-3、26S蛋白酶体(非ATP酶亚基12)和清道夫受体等已知蛋白有较高的同源性。【结论】利用建立的DDRT-PCR体系对供试材料进行扩增,扩增产物条带丰富且清晰,可用于分离差异表达基因,得到的触角差异显示基因可能参与了光肩星天牛的嗅觉识别过程。

光肩星天牛对5种槭树的40种挥发物EAG及嗅觉行为反应

通过EAD和“Y”型嗅觉仪测定光肩星天牛对5种槭树的40种挥发物及挥发物混合物的嗅觉电生理及嗅觉行为反应进行研究的结果表明:糖槭挥发物的主要成分为酯类、烯类、酸类和醇类,复叶槭为酯类、醛类和烷烃类,五角槭为酯类和醛类,华北五角槭为酯类、烯类和醇类,而挪威槭为酯类、醛类和醇类。光肩星天牛雌成虫对5种槭树的21种挥发物存在明显的的EAD反应,而雄成虫则对12种挥发物存在明显的EAD反应;其中雌雄成虫对11种挥发物均存明显的EAD反应。进一步嗅觉行为测定发现,光肩星天牛雌成虫对11种挥发物存在正趋向反应,雄成虫对7种挥发物存在显著的正趋向反应,具有明显的引诱作用,其中7种挥发物对雌雄均表现出引诱作用。雌成虫对8种挥发物存在负趋向反应,雄成虫对10种挥发物存在显著的负趋向反应,具明显的驱避作用,其中4种挥发物对雌雄成虫均表现出驱避作用。光肩星天牛雌雄成虫对5种槭树挥发物混合物的嗅觉反应都表现为正趋向反应,引诱率大小依次为糖槭、复叶槭、五角槭、挪威槭和华北五角槭。

光肩星天牛触角cDNA文库构建及质量分析

以新羽化的光肩星天牛(Anoplophora glabripennis)雄成虫触角为材料,采用RNA转录5'末端转换(SMART)技术构建全长c DNA文库。经验证,该文库容量为1.35×106pfu/m L,重组率为95%。从中随机挑取48个克隆进行表达序列标签(ESTs)测序,有效序列为45条,其中35条序列有相关同源信息,24条为全长序列,完整性比率为68.6%。

几种抗虫树种的化学成分分析及其抗虫机理的研究──Ⅲ驱避树种臭椿（Ailanthusaltissima）对天牛生长发育的影响

本文通过用不同比例的臭椿树皮粉饲喂天牛幼虫，发现臭椿对天牛幼虫的生长、化蛹、羽化及成虫产卵都有一定的影响，因此，臭椿作为驱避树种在营造抗虫林分时可大量栽植。

Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP （X is residue T, L, A or H） motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.