SeMNPV抑制HaSNPV诱导的Se—UCR细胞凋亡

在采用共感染和共转染的方法构建扩大杀虫范围的重组病毒的研究过程中发现棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)能诱导甜菜夜蛾细胞,Se-UCR发生典型凋亡,但不能诱导另一株甜菜夜蛾细胞Se-301产生凋亡。以5MOI的HaSNPV感染Se-UCR。在12h左右可以观测到少量细胞凋亡。24h能观察到明显的凋亡,凋亡细胞数量随时间不断增加,到72h基本上所有的细胞均发生凋亡,成为凋亡小体,基因组DNA片段化。同时发现HaSNPV诱导的甜菜夜蛾Se-UCR细胞凋亡能够被甜菜夜蛾多核衣壳核多角体病毒(Spodoptera exigua multicapsid nucleoplyhedrovirus,SeMNPV)所抑制,进一步点杂交试验发现SeMNPV和HaSNPV共同感染Se-UCR获得了HaSNPV在该细胞中的复制。     

The influence of greenhouse chrysanthemum on the interaction between the beet armyworm, Spodoptera exigua, and the baculovirus SeMNPV: parameter quantification for a process-based simulation model

During the building of a process-based simulation model for the epidemiology of the multicapsid nucleopolyhedrovirus of S. exigua (SeMNPV) in populations of Spodoptera exigua (Hübner) in greenhouse chrysanthemum, it was found that the effect of host plants had been under-rated. 'Missing links' included (i) the 'natural' background mortality of larvae of S. exigua in practical cropping conditions; (ii) the developmental rate of larvae of S. exigua on plant substrate in a glasshouse as compared to artificial medium in the laboratory; (iii) the validity of the results of dose-mortality and time-mortality bioassays conducted on artificial medium as compared to natural plant substrate; (iv) the distribution of inoculum released from deceased caterpillars over chrysanthemum leaves; and (v) the leaf visit rate of healthy caterpillars (as it affects horizontal transmission). Experiments were carried out to quantify these processes. Developmental rates of S. exigua larvae on greenhouse chrysanthemum were 36% lower than on an artificial diet. The fraction survival during the first, second, third and fourth instar S. exigua larvae in greenhouse chrysanthemum was 0.60, 0.80, 0.88 and 0.95, respectively. Forty percent of the first instar larvae reached the fifth larval stage. Second instar S. exigua larvae reared on chrysanthemum were significantly more susceptible to SeMNPV than larvae reared on an artificial diet. The food source had no effect on the time to kill S. exigua larvae. Cadavers of second, third and fourth instar S. exigua larvae contaminated on average 1.4, 2.5 and 3.3 chrysanthemum leaves. Second to fourth instar S. exigua larvae visited 2–3 leaves per day and spent 15–55% of the time on the underside of leaves. The above information is of critical importance for a trustworthy simulation of the epidemiology of SeMNPV in chrysanthemum.     

Effect of tinopal LPW on the insecticidal properties and genetic stability of the nucleopolyhedrovirus of Spodoptera exigua (Lepidoptera: Noctuidae)

The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.     

New cell lines from larval fat bodies of Spodoptera exigua: characterization and susceptibility to baculoviruses (Lepidoptera: Noctuidae)

Two new cell lines, designated IOZCAS-Spex-II and IOZCAS-Spex-III, were initiated from the fat bodies of larvae of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. The spherical cells were predominant among the various cell types and measures approximately 15 microm in diameter. The cell lines were mainly composed of tetraploid cells with chromosome numbers ranging from 116 to 131 (n=31). The cell lines were confirmed to have originated from the S. exigua by DAF-PCR technique. They were susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses from S. exigua.     

Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells

Successive viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and SeIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into nucleocapsids is hypothesized.     

甜菜夜蛾核多角体病毒sod基因的克隆及原核表达

甜菜夜蛾核型多角体病毒中国株(Spotoptera exigua MNPV—Z)超氧化物歧化酶基因(sod)业已被克隆及在大肠杆菌中进行了表达,证明了SeMNPV—Z的sod基因产物确有SOD活性,其活力单位约为291．19U／mL培养液。DNA测序结果表明SeMNPV-Z的sod基因编码151个氨基酸,与人的sodl基因的核苷酸的同源性为50％,与LdNPV、HaSNPV、HcNPV、AcNPV和BmNPV的sod基因的同源性分别为64％、63％、63％、65％、63％。     

甜菜夜蛾核多角体病毒BAC-TO-BAC外源基因表达系统的建立

用直接克隆法将miniF-lacZ-attFn7-kan 片段插入甜菜夜蛾核多角体病毒（Spodoptera exigua multicapsid nucleopolyhedrovirus, SeMNPV）〖JP〗美国分离株（SeUS1）基因组的多角体蛋白基因框内,miniF是大肠杆菌F因子复制子,携带miniF的重组病毒能够在大肠杆菌中低拷贝稳定复制,称为bacmid。由于SeUS1由不同的SeMNPV基因型组成,每个bacmid携带了一种病毒基因型,所有bacmid构成了SeUS1分离株的BAC文库。REN对111个bacmid分析表明,SeUS1分离株中除了包含具有完整SeMNPV遗传信息的基因型外,还包括不同类型的缺失基因型。将具有完整SeMNPV基因组的基因型SeBAC10转染昆虫细胞,可产生子代病毒,故SeBAC10是一种在真核细胞和原核细胞中均能复制的穿梭质粒。因为SeBAC10中多角体蛋白基因(Seph)被插入失活,将Seph作为报告基因通过位点特异性重组方式插入位于LacZ框内转座子Tn7的附着靶位点attTn7,得到重组SeBAC10 (即SeBAC10ph)转染甜菜夜蛾培养细胞Se301后,细胞出现典型的病理变化,核中出现多角体,证明SeMNPV BAC-TO-BAC外源基因表达载体系统构建成功。     

Biological activity of SeMNPV, AcMNPV, and three AcMNPV deletion mutants against Spodoptera exigua larvae (Lepidoptera: noctuidae)

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.     

甜菜夜蛾肽聚糖识别蛋白基因SePGRP-SA的克隆及功能鉴定

【目的】本研究旨在阐明甜菜夜蛾Spodoptera exigua幼虫体内肽聚糖识别蛋白(peptidoglycan recognition protein, PGRP)在响应苏云金芽胞杆菌Bacillus thuringiensis(Bt)感染过程中的功能。【方法】利用PCR方法扩增甜菜夜蛾幼虫肽聚糖识别蛋白基因SePGRP-SA全长cDNA;采用qRT-PCR分析SePGRP-SA在甜菜夜蛾不同发育阶段(卵、1-5龄幼虫、预蛹和蛹)及4龄幼虫不同组织(中肠、马氏管、围食膜、脂肪体、血淋巴和表皮)中的表达。通过RNAi技术沉默SePGRP-SA基因72 h后,qRT-PCR检测SePGRP-SA沉默效率及甜菜夜蛾4龄幼虫中肠抗菌肽相关基因(Ceropin, Attacin和Defensin)和细菌载量的变化。RNAi沉默SePGRP-SA 24 h后,以苏云金芽胞杆菌菌株Bt-GS57饲喂甜菜夜蛾4龄幼虫0, 24, 48, 72, 96和120 h,计算幼虫校正死亡率;饲喂甜菜夜蛾4龄幼虫Bt-GS57后0, 24, 48和72 h,利用qRT-PCR检测中肠SePGRP-SA, Ceropin, Attacin和Defensin的相对表达量。【结果】克隆获得甜菜夜蛾SePGRP-SA全长DNA(GenBank登录号：MW265930),开放阅读框长576 bp,编码191个氨基酸,其编码蛋白的预测分子量为21.59 kD。序列分析结果表明,SePGRP-SA具有典型的PGRP和Ami2保守结构域,信号肽为19个氨基酸,为分泌型蛋白;系统进化分析发现,SePGRP-SA与斜纹夜蛾Spodoptera litura的SlPGRP亲缘关系最近,氨基酸序列一致性达91.1%。发育表达谱结果表明SePGRP-SA在甜菜夜蛾4和5龄幼虫、预蛹和蛹中高表达;组织表达谱结果表明,SePGRP-SA在4龄幼虫各组织中均表达,其中以血淋巴中表达量最高。与注射dsEGFP(对照)相比,注射dsSePGRP-SA的甜菜夜蛾4龄幼虫在72 h时中肠SePGRP-SA基因表达量下调了95.26%,Cecropin, Attacin和Defensin表达量显著下调,中肠细菌载量显著升高。注射dsEGFP和dsSePGRP-SA的甜菜夜蛾4龄幼虫饲喂Bt-GS57,72 h时幼虫校正死亡率分别为50.00%和73.33%,表明幼虫对Bt-GS57的敏感性明显增加。甜菜夜蛾4龄幼虫取食Bt-GS57后,中肠SePGRP-SA, Cecropin, Attacin和Defensin表达量在48 h均显著增加,72 h时降低。【结论】Bt侵染能够引起甜菜夜蛾SePGRP SA基因激活抗菌肽相关基因Cecropin, Attacin和Defensin的表达。     

Biochemical characterization of three nucleopolyhedrovirus isolates of Spodoptera exigua and Mamestra brassicae

Three nucleopolyhedroviruses (NPVs), of different geographical origins, were biochemically characterized and compared. Two that were isolated from Spodoptera exigua (Se-UZB and Se-SP3) in Uzbekistan and Spain were SeMNPV type, and the third which was isolated from Mamestra brassicae (Mb-PL) in Poland was MbMNPV type. The Spanish isolate Se-SP3 showed restriction endonuclease (REN) profiles that were closely related to two previously described Spanish strains Se-SP1 and Se-SP2, but had some unique and characteristic REN fragments. On the other hand, comparison between the Uzbekian (Se-UZB) and the Spanish (Se-SP1, Se-SP2, and Se-SP3) isolates from S. exigua showed unrelated REN profiles. However, the Pst I and Bgl II profiles of Se-UZB and Mb-PL were identical, and very similar to the REN profiles of the MbMNPV strain, which constitutes the active component of Mamestrin^® (NPP, Nagueres, France), a commercial bioinsecticide. It is therefore very likely that the Se-UZB samples were cross-infected by the Polish strain (Mb-PL). This work presents two new strains of SeMNPV and MbMNPV, called Se-SP3 and Mb-PL, respectively, which were surveyed in two distant areas.     

A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.     

甜菜夜蛾半胱天冬酶基因的克隆及对细胞凋亡诱导剂和病原微生物感染的表达响应

[目的]本研究旨在明确甜菜夜蛾Spodoptera exigua半胱天冬酶(caspase)在细胞凋亡诱导剂诱导和病原微生物胁迫下的表达模式,为丰富鳞翅目昆虫细胞凋亡机制研究奠定基础.[方法]利用RT-PCR技术从甜菜夜蛾3龄幼虫体内扩增两个半胱天冬酶基因(SeCasp-3和SeCasp-4)编码区的全长;利用qPCR...     

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua

Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 +/- 0.45 microm and 2.84 +/- 0.14 microm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 +/- 0.21 microm x 2.00 +/- 0.11 microm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.     

SeMNPV持续性感染的甜菜夜蛾细胞株的建立

在昆虫种群中常常发生杆状病毒持续性感染,持续性感染可转变为增殖性感染而引发病毒流行病。本研究拟建立一个病毒-细胞模型,用于探讨杆状病毒持续性感染分子机制。将甜菜夜蛾核多角体病毒(Spodoptera exigua nucleopolyhedrovirus,SeMNPV)在其宿主细胞Se301中无稀释连续传代以弱化病毒毒力,用传至第8代的SeMNPV感染Se301细胞后,虽然大部分细胞因病毒感染而裂解,但仍有少量细胞存活并可传代培养,该传代细胞株命名为P8-Se301。P8-Se301细胞在对数生长期的群体生长倍增时间为58~65 h,慢于Se301细胞的生长速度。光镜和电镜观察表明,少部分P8-Se301细胞具有病毒发生基质、病毒粒子、多角体等病毒感染特征。终点稀释法和感染中心测定表明,4.14%±0.99%的P8-Se301细胞可持续释放感染性的子代病毒,但该子代病毒在Se301细胞中的复制速度较野生型SeMNPV慢。结果表明,P8-Se301细胞呈现典型的持续性感染特征。     

Activity and Persistence of Steinernema carpocapsae and Spodoptera exigua Nuclear Polyhedrosis Virus against S. exigua Larvae on Soybean

Experiments were conducted to assess the effects of the entomopathogenic nematode Steinernema carpocapsae and Spodoptera exigua multinucleocapsid nuclear polyhedrosis virus (SeMNPV), alone and in combinations, on mortality of the beet armyworm, S. exigua, larvae on soybean. In 1991 tests, field-grown soybean plants were treated with S. carpocapsae at 0.3 and 0.6 nematodes/cm² of leaflet, SeMNPV at 20 and 40 polyhedral inclusion bodies (PIB)/cm², and all possible combinations. Treated leaflets were collected from plants and bioassayed with 5-day-old larvae. The combination of S. carpocapsae at 0.6 nematodes/cm² + SeMNPV at 40 PIB/cm² produced significantly higher larval mortality (61.7%) compared with either S. carpocapsae (24.8-35.1%) or SeMNPV (26.5-33.7%) alone. In 1992, similar tests were repeated using S. carpocapsae at 0.2 and 0.5 nematodes/cm², and SeMNPV at 14 and 35 PIB/cm². The combination of 0.5 nematodes/cm² + 35 PIB/cm² resulted in significantly higher larval mortality (64.0%) than either pathogen alone (41.5-49.0%). Steinernema carpocapsae and SeMNPV produced additive effects on beet arlnyworm mortality. Persistence of S. carpocapsae was 12-24 hours and SeMNPV was 96-120 hours on soybean.     

Validation of a Comprehensive Process-Based Model for the Biological Control of Beet Armyworm,Spodoptera exigua,with Baculoviruses in Greenhouses

This paper describes the validation and sensitivity analysis of a process-based simulation model (BACSIM) for the control of beet armyworm, Spodoptera exigua, with baculoviruses. Model predictions are compared to results of independent greenhouse experiments in which second, third, or fourth instar larvae of S. exigua in chrysanthemum plots are treated with different concentrations of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and S. exigua MNPV (SeMNPV), two viruses with distinct differences in infectivity and mean time to kill. BACSIM provides robust predictions for the control of S. exigua populations in greenhouse chrysanthemum with both AcMNPV and SeMNPV. Mortality levels caused by AcMNPV and SeMNPV were generally predicted within a 25% margin of error compared to the observed values. None of the deviations was higher than 40%. All values of simulated foliage consumption, caused by S. exigua populations treated with AcMNPV or SeMNPV applications, fell within 95% confidence intervals of measurements. Simulated time to kill was, in general, lower than the measurements. This discrepancy may be caused by the behavior of S. exigua larvae which feed on the underside of chrysanthemum leaves where they are protected from polyhedra. This suggests that the larval foraging behavior may play an important role in the efficacy of baculovirus applications and should be further studied experimentally. This validated model can be used for the pretrial evaluation of the efficacy of genetically modified baculoviruses as biological control agents and for the optimization of spraying regimes in chrysanthemum cultivation.     

甜菜夜蛾HSP90基因克隆及高温胁迫下其表达量的变化

为阐明热激蛋白90（heat shock protein 90, HSP90）在甜菜夜蛾Spodoptera exigua (Hübner)幼虫抵抗高温过程中的作用, 克隆了其HSP90基因cDNA全长序列, 并检测了在系列高温胁迫下不同龄期幼虫体内其相对表达量。根据已报道的热激蛋白90基因序列同源性设计简并引物, 利用反转录聚合酶链式反应（RT-PCR）结合cDNA末端快速扩增（RACE）技术成功克隆了甜菜夜蛾HSP90基因全长cDNA（GenBank登录号FJ862050）。该cDNA序列开放阅读框长2 154 bp, 编码717个氨基酸, 预测的相对分子量和等电点分别为82.6 kD和5.0。该序列具有HSP90家族的典型特征和特殊的功能结构域, 并且与多种生物的HSP90基因序列有较高的同源性。为了研究HSP90抵抗高温的作用, 构建荧光定量RT-PCR体系, 检测了37, 39, 41, 43和45℃胁迫下甜菜夜蛾不同龄期幼虫体内HSP90表达量的变化。结果表明, 高温胁迫对甜菜夜蛾幼虫体内的HSP90表达具有明显的诱导作用。幼虫体内HSP90表达量随着温度升高呈增加的趋势。43℃和45℃胁迫下, 各龄幼虫体内HSP90的表达量均显著高于常温（P< 0.05）, 但不同龄期之间没有显著差异。这说明HSP90在甜菜夜蛾幼虫抗高温中起到重要作用。