A Baculovirus Expression System for Insect Cells

The review considers the biology of baculoviruses, construction of transfer vectors for the baculovirus expression system (BES), selection of recombinant baculoviruses, approaches to expression of multimeric proteins, and BES potentialities and prospects.     

虫酰肼对甜菜夜蛾多巴脱羧酶和酪氨酸羟化酶的抑制作用

虫酰肼模拟昆虫蜕皮激素的作用干扰新表皮的形成。为了探讨虫酰肼对昆虫新表皮形成的影响是否与抑制表皮形成相关酶的活性有关, 本研究应用高效液相色谱-荧光检测法（HPLC-RP）, 测定了甜菜夜蛾Spodoptera exigua 5龄幼虫用虫酰肼处理不同时间（24, 48和72 h）后多巴脱羧酶和酪氨酸羟化酶的活性。结果表明： 用LC₁₁ （28.41 μmol/L）和LC₃₃ （85.23 μmol/L）两个亚致死剂量的虫酰肼处理5龄幼虫后, 多巴脱羧酶和酪氨酸羟化酶的活性均受到明显抑制, 高浓度的抑制作用大于低浓度的抑制作用。随着处理时间的延长, 同一剂量的抑制作用逐渐增强。进一步测定虫酰肼处理24, 48和72 h后5龄幼虫血淋巴、 脂肪体、 中肠、 表皮和头部的多巴脱羧酶和酪氨酸羟化酶的活性, 可看出虫酰肼对幼虫不同组织的多巴脱羧酶和酪氨酸羟化酶的活性也具有相似的抑制作用。结果提示, 虫酰肼对甜菜夜蛾幼虫多巴脱羧酶和酪氨酸羟化酶活性具有明显抑制作用, 幼虫新表皮形成受阻可能与虫酰肼抑制多巴脱羧酶和酪氨酸羟化酶的活性有关。     

斜纹夜蛾线粒体复合物ⅢFe-S蛋白基因克隆、序列分析及在不同发育阶段的表达特征

Fe-S蛋白(rieske iron-sulfur protein, RISP)是线粒体复合物Ⅲ的关键蛋白亚基之一, 在呼吸链电子传递中起着重要作用。本文利用RT-PCR技术和RACE方法获得了斜纹夜蛾Spodoptera litura (Fab.)线粒体复合物ⅢRISP基因, 该基因序列全长4 413 bp, 含有3个内含子; 开放阅读框长816 bp, 编码271个氨基酸。经氨基酸序列比对, 与鳞翅目家蚕Bombyx mori和小菜蛾Plutella xylostella的Fe-S蛋白一致性较高, 分别为83%和79%, 与其他目昆虫的RISP一致性较低。qRT-PCR分析结果表明, SlitRISP基因在斜纹夜蛾不同发育阶段的表达水平存在显著差异, 即幼虫期和成虫期的表达量显著高于卵期和蛹期, 其中幼虫期又以第4和5龄幼虫表达水平显著高于初孵幼虫以及第1, 2, 3和6龄幼虫。斜纹夜蛾SlitRISP基因的克隆成功为今后对其功能研究以及作为靶标设计新型高效杀虫剂提供了基础。     

Food consumption, utilization and detoxification enzyme activity of larvae of three polyphagous noctuid moth species when fed the botanical insecticide rotenone

We assessed the effects of the isoflavonoid rotenone, an insecticidal allelochemical occurring in various legumes, on larval performance of three polyphagous noctuid species: the corn earworm (CEW), the fall armyworm (FAW) and the southern armyworm (SAW). As rotenone concentration was increased up to 1% fresh mass in an artificial diet, neither mortality (for all three species) nor food consumption (for SAW and FAW) was significantly affected, but developmental time of the latter two species was prolonged. In contrast, for CEW developmental time was shortened and food consumption declined, especially at the two highest rotenone concentrations. Final biomass of all three species declined as dietary rotenone increased.Analysis of covariance (ANCOVA) was used to evaluate larval food utilization analogous to, but without the potential statistical problems of, the traditional ratio-based indices approximate digestibility (AD) and efficiency of conversion of digested food (ECD). Frass output, statistically adjusted to account for differences in consumption, increased with rotenone concentration for all three species, suggesting that, when ingested, this allelochemical interfered with the digestion and/or absorption of ingested food, especially at the higher concentrations tested.Significant statistical interactions in the ANCOVA necessitated the use of utilization plots to examine the linear regressions between biomass gain and absorption at each rotenone concentration. At the two highest concentrations, rotenone tended to reduce the amount of food absorbed by the larvae, as well as their ability to convert the absorbed food to biomass.When piperonyl butoxide, an inhibitor of the polysubstrate monooxygenase (PSMO) system known to metabolize rotenone, was added to the diet along with 0.5 or 1% rotenone, mortality increased significantly only for FAW, approaching or exceeding 50%. The only apparent effects of dietary rotenone on PSMO (aldrin epoxidase) activity (adjusted by ANCOVA for the covariate, crude homogenate protein) were for CEW, where activity decreased in all rotenone treatments compared to the control, and for SAW, where activity at the 3 lowest rotenone concentrations (0.001–0.1% fm) declined about 50% below that of larvae on the control diet, whereas at 0.5 and 1% rotenone, it increased approximately 2-fold over that on the control diet.     

Lessons from Unsuccessful Attempts to Establish Spodoptera pectinicornis (Lepidoptera: Noctuidae), a Biological Control Agent of Waterlettuce

We released nearly 332 000 Spodoptera pectinicornis individuals for biological control of the floating aquatic weed Pistia stratiotes (waterlettuce) at 22 sites in Florida during December 1990-December 1997. Predation (by boat-tailed grackles, fire ants, and spiders) and Allee effects (resulting from rapid dispersal of adults) prevented populations from persisting during early attempts. These efforts consisted of small releases at multiple sites, with no nurturing of the introduced populations. Modification of our initial approach resulted in a series of release strategies that consisted of multiple releases at fewer sites and employed progressively more intensive nurturing of the introduced populations. The most successful strategy incorporated: (i) cages to restrict adult dispersal and exclude flying predators; (ii) plant-free zones around the cages to exclude crawling predators; (iii) enhancement of the nutritional quality of waterlettuce in the cages; and (iv) frequent inoculation of the cages with laboratory-reared insects. Use of this strategy resulted in several provisionally-established S. pectinicornis populations, numbering as much as 23.5 S. pectinicornis individuals m -2 , and produced up to seven post-release generations. Unfortunately, all of these incipient populations declined until S. pectinicornis was undetectable. We conclude that field populations of this moth no longer persist in Florida, and offer an introspective assessment of how this project might have been improved.     

斑痣悬茧蜂寄生、发育和生殖特征与寄主幼虫日龄的关系

【目的】“圆屋顶形”假说认为,对单寄生性姬蜂和茧蜂适合度而言,中间龄期幼虫寄主的品质高于更早和更晚龄期幼虫。该假说得到许多研究支持,但这些研究常以寄主幼虫脱皮划分虫龄,很少观测生殖特征,从而难以确切和全面描述适合度随寄主生长发育变化而变化的关系。本研究旨在检验“圆屋顶形”假说。【方法】本研究以斜纹夜蛾Spodoptera litura不同日龄幼虫为寄主,观测斑痣悬茧蜂Meteorus pulchricornis寄生和发育特征,并测定成蜂生殖力。【结果】线性回归分析表明,雌蜂对中间日龄寄主幼虫的寄生率大于对两端日龄寄主幼虫的寄生率;蜂卵至成虫的存活、成虫体型大小及其生殖力（产卵量）等适合度相关特征均表现出中间日龄寄主幼虫处理大于两端日龄幼虫处理。【结论】研究结果支持“圆屋顶形”假说。     

Localization and functional analysis of SeMNPV IE1 in mammalian cells

In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), belonging to group Ⅱ nucleopolyhedrovirus, was studied in mammalian cells.We amplified the SeMNPV ie1 gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn't influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.     

我国甜菜夜蛾大尺度暴发频度与广域温度和广域降雨量关系的预测模型

甜菜夜蛾Spodoptera exigua (Hübner)是我国多种农作物上的重要害虫, 在我国许多地区频繁暴发成灾。为探索甜菜夜蛾种群动态规律并建立种群数量发生趋势预测模型, 作者应用时间序列分析和逐步回归分析方法研究了我国广域（较大范围）温度和广域降雨量变化趋势对我国广域甜菜夜蛾年暴发频度的影响规律。结果表明: 甜菜夜蛾发生的长期趋势和年间波动状况均与广域温度和广域降雨量具有复杂的影响关系。在1979-2008年间, 我国甜菜夜蛾暴发频度呈现出波浪式上升趋势, 其暴发指数平均年递增率为0.076, 而我国广域温度（以27个省市级气象台数据统计为例）在1990-2008年间的平均年递升率为0.039℃, 即我国甜菜夜蛾暴发频度上升趋势与我国广域温度升高趋势同向而行。作者从52个因素（当年和上年1-12月各月及全年日均温和月均降雨量）中筛选出了具有显著回归影响（P<0.05或0.01）的10个因素进入回归模型, 初步找出了能够预测广域甜菜夜蛾暴发趋势指数的温度与降雨量或其组合因素, 并使其模型达到99%以上的历史符合率和预测准确度。作者认为, 广域温、 雨因素与广域甜菜夜蛾暴发趋势指数的这种密切相关性, 不是偶然的巧合, 而是必然的环境（温度和降雨量）作用于生物（甜菜夜蛾）的因果关系。     

斜纹夜蛾取食对Bt玉米挥发物组成和含量的影响

采用顶空吸附法和气相色谱-质谱法研究了两个Bt玉米品种(5422Bt1, 5422CBCL)和一个同源常规玉米品种(5422)在健康和经斜纹夜蛾取食危害后两种状态下释放的挥发物组成和含量差异.结果表明：健康状态下,5422释放了(E)-β-罗勒烯、芳樟醇、（3E）-4,8-二甲基-1,3,7-壬三烯(DMNT)和(E)-β-法尼烯4种挥发物;5422Bt1释放了芳樟醇和DMNT 2种挥发物;而5422CBCL只释放芳樟醇1种挥发物.虫害后,在5422和5422Bt1的挥发物中鉴定出了萜类、醇类、 酯类和吲哚等12种化合物;在5422CBCL中鉴定出了10种,缺少(Z)-3-己烯-1-醇乙酸酯和橙花叔醇;3个品种释放的萜烯类化合物种类和含量均明显增加,其中,5422和5422Bt1增加了8种,5422CBCL增加了7种;5422Bt1和5422CBCL 的芳樟醇和DMNT释放量高于5422,其余化合物的释放量均低于常规品种,尤其是(E)-β-法尼烯显著低于5422.可见,经斜纹叶蛾取食诱导后,Bt玉米释放的挥发物种类和含量与常规玉米不同,且不同的Bt玉米品种（Bt11与MON810）之间也存在差异.     

长柄木霉和泾阳链霉菌复配对烟苗生长及其抗病性的影响

以烟苗为试验材料,采用室内拮抗试验和活体筛选试验相结合的方法,研究了长柄木霉和泾阳链霉菌两种生防菌复配的亲和性和抑菌广谱性,并分析了生防菌及其组合对烟苗生长、诱导抗性和黑胫病防效的影响.结果表明：两生防菌株无相互抑制作用,具有较高的亲和性,并且两菌株本身及其代谢产物对不同生境下的病原菌(黑胫病、猝倒病和赤星病)均具有较高的抑菌活性和较广的抑菌谱.两菌株复配能影响根部和地上部的形态建成,促进烟苗根系和茎、叶的生长,显著增加地下部根系体积、根系总表面积、根长和根系平均直径以及地上部茎高、茎围、叶长、叶宽和生物量,具有显著的促生效能,并且两菌株复配的促生效应优于单独接种.生防菌组合能显著提高烟苗根系超氧化物歧化酶、过氧化物酶和过氧化氢酶等防御酶活性,对烟草黑胫病的相对防治效果达到了69.3%,具有显著的防病效能.研究表明长柄木霉和泾阳链霉菌是具有较高亲和性的增效组合,两菌株复配后在防病、促生方面能够发挥协同增效作用,可有效促进烟苗生长和防治烟草黑胫病.