宁波地区斜纹夜蛾幼虫寄生蜂调查研究

2007—2008年的6～11月对宁波地区重要害虫斜纹夜蛾的寄生性天敌进行了调查,发现寄生蜂有18种,主要种类有：马尼拉陡胸茧蜂Snellenius manilae、侧沟茧蜂Microplitis sp、棉铃虫齿唇姬蜂Carnpoletis chlorideae和斑痣悬茧蜂Meteorus pulchricornis,并对主要寄生蜂与斜纹夜蛾幼虫的田间消长动态进行了系统观察。     

甜菜夜蛾几丁质合成酶基因A和B启动子的克隆

几丁质不仅是昆虫的表皮和围食膜的主要成分,也是一个非常关键的害虫控制靶标,主要通过几丁质合成酶（chitin synthase,CHS）基因合成。本文在克隆甜菜夜蛾Spodoptera exigua的两个几丁质合成酶基因（SeCHSA和SeCHSB）cDNA和基因组序列的基础上,从基因的5′末端设计特异性引物和构建特定的基因组文库, 采用PCR的方法获得了5′端侧翼序列。通过5′RACE的方法确定SeCHSA和SeCHSB基因的转录起始位点后,获到了启动子序列。这为研究昆虫几丁质合成和转录调控奠定了基础。     

斜纹夜蛾种群控制的研究概况

斜纹夜蛾Spodoptera litura(Fabricius)是重要的农业害虫之一。为了控制斜纹夜蛾,使农作物免受危害,前人研究了化学防治、杀虫灯和性诱捕器诱杀、植物诱集和驱避、转基因抗虫品种以及生物防治对其种群控制作用。化学防治在压制斜纹夜蛾种群暴发、减轻农作物遭受损失方面作出了巨大贡献,但日益增强的抗药性、农药残留造成的环境污染、对天敌的杀伤和人畜安全的影响等问题不断受到关注,人们开始对该害虫的一些新的治理策略进行了探讨。在这些无公害的控制技术中,植物诱集和生物防治是2种值得提倡的有效措施。不少植物源提取物和昆虫生长调节剂对斜纹夜蛾幼虫具有极好的效果,是值得研发的2种新型生物制剂。香芋和蓖麻等诱集作物对斜纹夜蛾有较好的诱集效果,如果能将作物诱集和释放天敌昆虫或病原微生物制剂结合,使之成为一套有效的生态控制体系,将可现实斜纹夜蛾种群的持续控制。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

雷帕霉素对二种鳞翅目昆虫细胞自噬和凋亡的影响

以2种鳞翅目昆虫细胞为材料,采用雷帕霉素进行处理,初步研究自噬作用与昆虫细胞凋亡的关系。结果表明:雷帕霉素能够提高家蚕细胞系BMN-e细胞的自噬水平,并能诱导BMN-e细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤能抑制雷帕霉素诱导的BMN-e细胞凋亡。相反,雷帕霉素虽能诱导斜纹夜蛾细胞系SL-HP细胞的自噬水平提高,但不能诱导斜纹夜蛾Spodoptera litura(Fabricius)细胞发生凋亡;雷帕霉素的预处理能抑制放线菌素D诱导的斜纹夜蛾细胞系SL-HP细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤对放线菌素D诱导的细胞凋亡没有影响。因此家蚕Bombyx mori细胞自噬水平的提高与细胞凋亡具有正相关性,而斜纹夜蛾细胞自噬水平的提高与细胞凋亡不相关,相反还对细胞凋亡的诱导具有一定的抑制作用。     

Nitrogen and water affect direct and indirect plant systemic induced defense in cotton

Plants have direct and indirect constitutively produced and inducible defenses against herbivores and pathogens, which can substantially aid in their ability to defend themselves. However, very little is known about the influence of agronomic factors on such defenses. Here, we tested the effects of nitrogen levels and water availability on the ability of cotton plants to deter feeding by Spodoptera exigua through induction of anti-feedants, and to attract Microplitis croceipes through systemic induction of volatile emission. Cotton plants were grown with various nitrogen levels and were either exposed to water stress or normal water before being exposed to S. exigua for 48 h for induction of defenses. Dual choices of various nitrogen and water treatments were provided to M. croceipes in flight tunnel bioassays. Dual choices of leaf tissue from the various nitrogen and water treatments were provided to S. exigua larvae. Both water stress and nitrogen levels under and over the recommended levels increased leaf tissue consumption and decreased attraction of M. croceipes to the plants. Analyses of induced volatiles released from herbivore damaged plants indicate that their concentrations differ among the nitrogen levels tested with plants receiving no nitrogen or twice the recommended dose having amounts much lower than plants receiving the recommended dose. Because both direct and indirect plant defense mechanisms are negatively affected by improper nitrogen and insufficient water, we argue that these factors should be considered for a better natural control of pests in cotton and most probably in other crops.     

Variability in the behavioural responses of three generalist herbivores to the most abundant coumarin in Daphne laureola leaves

We evaluated the responses of three generalist herbivores (Lepidoptera: Noctuidae) to the most abundant coumarin in young leaves of spurge-laurel, Daphne laureola L. (Thymelaeaceae), a perennial shrub consistently fed upon by several noctuid species. Pseudenargia ulicis Staud and Noctua janthe Borkhausen are natural herbivores of the species in southeastern Spain, whereas Spodoptera littoralis Boisduval is a model species frequently used to address the antifeedant role of secondary compounds. Discrimination between control and coumarin-coated D. laureola leaves was investigated by short-term choice experiments. We found species-specific behavioural responses to the treatment: P. ulicis preferred the control leaves, whereas S. littoralis did not discriminate between leaf treatments, although it clearly avoided coumarin when incorporated into an artificial diet, and N. janthe preferred the coumarin-coated leaves. Furthermore, N. janthe larvae reduced consumption when the proportion of treated leaves ingested increased and consumption of S. littoralis larvae was also reduced in coumarin-containing diet under no-choice conditions. Our results highlight that different herbivore species feeding simultaneously on the same host plant respond differently to a single chemical defence compound, likely constraining a directional response of the plant to selection.     

Insecticidal and synergistic effects of Majorana hortensis essential oil and some of its major constituents

The essential oil from leaves of Majorana hortensis Moench (Lamiaceae) was isolated by hydrodistillation with a yield of 1.6% (wt/wt). The insecticidal activity of the oil was evaluated against fourth instars of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and adults of Aphis fabae L. (Hemiptera: Aphididae). The oil showed a remarkable toxic effect against S. littoralis in a topical application assay (LD₅₀ = 2.48 μg per larva) and in a residual film assay (LC₅₀ = 3.14 g/l). The oil of M. hortensis also exhibited a pronounced toxic effect against A. fabae adults with LC₅₀ values of 1.86 and 2.27 g/l in rapid dipping and residual film assays, respectively. Gas chromatography-mass spectrometry analyses of M. hortensis essential oils revealed the presence of 31 compounds and the main components were terpinen-4-ol (30.0%), γ-terpinene (11.3%), and trans -sabinene hydrate (10.8%). Repeated column chromatography of M. hortensis oil on silica gel led to the isolation of two major constituents, which were characterized based on ¹H-nuclear magnetic resonance and mass spectrometric data, as terpinen-4-ol and γ-terpinene. These two components were examined for their insecticidal and synergistic activities towards S. littoralis and A. fabae . Terpinen-4-ol and γ-terpinene exhibited a significant insecticidal activity against both insects, but γ-terpinene was more toxic than terpinen-4-ol. When tested in a binary mixture with the synthetic insecticides profenofos and methomyl, it was found that both compounds enhanced the insecticidal activity of these insecticides by two- to threefold. These results show that terpinen-4-ol and γ-terpinene have a synergistic effect on the insecticidal activities of synthetic insecticides profenofos and methomyl.     

Screening Spanish isolates of steinernematid nematodes for use as biological control agents through laboratory and greenhouse microcosm studies

Entomopathogenic nematodes (EPNs) are one of the best non-chemical alternatives for insect pest control, with native EPN strains that are adapted to local conditions considered to be ideal candidates for regional biological control programs. Virulence screening of 17 native Mediterranean EPN strains was performed to select the most promising strain for regional insect pest control. Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) Rioja strain produced 7%, 91% and 33% larval mortality for the insects Agriotes sordidus (Illiger) (Coleoptera: Elateridae), Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), respectively, and was selected as the most promising strain. The S. feltiae Rioja strain-S. littoralis combination was considered the most suitable to develop the Rioja strain as a biocontrol agent for soil applications. The effect of soil texture on the virulence of the Rioja strain against S. littoralis was determined through dose-response experiments. The estimated LC₉₀ to kill larvae in two days was 220, 753 and 4178 IJs/cm² for soils with a clay content of 5%, 14% and 24%, respectively, which indicates that heavy soils produced negative effects on the virulence of the Rioja strain. The nematode dose corresponding to the LC₉₀ for soils with a 5% and 14% clay content reduced insect damage to Capsicum annuum Linnaeus (Solanales: Solanaceae) plants under greenhouse microcosm conditions. The results of this research suggest that an accurate characterization of new EPN strains to select the most suitable combination of insect, nematode and soil texture might provide valuable data to obtain successful biological control under different ecological scenarios in future field applications.     

斜纹夜蛾泛素基因的克隆及表达

泛素介导的蛋白质降解途径对脑内蛋白的选择性降解起着重要作用。设计一对简并引物,从斜纹夜娥(Spodoptera litura)细胞中克隆了泛素基因的编码区,CenBank登录号AF436066。序列分析表明,该编码区的长度为228bp,编码由76个氨基酸组成的、分子质量为8．56kD的蛋白,其等电点为6．56。同源性比较发现,斜纹夜峨泛素基因不仅与其它真核生物的泛素基因在氨基酸水平上具有96％以上的相似性,而且与斜纹夜蛾核多角体病毒(SpltMNPV)泛素基因的同源性为84％。RT—PCR分析发现,泛素基因在所检测的斜纹夜蛾幼虫多种组织,尤其是脂肪体中均有表达。采用构建的原核表达载体pQEUB,在大肠杆菌M15中诱导并高效表达出了带有His—tag的重组融合蛋白,薄层扫描分析得知靶蛋白约占总蛋白的37％。利用Ni—NTA亲和层析胶纯化得到重组融合蛋白,经SDS—PAGE鉴定为单一区带,为进一步研究S．litura泛素在SpltMNPV感染中的作用打下了基础。