斜纹夜蛾Cecropin D成熟肽的原核表达及活性检测

采用RT-PCR方法从斜纹夜蛾Spodoptera litura脂肪体组织中扩增得到了Cecropin D成熟肽基因序列,分析发现Cecropin D成熟肽与斜纹夜蛾Cecropin B之间存在2个氨基酸残基的差异。将获得的基因序列连接入原核表达载体pGEX-4T-1,并在原核细胞中实现了该蛋白的融合表达。SDS-PAGE结果表明,诱导后的宿主菌比未诱导菌中多出了一条融合蛋白表达带,诱导后1 h就可以检测到该蛋白,从诱导后1 h到5 h该蛋白在表达量上没有明显的差异。生长曲线显示在IPTG诱导后宿主菌的生长受到明显的抑制,纯化后的蛋白对细菌具有一定的抑制作用。     

Binding properties of pheromone-binding protein 1 from the common cutworm Spodoptera litura

Pheromone-binding proteins (PBPs) were formerly thought to act as passive pheromone carriers. However, recent studies, particularly in Drosophila melanogaster, suggest that PBPs are involved in the recognition of semiochemicals, thus making ligand-binding studies more meaningful. Previously, we cloned three PBPs from Spodoptera litura (Slit), and showed that SlitPBP1 is much more abundant than the other two, particularly in male antennae. To investigate the ligand specificity of SlitPBP1, we expressed the protein in a bacterial system and performed binding experiments with the three components of the specific sex pheromones (Z9-14:Ac, Z9,E11-14:Ac and Z9,E12-14:Ac), as well as with 26 volatile ligands. The results indicated that SlitPBP1 bound all three sex pheromone components with dissociation constants between 0.6 and 1.1 μM. The same protein also bound with comparable affinities several pheromone analogs, but not plant volatiles. The presence of a double bond was the most important element for a strong binding, while its position and configuration also affected the affinity. Finally, the binding of pheromone components is strongly affected by pH, showing a critical pH value corresponding to isoelectric point of the protein. This suggests that a pH-dependent conformational mechanism might exist in SlitPBP1 for pheromone binding and release.     

Cloning and expression of apolipophorin-III from the common cutworm,Spodoptera litura

We have cloned apolipophorin-III (apoLp-III) cDNA from adult fat body of Spodoptera litura. The sequence encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. The circular dichroism spectrum from the purified apoLp-III indicated a considerable content of α-helix. Sequence alignment showed that S. Litura apoLp-III has a relatively high degree of sequence identity with the apoLps-III of lepidopteran, Manduca sexta (72%), Galleria mellonella (67%), Bombyx mori (60%). These alignments with four lepidopteran apoLps-III showed highly identical residues and conservative replacements at a degree of 86%. Levels of mRNA from last instar larval fat body and adult fat body were compared through Northern blot analysis using ³²P-labeled 704 bp apoLp-III cDNA probe. A 850 bp mRNA was detected in both stages and mRNA level of day 1 adult fat body was much higher than that of last instar larval fat body. The tissue-distribution of apoLp-III mRNA in adult ovary and testis was also examined and we confirmed the presence of apoLp-III mRNA in ovary and testis although apoLp-III was expressed in these tissues at very low levels compared with the adult fat body. Arch. Insect Biochem. Physiol. 39:166–173, 1998. © 1998 Wiley-Liss, Inc.     

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm,Spodoptera litura

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.     

幼虫密度对斜纹夜蛾抗病能力的影响

为了阐明斜纹夜蛾Spodoptera litura Fabricius幼虫密度对其抗病能力的影响,在室内条件下(温度23℃±1℃,相对湿度75%)对不同幼虫密度(1、2、5、10、15头/皿(直径为12cm))饲养的斜纹夜蛾幼虫抵抗斜纹夜蛾核型多角体病毒侵染的能力及其免疫指标进行了研究。结果表明:幼虫密度对斜纹夜蛾幼虫接种核型多角体病毒后的存活率、存活时间及血淋巴酚氧化酶活性影响显著。随着幼虫密度的增加,接种核型多角体病毒后幼虫的存活率降低,存活时间缩短。当幼虫密度达到15头/皿时,幼虫存活率显著低于其它幼虫密度。不同幼虫密度幼虫的存活时间以1头/皿的最高,15头/皿的最低,且二者之间差异显著。幼虫血淋巴中酚氧化酶活性随幼虫密度的增加而明显降低,当幼虫密度达到5头/皿时,幼虫酚氧化酶活性显著低于1头/皿的。另外,幼虫溶菌酶活性和血细胞总数受幼虫密度影响不显著。不同密度幼虫抗病性的变化与其血淋巴中酚氧化酶活性的变化趋势较为一致。所以斜纹夜蛾幼虫抗病能力的降低可能与幼虫酚氧化酶活性的下降有关。因此,幼虫密度是影响斜纹夜蛾幼虫抗病性变化的重要因子。     

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。     

Pathogenicity of entomopathogenic fungi to eggs and larvae of Spodoptera litura,the common cutworm

The eggs and second instar larvae of Spodoptera litura were treated with different concentrations of conidial suspensions of six isolates of fungi belonging to five species, Metarhizium anisopliae var. anisopliae (Metschnikov) Sorokin (ARSEF 7487), Lecanicillium muscarium (Petch) Zare & W Gams (ARSEF 7037 and ARSEF 6118), Cordyceps cardinalis (ARSEF 7193), Fusarium lateritium Nees (ARSEF 8291/MTCC 9050) and Aspergillus sp. (ARSEF 8519). In bioassay with unscaled eggs, M. anisopliae, C. cardinalis, F. lateritium and Aspergillus sp. resulted in 100% mortality above 10⁶ conidia/mL. However, with scaled egg masses, the highest hatching rate (56%) was observed with L. muscarium (ARSEF 6118) whereas the lowest hatchings were observed in the case of M. anisopliae followed by L. muscarium (ARSEF 7037), Aspergillus sp., F. lateritium, and C. cardinalis. The larvae were also found susceptible to all isolates in a dose dependent manner. Three promising isolates against larvae, viz. M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) resulted in average percent mortalities of 88, 89 and 77%, respectively at ≈10⁸ conidia/mL. When both larvae and the leaves (provided as food) were treated with ≈10⁸ conidia/mL, mortality further increased for these isolates. Based on the mortality data and the LC₅₀ values, we suggest that M. anisopliae, F. lateritium and L. muscarium (ARSEF 7037) could be developed as potential biocontrol agents against rice cutworm in IPM programs.     

寄主植物对斜纹夜蛾酯酶和乙酰胆碱酯酶活性的影响

利用生物测定与生物化学的方法,就取食不同寄主植物的2个斜纹夜蛾[Spodoptera litura(Fab.)]品系对丙溴磷和灭多威的敏感性及其体内的酯酶与乙酰胆碱酯酶的活性变化进行了研究。结果表明,取食不同寄主植物的斜纹夜蛾对丙溴磷和灭多威的敏感性不同。在敏感品系中,取食不同食料的斜纹夜蛾后代对灭多威的敏感性顺序为:烟草<棉花<大豆<人工饲料;对丙溴磷而言,敏感性顺序则为:棉花<烟草<大豆<人工饲料。而在田间抗性品系中,对丙溴磷的敏感性顺序为:棉花<烟草<人工饲料<大豆;对灭多威的敏感性顺序为:棉花<烟草<大豆<人工饲料。此外,在田间抗性和敏感品系中,取食不同食料的斜纹夜蛾体内的酯酶活性之间均存在显著差异,对其乙酰胆碱酯酶的活性也有程度不同的诱导效应,但并不会引起乙酰胆碱酯酶的质变。     

斜纹夜蛾抗药性及其防治对策的研究进展

斜纹夜蛾Spodopteralitura是多种作物的重要害虫 ,对有机氯、有机磷、氨基甲酸酯类、拟除虫菊酯类以及Bt制剂等杀虫剂均产生抗药性。本文对斜纹夜蛾抗药性的形成与发展、抗性机理及其防治对策等方面进行了综述     

Genomic sequence analysis of granulovirus isolated from the tobacco cutworm, Spodoptera litura

Background

Spodoptera litura is a noctuid moth that is considered an agricultural pest. The larvae feed on a wide range of plants and have been recorded on plants from 40 plant families (mostly dicotyledons). It is a major pest of many crops. To better understand Spodoptera litura granulovirus (SpliGV), the nucleotide sequence of the SpliGV DNA genome was determined and analyzed.

Methodology/Principal Findings

The genome of the SpliGV was completely sequenced. The nucleotide sequence of the SpliGV genome was 124,121 bp long with 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 or more nucleotides. The 133 putative ORFs covered 86.3% of the genome. Among these, 31 ORFs were conserved in most completely sequenced baculovirus genomes, 38 were granulovirus (GV)-specific, and 64 were present in some nucleopolyhedroviruses (NPVs) and/or GVs. We proved that 9 of the ORFs were SpliGV specific.

Conclusions/Significance

The genome of SpliGV is 124,121 bp in size. One hundred thirty-three ORFs that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. No chitinase or cathepsin genes, which are involved in the liquefaction of the infected host, were found in the SpliGV genome, explaining why SpliGV-infected insects do not degrade in a typical manner. The DNA photolyase gene was first found in the genus Granulovirus. When phylogenic relationships were analyzed, the SpliGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XecnGV), which belong to the Type I-granuloviruses (Type I-GV).     

Laboratory evaluation of flight activity of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae)

Abstract  The flight activity of Spodoptera litura in tethered conditions is evaluated using a computer-mediated flight-mill in the laboratory. The 3–4-day-old moths fly longer and farther than those of other ages. Male and female moths exhibit similar flight activity. Mating status does not influence the flight duration and distance of 2-day-old females. However, these two flight parameters with a 6-day-old mated female is significantly lower than that of unmated ones. The optimum temperature for flight ranged from 16–24°C, whereas the optimum RH ranged from 60%–100%. During 72-h period, the total flight duration and distance of 1-day-old male and female moths were 19.6 h (± 5.8) and 83.3 km (± 28.4), and 24.0 h (± 7.0) and 105.4 km (± 37.4), respectively. These results indicate that S. litura has a great potential to undertake long-distance migratory flights.     

Design, synthesis and antibacterial activity of cecropin-like model peptides

In order to investigate structure-activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C18-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.     

Effects of sublethal dose of chlorfluazuron on testicular development and spermatogenesis in the common cutworm, Spodoptera litura

This paper describes the physiological mechanism of action of chlorfluazuron on testicular development and spermatogenesis when sublethal doses (LD₁₀: 1.00 ng/larva or LD₃₀: 3.75 ng/larva) are applied topically to the cuticle of newly moulted fifth instars of the common cutworm Spodoptera litura (F.) (Lepidoptera, Noctuidae). These doses disrupt the growth and development of testes by decreasing the volume and weight of testes and thickness of testes sheath as compared with that of the controls. Sublethal doses of chlorfluazuron also significantly reduce the protein content of the testis, but do not affect the carbohydrate and lipid contents in newly emerged treated males when measured in μg/mg of testis as compared with that of the controls. Additionally, such doses disrupt spermatogenesis by reducing the number and size of eupyrene and apyrene sperm bundles in the testis. Very few or no eupyrene sperm bundles are observed in vas deferens of pre‐ and newly moulted adults compared with controls. This result shows that the transfer of sperm bundles from testes to vas deferens is delayed in treated males. The effects of chlorfluazuron on testicular development and spermatogenesis is thought to be one of the factors responsible for the reduction in fecundity, fertility and hatchability caused by sublethal doses of chlorfluazuron.     

Effect of plant secondary metabolites on common cutworm,Spodoptera litura (Lepidoptera: Noctuidae)

The effect of various flavonoids, lectins and phenyl β‐_D‐glucoside on larval survival, weights and the activities of digestive (total serine protease and trypsin) and detoxifying (esterase and glutathione‐S‐transferase) enzymes of Spodoptera litura larvae at 7 days after treatment was studied through diet incorporation assay. Flavonoids (rutin, chlorogenic acid, quinic acid, caffeic acid, naringenin, quercitin, kaempferol, myricetin, catechin, and ferulic acid) were incorporated in artificial diet at 100, 500 and 1000 ppm, lectins: groundnut leaf lectin (GLL), concavalin A (ConA) and phenyl β‐_D‐glucoside at 1, 2 and 5 μg/mL. Flavonoids such as rutin, quercitin and kaempferol at 1000 ppm were more toxic to S. litura larvae than quinic acid, caffeic acid, naringenin, myricetin, catechin, and ferulic acid. Larval growth and development were significantly reduced in S. litura larvae fed on a diet with GLL and ConA at 5 μg/mL compared to the larvae fed at 2 and 1 μg/mL concentrations. The larvae fed on flavonoid‐treated diets showed significant reduction in serine protease, trypsin and esterase activities. The flavonoids such as rutin, chlorogenic acid, quinic acid, naringenin, quercitin, kaempferol and myricetin, and lectins, GLL and ConA can be utilized in insect control programs.     

斜纹夜蛾铁硫亚基蛋白的表达及功能鉴定

铁硫亚基蛋白(rieske iron-sulfur protein, RISP) 是线粒体复合物Ⅲ的关键蛋白亚基之一, 在呼吸链电子传递过程中起到重要作用。本研究通过RT-PCR克隆得到斜纹夜蛾Spodoptera litura RISP基因SlitRISP的ORF, 构建了pET32a-SlitRISP原核表达载体, SDS-PAGE和Western blot检测结果显示, SlitRISP原核表达蛋白以包涵体的形式存在于菌体沉淀中, 且RISP抗体可成功用于该蛋白的免疫印迹检测。为了进一步鉴定SlitRISP在斜纹夜蛾离体细胞系SL-1中的功能, 通过向细胞内转染siRNA, 利用RNAi技术沉默SL-1中的SlitRISP。qRT-PCR结果表明, 分别经50 nmol/L和100 nmol/L siRNA处理48 h后, SL-1中SlitRISP的表达均几乎完全被抑制; Western blot结果显示, SL-1中SlitRISP含量显著低于CK。当SL-1 SlitRISP被成功沉默后, 通过检测SL-1线粒体膜电位、 细胞ATP含量和细胞增殖抑制率鉴定RISP在线粒体电子传递过程中的重要作用。流式细胞仪测定结果表明, 经50 nmol/L和100 nmol/L siRNA处理24 h后, SL-1线粒体膜电位相对于CK分别降低23.52%和11.32%, 而处理48 h后, SL-1线粒体膜电位则分别升高5.58%和27.66%; siRNA处理24 h和48 h后, SL-1 ATP含量相对于CK分别降低82.71%和84.50%, 最终导致SL-1细胞增殖抑制率分别为53.64%和67.94%。这些结果表明SlitRISP在SL-1中参与线粒体膜电位的形成和细胞ATP的合成。介于RISP在线粒体电子传递链中的重要作用, 其可能成为新型杀虫作用靶标, 这可为研制新型呼吸抑制剂提供参考。     

Cloning and expression analysis of six small heat shock protein genes in the common cutworm, Spodoptera litura

Small heat shock proteins (sHsps) are probably the most diverse in structure and function among the various superfamilies of stress proteins. To explore the diverse functions of insect sHsps, six sHsp cDNAs were cloned from the midgut cDNA library of Spodoptera litura, and a phylogenetic tree was constructed based on the conserved α-crystalline domains. The expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) induction, were studied by real-time quantitative PCR. Based on sequence characteristics and phylogenetic relationships, the six SlHsps were classified into three independent groups: BmHsp20.4 like proteins (SlHsp19.7, 20.4, 20.7, 20.8), BmHsp26.6 like protein (SlHsp20), and BmHsp21.4 like protein (SlHsp21.4). All the SlHsps showed highest expression in the Malpighian tubules. The four BmHsp20.4 like protein genes were up-regulated by thermal stress and showed expression variation with development. SlHsp20 exhibited lower expression levels in both egg and larval stages than in pupal and adult stages. SlHsp21.4 retained a constant expression level during all life stages. The expression of both SlHsp20.4 and SlHsp20.8 was significantly up-regulated by 20E. These results indicate that sHsps play diverse functions in S. litura: the BmHsp20.4 like proteins are involved in both thermal adaptation and development; SlHsp20 does not respond to temperature stress but possibly plays a role in metamorphosis; SlHsp21.4 may have no direct relationship with either thermal response or development.     

斜纹夜蛾两个天蚕素D基因的克隆及序列分析

天蚕素是昆虫抵御病菌入侵的一类抗菌肽家族。根据斜纹夜蛾Spodoptera litura天蚕素B基因设计特异引物,通过PCR扩增得到2个新的天蚕素基因部分序列,分别命名为cecD1和cecD2（GenBank登录号分别为EF555567和EF555568）。2个基因编码同一个天蚕素D蛋白,该蛋白的成熟肽与天蚕素B存在2个氨基酸残基差异。序列分析发现cecD1和cecD2中分别包含568 bp和377 bp的内含子序列,它们有相同的5′和3′拼接位点,A+T含量分别为59.7%和69.8%,符合大多数真核生物内含子高A+T含量的特征。     

Flight capability and fatty acid level in triacylglycerol of long-distance migratory adults of the common cutworm, Spodoptera litura

The larvae of Spodoptera litura were reared on an artificial diet, and the flight capability, and triacylglycerol (TG) level plus its fatty acid composition in 3-day-old sexually mature and non-fed adults were compared. In males, during 3 hr of tethered flight, the levels of abdominal TG and its fatty acid components did not change. But thereafter the TG and fatty acids, significantly unsaturated fatty acids in TG declined in their levels with the prolongation of flight, unsaturated fatty acids being exhausted preceding saturated fatty acid decline. When males were tested by tethered flight for 20 hr, some could fly for nearly the whole period, and were judged to be able to fly for approximately 24 hr, depending on the level of residual TG. Fatty aids in TG decreased in females similarly to males during tethered flight and some females with fully developed ovaries deposited eggs after 12 hr of flight similarly to non-flown individuals, which supports the long-distance flight capability even in sexually mature females. These results are discussed with regard to the overseas migration of this moth.     

Overseas migration of the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae), from May to mid-July in East Asia

Spodoptera litura (Fabricius) males were monitored with sex-pheromone traps from spring to early summer in 1999, 2002, 2003, 2009, and 2010 in four regions in East Asia: western Japan, South Korea, China, and Taiwan. The backward trajectory analysis was conducted to investigate whether any air current arrived over the monitoring sites around the dates when distinguished increases in catches of >100 insects per day occurred. The catch data showed abrupt catch increases. Some of the catch increases coincidentally appeared among monitoring sites several hundreds of kilometers apart. These catch increases were found to coincide with the arrival of southwesterly air currents within 24–36 h from southern China and/or Taiwan. Other evidence related to the overseas migration are discussed in detail. The results of this and previous studies suggest that S. litura males immigrate into western Japan and South Korea in spring to early summer using air currents from southern China and/or Taiwan.