保幼激素类似物对斜纹夜蛾核型多角体病毒在宿主血淋巴中增殖动态的影响

通过研究保幼激素类似物(juvenile hormone analogues, JHA) methoprene对斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）在宿主血淋巴中增殖的影响,以探明JHA促进 SpltNPV增殖的初步机制,为阐明JHA促进病毒增殖提供更全面的理论依据。应用SDS PAGE及Western blot法,分析了methoprene对SpltNPV多角体蛋白(polyhedron, POLH)在宿主斜纹夜蛾6龄幼虫血淋巴中合成的影响。结果表明：经methoprene处理后2～3天可明显促进幼虫血淋巴液中POLH的合成。在此基础上,通过荧光定量PCR检测,发现methoprene对SpltNPV在幼虫血淋巴液复制的影响主要发生在处理后的第4和第5天,该期间polh基因的拷贝数比对照显著增加,拷贝数的峰值达1.22×10¹⁰/mL。     

OB增效剂对斜纹夜蛾核多角体病毒的增效作用

本文以OB作为斜纹夜蛾核多角体病毒（Spodoptera litura nucleopolyhedrovirus,SpltNPV）的增效剂,对斜纹夜蛾Spodoptera litura幼虫进行生物测定,结果表明,在0．25％-1．00％的浓度范围内,随着OB增效剂浓度的提高,其对斜纹夜蛾核多角体病毒的增效作用也随着提高,最高增效倍数达85．1倍;在2～4龄幼虫范围内,随着虫龄的增大,OB增效剂对斜纹夜蛾核多角体病毒的增效作用也增加;而随着温度的升高,增效剂的增效作用无显著提高。     

斜纹夜蛾核多角体病流行的时间动态

通过对病病幼虫进行跟踪观察,观察了病毒不同浓度处理下,斜纹夜蛾核多角体病的田间流行动态结果表明,在试验病毒浓度范围（３．１＊１０＾５－３．１＾１０＾８ＰＩＢｓ．ｍｌ＾－１）内,幼虫大多在喷施病毒后第４天开始发病,第５－７天为发病高峰,第５－６天开始病死,第６－天为病死高峰,宿主现患高峰民发病高峰基本一致。宿主种群发病和病死时间分布可用时间－剂量－死亡率模型较好地拟合,模型模拟与实测值有较好的吻合（     

斜纹夜蛾核多角体病毒几丁质酶基因的克隆及序列分析

以苜蓿丫纹夜蛾核多角体病毒（Ａｕｔｏｇｒａｐｈａ ｃａｌｉｔｉｒｎｉｃａ ｍｕｌｔｉｎｕｃｌｅｏｃａｐｓｉｄ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＡｃＭＮ－ＰＶ）基因组含几丁质酶基因（ｃｈｉＡ）的ｐｓｔＩ Ｍ片段为探针,通过Ｓｏｕｔｈｅｒｎ杂交将斜纹夜蛾核多角体病毒（Ｓｐｏｄｏｐｔｅｒａ ｌｉｔｕｒａ ｎｕｃｌｅａｒ ｐｏｌｙｈｅｄｒｏｓｉｓ ｖｉｒｕｓ,ＳｐｌｔＮＰＶ）的ｃｈｉ     

斜纹夜蛾核多角体病毒抑制苜蓿丫纹夜蛾核多角体病毒诱导的斜纹夜蛾细胞凋亡

野生型苜蓿丫纹夜蛾核多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)感染斜纹夜蛾(Spodoptera litura)细胞系Sl-zsu-1,可引起典型的细胞凋亡;但可以在草地夜蛾(Spodoptera frugiperda)细胞Sf-9中复制并形成多角体.比较了AcMNPV p35基因在病毒感染两种细胞的复制和转录情况,认为p35在非受纳细胞中及时有效的表达能阻止细胞发生凋亡;共感染实验结果表明,斜纹夜蛾核多角体病毒(Spodoptera litura multicapsid nucleopolyhedrovirus, SpltMNPV)可以抑制AcMNPV诱导的细胞凋亡并可帮助病毒进行复制,推测SpltMNPV基因组中与p35同源的p49基因挽救了细胞的自杀行为.     

斜纹夜蛾核多角体病毒几丁质酶基因上游4.0kb的序列分析

本文报道了斜 诳蛾核多角体病毒几丁质酶基因（chiA）上游约4.0kb范围内的序列,它包括了六个读码框（ORF1－6）,其长度分别为156bp、279bp、540bp、369bp、1281bp和228bp,可编码的氨基酸长度分别为51、98、179、122、426和75个,分子量分别为6.15kD、11.46kD、21.70kD、14.69kD 、47.59kD和9.09kD。在ORF1ORF2、ORF3起始密码前分别有一个、二个及一个杆状病毒早期启动子基序CAGT;在ORF4、ORF5起始密码前各有一个及二个杆状病毒晚期启动子基序TAAG。在ORF1、ORF4、ORF5终止密码下游有真核生物mRNA转录poly（A)加尾信号。ORF4为AcMNPV ORF53、BmNPV ORF42、OpMNPV ORF56、LdMNPV ORF54的同源基因。ORF1、ORF2、ORF6与已知的杆状病毒基因沿有同源性,可能为三个新的基因。     

斜纹夜蛾核型多角体病毒分子生物学研究进展

斜纹夜蛾核型多角体病毒(Spodoptera lituramulticapsid nucleopolyhedrovirus,SpltMNPV)属NPV科A亚群。近几年有关该病毒的序列测定、基因结构、功能和表达调控等系统的分子生物学研究工作进展迅速,特别是对一些重要基因的结构分析,有助于筛选毒力较强的杀虫毒株,并为这一病毒杀虫剂的改良和发展以及组建昆虫杆状病毒表达载体奠定基础。综述了与SpltMNPV相关的分子生物学领域的研究进展。     

“三明治”杂交法对斜纹夜蛾核型多角体病毒DNA克隆株的鉴定

用限制性内切酶HindⅢ将斜纹夜蛾核型多角体病毒DNA切割为A、B、C、D_1、D_2、E、F七个片段,与载体pUC18体外连接后转化大肠杆菌TG_1。通过“三明治“杂交法鉴定,分别获得七个片段的克隆株。但A片段克隆株中外源DNA片段发生了缺失。     

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone (JH) is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA (cDNA) was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate‐binding motifs were predicted in the protein. Modeling of the 3‐D structure showed that the protein consisted of eight α‐helixes linked with loops, with no β‐sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae of S. litura.     

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.     

自然界斜纹夜蛾核型多角体病毒的基因型多态性

【目的】从基因组序列角度进一步揭示自然界斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）的基因型多态性。【方法】病毒克隆A5, F1, X3 和 X15分别以活体克隆法分离自SpltNPV埃及株、 日本福冈株和日本小笠原株。根据SpltNPV基因组全序列（GenBank登录号: AF325155）和海灰翅夜蛾核型多角体病毒（S. littoralis NPV, SpliNPV）部分基因序列（GenBank登录号: X99377, X99376 和X98924）设计引物, PCR扩增获得A5, F1, X3 和 X15的多角体蛋白（polyhedrin, polh）基因和ORF18~ORF23序列。【结果】根据多角体蛋白基因序列, X3和X15属于SpltNPV型, 而A5和F1属于SpliNPV型。将A5, F1, X3 和 X15的ORF18~ORF23与SpltNPV和SpliNPV相应的基因序列进行同源性比较。结果发现, F1与SpliNPV以及X3与SpltNPV的核苷酸序列相似性高, 但X3的ORF20在172~558 nt处缺失387 bp。尽管依据多角体蛋白基因序列X15属于SpltNPV型, 但对于ORF18~ORF23序列, X15与SpliNPV的相似性高于与SpltNPV的相似性。同样, A5属于SpliNPV型, ORF18~ORF20与SpliNPV相应的核苷酸序列相似性高, 但ORF21与SpltNPV相应的核苷酸序列一致性为100%, 特别是ORF22, SpltNPV的特有序列出现在A5的基因组中, 而且与SpltNPV的ORF22一致性为100%; 反过来, ORF23又与SpliNPV相应的核苷酸序列相似性高。【结论】所有这些都表明, SpltNPV在自然界不仅存在基因型多态性, 而且即使属于同一基因型, 它们的基因组序列也有显著差异。可利用SpltNPV在自然界的这种异质性筛选适宜防治斜纹夜蛾幼虫的株系。     

Biological and genetic characterization of a Pakistani isolate of Spodoptera litura nucleopolyhedrovirus

Spodoptera litura is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to Bacillus thuringiensis toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of S. litura nucleopolyhedrovirus (SpltNPV, Baculoviridae), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD₅₀) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT₅₀) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes polyhedrin, DNApol, pif-2 and lef-8 confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of S. litura in Pakistan.     

Effect of nucleopolyhedrovirus infection of Spodoptera litura larvae on host discrimination by Microplitis pallidipes

The behaviour and oviposition of solitary endoparasitoid Microplitis pallidipes Szepligeti (Hymenoptera: Braconidae) were monitored to investigate the ability of the parasitoids to distinguish between nucleopolyhedrovirus (NPV)-infected and noninfected Spodoptera litura Fabricius (Lepidoptera: Noctuidae) larvae. The results indicated that the parasitoid searching time and the time until the first parasitoid attack on infected larvae were greater than those recorded on noninfected larvae; the number of infected larvae attacked by parasitoids, the percent of first attacks and parasitism rate in infected larvae were lower than those on noninfected larvae; and these differences were all significant 3 to 5 days postexposure of the larvae to a dose of 1.6 × 10⁸ occlusion bodies (OB)· ml^?1 and significant 4 and 5 days postexposure of the larvae to a dose of 1.6 × 10⁷ OB·ml^?1. The lowest dosage (1.6 × 10⁶ OB·ml^?1) had no significant effect on the above index values. In a field cage experiment, we found that the percentage of infected larvae parasitized by M. pallidipes gradually decreased as the time after NPV inoculation (1.6 × 10⁸ OB·ml^?1) increased, and that M. pallidipes significantly preferred to oviposit in healthy larvae from day 3 to day 5 after virus inoculation. Our research concluded that this parasitoid's ability to discriminate between healthy and infected hosts increased as virus concentration increased and as the time between exposure of hosts to virus and subsequent exposure to parasitoids increased.     

Effect of juvenile hormone and pyriproxyfen treatments on the production of Spodoptera litura nuclear polyhedrosis virus

Efficient production is essential for developing baculoviruses into marketable bioinsecticides. In this study, we evaluated how juvenile hormone (JH) III and the JH analog pyriproxyfen affected the production efficiency of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (SpltNPV) in SpltNPV‐inoculated early sixth instars. Both the JH III and pyriproxyfen treatments significantly enhanced the body size and weight of S. litura, which resulted in a 1.5‐ to 2‐fold increase in the production of occlusion bodies (OBs). The OBs obtained from JH‐ and pyriproxyfen‐treated larvae were as pathogenic as those produced in untreated larvae. More importantly, JH‐ or pyriproxy‐fen‐treated larvae consumed less than twice the amount of food consumed by two untreated larvae. These results indicate that JH and pyriproxyfen treatments improve the production efficiency of SpltNPV in S. litura. SpltNPV infection affected the development of S. litura not only as larvae, but also during subsequent growth stages and generations.     

斜纹夜蛾（Spodoptera litura）的胚胎发育

本文利用实验胚胎学方法详细研究了斜纹夜蛾胚胎发育的全过程。结果表明：在２４±１℃的恒温条件下,仅需８７小时即可孵化出幼虫,同时证明了马氏管起源于外胚层。     

斜纹夜蛾核型多角体病毒不同分离株基因序列的同源性分析

研究SpltMNPV不同分离株及SpltMNPV分离株与SpliNPV间基因序列的同源性,为SpltMNPV分离株的利用提供理论基础。根据已发表的斜纹夜蛾核型多角体病毒（SpltMNPV）中国株（Zh）基因组全序列（AF527603）和海灰翅夜蛾核型多角体病毒（SpliNPV）Not I-D片段序列（AF527603）设计引物,PCR方法扩增得到SpltMNPV日本福冈株（Fu）、埃及株（Eg）和小笠原株（Og）的ORF39～ORF42和ORF119～ORF124编码区全序列。SpltMNPV不同分离株及SpltMNPV分离株与SpliNPV间基因序列的相似性比较, Zh株和Og株,Eg株、Fu株和SpliNPV的相似性高,而Zh株和Eg株、Fu株或SpliNPV,Og株和Eg株、Fu株或SpliNPV的相似性都比较低。亦即SpltMNPV 3种基因型,B型和C型的同源性高,A型与B型或C型的同源性比较低,但A型与SpliNPV的同源性高;同一基因型内不同分离株（Eg株和Fu株）的同源性高。ETG分子进化分析表明Eg株、Fu株和SpliNPV处于一个分支,而Eg株、Fu株和SpliNPV与Zh株和Og株则处于不同的分支。因此推断Eg株和Fu株为SpliNPV的分离株,而Og株为SpltMNPV的分离株。