The complete genome sequence of a single-stranded RNA virus from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois)

The complete genome sequence of a single-stranded RNA virus infecting the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by sequencing cDNA prepared from insects collected from the Mississippi Delta. The 9655 nucleotide positive-sense single-stranded RNA genome of the L. lineolaris single-stranded RNA virus (LyLV-1) contained a single open reading frame of 8958 nucleotides encoding a 2986 amino acid genome polypeptide. The open reading frame was flanked by untranslated regions of 603 and 69 nucleotides at the 5'- and 3'- ends of the genome, respectively. Database searches and homology based modeling was used to identify four capsid proteins (VP1-VP4), helicase/AAA-ATPase, cysteine protease (C3P), protease 2A, and the RNA-directed RNA polymerase (RdRp). In addition, a region with weak similarity to the eukaryotic structural maintenance of chromosome (SMC) domain was identified near the amino-terminal of the polyprotein and adjacent to the VP1 domain. The amino acid sequence of LyLV-1 was approximately 44.4% similar to that of sacbrood virus (SBV) of the honey bee. The genomic organization of both viruses showed remarkable similarity with the exception of highly divergent amino acid regions flanking fairly conserved structural and non-structural polypeptide regions. High similarity to the SBV genome and similarities in the genome organization and amino acid sequence with the viruses of the family Iflaviridae suggested that LyLV-1 was a novel member of this family. Virus particles were 39 nm in diameter and appeared to transmit vertically via eggs. Although this virus may only cause covert infections under normal conditions, the potential for using this virus in biological control of L. lineolaris is discussed.     

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。     

Morphological, phylogenetic and biological characteristics of Ectropis obliqua single-nucleocapsid nucleopolyhedrovirus

The tea looper caterpillar, Ectropis obliqua, is one of the major pests of tea bushes. E. obliqua single-nucleocapsid nucleopolyhedrovirus (EcobSNPV) has been used as a commercial pesticide for biocontrol of this insect. However only limited genetic analysis for this important virus has been done up to now. EcobSNPV was characterized in this study. Electron microscopy analysis of the occlusion body showed polyhedra of 0.7 to 1.7 mum in diameter containing a single nucleocapsid per envelope of the virion. A 15.5 kb genomic fragment containing EcoRI-L, EcoRI-N and HindIII-F fragments, was sequenced. Analysis of the sequence revealed that the fragment contained eleven potential open reading frames (ORFs): lef-1, egt, 38.7k, rr1, polyhedrin, orf1629, pk-1, hoar and homologues to Spodoptera exigua multicapsid NPV (SeMNPV) ORFs 15, 28, and 29. Gene arrangement and phylogeny analysis suggest that EcobSNPV is closely related to the previously described Group II NPV. Bioassays on lethal concentration (LC(50) and LC(90)) and lethal time (LT(50) and LT(90)) were conducted to test the susceptibility of E. obliqua larvae to the virus.     

REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua

Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringiensis Cry1Ca toxin and non-exposed insects. Based on the SSH results, full cDNA sequences coding for four homologous proteins were obtained. Quantitative and semi-quantitative RT-PCR showed the increased expression of the genes coding for these proteins after exposure to different B. thuringiensis toxins as well as after infection with baculovirus. The proteins were named REPAT after their increased expression in Response to Pathogen. REPAT1, a member of this family, was recombinantly expressed using the baculovirus expression system, revealing the glycosylated nature of the protein. Recombinant baculoviruses expressing REPAT1 were used to infect larvae from S. exigua, showing that expression of REPAT1 was reducing the virulence of baculovirus to the infected larvae. Together, these results suggest a role for REPAT1 in mitigating pathological effects.     

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.     

A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV)

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.     

Epizootics caused by a nuclear polyhedrosis virus in populations of spodoptera exigua in southern Spain

A nuclear polyhedrosis virus (NPV) has been identified as the etiological agent causing epizootics in natural populations of Spodoptera exigua in the south of Spain. The incidence of this NPV has been determined from samples of larvae collected from sunflower fields and vegetable greenhouses at two sites located 500 km apart. The NPV was the only disease agent identified at both sites. Through the period surveyed the percentage of NPV‐infected larvae increased from 4.3% to 23% and from 22.5% to 100% in S. exigua larvae collected from sunflower fields and vegetable greenhouses respectively. No mortality attributed to the NPV was found in Spodoptera littoralis, Heliothis armigera, Peridroma saucia and Plusia gamma larvae collected in the same survey from the same sites. This is the first report of epizootics caused by NPV in S. exigua populations in field and greenhouse crops in Europe.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

不同方式诱导植物抗性对甜菜夜蛾幼虫及其寄生蜂斑痣悬茧蜂发育表现的影响

【目的】明确诱导植物抗性对植食性昆虫甜菜夜蛾 Spodoptera exigua (Hübner) 及内寄生蜂斑痣悬茧蜂 Meteorus pulchricornis (Wesmael) 生长发育的影响。【方法】采用取食损伤、机械损伤及外源喷施3种浓度 (0.01, 0.1和1 mmol/L) 茉莉酸甲酯 (MeJA) 3种方式来诱导菜薹抗性,观察甜菜夜蛾初孵幼虫及其寄生蜂斑痣悬茧蜂的发育表现。【结果】取食取食损伤和茉莉酸甲酯处理的菜薹后,甜菜夜蛾幼虫存活率显著降低,茉莉酸甲酯浓度与幼虫存活呈负相关; 取食0.1 mmol/L MeJA处理的菜薹后,甜菜夜蛾幼虫体重显著低于清水对照。甜菜夜蛾取食取食损伤和0.1 mmol/L MeJA处理的菜薹,其寄生蜂斑痣悬茧蜂幼虫的死亡率显著增加,同时结茧率显著降低;0.1 mmol/L MeJA处理的菜薹使成蜂体重显著低于对照。不同处理间斑痣悬茧蜂的卵巢发育无明显差异,卵巢由8~10根卵巢管组成,羽化24 h后成熟卵数量为7~10个,成熟卵的长度和宽度分别为350和78 μm。【结论】结果说明,取食损伤和茉莉酸甲酯处理诱导的菜薹反应会影响甜菜夜蛾的存活和大小,进而抑制斑痣悬茧蜂生长。     

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.     

甜菜夜蛾对氯氟氰菊酯抗性的表皮穿透机理

用三种方法测定了采自南京江浦的甜菜夜蛾Spodoptera exigua对氯氟氰菊酯的抗性。结果表明,抗性水平的次序为3龄幼虫点滴法（5 499.5倍）和5龄幼虫点滴法（3 973.2倍）＞3龄幼虫浸叶法（1 041.6倍）＞5龄幼虫叶片夹毒法（24.7倍）,因此该品系触杀毒力的抗性水平至少为胃毒毒力LD50的160倍。用¹⁴C标记氯氟氰菊酯测定甜菜夜蛾抗性和敏感品系5龄幼虫表皮穿透率结果表明,处理后8h,抗性品系5龄幼虫的表皮穿透率仅为敏感品系幼虫表皮穿透率的55.5%。证实表皮穿透率的降低是产生抗性的一个重要机理。     

甜菜夜蛾虫酰肼抗性品系选育及相对适合度研究

本试验在室内对甜菜夜蛾Spodoptera exigua(Hübner)武汉敏感品系进行了8代群体汰选后分别于9、10、12代采用了单对汰选。结果表明虫酰肼处理后各单对品系后代幼虫存活率发生明显分离,F9(s1)、F10(s2)和F12(s3)代幼虫平均存活率分别为48.34%、11.72%和2.37%,筛选后分别为84.00%、83.33%和7.29%。经过12代汰选后,汰选品系抗性倍数为敏感品系的5.24倍,且单对汰选世代对虫酰肼的抗性发展较快,其抗性倍数分别是群体汰选的1.27、1.67和1.38倍。比较单对汰选世代与群体汰选世代某些生物学特性,显示单对汰选世代并不存在生长发育和生殖不利性,表明在虫酰肼群体汰选中穿插几代单对汰选可以加速抗性品系的选育。汰选品系相对于敏感品系具有0.31的适合度,表明甜菜夜蛾对虫酰肼产生抗性后存在适合度缺陷。     

Stimulation and attenuation of induced resistance by elicitors and inhibitors of chemical induction in tomato (Lycopersicon esculentum) foliage

Elicitors and inhibitors of chemical induction were used to manipulate the activities of several putative defense-related proteins in leaves of the tomato, Lycopersicon esculentum Mill. The four presumptive defenses manipulated were proteinase inhibitors, polyphenol oxidase, peroxidase, and lipoxygenase. The elicitors used were jasmonic acid, methyl jasmonate, ultraviolet light, and feeding by larvae of the noctuid, Helicoverpa zea Boddie; the inhibitors used were salicylic acid and acetylsalicylic acid. These chemical manipulations were combined with short-term growth assays using larvae of the generalist noctuid, Spodoptera exigua Hubner, in order to assess the relative roles of the proteins in induced resistance to S. exigua. When activities of proteinase inhibitors and/or polyphenol oxidase in leaf tissue were high (e.g., in damaged or elicited plants), growth rates of larvae of S. exigua were low; when activities of polyphenol oxidase and proteinase inhibitors were low (e.g., in undamaged or damaged, inhibited plants), growth rates of larvae were high. In contrast, high activities of peroxidase and lipoxygenase were not associated with decreases in suitability of leaf tissue for S. exigua. The association of high levels of proteinase inhibitors and polyphenol oxidase with resistance to S. exigua – irrespective of the presence or absence of damage – strongly implicates these proteins as causal agents in induced resistance to S. exigua.     

Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC delta-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.     

Isolation and characterization of strain of Bacillus thuringiensis subsp. kenyae containing two novel cry1-type toxin genes

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cry1-5 and cry1-12, in addition to the cry1Ja1 gene. The deduced amino acid sequences of cry1-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and Cry1Ja crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.     

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.