Comparative study on the susceptibility of cutworms (Lepidoptera: Noctuidae) to Agrotis segetum nucleopolyhedrovirus and Agrotis ipsilon nucleopolyhedrovirus

The common cutworm (Agrotis segetum) and the black cutworm (Agrotis ipsilon) are serious soil pests of many vegetable and field crops all over the world. We have demonstrated the cross-infectivity of two baculoviruses, A. segetum nucleopolyhedrovirus (AgseNPV) and A. ipsilon nucleopolyhedrovirus (AgipNPV) for these two insect pests. The susceptibility of A. segetum to AgipNPV was confirmed by DNA restriction endonuclease analyses of DNA isolated from virus harvested from infected A. segetum larvae. For an initial comparison of both viruses, partial polyhedrin sequences were amplified by PCR, cloned, and sequenced. Both viruses shared a very similar polyhedrin gene sequence resulting in only three amino acid substitutions. Phylogenetic analyses clearly demonstrated that both viruses belong to NPV group II and are most closely related to a clade consisting of Spodoptera exigua NPV, Spodoptera frugiperda NPV, and Spodoptera littoralis NPV. Since AgipNPV shows high virulence for both cutworm species, it appears to be a suitable candidate as a single biological control agent of A. segetum and A. ipsilon.     

A simplified method for the characterization of nuclear polyhedrosis virus genomes

Abstract A simplified restriction endonuclease analysis procedure is described which allows the characterization of baculovirus DNA obtained directly from a single larvae without purification of virus. This rapid method was used to demonstrate the genomic stability of nuclear polyhedrosis viruses (NPVs) from Agrotis segetum, Euxoa messoria and Mamestra brassicae after several passages in Euxoa scandens     

A simplified method for the characterization of nuclear polyhedrosis virus genomes

A simplified restriction endonuclease analysis procedure is described which allows the characterization of baculovirus DNA obtained directly from a single larvae without purification of virus. This rapid method was used to demonstrate the genomic stability of nuclear polyhedrosis viruses (NPVs) from Agrotis segetum, Euxoa messoria and Mamestra brassicae after several passages in Euxoa scandens.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.     

用免疫学技术研究两种颗粒体病毒的亲缘关系

从新疆分离的黄地老虎颗粒体病毒（ＡｓＧＶ）和惊纹鸣夜蛾颗粒体病毒（ＡｅＧＶ）除感染各自的分离寄主外,均能感染对方的分离寄主。用琼脂糖双扩散、对流免疫电泳,斑点－酶联免疫吸附和线免疫电泳等免疫学技术检测ＡｓＧＶ和ＡｅＧＶ的免疫学亲缘关系,结果表明二者间存在非常密切的亲缘关系。并对线免疫电泳技术的效用进行了讨论。     

Mortality of Cutworm Larvae Is Not Enhanced by Agrotis segetum Granulovirus and Agrotis segetum Nucleopolyhedrovirus B Coinfection Relative to Single Infection by Either Virus

Mixed infections of insect larvae with different baculoviruses are occasionally found. They are of interest from an evolutionary as well as from a practical point of view when baculoviruses are applied as biocontrol agents. Here, we report mixed-infection studies of neonate larvae of the common cutworm, Agrotis segetum, with two baculoviruses, Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV). By applying quantitative PCR (qPCR) analysis, coinfections of individual larvae were demonstrated, and occlusion body (OB) production within singly infected and coinfected larvae was determined in individual larvae. Mixtures of viruses did not lead to changes in mortality rates compared with rates of single-virus treatments, indicating an independent action within host larvae under our experimental conditions. AgseNPV-B-infected larvae showed an increase in OB production during 2 weeks of infection, whereas the number of AgseGV OBs did not change from the first week to the second week. Fewer OBs of both viruses were produced in coinfections than in singly infected larvae, suggesting a competition of the two viruses for larval resources. Hence, no functional or economic advantage could be inferred from larval mortality and OB production from mixed infections of A. segetum larvae with AgseNPV-B and AgseGV.     

Polyhedrin gene sequence and phylogenetic analysis of a nucleopolyhedrovirus isolated from Orgyia ericae Germar.

Molecular characterization of a nucleopolyhedrovirus (NPV) isolated from the diseased larva of Orgyia ericae Germar was firstly analyzed. The genomic size of O. ericae NPV was estimated to be 134.6 kb by restriction endonuclease analysis. The gene encoding the major structural protein, polyhedrin, was cloned and sequenced. Phylogenetic analyses using polyhedrin sequences revealed that O. ericae NPV (OeNPV) was a member of the Group II NPVs and was closely related to the BusuSNPV and OpSNPV cluster. Electron microscopic observations confirmed that OeNPV was a single nucleocapsid type virus (SNPV).     

Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction.

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. The amino acid sequences of the polyhedrin gene were compared in twenty-six NPVs. A highly conserved DNA sequence within the coding region of the polyhedrin gene was targeted for amplification. One pair of degenerate PCR primers was designed to produce fragments of about 430 bp. The NPVs detected by this technique were Autographa californica NPV, Bombyx mori NPV, Hyphantria cunea NPV, Spodoptera exigua NPV, S. litura NPV, and Lymantria dispar NPV. This technique would be useful in monitoring the distribution of NPVs and release of the wild type and recombinant NPVs.     

Continuous single sensillum recording as a detection method for moth pheromone components in the effluent of a gas chromatograph

ABSTRACT. Single sensillum recordings were made from male antennal pheromone receptors of Agrotis segetum (Schiff.) (Noctuidae) and Adoxophyes orana (F.v.R.) (Tortricidae). A gas chromatograph (GC) was used to prepare and to deliver the odour stimuli. Samples of female abdominal gland extracts were injected into the column of the GC and the responses of the receptor cells to the effluent were recorded continuously. The receptor cells responded with an increase of their action potential frequency during elution of the major pheromone components. The pheromone receptors of Agrotis showed a much higher sensitivity than those of Adoxophyes. In the extract of female glands from Agrotis , compounds were detected which have not been identified in previous studies of the sex pheromone of this species. It is suggested that the combination of GC techniques with direct single sensillum recording may serve as a valuable supplement to electro-antennographic techniques.     

黄地老虎种群动态与积雪的关系

利用新疆北部地区黑光灯下黄地老虎越（Agrotis segetum）冬代种群数据（1992～2001年）,分析了越冬代种群数量与冬季积雪最大厚度和天数的关系。结果表明,黄地老虎越冬代发蛾的时间为4月30日～6月15日,越冬代种群数量年际间存在很大波动性。黄地老虎越冬代种群数量与当年度覆雪天数（1月1日～4月初）呈显著负相关（R^2=0．6022,P〈0．0083）,而与冬季最大积雪厚度（R^2=0．23,P=0．1577）和秋冬季（11月～翌年4月）积雪天数（R^2=0．099,P〈0．3748）相关不显著。当年度覆雪天数可以作为黄地老虎预测中的一项重要的参考指标。     

2009年云南省边境地区健康儿童肠道病毒测序定型和ECHO7、ECHO13病毒的基因特征分析

了解2009年缅甸入境健康儿童和云南省口岸地区健康儿童肠道病毒(enterovirus,EV)带毒情况并对埃柯病毒7型(ECHO7)和埃柯病毒13型(ECHO13)的基因特征进行了描述。采集9个边境口岸小于15岁的健康儿童粪便标本271份,进行病毒分离和基因测序定型。271份便标本中共检测到EV30株(带毒率为11.1%),其中脊髓灰质炎病毒(poliovirus,PV)6株(阳性率2.8%),均为疫苗株,未发现脊灰野病毒;检测到非脊灰病毒(NPV)24株(阳性率8.9%)。经VP1区核苷酸序列测定,24株NPV全部为人类肠道病毒B组(HEV-B,6个血清型),其中13株为埃柯病毒7型(echovirus 7,ECHO7,占54.17%),5株为ECHO13(占20.83%)。未分离到HEV-A组,HEV-C组和HEV-D组病毒。2009年缅甸入境健康儿童和云南省口岸地区健康儿童中肠道病毒携带率较高,且均为HEV-B组病毒,其中主要型别为ECHO7和ECHO13,这两种病毒存在基因多样性特点(即存在不同的基因型)。     

Host range and virulence of five baculoviruses from lepidopterous hosts

The host range and virulence of five insect baculoviruses (two multiply-enveloped nuclear polyhedrosis viruses (MNPVs) from Agrotis segetum and Mamestra brassicae; one singly-enveloped NPV from Plusia gamma and two granulosis viruses (GVs) from A. segetum and Pieris brassicae) were studied for seven lepidopterous pests of temperate agriculture (A. segetum, Agrotis exclamationis, Lacanobia oleracea, M. brassicae, Noctua pronuba, P. gamma and Pieris rapae). None of the viruses killed all species but M. brassicae MNPV failed to infect only P. rapae. The other viruses were restricted to the homologous host, or members of its genus or subfamily. In all examples except A. segetum GV, the median lethal dose for the most susceptible host, was less than 22 virus inclusion bodies and median lethal times for all infections ranged from 5·5 to 16·6 days. The low susceptibility of A. segetum and other noctuids to GV infections is discussed in relation to the structure of inclusion bodies and the nature of the infectious unit in baculoviruses.     

混合感染后杆状病毒间的增效作用——病毒蛋白及核酸的初步分析

AsNPV+HasNPV、AsNPV+HaNPV、AsNPV+PsNPV分别感染烟青虫、棉铃虫和粘虫幼虫,对分离到的核型多角体病毒(AsNPV+HasNPV)-Helicoverpa assulta、(AsNPV+HaNPV)- H.Armigera和(AsNPV+PsNPV)-Pseudaletia separata,经电镜观察,多角体蛋白及病毒粒子蛋白SDS-PAGE电泳,病毒核酸的限制性内切酶酶解分析等研究,证明各病毒的多角体形态不规则,大小差异极大,病毒粒子为杆状,(AsNPV+HasNPV)-H.Assulta 和(AsNPV+HaNPV)-H.Armigcra病毒粒子有单粒和多粒包埋类型, (AsNPV+PsNPV)-P.Separata为多粒包埋型。各病毒的多角体蛋白基本上只有一种多肽,分子量为25 000道尔顿左右。(AsNPV+HasNPV)-H.Assulta、(AsNPV+HaNPV)-H.armigera和(AsNPV+PsNPV)-P.Separata的病毒粒子分别有10、14、5条多肽,分子量大小在13 500～98 000,13 000～88 000,18 500～52 000道尔顿之间。病毒核酸经EcoRI,HindIII,HindIII+BamHI酶解,其DNA的酶切位点,大小及DNA的总分子量与AsNPV和原寄主的Has-NPV,HaNPV和PsNPVDNA的酶切图谱存在一定差异,混合病毒侵染昆虫后新复制的病毒核酸发生一定的变化,从而导致病毒蛋白和病毒形态的变化。混合感染后AsNPV对Has-NPV、HaNPV和PsNPV的侵染有明显的增效作用,其机理有待深入研究。     

黄地老虎幼虫取食诱导的棉花植株挥发物驱避雌蛾产卵

[目的]评价黄地老虎Agrotis segetum幼虫取食危害诱导棉花植株产生的虫害诱导植物挥发物(herbivore-induced plant volatiles,HIPVs)对雌蛾产卵选择的影响,并鉴定HIPVs的主要组分及其功能,以筛选对雌蛾产卵有吸引或驱避作用的活性成分.[方法]利用气相色谱-质谱联用仪(GC...     

Construction of a Genetic Map of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus by Marker Rescue of Temperature-Sensitive Mutants

Mutations of seven temperature-sensitive mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (NPV) were mapped with respect to the physical restriction map of the A. californica NPV DNA by marker rescue. DNAs from two distantly related NPVs of the multiply embedded type and two NPVs of the singly embedded type were unable to rescue two A. californica NPV mutants.     

Molecular characterization and evolution of pheromone binding protein genes in Agrotis moths

Pheromone-binding proteins (PBPs) are soluble transporter proteins that increase the capture and the solubilization of pheromone molecules in the lymph surrounding the olfactory receptors. A polymerase chain reaction-based method was used to identify PBP genes in Agrotis species for an evolutionary genomic study of noctuid moth PBPs. From genomic DNA we determined the structure of different PBP genes in the two closely related species, Agrotis ipsilon and A. segetum. In all, we clearly identified four genes (Aips-1, Aips-2, Aseg-1 and Aseg-2) that represent two distinct PBP orthology groups. We found that the four genes have the same exon-intron structure and that they comprise three exons and two introns but differ in length mainly in the second intron. The three exons of Aseg-2 and Aips-2 have the same lengths but both intron 1 and intron 2 differ in length between the genes. In contrast, Aips-1 and Aseg-1 show dissimilarity only in the length of intron 2. Interestingly, introns 1 and 2 are inserted in the same positions in the Aips-1, Aips-2, Aseg-1 and Aseg-2 genes. These findings show that the Agrotis PBP genes have common ancestry and probably originate from gene duplication before the speciation of ipsilon and segetum. We found that expression of Aips-1/Aseg-1 and Aips-2/Aseg-2 is antennal-specific, but expression is not restricted to the male antennae.     

Characterisation of a nucleopolyhedrovirus and Spiroplasma sp. bacterium associated with outbreaking populations of the Antler moth Cerapteryx graminis

A broad survey was undertaken to characterise microbes associated with larval outbreaks of the Antler moth Cerapteryx graminis in Cumbria, United Kingdom. A nucleopolyhedrovirus present in all sampled populations at ?5% prevalence, was characterised via restriction fragment length polymorphism and partial sequencing the Polyhedrin, Lef-8 and Lef-9 genes; indicating a previously uncharacterised species most closely related to Agrotis ipsilon NPV. A survey of the host-associated bacterial community detected a species phylogenetically related to Spiroplasma sp., a male-killing phenotype previously isolated from Lepidoptera and Coleoptera, present at <63% prevalence in larvae. The implications of these associated microbes for host population dynamics are discussed.     

Characterization of a baculovirus newly isolated from the tea slug moth, <Emphasis Type="Italic">Iragoidae fasciata</Emphasis>

The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC₅₀) and lethal time (LT₅₀) were conducted to test the susceptibility of I. fasciata larvae to the virus.     

A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.     

Group Ⅰ but not Group Ⅱ NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group Ⅰ and Group Ⅱ based on the phylogenetic analysis. It has been reported that Group Ⅰ NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group Ⅱ NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group Ⅱ had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.