Biological Comparison of Two Genotypes of Helicoverpa armigera Single-Nucleocapsid Nucleopolyhedrovirus

Baculovirus isolates from the same host species often show a considerable degree of variation on phenotypes. The completely sequenced genotypes C1 and G4 of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) were compared. Bioassay studies suggested that nearly double of HaSNPV G4 virus was required compared with HaSNPV C1 to achieve a similar LD₅₀, and at the LD₉₀ level the insect-killing speed for HaSNPV C1 was quicker than that of HaSNPV G4. The budded virus (BV) production of HaSNPV C1 was nearly two- to threefold higher at 24 and 48 h post-infection (p.i.) than that of HaSNPV G4. However, the kinetics of polyhedral inclusion body (PIB) formation in HzAM1 cells was similar in both the genotypes, which implied that the insect-killing speed was not influenced by PIB formation, but by the kinetics of BV production. The results suggested that the HaSNPV C1 isolate was a better choice than HaSNPV G4 virus for controlling H. armigera.     

Genomic sequencing and analyses of HearMNPV-a new Multinucleocapsid nucleopolyhedrovirus isolated from Helicoverpa armigera

ABSTRACT: BACKGROUND: HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed. METHODS: The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the "partial filling-in" method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses. RESULTS: Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome. CONCLUSIONS: HearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.     

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth (growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five™ cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium (LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Systematic differences in the population density of green spruce aphid, Elatobium abietinum in a provenance trial of Sitka spruce, Picea sitchensis

Quantitative bioassay techniques were used to measure the susceptibility of Heliothis armigera to three nuclear polyhedrosis viruses (NPVs): H. armigera singly-enveloped NPV (HaSNPV), H. zea SNPV (HzSNPV) and H. armigera multiply-enveloped NPV (HaMNPV). Viruses were identified by EcoRI restriction endonuclease analysis. Electrophoretic profiles of DNA fragments revealed that the HaSNPV isolate was a previously undescribed genotypic variant. Bioassays with neonate and 6-day-old larvae measured small but significant differences in virulence between the three viruses. HzSNPV was the most virulent for neonate larvae with a median lethal dose (LD₅₀) of five polyhedra. HaMNPV was least virulent for 6-day-old larvae, with a LD₅₀ of 1400 polyhedra compared with 640–670 polyhedra for HaSNPV and HzSNPV. In addition, the median lethal time (LT₅₀) for infection with HaMNPV in neonate larvae was approximately 1·7 days longer than for the other viruses. Although they varied in virulence, each of the three viruses was sufficiently virulent to have considerable potential as a microbial control agent of H. armigera.     

A host-plant specialist, Helicoverpa assulta, is more tolerant to capsaicin from Capsicum annuum than other noctuid species

Plant secondary compounds not only play an important role in plant defense, but have been a driving force for host adaptation by herbivores. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), an alkaloid found in the fruit of Capsicum spp. (Solanaceae), is responsible for the pungency of hot pepper fruits and is unique to the genus. The oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), is a specialist herbivore feeding on solanaceous plants including Capsicum annuum, and is one of a very few insect herbivores worldwide capable of feeding on hot pepper fruits. To determine whether this is due in part to an increased physiological tolerance of capsaicin, we compared H. assulta with another specialist on Solanaceae, Heliothis subflexa, and four generalist species, Spodoptera frugiperda, Heliothis virescens, Helicoverpa armigera, and Helicoverpa zea, all belonging to the family Noctuidae. When larvae were fed capsaicin-spiked artificial diet for the entire larval period, larval mortality increased in H. subflexa and H. zea but decreased in H. assulta. Larval growth decreased on the capsaicin-spiked diet in four of the species, was unaffected in H. armigera and increased in H. assulta. Food consumption and utilization experiments showed that capsaicin decreased relative consumption rate (RCR), relative growth rate (RGR) and approximate digestibility (AD) in H. zea, and increased AD and the efficiency of conversion of ingested food (ECI) in H. armigera; whereas it did not significantly change any of these nutritional indices in H. assulta. The acute toxicity of capsaicin measured by injection into early fifth instar larvae was less in H. assulta than in H. armigera and H. zea. Injection of high concentrations produced abdominal paralysis and self-cannibalism. Injection of sub-lethal doses of capsaicin resulted in reduced pupal weights in H. armigera and H. zea, but not in H. assulta. The results indicate that H. assulta is more tolerant to capsaicin than the other insects tested, suggesting that this has facilitated expansion of its host range within Solanaceae to Capsicum after introduction of the latter to the Old World about 500 years ago. The increased larval survival and growth due to chronic dietary exposure to capsaicin suggests further adaptation of H. assulta to that compound, the mechanisms of which remain to be investigated.     

棉铃虫核型多角体病毒几丁质酶基因及杆状病毒 几丁质酶基因的分子进化

用PCR方法扩增了棉铃虫Helicoverpa armigera单粒包埋型核型多角体病毒（HaSNPV）几丁质酶基因,测定了基因编码区的核苷酸全序列。基因编码区全长1.713 bp,可编码570个氨基酸残基组成的多肽,预计分子量为63.6 kD。将所推导的HaSNPV几丁质酶氨基酸序列与其它已知杆状病毒几丁质酶氨基酸序列进行联配比较,结果表明HaSNPV 与谷实夜蛾H.zea单粒包埋型核型多角体病毒（HzSNPV）的氨基酸序列非常相似,同源性高达90.7%,与苜蓿丫纹夜蛾Autographa californica多粒包埋型核型多角体病毒（AcMNPV）、家蚕Bombyx mori核型多角体病毒（BmNPV）、美国白蛾Hyphantria cunea核型多角体病毒（HcNPV）、舞毒蛾Lymantria dispar多粒包埋型核型多角体病毒（LdMNPV）、黄杉毒蛾Orgyia pseudotsugata多粒包埋型核型多角体病毒（OpMNPV）和云杉卷叶蛾Choristoneura fumiferana核型多角体病毒（CfMNPV）氨基酸序列同源性分别为64.4%、64.9%、64.2%、62.9%、66.2%和61.5%。根据氨基酸序列用PC＼GENE程序绘制已知杆状病毒几丁质酶的分子系统树,并与杆状病毒中最为保守的多角体蛋白基因系统树作了比较,结果表明几丁质酶基因和多角体蛋白基因的进化速率是不尽相同的。     

Pseudoplusia includens single nucleopolyhedrovirus: Genetic diversity,phylogeny and hypervariability of the pif-2 gene

The soybean looper (Pseudoplusia includens Walker, 1857) has become a major pest of soybean crops in Brazil. In order to determine the genetic diversity and phylogeny of variants of Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IA to -IG), partial sequences of the genes lef-8, lef-9, pif-2, phr and polh were obtained following degenerate PCR and phylogenetic trees constructed using maximum parsimony and Bayesian methods. The aligned sequences showed polymorphisms among the isolates, where the pif-2 gene was by far the most variable and is predicted to be under positive selection. Furthermore, some of the pif-2 DNA sequence mutations are predicted to result in significant amino acid substitutions, possibly leading to changes in oral infectivity of this baculovirus. Cladistic analysis revealed two closely related monophyletic groups, one containing PsinNPV isolates IB, IC and ID and another containing isolates IA, IE, IF and IG. The phylogeny of PsinSNPV in relation to 56 other baculoviruses was also determined from the concatenated partial LEF-8, LEF-9, PIF-2 and POLH/GRAN deduced amino acid sequences, using maximum-parsimony and Bayesian methods. This analysis clearly places PsinSNPV with the Group II Alphabaculovirus, where PsinSNPV is most closely related to Chrysodeixis chalcites NPV and Trichoplusia ni SNPV.     

一种棉铃虫多角体病毒的纯化与鉴定

对一株纯化的棉铃虫多角体病毒进行电镜观察,形态呈不规则型,直径约2 μm,命名为棉铃虫多角体-B(Helicoverpa armigera nucleocapsid nucleopolyhedrovirus-B,HaNPV-B),它对东方粘虫有较好的感染性,与棉铃虫单核型多角体病毒(Helicoverpa armigera single nucleocapsid NPV,HaSNPV)有不同的宿主域.通过半补齐法建立HaNPV-B基因组文库,拼接了一个6 035 bp的核苷酸片段,推测包含6个ORF,其包含基因内容及基因结构与HaSNPV不同,分析表明两者是不同种病毒,这为杆状病毒与宿主的相互作用及共同进化提供了信息.     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno

Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.     

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO₂ production (VCO₂), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD₂₅ or LD₇₅ survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO₂ values of larvae that survived inoculation (0.0288 ml h⁻¹), the uninoculated controls (0.0250 ml h⁻¹), and the larvae that did not survive inoculation (0.0199 ml h⁻¹) differed significantly from one another. At 4 dpi, the VCO₂ of the uninoculated controls were significantly lower than the VCO₂ of inoculation survivors, but significantly higher than the VCO₂ of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4 dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.     

Identification of mariner‐like elements belonging to the cecropia subfamily in two closely related Helicoverpa species

Abstract Mariner transposons are widespread in eukaryote genomes and have been used as transposon vectors in insect transgenesis. We examined two closely related Helicoverpa species, the cotton bollworm Helicoverpa armigera and corn earworm Helicoverpa zea, for the presence of mariner‐like elements (MLEs). Multiple copies of two distinct MLEs, Hamar1 and Hamar2, were isolated in H. armigera, and a MLE showing a high degree of conservation to Hamar1 was detected in H. zea and was named Hzmar1. These MLEs belong to the cecropia subfamily, containing indels in the transposase coding region. Sequence analysis indicated the earlier invasion of Hamar1 and relatively recent activity of Hamar2.