The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus (AgMNPV)

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.     

柞蚕核型多角体病毒载体在培养细胞和休眠蛹中的基因表达效果

柞蚕核型多角体病毒（AnpeNPV）作为基因表达载体在柞蚕培养细胞（AnPe细胞）和柞蚕蛹中已经成功地表达出了外来基因,并生产出了大量蛋白质。本文比较了AnpeNPV与苜蓿尺蠖核型多角体病毒(AcMNPV)、家蚕核型多角体病毒(BmNPV)和美国白蛾核型多角体病毒(HycuNPV)基因表达载体在培养细胞和昆虫活体组织内的β-半乳糖苷酶基因表达效果。结果显示,5×105个细胞中β-半乳糖苷酶的最高酶活性分别是AnpeNPV在AnPe细胞为40.9 units/ml （TC-100培养液,FBS10%）和59.9 units/ml（SF-900Ⅱ培养液）,AcMNPV在Sf9细胞为72.4 units/ml（TC-100,FBS10%）和66.4 units/ml（SF-900Ⅱ）、在High5细胞为326 units/ml（EX-CELL 405培养液）,BmNPV在Bm4细胞为15.1 units/ml（TC-100,FBS10%）,HycuNPV在SpIm细胞为68.6 units/ml（SF-900Ⅱ）。活体组织内β-半乳糖苷酶的最高酶活性分别是柞蚕雌蛹为14.3 units/g、雄蛹为11.7 units/g,家蚕幼虫是10.1 units/g。实验证明AnpeNPV/AnPe的外来基因表达水平与AcMNPV/ Sf9和HycuNPV/SpIm相似、比BmNPV/ Bm4高、不及AcMNPV/ High5;AnpeNPV/柞蚕蛹,其雌蛹比BmNPV/家蚕5龄幼虫的外来基因表达效果好、雄蛹与之无明显差异,说明AnpeNPV基因表达载体无论是在培养细胞还是昆虫活体组织中均可与其他NPV基因表达载体相媲美。柞蚕蛹由于可以机械化、大规模地操作,显示对于大量生产蛋白质具有更好的应用前景。     

Characterization of Adoxophyes honmai single-nucleocapsid nucleopolyhedrovirus: morphology,structure, and effects on larvae

A nucleopolyhedrovirus (NPV) was isolated from a diseased larva of the smaller tea tortrix, Adoxophyes honmai, collected from a tea field in Tsukuba, Ibaraki, Japan. Electron microscopic observations confirmed that A. honmai NPV (AdhoNPV) was a single-nucleocapsid type virus. The genome size of AdhoNPV was estimated to be 111.6 +/- 0.9kb (mean +/- SE) by restriction endonuclease analysis. AdhoNPV was also infectious to two other Adoxophyes species, the summer fruit tortrix Adoxophyes orana and Adoxophyes dubia. The LD50 values for neonatal, second, third, fourth, and fifth (final) instar larvae of A. honmai were determined as 61, 107, 688, 1,961, and 4,085 occlusion bodies/insect, respectively. Most of the infected larvae died 5-9 days after molting to the final instar, regardless of the timing of inoculation. However, when neonates were exposed to extremely high doses of AdhoNPV (greater than 100 x LD90), larval development was prevented and most of the larvae died in the first instar.     

A cell line (NTU-MV) established from Maruca vitrata (Lepidoptera: Pyralidae): Characterization, viral susceptibility, and polyhedra production

Here we describe the establishment of a new cell line, NTU-MV, derived from pupal tissues of an economically important pest, the legume pod borer Maruca vitrata. This cell line contained four major cell types: polymorphic cells, round cells, spindle-shaped cells, and comma cells. The doubling time of MV cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 27h. The chromosome numbers of MV cells varied widely from 16 to 268. Compared to other insect cell lines, the MV cell line produced distinct isozyme patterns with esterase, malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Confirmation that NTU-MV was derived from M. vitrata was demonstrated by showing that the sequence of the internal transcribed spacer regions (ITS) of the MV cells was 98% identical to that of M. vitrata larvae. Two NTU-MV cell strains, NTU-MV1 and NTU-MV56, were selected based on susceptibility to MaviMNPV (M. vitrata multiple nucleopolyhedrovirus). NTU-MV, MV1, and MV56 cells showed a high susceptibility to MaviMNPV and produced high yields of polyhedra (47-50OBs/cell, 4x10(7)-5.96x10(7)OBs/ml) after 2 weeks of MaviMNPV infection. We conclude that the NTU-MV cell line will be a useful tool for studying MaviMNPV as well as for the mass production of MaviMNPV polyhedra for the biocontrol of M. vitrata.     

Relative activity of baculoviruses of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.) (Lepidoptera: Plutellidae)

The relative activities of the granulovirus (PxGV) and the nucleopolyhedrovirus (PxMNPV) of the diamondback moth, Plutella xylostella (L.), against this insect were evaluated in the laboratory. Similar numbers of occlusion bodies (OBs) of PxGV and PxMNPV were required to kill P. xylostella. However, cadavers of insects infected with PxGV contained more OBs per unit weight than did cadavers infected with PxMNPV. Less cadaver material was thus required to kill insects with PxGV. Larvae that were killed by PxGV survived approximately 2 d longer than did those killed by PxMNPV. When P. xylostella larvae were fed both viruses together, mortality was higher than that caused by either virus alone. The virulence of PxMNPV produced in P.␣xylostella did not differ from that of PxMNPV produced in the beet armyworm, Spodoptera exigua (Hübner). S. exigua was less susceptible to PxMNPV than to its homologous nucleopolyhedrovirus, SeMNPV. Use of trade names does not imply endorsements by USDA-ARS of products named nor criticism of similar ones not mentioned.     

Population fluctuations of the western tent caterpillar in southwestern British Columbia

Cyclic populations of western tent caterpillars fluctuate with a periodicity of 6–11 years in southwestern British Columbia, Canada. Typically, larval survival is high in early stages of the population increase, begins to decline midway through the increase phase, and is low through several generations of the population decline. Fecundity is generally high in increasing and in peak populations but is also reduced during the population decline. Poor survival and low fecundity for several generations cause the lag in recovery of populations that is necessary for cyclic dynamics. The dynamics of tent caterpillar populations vary among sites, which suggests a metapopulation structure; island populations in the rainshadow of Vancouver Island have more consistent cyclic dynamics than mainland populations in British Columbia. Sudden outbreaks of populations that last a single year suggest that dispersal from source to sink populations may occur late in the phase of population increase. Wellington earlier discussed qualitative variation among tent caterpillar individuals as an aspect of population fluctuations. The variation in caterpillar activity he observed was largely statistically nonsignificant. Recent observations show that the frequency of elongate tents as described by Wellington to characterize active caterpillars varies among populations but does not change in a consistent pattern with population density. The level of infection from nucleopolyhedrovirus (NPV) was high in some populations at peak density but was not associated with all population declines. Sublethal infection can reduce the fecundity of surviving moths, and there is a weak association between viral infection and egg mass size in field populations. The impact of weather in synchronizing or desynchronizing populations is a factor to be investigated further. Received: May 25, 1999 / Accepted: March 28, 2000     

Sequence comparison between three geographically distinct Spodoptera frugiperda multiple nucleopolyhedrovirus isolates: Detecting positively selected genes

The complete genomic sequence of a Nicaraguan plaque purified Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genotype SfMNPV-B was determined and compared to previously sequenced isolates from United States (SfMNPV-3AP2) and Brazil (SfMNPV-19). The genome of SfMNPV-B (132,954 bp) was 1623 bp and 389 bp larger than that of SfMNPV-3AP2 and SfMNPV-19, respectively. Genome size differences were mainly due to a deletion located in the SfMNPV-3AP2 egt region and small deletions and point mutations in SfMNPV-19. Nucleotide sequences were strongly conserved (99.35% identity) and a high degree of predicted amino acid sequence identity was observed. A total of 145 open reading frames (ORFs) were identified in SfMNPV-B, two of them (sf39a and sf110a) had not been previously identified in the SfMNPV-3AP2 and SfMNPV-19 genomes and one (sf57a) was absent in both these genomes. In addition, sf6 was not previously identified in the SfMNPV-19 genome. In contrast, SfMNPV-B and SfMNPV-19 both lacked sf129 that had been reported in SfMNPV-3AP2. In an effort to identify genes potentially involved in virulence or in determining population adaptations, selection pressure analysis was performed. Three ORFs were identified undergoing positive selection: sf49 (pif-3), sf57 (odv-e66b) and sf122 (unknown function). Strong selection for ODV envelope protein genes indicates that the initial infection process in the insect midgut is one critical point at which adaptation acts during the transmission of these viruses in geographically distant populations. The function of ORF sf122 is being examined.     

Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney

Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles in order to investigate the prevalence of virus pathogens. Reoviruses were isolated in 11 of the 13 winter moth populations sampled, with 3 novel species being detected. Two species of Cypoviridae (CPV) were isolated, Operophtera brumata CPV18 and O. brumata CPV19, with one host population suffering 46% infection prevalence of OpbuCPV19. A third virus, O. brumata Reovirus (OpbuRV), was isolated from both winter moth and a hymenopteran parasitoid wasp, Phobocampe tempestiva, which is abundant in these populations. This was identified as a non-occluded reovirus, which was clearly able to infect and persist in both the lepidopteran and the hymenopteran host. The genomes of the three viruses were characterised using gel electrophoresis and the virus structure was investigated using transmission electron microscopy. The relationship of these viruses with a baculovirus that also infects winter moth, OpbuNPV, was investigated, as well as the association of OpbuRV with P. tempestiva. The detection of such viruses is discussed with reference to studies of similar viruses in other lepidopteran and hymenopteran host systems.     

High levels of genetic diversity in Spodoptera exempta NPV from Tanzania

The African armyworm, Spodoptera exempta, is a major pest in sub-Saharan Africa. A nucleopolyhedrovirus (NPV) is often recorded in later population outbreaks and can cause very high levels of mortality. Research has been addressing whether this NPV can be developed into a strategic biological control agent. As part of this study, the variation in natural populations of NPV is being studied. An isolate of S. exempta NPV was cloned in vivo and found to contain at least 17 genetically-distinct genotypes. These genotypes varied in size from approximately 115 to 153 kb.