Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production

In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Antifeedant and larvicidal activities of Rhein isolated from the flowers of Cassia fistula L.

Antifeedant and larvicidal activities of rhein (1,8-dihydroxyanthraquinone-3-carboxylic acid) isolated from the ethyl acetate extract of Cassia fistula flower were studied against lepidopteron pests Spodoptera litura and Helicoverpa armigera. Significant antifeedant activity was observed against H. armigera (76.13%) at 1000 ppm concentration. Rhein exhibited larvicidal activity against H. armigera (67.5), S. litura (36.25%) and the LC₅₀ values was 606.50 ppm for H. armigera and 1192.55 ppm for S.litura. The survived larvae produced malformed adults.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

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.     

In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors

We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera.     

Construction of two vectors for gene expression in Trichoderma reesei

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Biocontrol Potential of Steinernema thermophilum and Its Symbiont Xenorhabdus indica Against Lepidopteran Pests: Virulence to Egg and Larval Stages

Under laboratory conditions, the biocontrol potential of Steinernema thermophilum was tested against eggs and larval stages of two important lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura (polyphagous pests), as well as Galleria mellonella (used as a model host). In terms of host susceptibility of lepidopteran larvae to S. thermophilum, based on the LC₅₀ 36 hr after treatment, G. mellonella (LC₅₀ = 16.28 IJ/larva) was found to be more susceptible than S. litura (LC₅₀ = 85 IJ/larva), whereas neither host was found to be significantly different from H. armigera (LC₅₀ = 54.68 IJ/larva). In addition to virulence to the larval stages, ovicidal activity up to 84% was observed at 200 IJ/50 and 100 eggs of H. armigera and S. litura, respectively. To our knowledge this is the first report of entomopathogenic nematode pathogenicity to lepidopteran eggs. Production of infective juvenile (IJ) nematodes/insect larva was also measured and found to be positively correlated with rate of IJ for H. armigera (r = 0.990), S. litura (r = 0.892), as well as G. mellonella (r = 0.834). Both Phase I and Phase II of symbiotic bacteria Xenorhabdus indica were tested separately against neonates of H. armigera and S. litura by feeding assays and found to be virulent to the target pests; phase variation did not affect the level of virulence. Thus S. thermophilum as well as the nematode’s symbiotic bacteria applied separately have the potential to be developed as biocontrol agents for key lepidopteran pests.     

Establishment of a cell line from Chrysodeixis chalcites permissive for Chrysodeixis chalcites and Trichoplusia ni nucleopolyhedrovirus

A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.