Soo-Dong Woo

Sequence and gene organization of 24 circles from the Cotesia plutellae bracovirus genome

Twenty-four genomic segments of Cotesia plutellae bracovirus (CpBV) were completely sequenced, and their genomic structures were analyzed. The aggregated genome size is 351,299xa0bp long and exhibits an average GC content of approximately 34.6%. Average coding density is about 32.3%, and 125 putative open reading frames (ORFs) are predicted. More than half (52.5%) of predicted genes are annotated as hypothetical, but they share sequence similarities with those of other bracoviral genomes. The annotated ORFs can be classified into the known bracoviral families, in which a family of protein tyrosine phosphatase is the largest, including 36 ORFs, suggesting a significant role during parasitization. In addition, 8 and 7 ORFs encode ankyrin-like and EP1-like genes, respectively. Some predicted genes are known only in Cotesia-associated bracoviral genomes. Phylogenetic analyses based on PTP, ankyrin and EP1-like gene groups revealed no correlation between bracoviruses.

Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

Analysis of promoter activity of selected Cotesia plutellae bracovirus genes.

In a previous study, we cloned 27 discrete genome segments of Cotesia plutellae bracovirus (CpBV) and provided the complete nucleotide sequences and annotation. Seven putative coding regions were predicted from one of the largest segments, CpBV-S30. The activity of promoters associated with six predicted ORFs from this segment were investigated using both transient and baculovirus expression assays with enhanced green fluorescent protein as a reporter gene. CpBV promoters showed activity earlier than the polyhedrin promoter and the activity of some of these promoters was superior to that of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ie-1 promoter in the baculovirus expression assays. The promoter of ORF3004 showed the highest level of activity in insect cells, exhibiting 24 % of the activity obtained with the AcMNPV polyhedrin promoter in Sf9 cells. In Spodoptera exigua larvae, the ORF3006 promoter showed the highest activity, with about 35 % of the activity measured with the polyhedrin promoter. In addition, analysis of the ORF3006 promoter revealed that the region between -382 and -422 from the translation start point was critical for activity of this promoter. These results suggest that the CpBV-S30 promoters characterized here could be useful tools in a variety of biotechnological applications, such as gene expression analyses and insecticide development.

Characterization of the Helicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene region

A local strain ofHelicoverpa assulta nucleopolyhedrovirus (HasNPV) was isolated from infectedH. assulta larvae in Korea. Restriction endonuclease fragment analysis, using 4 restriction enzymes, estimated that the total genome size of HasNPV is about 138 kb. A degenerate polymerase chain reaction (PCR) primer set for the polyhedrin gene successfully amplified the partial polyhedrin gene of HasNPV. The sequencing results showed that the about 430 bp PCR product was a fragment of the corresponding polyhedrin gene. Using HasNPV partial predicted polyhedrin to probe the Southern blots, we identified the location of the polyhedrin gene within the 6 kbEcoRI, 15 kbNcoI, 20 kbXhoI, 17 kbBgl II and 3 kbClaI fragments, respectively. The 3 kbClaI fragment was cloned and the nucleotide sequences of the polyhedrin coding region and its flaking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame of 735 nucleotides which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of HasNPV polyhedrin shared 73.7% identity with the polyhedrin gene fromAutographa californica NPV but were most closely related toHelicoverpa andHeliothis species NPVs with over 99% sequence identity.

Entomopathogenic Fungi as Dual Control Agents against Both the Pest Myzus persicae and Phytopathogen Botrytis cinerea

Abstract The green peach aphid (Myzus persicae), a plant pest, and gray mold disease, caused by Botrytis cinerea, affect vegetables and fruit crops all over the world. To control this aphid and mold, farmers typically rely on the use of chemical insecticides or fungicides. However, intensive use of these chemicals over many years has led to the development of resistance. To overcome this problem, there is a need to develop alternative control methods to suppress populations of this plant pest and pathogen. Recently, potential roles have been demonstrated for entomopathogenic fungi in endophytism, phytopathogen antagonism, plant growth promotion, and rhizosphere colonization. Here, the antifungal activities of selected fungi with high virulence against green peach aphids were tested to explore their potential for the dual control of B. cinerea and M. persicae. Antifungal activities against B. cinerea were evaluated by dual culture assays using both aerial conidia and cultural filtrates of entomopathogenic fungi. Two fungal isolates, Beauveria bassiana SD15 and Metarhizium anisopliae SD3, were identified as having both virulence against aphids and antifungal activity. The virulence of these isolates against aphids was further tested using cultural filtrates, blastospores, and aerial conidia. The most virulence was observed in the simultaneous treatment with blastospores and cultural filtrate. These results suggest that the two fungal isolates selected in this study could be used effectively for the dual control of green peach aphids and gray mold for crop protection.

Effects of an insect-mediated mental healthcare program for mentally disordered children

Insects are the most diverse groups of animals on the planet, representing more than one‐half of all known living organisms, and are found in nearly every environment. Recently, the importance of insects as food sources or as pets has increased in many countries, including Korea. In addition, several insects have been shown to exert a strong influence on peoples emotions. Our insect‐mediated mental healthcare program is designed to help meet the physical, behavioral and developmental needs of people with mental disorders. Children with mental disorders, the experimental group, were provided with an insect‐mediated mental healthcare program for a total of eight sessions, one session per week, at 1–2u2009h per session, accompanied by a pre‐test and post‐test. The overall, gender, education level, and mental disease profiles of the participants in this study were balanced. Our results indicated that children who participated in the insect‐mediated healthcare program group once showed significant improvement in their emotional health and insect awareness. Additionally, paired education levels t‐tests showed that the outcomes of the participants in treatment group were significantly improved (α < 0.05). However, participants satisfaction with their school life (middle school) was not influenced. These results suggest that insects positively influence childrens emotional health through an insect‐mediated healthcare program. Further research on the basis of this study is expected to help children with emotional therapy in other areas.

Characterization of the Polyhedrin Gene of Spodoptera litura Nucleopolyhedrovirus Isolated in Korea

Abstract A local strain of Spodoptera litura nucleopolyhedrovirus (SINPV-K1) was isolated from infected larvae of a Korean population of S. litura. Degenerate PCR primer set for the polyhedrin gene successfully amplified the partial polyhedrin gene of SINPV-K1. The sequencing results showed that the about 430 bp PCR product was a fragment of Corresponding polyhedrin gene. Southern blot analysis of SINPV-K1 restriction fragments was performed by using 430 bp polyhedrin PCR product of SINPV-Kl as a probe. As the result, we identified the location of the polyhedrin gene within the approximately 6 kb EcoR I-, 3.5 kb Hind III-, 20 kb Xho I- and 4 kb Cla I-digested fragments, respectively. The Hind III 3.5kb fragment was cloned for sequencing the complete polyhedrin gene. Nucleotide sequence analysis indicated the presence of an open reading frame of 747 nucleotides, which could encode 249 amino acids with a predicted molecular mass of 31 kDa. The nucleotide sequences within the coding region of SINPV-K1 polyhedrin shared maximum 94.0% similarity with the polyhedrin gene from previous reported other SINPVs but were most closely related to Spodoptera littoralis NPV with 99.0% sequence identity. These suggest that the SINPV isolate from Korea is a different SINPV strain.

Efficient Method for the Rapid Purification of Nosema ceranae Spores

Abstract Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an 8–11 μm pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.

Growth Characteristics of Paecilomyces tenuipes by the Passage in Liquid Media

The growth characteristics of Paecilomyces tenuipes according to the passage in the two kind of liquid media were investigated by comparing the mycelium and conidium formation degrees. The potato dextrose broth medium and the silkworm larvae medium containing the silkworm powder were used as the liquid media, and the potato dextrose agar medium and the brown rice medium containing the powder of silkworm pupa were used as the solid media. The conidium formation degree in liquid media differed by the passages but that in solid media was not. This suggested that the passage in liquid media did not affect significantly the conidium formation in solid media. When the brown rice media were inoculated with the concentration of conidia/ml, conidia/ml and conidia/ml, respectively, the conidium formation degrees were similar. This indicated that the optimal inoculation concentration of conidium to the brown rice media is conidia/ml.

Determination of the Optimal Concentration of Fetal Bovine Serum for the Growth of Two Insect Cell and Viruses

To determine the optimal concentration of fetal bovine serum (FBS) on the growth of insect cells and the multiplicity of viruses, the growth of cells (Sf21 and Bm5) and viruses were examined on the various concentrations of FBS. In view of the viability, growth speed, proliferation of cells and the amount of FBS, the most proper concentration for the cell culture were 7% and 5% for Sf21 and Bm5, respectively. The multiplicity of viruses at the various concentrations of FBS was similar in both cell lines at 5 days post-infection (p.i.). However, it differed significantly at 2 and 3 days p.i. The proper concentration of FBS were 10% and 3% for Sf21 at 2 and 3 days p.i., respectively, and 5% for Bm5 at both 2 and 3 days p.i. These results suggested that the optimal concentration of FBS should be determined according to the used cell lines and viruses for their optimum production.