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Higashimurayama-shi, Japan

Xu J.,Kyushu University | Mon H.,Kyushu University | Kusakabe T.,Kyushu University | Li Z.,Kyushu University | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2013

The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAimediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV-Bme21 BEVS. © Springer-Verlag Berlin Heidelberg 2013.


Bernd Blossey, associate professor of natural resources, has a better idea: Let bugs do the work. Blossey is nearing the end of a research program that has identified a leaf beetle, Galerucella birmanica, that feasts on water chestnuts in its native China, as the perfect predator to help clear New York's waters. Water chestnut (Trapa natans) is native to Europe, Asia and Africa. It was introduced to North America through the Botanic Gardens in Cambridge, Massachusetts, in the 1870s. Since then, water chestnut has spread across the Northeast, into Canada and as far south as Virginia. With no natural predators, water chestnut overtakes its environment, forming floating mats of vegetation that make rivers and lakes impassable for boats and swimmers, and prevents light and oxygen from reaching plants and creatures below. If the beetle proves to be an effective predator against water chestnut, the benefit could be felt in millions of dollars in savings for wildlife agencies and provide a more sustainable approach to using herbicide to knock back the invasive plant. In a carefully controlled, quarantined environment, Blossey and his research team have found that G. birmanica is "very, very specific" in eating water chestnut plants and very little else. Blossey obtained the leaf beetles from the Wuhan Botanical Garden in Hubei province, China. "We had to get permission from the Chinese authorities to bring them out and the U.S. authorities to bring them in," Blossey said. "We've tried 50 different plant species, and the insects are as specific as we'd hoped they were. We're really, really pleased with the progress we have made." Water chestnut is found throughout much of the Finger Lakes, central New York and tributaries to Lake Ontario. The species has invaded eastern coastal states and, Blossey warns, could be ready for a rapid westward expansion through the Great Lakes human and cargo transportation system. Millions of dollars have been spent to thwart water chestnut's spread. Each year $500,000 is spent on eradication efforts on Lake Champlain alone. This year, the Finger Lakes Partnership for Regional Invasive Species Management received a $750,000 grant from the Great Lakes Restoration Initiative for hand pulling of the invasive plant. Blossey also studies invasiveness in plant species generally. Very few introduced plant species become problematic: Of all plant species moved outside their natural habitats, only 10 percent become established at all, and only 1 percent become invasive. In 1995, Blossey and Rolf Nötzold, then of the international Institute of Biological Control, European Station, proposed a hypothesis to explain why some species become invasive while others don't: the evolution of increased competitive ability hypothesis. "Some of the species that are serious invaders outside the home range are hammered inside the home range by predators or diseases. If you have a lot of natural enemies, you develop a lot of defenses. So when they leave the home range, they have less competition and they thrive," Blossey said. This isn't the first time Blossey has helped address an out-of-control invasive species through biological control. In 1992 he led a team that tested insects as biological controls for the invasive purple loosestrife, a European wetland plant that has spread throughout North America. The team gained federal and state approval to release four insects that attack the plant: two leaf beetles and two weevils, which jointly go after the plant's roots, leaves and flowers. Another project, on which Blossey has worked for 18 years with collaborators in Europe and Rhode Island, involves controlling common reed, Phragmites australis, another wetland plant invader. The team is investigating stem-eating moths, which are "almost 100 percent specific" to the invasive plant, he said. As for the water chestnuts, there are still some questions to be answered and additional tests that Blossey plans to run early next spring. If all goes well, he would then submit a petition to a U.S. Department of Agriculture oversight group, asking for permission to release the insects. "We hope to release them in 2018," Blossey said. "That's an incredibly quick timeline, but we've had a good work plan, and the insects were really, really cooperative." Blossey's work on water chestnuts has been supported in part by the New York State Department of Environmental Conservation through the New York Invasive Species Research Institute at Cornell University. Explore further: New study in US and Europe shows how invasive plant species fare better than natives


Lee Y.K.,Institute of Biological Control | Lee Y.K.,Chungnam National University | Jin N.Y.,Institute of Biological Control | Jin N.Y.,Chungnam National University | And 11 more authors.
Journal of the Faculty of Agriculture, Kyushu University | Year: 2015

Strains were isolated from soil samples collected from mountains and fields of Yeong-Dong and Ok-cheon, ChungBuk province in order to select B. thuringiensis strains with high insecticidal activities against lepidopteran pests. Eight B. thuringiensis strains producing spores and endotoxin proteins were selected from a total of 26 soil samples. Tests of biological activities in Spodoptera litura, Spodoptera exi-gua, Plutella xylostella, and Aedes aegypti were performed with 8 isolates of B. thuringiensis. CAB565 and CAB566 strains were selected, because such strains showed more than 80% of insecticidal activities against four kinds of pests. Selected two strains formed endotoxin proteins in a typical bipyramidal type. CAB565 and CAB566 strains were identified as thuringiensis subsp. kurstaki, and thuringiensis subsp. aizawai, respectively. LC50, value of CAB566 strain against larvae of S. litura at the fourth stage was 7.7×105 (cfu/ml), which was the highest activity. LC50 value of CAB565 strain against larvae of S. exigua at the fifth stage was 3.0×106 (cfu/ml), which was high activity. Through SDS-PAGE, CAB565 and CAB566 strains showed a band at 130 kDa. After it reacted with midgut juice of S. litura and S. exigua, a band of toxin protein was shown at 65 kDa. According to results of PCR analysis, it was found that cry1Aa, cry1Ac and cryII genes were present in CAB565 strain and it was different from B. thuringiensis subsp. kurstaki. cry1Aa, cry1C, cry 1D and cry1I genes were present in CAB566 strain and it was different from B. thuringiensis subsp. aizawai. As a result of plasmid DNA patterns, B. thuringiensis subsp. kurstaki KB099 strain and CAB565 strain had 5 and 10 plasmid DNAs, respectively. B. thuringiensis subsp. aizawai KB098 strain and CAB566 strain had 9 and 7 plasmid DNAs, respectively.


Jin N.Y.,Institute of Biological Control | Jin N.Y.,Chungnam National University | Lee Y.K.,Institute of Biological Control | Lee Y.K.,Chungnam National University | And 13 more authors.
Journal of the Faculty of Agriculture, Kyushu University | Year: 2015

Insecticidal activity was enhanced when Bacillus thuringiensis subsp. kurstaki KB100 strain containing insecticidal activity against Spodoptera exigua was mixed with tannic acid, a protease inhibitor. To investigate the cause of this result, inhibition rate of tannic acid against protease activity in midgut juice of S. exigua was measured. As a result, it was found that protease activity in midgut juice of 5. exigua was about 83.1%, 77.6%, 68.0%, and 40.1% at concentrations of 10, 20, 40 and 80 mM, respectively. Such reduction of protease activity was measured with increasing concentration. Analysis of substrate reaction in several kinds of proteases was performed. Trypsin exhibited 91.4% and 89.4% of proteolytic activity in BApNA and BPVApNA substrates, respectively. Thus, when Trypsin was treated with tannic acid, proteolytic activity was 62.2% and 54.5% in BApNA and BPVApNA substrates, respectively. Trypsin's proteolytic activity was inhibited by 29.2% and 34.9% when mixed with tannic acid. However, proteolytic activity of chymotrypsin and elastase was not inhibited by tannic acid. Digestion of protoxin produced in B. thuringiensis KB100 strain by Trypsin was analyzed using SDS-PAGE. As a result, a band appeared at about 60 kDa-70 kDa and it seemed to be inhibited by tannic acid. Digestion patterns of protoxin were measured over time. As it was over-digested in a group of Trypsin treatment, a band of protoxin at 60 kDa completely disappeared. On the other hand, when it was treated with tannic acid, a digestion inhibitor, protoxin was maintained for up to 24 hours.

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