The lycanid butterfly, Shijimiaeoides divina (Lepidoptera: Lycaenidae) is listed as the second-degree endangered wild animal in Korea from 2012. The 15,259-bp long complete mitochondrial genome of the species consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in majority of Lepidoptera. The 15,259-bp long S. divina mitogenome is well within the range found in Lycaenidae and has typical sets of 37 genes and a major non-coding A+T-rich region as 379 bp. As other lycanid butterflies S. divina COI also started with CGA. The gene arrangement of S. divina is identical to that of the Ditrysia in Lepidoptera that has the order trnM-trnI-trnQ (underline for inverted gene) between the A+T-rich region and ND2. Comparison of the skewness between the PCGs encoded in major and minor strand indicates a substantial difference between them in GC skewness (0.261 ~ 0.340 in minor strand vs. -0.081 ~ -0.115 in major strand). The 151-bp intergenic spacer sequence of the S. divina mitogenome is spread over 16 regions ranging in size from 1-53 bp. The longest one (53 bp) located between trnQ and ND2 shows substantially high sequence homology to neighboring ND2 may indicating the origination of the region by a partial duplication of the ND2 gene. One of the unusual features of the S. divina mitogenome is the presence of a trnK-like sequence that is encoded at the major strand of the genome in the A+T-rich region.

During the last century, the overuse of synthetic chemical insecticides led to problems associated with natural environment such as harmful effects on nontarget organisms and development of resistance. Therefore, the application of insect pathogenic microorganisms including Bacillus thuringiensis (Bt), baculoviruses, and entomopathogenic fungi has been an environmentally favorable methods for insect control. However, their use has been limited by several factors such as narrow host spectrum and slow speed of insecticidal activities. A number of approaches have been taken to overcome these defects of microbial insecticides. A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of the NeuroBactrus, the Bt cry1-5 crystal protein gene was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but also activated by treatment with trypsin, resulting in an approximately 65-kDa active toxin. The NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Expression of the foreign proteins (Bt toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. These results suggested that the NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging. The cry gene from Bt, encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. Through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of S. exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 34 mutant cry genes. These mutant cry genes were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

To improve its textural properties as a support for platinum catalyst, carbon aerogel was chemically activated with KOH as a chemical agent. Carbon-supported platinum catalyst was subsequently prepared using the prepared carbon supports(carbon aerogel(CA), activated carbon aerogel(ACA), and commercial activated carbon(AC)) by an incipient wetness impregnation. The prepared carbon-supported platinum catalysts were applied to decalin dehydrogenation for hydrogen production. Both initial hydrogen evolution rate and total hydrogen evolution amount were increased in the order of Pt/CA < Pt/AC < Pt/ACA. This means that the chemical activation process served to improve the catalytic activity of carbon-supported platinum catalyst in this reaction. The high surface area and the well-developed mesoporous structure of activated carbon aerogel obtained from the activation process facilitated the high dispersion of platinum in the Pt/ACA catalyst. Therefore, it is concluded that the enhanced catalytic activity of Pt/ACA catalyst in decalin dehydrogenation was due to the high platinum surface area that originated from the high dispersion of platinum.

Insect and natural enemy fauna were surveyed at conventional (CV) and organic-farming persimmon orchards (OF) at Jinju, Korea, using sweeping net and naked eyes in 2013 and 2014. Using sweeping net, 49 species at an OF and 18 species at a CV were observed in 2013. In 2014 too, more species were observed at OF (66 species, 33 families, 7 orders) than at a CV (25 species, 15 families, 5 orders). During both the years, dominant species were all hemipteran insects like Nysius plebejus (Lygaeidae) and Cletus punctiger (Coreidae) in both types of orchards, followed by Rhopalus maculatus (Rhopalidae), Riptortus pedestris (Alydidae). Similarly, naked eye inspection also found more species at OF (192 species, 72 families, 11 orders) than at CV (104 species, 52 families, 10 orders) in 2014. Dominant species in naked eye inspection were Lycorma emelianovi (Hemiptera: Fulgoridae), Apis mellifera (Hymenoptera: Apidae), Uroleucon cephalonopli (Hemiptera: Aphididae) at OF, and A. mellifera and R. maculatus at CV in 2014. Using naked eye inspection or sweeping net in 2014, more species of natural enemies were observed at OF (31 and 9 species, respectively) than at CV (18 and 5 species, respectively).

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

We have used bulked segregant analysis to screen the strain-specific DNA marker associated thermophilic strain of Pleurotus eryngii. Bulked genomic DNAs of Pleurotus eryngii were amplified by randomly amplified polymorphic DNA (RAPD) using OP-A, OP-B, OP-L, OP-P, OP-R and OP-S primers to screen the strain-specific DNA marker. A unique DNA fragment of 550 bp was amplified with OP-S07 primer from the thermophilic strain and sequenced. A sequence characterized amplified region (SCAR) marker was designed on the basis of the determined sequence and named as OP-S07-1. The PCR analysis with the OP-S07-1 primer showed that this SCAR marker clearly distinguish the thermophilic strains from the control strains.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of the NeuroBactrus, the Bacillus thuringiensis cry1-5 crystal protein gene was introduced into the Autographa californica nucleopolyhedrovirus(AcMNPV) genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an approximately 65-kDa active toxin. In addition, qPCR revealed that the neurotoxin was expressed from the early phase of infection. The NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time(LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Re-recombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins(Bt toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passed NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that the NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-κB, NF-κB-related genes, inflammatory cytokines, TNF-α and IL-1β in RAW 264.7 cells. NF-κB inhibitor pretreatment significantly reduced the levels of TNF-α and IL-1β mRNA and protein. In addition, the Aa LPS-induced TNF-α and IL-1β expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-α and IL-1β expression through NF-κB and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)- activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm (Bombyx mori) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, and porPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or typsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, B. thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.