Varroa destructor is a devastating ectoparasitic mite which attacks Honeybee, Apis mellifera. V. destructor feeds on honeybee hemolymph, and often harbors small RNA viruses such as the deformed wing virus to transmit these viruses in the infested bee hive. To survey the genes of V. destructor, total RNA was subjected to high-throughput transcriptome sequencing to construct in silico cDNA library by using the Illumina HiSeq 2000 platform. Total of 2×107,748,792 paired-end short reads were obtained and quality filtered reads were subjected to Trinity de novo assembler followed by TransDecoder, and CD-HIT program to make a V. destructor reference cDNA library containing 28,023 of clustered contigs with protein coding capacity. These cDNA sequences will help us to understand the molecular biology of V. destructor.

Crystals of proteinaceous insecticidal proteins, Cry proteins, produced by Bacill us thuringiensis (Bt) have been generally used used to control insect pests. In this st udy, 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 Plutella xylostella, Spodopt era exigua and Ostrinia furnacalis were identified. To construct novel cry genes wi th enhanced insecticidal activity, we randomly mutated these 24 amino acid sequen ces by in vitro muti site-directed mutagenesis, resulting in totally 34 mutant cry gen es. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded in to polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activit ies 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 compar ed to those of Cry1Ac and Cry1C. Especially, among them Mutant-N16 showed th e highest insecticidal activity against to both of P. xylostella, S. exigua and Ostrinia furnacalis. Therefore, Mutant-N16 is estimated to have the potential for the efficac ious bioagent.

The Asian honeybee, Apis cerana, is a native honeybee species in Korea which is important in agriculture for pollination and honey production. For better understanding of the physiology of A. cerana, high-throughput Illumina transcriptome sequencing was performed to analyze the gene expression profiles of queen, worker and larva. A total of 219,799,682 clean reads corresponding to 22.2Gb of nucleotide sequences was obtained from the whole body total RNA samples. The Apis mellifera reference mRNA sequence database was used to measure the gene expression level with Bowtie2 and eXpress software and the Illumina short reads were mapped to 11,459 out of 11,736 A. mellifera reference genes. Total of 9,221 genes with FPKM value greater than 5 of each sample group were subjected to evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) with BLASTX for gene ontology analysis. The differential gene expression between queen and worker, and worker and larva were analyzed to screen the overexpressed genes in each sample group. in the queen and worker sample group, total of 1,766 genes were differentially expressed with 887 and 879 genes overexpressed over two folds in queen and worker, respectively. In the worker and larva sample group, total of 1,410 genes were differentially expressed with 1,009 and 401 genes overexpressed over two folds in worker and larva, respectively.

Rice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice and mediated by small brown planthopper, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among Rice stripe virus, rice and small brown planthopper, transcriptomes of the RSV-viruliferous (RVLS) and non-viruliferous L. striatellus (NVLS) were comparatively analysed. For this, non-viruliferous L. striatellus were collected from non-infected rice field and fed RSV-infected rice for 5 days. With the RNAs prepared from the RSV-viruliferous and the non-viruliferous small brown planthoppers, we conducted Illumina RNA sequencing (Hiseq 2000) and then two transcriptome databases were generated from RVLS and NVLS, respectively. The transcriptome of RVLS and NVLS were campared to figure out how the gene expression of the insects affected by Rice Stripe Virus. RSV-dependently regulated genes analysed from this study may have important functions in the transmission and replication of RSV.

Sacbrood virus (SBV) is one of the most fatal pathogens against Asian honeybee, Apis cerana. This virus cause failure of the insect larvae to pupate and death of the adult insects. This study has analyzed the host genes affected by viral infection, by comparing the expression level of host transcripts infected with or without SBV. As a first step, we sequenced the cDNA libraries of Asian honeybee by using illumina RNA sequencing. The sequences were de novo assembled to acquire honeybee transcriptome sequences. The transcriptome was annotated by the sequence comparison to known protein sequences by BLASTX and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database with functional categories and description. By mapping the RNA-seq data to de novo assembled transcripts, we characterized the differentially expressed transcripts between SBV-infected and non-infected Asian honeybee.

The novel serogroup of Bacillus thuringiensis serovar mogi (H3a3b3d) was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. Plasmids from B. thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. In this study, the genome sequence of the strain was determined. The 6.0-Mb genome of B. thuringiensis mogi contains three replicons: a circular chromosome (5.40-Mb) encoding 5,652 predicted open reading frames (ORFs), and two megaplasmids, pMOGI364 (364 564 bp) and pMOGI222 (222 348 bp). The G+C contents of these replicons ranged from 31.3% to 34.2% for pMOGI364 and pMOGI222, respectively. There are six putative cry genes, cry19Bb1, cry73Aa, cry20Bb1, cry27Ab1, cry4Aa and cry56Ba1, distributed on these two megaplasmids. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. Also, these cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization.

Rice stripe virus disease (RSVD), one of the most serious disease of rice is mediated through the sucking by small brown planthopper, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among the host plant, vector insect and plant-pathogenic virus, we investigated transcriptome of the vector insect and the differences between viruliferous and naïve L.striatellus. For this, naïve L. striatellus were collected from non-infected rice field and 50 L.striatellus of them were fed RSV-infected rice for 5 days. With the RSV-viruliferous and the naïve insects, we conducted Illumina RNA sequencing (Hiseq 2000) and obtained 175,243,488 and 146,031,348 reads from viruliferous and naïve L.striatellus, respectively. These reads were assembled into contigs and two transcriptome databases were generated. The transcriptome of naïve and RSV-viruliferous L. striatellus were campared to figure out up-regulated or down-regulated genes. These RSV-dependently regulated genes may have important function in the behavior of planthoppers or the transmission of RSV.

Proteinaceous insecticidal proteins, Cry proteins, from Bacillus thuringiensis (Bt) are insecticidal proteins that are highly active against several species of Lepidoptera. Thus, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, 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 Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes encodes potent insecticidal proteins in the form of crystalline protoxins of 95 kDa. 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.

Plasmids are crucial for determining the pathogenicity and host range of organisms of the Bacillus thuringiensis strains. In this research, a novel serogroup of B. thuringiensis serovar mogi (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained two megaplasmids (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that there are 7 putative cry genes, cry19Bb1, cry73Aa, cry40orf2, cry20Bb1, cry27Ab1, cry56Ba1 and cry39orf2, distributed on the two different megaplasmids, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and p1KSD, which is a recombinant expression vector containing cyt1Aa promoter combined with the STAB-SD sequence, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. These results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.