The development of entomopathogenic fungi has received increasing interest as part of integrated pest management strategies as biocontrol agents. It is reasonable to assume that entomopathogenic fungi might produce secondary metabolites modulating juvenile hormone for their survival against defense mechanisms of host insects. In this study, acetone extracts of 189 entomopathogenic fungi cultured on unpolished rice medium were screened for their juvenile hormone antagonist (JHAN) activities using the yeast-two hybrid system. Among 14 extracts showing JHAN activities, extract of the F-145 showed high level of insecticidal activities against both Plutella xylostella and Aedes albopictus. This isolate was identified as Lecanicillium attenuatum. These results suggested that the Lecanicillium attenuatum could be useful for development of eco-friendly insecticides.

The virus-like particle (VLP) is similar to a pathogenic virus and has a high immunogenicity. However, in the selection of various structural proteins to form VLP, all expression systems commonly consume most of the time and suffer from various difficulties. Therefore, there is a need for a system that can rapidly determine the structural proteins required for effective VLP production. This study aims to construct a transient expression platform using insect cells to solve this problem. Plasmid-based expression vectors and baculovirus-inducible expression vectors were constructed. The vectors were evaluated for overexpression using EGFP. We also confirmed the formation of virus like particles through overexpression of virus structural proteins.

The population of managed honeybees has been dramatically declining the recent past in worldwide. N. ceranae causessignificant detriment to honey production and results in economic losses critically. In our knowledge, Fumagillin is theonly antibiotic approved for control of nosemosis in honeybees. In this study, to select isolate with anti-Nosema activityagainst N. ceranae. Entomopathogenic fungi cultural filtrates were screened using in vitro polar tube germination assay.These fungi cultural filtrates were used to evaluate the safety of honeybees and their inhibition of nosemosis. As a result,P. marquandii 364 and Pochonia sp. 60 showed inhibitory activity on the growth of N. ceranae in honeybees and didnot significantly affect the survival rate of honeybees. These may be employed as antibiotic agents and a good featureto be used in the development of new biocontrol agents of nosemosis.

The baculovirus expression vector system (BEVS) is an effective and widely used system for the production of recombinantproteins in insect cells or larvae. However, the expression efficiency of recombinant proteins using the polyhedrin promotercould not acquire the protein yields observed for native polyhedrin. In this study, we tried to develop hyper expressionvector by the optimal combination of previously reported various enhancer factors. The selected enhancer factors for optimalexpression consists homologous region5 (hr5), VP39 promoter and burst sequences. Seven recombinant viruses were madeto compare EGFP expression level. Each recombinant viruses showed different expression levels respectively, and themost of expression level was observed with higher than those of the previous vectors. This study suggests a new optionfor hyper expression of useful recombinant protein using the BEVS.

The two-spotted spider mite, Tetranychus urticae, and gray mold disease caused by Botrytis cinerea, are importantpest and plant disease. To control these, many people have been relied on chemical methods for a long time. However,their continuous use has resulted in resistance of pest and disease. To surmount or avoid these problems, we evaluatedantifungal activity of the selected fungi with high virulence to mite to explore the potential for the dual control of notonly T. urticae but also B. cinerea. To maximize the use of spores and cultural filtrates, the virulence to mite was evaluatedusing culture products, and effective culture condition was investigated against blastospore production and antifungal activity.Consequently, the 2 fungal isolate selected in this study were confirmed to have diverse potential and would be usedeffectively for dual control agents against the two-spotted spider mite and plant diseases

Entomopathogenic fungi have been widely studied for their potential as the effective biological control agents. Theyproduce variety of secondary metabolites with insecticidal activities, and it is reasonable to assume that entomopathogenicfungi might produce secondary metabolites modulating juvenile hormone for their survival against defense mechanismsof host insect. In this study, Acetone extracts of 189 entomopathogenic fungi cultured on unpolished rice medium werescreened for their juvenile hormone antagonist (JHAN) activities using the yeast-two hybrid system. Among them, 14extracts showed high level of JHAN activities and their insecticidal activities against Aedes albopictus were investigated.

Viral particles of Porcine epidemic diarrhea virus (PEDV) consist of a four structural proteins. Among them Spikeprotein mediated responsible for receptor binding and membrane fusion during viral infection and therefore the main targetof neutralizing antibodies. Virus-like particles (VLPs) are consisted of one or more viral structural proteins, and theirmorphologies closely resemble those of the native virus. VLPs have no virulence and can elicit robust immune responsesas compared with inactivated or live-attenuated virus vaccines. Thus, in this study, we tried two methods for VLP constructionin Bombyx mori, one is traditional method and the other is chimeric VLP method using the influenza matrix protein.Both methods could produce successfully PEDV VLPs.

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various enhancer factor were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). As a hyper expression factor, the optimal hyper BEVS was constructed by combining Hr5 sequence, VP39 promoter and Burst sequences. Additionally, the proteins expressed by the hyper expression system was markedly increased. This study suggests a new option for higher expression of useful foreign recombinant protein using the BEVS.

The green peach aphid, Myzus persicae, is one of the most important pests of vegetable crops worldwide. This species cause direct damage by feeding on plant nutrients and indirect damage as transmits many virus vectors. The control of aphid has relied on the use of chemical insecticides and their intensive use over many years has led to the development of resistance. To surmount or avoid these problems, we screened the entomopathogenic fungi against green peach aphid and evaluated their virulences. Several entomopathogenic fungal isolates were selected with high virulence to green peach aphid. Additionally, the antimicrobial activity of the selected fungal isolates was tested and analyzed to phytopathogens. Consequently, these entomopathogenic fungi would be used effectively for dual control agents for the green peach aphid and phytopathogen.

Recombinant proteins including a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. However, the affinity tag must be removed from the target after the purification process. Recently, the Synechocystis sp. PCC6803 DnaB mini-intein (Ssp DnaB mini-intein) is widely used in Escherichia coli expression systems as the solution of this problem. The Ssp DnaB mini-intein can be induced simply by shifting of pH and temperature, offering a benefit to cleave a peptide bond without using a protease or chemical reagent. Although the utility of this novel tag is widely studied in E. coli, there is no report yet in baculovirus expression vector system (BEVS). In this study, we generated several recombinant baculoviruses to express foreign proteins with Ssp DnaB mini-intein. In conclusion Ssp DnaB mini-intein was good tag also in BEVS with more advantages.

Recombinant baculovirus genome that absence part of ORF1629 has used to enhance efficiency of recombinant virus selection because a role of ORF1629 is believed to essential for viral replication in insect cells. It can be recovered by recombination with a transfer vector containing a complete ORF1629. Some recombinant bacmids were generated for this purpose. Recombinant bacmid GOZA, one of them, has a mini-F replicon for Escherichia coli and the partial ORF1629 gene. By accident, we could observe the replication of ApGOZA singly in Sf9 cells. Produced virus from ApGOZA was investigated for the existence of truncated ORF1629 not intact ORF1629 by PCR amplification and genomic sequencing. Also, we could observe the replication of AcGOZA alone in Sf9 cells. To analyze the influence of truncated ORF1629, the viral growth, BV production and polyhedral formation were conducted comparing to wild type virus and recombinat virus containing intact ORF1629. The results suggested the probability that ORF1629 is not essential for replication.

Canine parvovirus (CPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in Canine reproductive failure, causing serious economic losses in the pet industry. The major capsid protein, VP2 is the main target protein for neutralizing antibodies in CPV. When VP2 was expressed using baculovirus, it was produced abundantly and assembled into virus-like particles (VLPs) similar in size and morphology to the original virions. It was named as CPV-VLP. Additionally, p35 sequences of canine distemper virus (CDV) as T-helper epitope were fusion-expressed with each of N-term, C-term or both sides of CPV-VP2. Production of double antigenic recombinant protein and formation of VLPs were analyzed by SDS-PAGE and transmission electron microscopy, respectively.

Honeybees have faced many diseases which treaten bee colony including a serious population decline phenomenon called Colony Collapse Disorder (CCD). Nosema ceranae is a pathogen cause nosemosis, which is now wide spread around the world. According to the genome sequencing for N. ceranae, it has been identified that the presence of machinery for RNA silencing. Microsporidia N. ceranae that are obligated intracellular parasites depend on their host for energetic and metabolic needs. Here we selected the several genes from mitosome of N. ceranae to develop RNAi for the control of Nosema. Especially, TOM40, FNR1, FNR2 and Nar1 were chosen. After infection of N. ceranae, the Honeybee were treated with RNAi either by using only one or combining two or more. The infection rate and specific gene silecing in Nosema were analyzed.

The two-spotted spider mite, Tetranychus urticae, is one of the most important pests of crops. However, chemical acaricides are not always effective and their continuous use has resulted in resistance among two-spotted spider mite populations. To reduce or replace use of chemical acaricides, the entomopathogenic fungi having acaricidal activity were investigated. As results, 12 isolates could be selected from 342 isolates of fungi against two-spotted spider mite. To show the multiple roles of entomopathogenic fungi against plant pathogen, the antimicrobial activity of selected 12 isolates were evaluated to plant pathogenic bacterium Ralstonia solanacearum and fungus Botrytis cinerea. Various antimicrobial activities were observed against these plant pathogens. Consequently, these entomopathogenic fungi would be offered an alternative for dual control agents against the two-spotted spider mite and plant diseases.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs with high mortality. PEDV is belong to Coronavirus, enveloped, single-stranded, positive-sense RNA virus. PEDV particles were composed of four structure proteins such as a glycosylated peplomer (spike, S) protein, envelope (E), glycosylated membrane (M) protein, and unglycosylated RNA-binding nucleocapsid (N) protein. Many of previous studies talk about this four structure proteins have a great potential to diagnosis and prevent PEDV. In this study we investigated expression of these structure proteins using the bacterial and baculovirus expression system. In bacterial expression system, our results showed that structure proteins fused polyhedrin and intein gene were expressed higher than non-fusion structure proteins. The expressed fusion proteins were used to immune mice for generating a polyclonal antibodies. In baculovirus expression system, co-infection of insect cells with these four recombinant baculoviruses led to self-assembly of virus-like particles as demonstrated by Transmission electron microscopy. They were confirmed by western blot analysis using pre-made polyclonal antibodies. Finding in this study may provide important information for vaccine and diagnostic development.

Insects are among the most diverse groups of animals on the planet, representing more than half of all known living organisms. These insects are found in nearly every environment. Although humans regard certain insects as pests and attempt to control them using insecticides, most insects perform complex ecological functions, and provide either direct or indirect economic benefits to humans. Recently, the importance of insects used as food sources or as pets has increased in many countries, including Korea. In addition, several insects have a strong influence on people's emotion. Insect-mediated mental healthcare program is designed to help people who have disorders with physical, behavior and development. Children who have mental disorder, the experimental group that was provided with an insect-mediated mental healthcare program over a total of 8 sections, one section per week, 60 minutes per section, followed by pre-test and post-test. They responded to therapeutic effect after the completion of the program. Further research on the basis of this study is expected to help children with emotional therapy in other areas.

The population of managed honey bees has been dramatically declining the recent past in worldwide. The one of most common disease of bees is nosemosis, the nosemosis is caused by microsporidia in the genus Nosema. Nosema apis and N. ceranae have been described as honeybee pathogens. These microsporidia are highly evoloved fungi with an obligately intracellular parasitic lifstyle. The disease causes significant detriment to honey production and results in economic losses. In our knowledge, Fumagillin is the only antibiotic approved for control of nosemosis in honey bees, however this antibiotic may have unintended effects on the honey bee host, ultimately contributing to increased prevalence and pathogenicity of Nosema. Therefore, we screened anti-Nosema substances from entomopathogenic fungal culture filtrates using in vitro polar tube germination assay. These fungal metabolites are employed as antibiotic agents. As results, Total 3 samples (23% of 13 total samples) showing the germinating inhibition against N. ceranae. This screening method may be useful for the detection of anti-Nosema substances from various samples and selected samples in this study may be a good feature to be used in the development of a new biocontrol method of nosemosis.

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Additionally, the several proteins expressed by the partial polyhedrin-fused expression system was markedly increased. However, we identified that hyper-expression of target protein varied depending on the partial polyhedrin. Therefore, we constructed the virus inducible partial polyhedrin fusion transient expression system. This system amenable for screening of suitable partial polyhedrin to produce the target protein. The present study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a non-enveloped virus and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2 is the main target for neutralizing antibodies in PPV. When VP2 was expressed in large amounts, it assembled into virus-like particles (VLPs) similar in size and morphology to the original virions. In this study, we generated the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) to express the VP2 protein. Expression of the VP2 protein was analyzed by SDS-PAGE and Western blot. The recombinant VP2 protein of approximately 64 kDa was detected by both analyses. The formation of VLP by recombinant VP2 was confirmed through transmission electron microscopy examination. The purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm.

Insects constitute the largest and most diverse group of animals on Earth. They also serve as the hosts or nutrient sources for an immense assemblage of parasites, pathogens, and predators, ranging from bacteria and fungi to plant and animals. More than 700 known fungal species from 100 genera have adopted an entomopathogenic lifestyle. These fungi are widely distributed, occurring in aquatic, forest, and agricultural habitats, and are often used as active ingredients in microbial insect pest control agents. Their mode of action against insects involves the attachment of conidia to the insect cuticle, followed by germination, cuticle penetration, and internal dissemination throughout the insect. During this process, secreted enzymes, protein toxins, and secondary metabolites can be used by the fungus to overcome the host immune system, modify host behavior, and defend host resources against competing pathogens and saprophytes. In some cases, the host insect relationship has been found to be associated with bioactive fungal metabolites. These metabolites exhibit a wide variety of insecticidal, antibacterial, antifungal, anticancer, antioxidant, and antiviral activities. Using molecular techniques and phylogenetic analyses, both the asexual (anamorphic) stages and sexual (teleomorphic) stages of entomopathogenic fungi have recently been shown as linked together. Therefore, entomopathogenic fungi, especially in complex with the host insect, might be a promising source of bioactive molecules of pharmaceutical and various industrial interests. Here, we evaluated the antimicrobial activity of entomopathogenic fungi metabolites against plant pathogenic bacteria and fungi for the use in agriculture. The radicals scavenging activity and the anticancer activity were also evaluated for pharmaceutical interests.