폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus) 바이러스 게놈에 포함된 시스타틴(CpBV-CST1) 유전자의 과발현이 곤 충의 면역 및 발육을 교란한다. 이 연구는 바이러스 유래 시스타틴의 생물적 기능의 심화 연구와 해충저항성 작물 개발을 위해 담배형질전환체를 구축하는 데 목적을 두었다. 이를 위해 형질전환체를 대상으로 목표유전자의 발현분석과 곤충에 대한 발육억제에 대한 생물검정을 수행했다. 시 스타틴 유전자를 pBI121 운반체에 재조합한 pBI121-CST를 제작하고, 이를 아그로박테리움(Agrobacterium tumefasciens) 세균 매개에 의한 담 배 형질전환 및 재분화를 유도하여 약 92%의 높은 신초 재분화율을 나타냈다. 이들 재분화된 개체 가운데 담배 genomic DNA에 시스타틴 유전 자가 삽입된 형질전환 추정 개체를 PCR 분석법으로 선발하였다. 다시 quantitative real-time PCR (qRT-PCR) 분석을 통해 이들 목표유전자 의 발현을 분석하였다. qRT-PCR 결과는 형질전환 추정 개체가 비형질전환체에 비해 유전자 발현이 약 17 배 높게 나타나 형질전환계통에서 목 표유전자가 안정적으로 발현되고 있음을 확인했다. 선발된 형질전환담배를 대상으로 갓 부화한 담배나방(Helicoverpa assulta) 1령 유충에 대한 살충효과를 확인하였다. 살충력에 있어서 형질전환계통간의 차이가 있었다. 특히 T9와 T12계통은 섭식 후 7 일차 조사에서 95% 이상의 살충효 과를 보였다. 이상의 결과들은 CpBV-CST1이 해충저항성 작물 개발에 필요한 유용 유전자 자원으로서 활용될 수 있음을 제시하고 있다.

배추좀나방(Plutella xylostella)이 국내에서 월동이 가능한 지 명확하지 않았다. 본 연구는 배추좀나방의 내한성에 기초한 야외 노출 실험 을 실시하여 월동 환경 조건을 결정하고, 동계 야외 지역의 배추좀나방 유충 서식지 관찰 및 성페로몬을 이용한 성충 모니터링을 통해 월동이 가 능한 지 조사하였다. 또한 이들 월동집단의 유래를 추적하기 위해 다형유전좌위를 이용한 집단 분석을 실시하였다. 배추좀나방의 체내빙결점 보 다 높은 -5℃로 처리한 결과 모든 미성숙 발육태에서 뚜렷한 생존력 저하를 보여 직접적 냉해 피해를 주었다. 여기서 유충발육태는 가장 낮은 냉 해 피해를 받았다. 그러나 5℃로 장기간(4 주) 처리한 결과 냉해 피해는 없었지만, 유충의 경우 먹이가 없는 상태에서 치사율이 증가했다. 모든 발 육태의 배추좀나방을 대상으로 겨울 기간 동안 야외조건에 노출시킨 결과 모든 발육태에서 생존력 저하를 나타냈다. 그러나 비가온 실내조건에서 저온 피해를 줄였으며 유충의 경우 먹이가 공급되면 생존력이 뚜렷하게 증가하였다. 동계 성페로몬 모니터링 결과 2014년도 최초의 성충발생일 은 유사한 시기에 서로 다른 지역(약 260 Km 거리)에서 나타났으며 월동집단의 성충은 4월 상순까지 포획되었다. 지역간 이들 월동집단의 유전 적 거리는 다형분자마커를 이용하여 분석되었으며 이들 월동집단들 사이에 뚜렷한 유전적 분화가 있는 것을 나타냈다. 본 연구는 배추좀나방의 국내 월동이 남쪽 지역 또는 기주 식물이 있는 시설재배지에서 가능한 것으로 제시하고 있다.

Six of cadherins have been selected from the P. xylostella genome 52 open reading frames are annotated as cadherin-like genes. Compared to other 5 cadherins of P. xylostella (PxCads), PxCad1 has the highest homology with other lepidopteran insect cadherins and PxCad1 was expressed in all developmental stages specially in gut tissue. Expression of PxCad1 was suppressed by feeding its specific double-stranded RNA (dsRNA, 150ng/larva) and treatment of dsPxCad1 significantly reduced susceptibility to Bt Cry1Ac toxin. To confirm the specific interaction between PxCad1 and Cry1Ac, a toxin-binding assay was performed using enzyme-linked immunosorbent assay (ELISA). The ELISA indicates that BBMV extracted from PxCad1-silenced P. xylostella have significantly lower binding activity to active form of Cry1Ac than control BBMV. Moreover, the analysis of the binding parameters showed that the toxin affinity (Kd) of the control BBMV extract (BBMV-dsCON) was 6.08 ± 0.84 nM, which was not much different to the affinity value (6.72 ± 0.81 nM) of the dsPxCad1 treatment. However, there was a remarkable difference in number of binding sites (Bmax), in which BBMV-dsCON extract had 1.61 ± 0.04, but the BBMV-dsPxCad1 extract had 0.88 ± 0.02. Taken together, these results are suggest that PxCad1 is a functional receptor for Cry1Ac toxicity against P. xylostella larva.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.

Glycerol is a polyol that is responsible for the cold hardiness of insects. Glycerol kinase gene, which is an important key enzyme for glycerol biosynthesis, was predicted from whole genome sequencing data from the diamondback moth, Plutella xylostella. Four of P. xylostella glycerol kinase genes (PxGKs) were determined as a functional glycerol kinase through in silico study. Pre-exposure of P. xylostella larvae to 4°C for 7 h significantly enhanced survival (rapid cold hardiness: RCH) under a freezing temperature (-10°C) and increased glycerol titers. To determination of functional GK gene, expressions of all GK genes were measured by RT-PCR analysis. All GK genes were expressed in all larval stage and tissues (gut, hemocyte, and fat body). Expressions of all GK genes were suppressed by its specific dsRNA treatment into 4 th instar larva. Each 150 ng of dsRNA PxGK2 treatment significantly decreased glycerol amount in hemolymph by HPLC analysis. Larval treated by dsRNA PxGK2 also significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by a specific PxGK2 gene among GK gene isoforms in P. xylostella. In addition, the beet armyworm, Spodpotera exigua, encodes RCHassociated SeGK1, which has been functionally identified by RNA interference.

Putative cadherin genes, which are a receptor of the Bacillus thuringinesis toxins, were predicted from a whole genome sequencing data from the diamondback moth, Plutella xylostella. After the sequence and expression analysis, a Bt receptor cadherin gene was selected. The P. xylostella cadherin gene (PxCad1, GenBank Accession no. GU901158.1) encodes 11 cadherin repeats and a transmembrane domain. The PxCad1 gene was expressed in all developmental stage specifically in gut tissue by RT-PCR analysis. Expression of PxCad1 gene was suppressed by feeding of its specific dsRNA PxCad1 in 4th instar larval stage. The suppression of PxCad1 expression did not significantly feeding of its specific dsRNA PxCad1 in 4th instar larval stage. The suppression of PxCad1 expression did not significantly influence on pupal and adult development of P. xylostella. However, the larval treated with dsRNA PeCad1 (150 ng/larva) significantly reduced susceptibility to B. thuringiensis Cry1Ac (4.83 μg/ml). By contrast, the dsRNA PxCad1 -treated larvae did not show any change in susceptibility to B. thuringiensis Cry1Ca (0.24 μg/ml). These results suggest that PxCad1 is a specific receptor of Cry1Ac toxin from B. thuringiensis in P. xylostella.

Cold tolerance of the palm thrips, Thrips palmi Karny, was investigated to predict its survival in field during winter. Supercooling temperatures of T. palmi ranged from -26.4 to -18.4°C. However, exposure to subzero temperatures (from -5°C to -15°C) gave significant mortality to all developmental stages of T. palmi. Thus, T. palmi was determined to be a freeze-susceptible and suffered with cold injury. A brief pre-exposure to a low temperature (4°C) for 7 h significantly increase the cold tolerance of all stages of T. palmi with respect to survival at -10°C and supercooling capacity. A pre-exposure of T. palmi at 4°C significantly increased the survival rate on all developmental stages at -10°C. The rapid cold hardiness (RCH) was dependent on the duration of the pre-exposure period at 4°C in adult stage. Cryoprotectant analysis using an HPLC showed that the pre-exposure treatment increased the adult to synthesize glycerol, trehalose, mannitol, and mannose, at which trehalose represented the highest content. This study suggests that all stages of T. palmi are able to become cold-hardy by RCH, in which several polyols may play crucial roles as cryoprotectant.

A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared to a wild type primary form, a secondary form usually lose several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in production of eicosanoid-biosynthesis inhibitors (EBIs) compared to the primary form of X. nematophila. Production of oxindole and p-hydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on a cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila significantly more enhanced the pathogenicity of Bacillus thuringiensis (Bt) than the culture broth of the secondary form. Furthermore, this study developed a high efficient ‘Dual Bt-Plus’ to control both lepidopteran insect pests of Plutella xylostella and Spodoptera exigua by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.

An entomopathogenic bacterium, Xenorhabdus nematophila (Xn), is symbiotic to a nematode, Steinernema carpocapsae, and exhibits high pathogenicity to lepidoptera insects. Its metabolites released into the bacterial culture broth and also virulent in oral especially when they are treated with Bacillus thuringiensis (Bt). This study devised a high efficacy microbial insecticide by combining Xn culture broth and Bt. Bt kurstaki (Btk) exhibited relatively higher pathogenicity to Plutella xylostella than Spodoptera exigua larvae. By contrast, Bt aizawai (Bta) showed a reverse pathogenicity pattern. Phase Ⅰ type of Xn (XnK1) was isolated from S. carpocapsae Pochun and exhibited high pathogenicity than phase Ⅱ bacteria. Three bacterial mixtures of Bta+XnK1, Btk+XnK1, and Bta+Btk+XnK1 were prepared and analyzed in their target insects. Bta+XnK1 showed higher pathogenicity than those of Bta alone or Btk+XnK1 in P. xylostella. Btk+XnK1 showed higher pathogenicity than those of Btk alone or Bta+XnK1 in S. exigua. Bta+BtK+XnK1 showed high pathogenicity against both P. xylostella and S. exigua.

Cry3 toxins from Bacillus thuringiensis are used as biopesticides and the transgenic crops to control of leaf-feeding beetles. Cadherin in insect midgut epithelium is identified as receptor for Cry toxins in several insects and some domains of it synergizes Cry toxicity. Cadherin (DvCad1-CR8-10) fragment of Diabrotica virgifera virgifera enhanced Cry3Bb toxicity to Colorado potato beetle (CPB), Leptinotarsa decemlineata. Single cadherin repeat (CR) fragment of DvCad1-CR8-10, have a strong binding affinity to the active Cry3Bb toxin. The dissociation constant Kd value of CR8, CR9, and CR10 were 4.9 nM, 28.2 nM, and 4.6 nM, respectively. Interestingly, CR8 and CR10 enhanced Cry3Bb toxicity against CPB and Lesser mealworm (LMW), Alphitobius diaperinus, neonates in up to 2-folds. The DvCad1-CR10 peptide is further analyzed by in-frame deletion to determine the active site for Cry3Bb toxin. The active site is narrowed down to a 26 amino acid locating in the N-terminal region of DvCad1-CR10 that either synergized Cry3Bb toxicity on the CPB and LMW neonates in 3-folds or bound to the toxin with high affinity. The extent of Cry3Bb toxin enhancement by the activie site in DvCad1-CR10 may have practical application for control of CPB and LMW.

Cadherin gene, which is a receptor of the Bacillus thuringiensis toxins, was predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, Spodoptera exigua. The S. exigua cadherin gene (SeCad1) encodes 9 cadherin repeats and a tranmembrane domain. The SeCad1 gene was expressed in all developmental stage specifically in gut tissue by RT-PCR analysis. Expression of SeCad1 gene was suppressed by both injection and feeding of its specific dsRNASeCad1 in 5th instar larval stage. The suppression of SeCad1 expression did not significantly influence on pupal and adult development of S. exigua. However, the larval treated with dsRNASeCad1 (100 ng/larva) significantly reduced susceptibility to B. thuringiensis ssp. aizawai (3 × 106 CFU/larva). By contrast, the dsRNASeCad1-treated larvae did not show any change in susceptibility to B. thuringiensis ssp. krustaki (4 × 107 CFU/larva). These results suggest that SeCad1 is a specific receptor of Cry1A toxin from B. thuringiensis in S. exigua, but not Cry1C toxin.

The beet armyworm, Spodoptera exigua, is a freeze-susceptible species and overwinters without diapause in temperate zone. Depression of supercooling point (SCP) and rapid cold hardiness (RCH) allow S. exigua to survive at low temperatures. This study reports a polyol which is responsible for the cold hardiness of S. exigua. Pre-exposure of S. exigua larvae to 4°C for 6 h significantly enhanced survival under a freezing temperature (-10°C). This pre-exposure treatment also significantly depressed larval SCPs. Analysis of polyols indicated that glycerol titers significantly increase with increase of pre-exposure time. Glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) are involved in glycolysis pathway of insect. The S. exigua GK (SeGK1) and G3PDH (SeG3PDH1) genes were predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, S. exigua. The SeGK1 and SeG3PDH1 genes both were expressed in all larval stage by RT-PCR analysis. Expression of SeGK1 and SeG3PDH1 genes were suppressed by its specific dsRNASeGK1 or dsRNASeG3PDH1 injection into hemocoel of 5th instar larva. Each 200 ng of dsRNASeGK1 or dsRNASeG3PDH1 injection also significantly decreased glycerol amount in hemolymph. Larval treated by either dsRNASeGK1 or dsRNASeG3PDH1 significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by SeGK1 and SeG3PDH1 genes in S. exigua.

An entomopathogenic bacterium, Xenorhabdus nematophila, secretes at least eight bacterial metabolites, which have suppressive effects on insect immunity. This study quantified their sequential production during bacterial growth and analyzed their individual immunosuppressive activities against an insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth in both insect host and culture medium, in which eight metabolites were secreted in different time points. At early growth phase (6 to 12 h), Ac-FGV, Cis-cPY, PHPP and indole metabolites were detected in the culture broth. During early growth phase, PHPP was highly potent to inhibit phenoloxidase activity as well as nodule formation. At late growth phase (24 to 48 h), BZA, HPA, PY were detected at 10 – 140 ppm in the culture broth, their metabolites were highly potent to inhibit phospholipase A2 and to induce cytotoxicity to hemocytes. These results suggest that X. nematophila sequentially produces the immune suppressive metabolites, which cooperatively inhibit different steps of insect immune responses.