Translational control is a strategy for various viruses to manipulate their hosts to suppress any acute antiviral activity. Some cys-motif genes encoded in polydnaviruses or teratocytes act as host translation inhibitory factor (HTIF) to defend the host antiviral activity. A novel cys-motif gene, TSP13, was encoded in the genome of an endoparasitoid wasp, Cotesia plutellae. TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value at 7.928. Genomic DNA region encoding open reading frame is interrupted with three introns. TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected to nonparasitized P. xylostella. In the virus-injected P. xylostella, TSP13 was shown to be expressed by RT-PCR analysis. Thus, TSP13 was turned out to be encoded in the proviral CpBV genome. TSP13 was cloned into a eukaryotic expression vector, which was then used to infect Sf9 cells to transiently express TSP13. The synthesized TSP13 was detected in the culture broth. Purified TSP13 significantly inhibited cellular immune responses. Furthermore, TSP13 entered the target cells and was localized in the cytosol. This study reports a novel cys-motif gene, which is encoded in CpBV genome localized on chromosome(s) of C. plutellae and replicated to be encapsidated in the episomal viral particles during parasitization.

Baculovirus expression system has been used to produce functional proteins of various eukaryotic genes. A polydnaviral gene, CpBV-ELP1, was cloned in an alpha-baculovirus, Autographa californica multiple nuclear polyhedral virus (AcNPV), and expressed in Sf9 cells. CpBV-ELP1 protein was released into the culture medium due to its signal peptide. The culture broth containing CpBV-ELP1 was collected and fractionated with different concentrations of ammonium sulfate. Most CpBV-ELP1 was precipitated in 25-100% ammonium sulfate. The precipitate proteins were separated with a size exclusion chromatography sieving 100 kDa size. CpBV-ELP1 was eluted after relatively high molecular weight protein peaks. The fractions rich in CpBV-ELP1 were collected and further fractionated with an anion exchange chromatography. The purified CpBV-ELP1 was toxic to both larvae of Plutella xylostella and Spodoptera exigua by oral test used as leaf dipping method. A lethal median concentrations (LC50) were 7.5 ㎍/mL (95% CI: 1.2-24.3 ㎍/mL) for 2nd instar larvae of P. xylostella and 4.4 ㎍/mL (95% CI: 1.9-8.4 ㎍/mL) for 3rd instar larvae of S. exigua. These results suggest that CpBV-ELP1 may be applied to develop novel transgenic crops.

Insect immunity is innate and consists of cellular and humoral immune responses. Cellular immune response usually requires hemocyte-spreading behavior, which is accompanied by cytoskeletal rearrangement. A glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), catalyzes an oxidation reaction of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate in the cytosol. Another function of GAPDH in mammalian cell is to bind C-terminal α-tubulin to facilitate cytoskeletal arrangement. An immunoprecipitation (IP) of viral protein, CpBV-CrV1, against hemocyte protein lysate revealed that CpBV-CrV1 binds to GAPDH, identified by MALDI-TOF analysis. RNA interference (RNAi) of GAPDH significantly suppressed cellular immune response, but neither RNAi of hexokinase nor aldolase suppressed the cellular immune response. A common molecular motif of CpBV-CrV1 and a-tubulin at C-terminal region supported the IP analysis. To test the role of α-tubulin motif in CpBV-CrV1, point mutations of CpBV-CrV1 were applied and resulted in loss of the biological activity of CpBV-CrV1. Furthermore, an immunofluorescence assay indicates CpBV-CrV1 colocalized with a-tubulin in hemocytes collected from Plutella xylostella parasitized by Cotesia plutellae possessing C. plutellae bracovirus (CpBV). This result suggests that GAPDH plays a critical role in hemocyte-spreading behavior during immune challenge, and it is a molecular target of the pathogenic virus.

The red-spotted apollo butterfly, Parnassius bremeri, immatures grow during winter and spring. Supercooling point of larvae during January goes much below -20℃. Morphologically, the larvae appear to be adapted to cold temperatures. Dark-colored body surface is useful to absorb solar energy and spiny integument may prevent any external ice formation on the body surface. Biochemically, P. bremeri larvae elevate glycerol as a cryoprotectant. This study reports two genes associated with glycerol biosynthesis in P. bremeri. Larval transcripts were analyzed using RNA-Seq technique. A total of 14 Gb transcripts were read by Illumina HiSeq and assembled to be 127,279 contigs. To specify the the genes associated with glycerol biosynthesis, a biosynthetic pathway to synthesize glycerol from dihydroxyacetone-3-phosphate was predicted with two genes of glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK). Both genes were annotated in the transcriptome of P. bremeri. Pb-GPDH encodes 166 amino acid residues containing NAD+-binding region, catalytic site, and calcium binding region. The predicted amino acid sequence was clustered with other lepidpopteran GPDH genes. Three Pb-GK genes were annotated from the transcriptome. Pb-GK1 encodes a full open reading frame of 514 amino acid residues. A ohylogenetic analysis showed that these three GKs were separately clustered. Interestingly, Pb-GK1 was clustered with other GKs that were known to be associated with rapid cold hardiness.

A fumigant, methyl bromide (MB), has been widely used to control insect pests on various post-harvest agricultural products. Due to its negative side effects, such as ozone deplete and phytotoxicity, MB should be replaced and alternative control technique ought to be developed. Furthermore, organic agricultural products need to be treated with non-chemical methods. To meet these needs, controlled atmosphere and temperature treatment technique (CATTS) has been developed to control insect and mite pests infesting various post-harvest fruits. CATTS uses heat treatment at 46℃with a gradual increase of temperature program under ananoxia condition (less than 1% O2 and 15% CO2). The heating rate at ramping step is crucial to prevent any heat damage to fruits by mimicking natural conditions during summer period experienced by fruits in orchards. The exposure period to the high temperature is minimized by anoxia condition, which prevents any recovery metabolism occurring in the target insect/mite pests after the heat treatment. To develop MB alternative technique for exporting apples, CATTS conditions have been determined against several quarantine insect/mite pest, which are restricted from Korean apple import countries. Target insect pests include Grapholita molesta, Adoxophyes paraorana, Tetranychus viennensis, Conogethes punctiferalis, and Carposina sasakii. In each insect species, differences in susceptibility to high temperature were analyzed in all developmental stages. The highest tolerant developmental stages were adults in T. viennensis, but the fifth instar larvae in G. molesta, C. punctiferalis, C. sasakii, and A. paraorana. Gas conditions of CATTS kept 1% O2 and 15% CO2. After the internal fruit temperature increase from 25℃ to 44℃ during ramping stage (120-150 min), CATTS treatment lasted for 1-2 h. Under 1 h CATTS treatment, 100% mortality was recorded in all assays using more than 3,000 individuals of each species of T. viennensis adults, G. molesta fifth instar, C. sasakii fifth instar, and A. paraorana fifth instar. With 2 h CATTS treatment, 100% mortality was recorded in C. punctiferalis assay using 3,022 fifth instar larvae. Under 1 h CATTS treatment, no fruit damage was recorded in Fuji variety of apples. Treatment cost of CATTS is higher than that of methyl bromide (MB) treatment. However, once CATTS is equipped with nitrogen generator, it would be cheaper than MB in treatment cost. CATTS is a promising post-harvest pest control technique to replace MB treatment. Especially, CATTS would be an ideal control technique to disinfect any pests against organic agricultural products.

Evolution of resistance to entomopathogenic bacterium, Bacillus thuringiensis (Bt), can potentially reduce the efficacy of insecticidal proteins from Bt to insect pests in fields. Bt resistance is involved in modification of the toxin binding to its specific midgut membrane receptors, such as cadherin, aminopeptidase N, alkaline phosphatase, and ABC transporters. The beet armyworm, Spodoptera exigua, is one of major lepidopteran insect pest in Korea and showed highly susceptible to Cry1Ca. We investigated the Cry1Ca toxicity with respect to its binding affinity to a Bt receptor, cadherin compared with Cry1Ac. RNA interference (RNAi) of a cadherin of S. exigua (SeCad1) significantly suppressed the Cry1Ca to the toxic level of Cry1Ac. Binding affinity of Cry1Ca to brush border membrane vesicle (BBMV) of S. exigua midgut was significantly lost after SeCad1 RNAi. Binding affinity of Cry1Ac to BBMV was much low compared to that of Cry1Ca and less sensitive to SeCad1 RNAi. Direct binding assay of Cry toxins to SeCad1 was assessed using a recombinant cadherin repeat 10-11 (rCR10-11) of SeCad1. The addition to rCR10-11 to Cry1Ca significantly enhanced the toxicity under SeCad1 RNAi. However, the synergistic effect of rCR10-11 on toxicity of Cry1Ac was not much significant with poor binding affinity of Cry1Ac compared to Cry1Ca. These results indicate that the differential toxicity of Cry toxins against S. exigua is caused by the different affinities to the Bt receptor, cadherin.

Insect growth includes a mixture of continuous increase of body weight and discontinuous increase of exoskeleton size. Like other poikilothermic animals, insect growth varies with ambient temperatures, which determine the rates of various biochemical reactions of internal body metabolism. In addition, nutrient supply from food diet is necessary for insects to grow. The nutrient signal in insects is delivered to all internal tissues via insulin-like peptide (ILP). ILP signal transduction pathway in the target cells is highly similar to that of vertebrates. Insulin receptor (InR) specifically binds to ILP and activates PI3K to increase PIP3 intracellular level. The secondary messenger activates a specific serine-threonine kinase (Akt). Akt phosphorylates a nuclear receptor, FOXO, to prevent its translocation into nucleus and activates R6 kinase to upregulate protein synthesis. Insect exoskeleton is a physical barrier for growth and should be replaced with a new and greater size cuticle by molting at every growth. Upon reaching a critical body size for molting, the brain releases PTTH to stimulate 20-hydroxyecdysone (20E) biosynthesis in the PTG. The secreted 20E binds to EcR in the epidermal cells and forms a heterodimer receptor complex with USP. The active receptor enters the nucleus and activates molting-associated genes by inducing a specific upstream transcription factors. Decrease of 20E levels triggers apolysis to detach the old cuticle from the newly synthesized cuticle. With a series of neuropeptides, such as corazonin, ETH, EH, and CCAP, a sterotyped ecdysis behavior is released. The newly molted cuticle is then sclerotized by quinone molecules via bursicon signal. Metamorphosis is one of the most remarkable and characteristic physiological phenomena in insects among animals. Amphibians like frogs also exhibit the metamorphic development from an immature tadpole to a matured adult via two antagonistic developmental hormones, prolactin and thyroxine. In insects, juvenile hormone and 20E regulate the metamorphic developments, such as nymph-to-adult, larva-to-pupa, and pupa-to-adult. Insects are useful to monitor climate change by analyzing annual variation in their season phenology. Day-degree model has been widely used to predict insect occurrence based on climate change scenarios. However, insect growth depends on nutrient availability and quality as well as ambient temperature. Furthermore, insects also evolve in their growth and metamorphic developmental patterns according to climate change probably via epigenetic mode followed by genetic differentiation. Thus, we need to refine the prediction model of insect occurrence based on day-degree model with a fixed developmental threshold temperature.

국내 사과를 외국으로 수출하기 위해서는 수입국의 검역 대상 해충을 완전히 제거하는 것이 필수적이다. 이를 위한 수확 후 해충 사멸 기술 로서 환경조절열처리(controlled atmosphere and temperature treatment system: CATTS) 기술이 개발되고 있다. 본 연구는 상이한 가해 습 성으로 사과 과실에 피해를 주는 두 해충에 대해 CATTS의 사멸조건을 결정하였다. 사과애모무늬잎말이나방(Adoxophyes paraorana)은 사과 과 실 표면을 가해하는 해충인 반면 복숭아명나방(Dichocrocis punctiferalis)은 과실의 내부를 가해한다. 여러 가해 발육태 가운데 두 곤충 모두 5령 유충이 열에 대한 내성이 가장 높았다. 고농도(15%)의 이산화탄소, 저농도(1%) 산소 및 46℃ 온도 조건에서 1 시간 동안 처리하는 CATTS 처리 조건은 사과애모무늬잎말이나방 5령 유충을 100% 사멸시켰다. 그러나 동일한 CATTS 조건에서 복숭아명나방 5령 유충은 88%의 사멸 효과를 보였으며, 100% 사멸 효과를 나타내기 위해서는 2 시간의 열처리를 요구했다. 이를 바탕으로 사과를 가해하고 있는 두 종의 5령 유충 3,000 마리 이상에 대해서 각각 CATTS 처리 효과를 확증하였다. 본 연구는 완전 사멸을 위해서는 심식충이 비심식충에 비해 장기간 CATTS 처리가 요구 된다는 것을 보여 주었다.

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.

담배가루이(Bemisia tabaci)는 바이러스 매개 역할과 함께 참외에 심각한 경제적 피해를 주고 있다. 담배가루이는 기주 작물과 농약 감수성 정도에 따라 다양한 생태형으로 분류되고 있다. 본 연구는 안동시 풍천면에 소재한 참외밭에서 성충을 채집하여 PCR 분자진단기법으로 동정하 였다. 전체 11 곳의 채집 장소에서 Q 생태형 담배가루이를 진단하였고, 이 가운데 4 곳의 채집 장소에서 B 생태형도 검출되었다. 이러한 결과는 경북지역 참외 재배지에서 담배가루이가 발생한다는 최초의 보고이며, 특히 두 생태형이 동일한 재배지에 혼재한다는 것을 나타낸다.

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.

시스타틴(cystatin: CST)은 C1A류 시스테인 단백질분해효소에 대한 경쟁적 가역억제자로서 동식물류에서 파파인과 같은 캐셉신을 억제 대상으로 작용하게 된다. 바이러스 유래 CST (CpBV-CST1)이 폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus)에서 동정되었 다. 기존 연구는 이 유전자의 과발현이 배추좀나방(Plutella xylostella) 유충의 면역 및 발육을 교란한다는 것을 보여 주었다. 본 연구는 이 유전자 의 단백질 기능을 분석하기 위해 세균발현시스템을 이용하여 재조합단백질(rCpBV-CST1)을 형성하여 단백질분해효소에 대한 활성억제효과를 결정하고, 곤충의 면역과 발육에 대한 생리적 억제효과를 분석했다. 이 유전자 번역부위는 138 개 아미노산으로 약 15 kDa 크기의 단백질로 추 정되었다. CpBV-CST1이 먼저 pGEX 발현벡터에 재조합되고, BL21 STAR (DE3) competent cells에 형질전환된 후 0.5 mM IPTG로 4 시 간동안 과발현되었다. 분리된 재조합단백질은 파파인에 대한 뚜렷한 억제효과를 나타냈다. 이 재조합단백질은 파밤나방(Spodoptera exigua)에 대 해서 혈구소낭형성의 세포성 면역반응을 억제하고, 경구로 처리할 때 배추좀나방의 유충발육을 처리 농도에 비례하여 제한시켰다. 이상의 결과 는 CpBV-CST1이 해충 밀도 억제에 응용될 수 있음을 제시하고 있다.

Cotesia plutellae, an endoparasitoids braconid wasp, possesses a polydnaviruses (PDVs) called Cotesia plutellae bracovirus (CpBV) that encodes viral histone H4 (= CpBV-H4). This viral histone H4 shares high sequence homology (82.5%) with host`s H4 of P.xylostella, except an extended N-terminal tail consisting of 38 amino acid residues with nine lysines. Its extended N-terminal tail has been postulated to play a crucial role in suppressing host immunity, growth and development-associated genes, presumably through an epigenetic control mechanism. A suppression subtractive hybridization (SSH) analysis was analyzed in transcriptome by short-read sequencing technology and provided several target and non-target genes of a viral histone H4. In this study, we analyzed the effect CpBV-H4 on the expression of two target genes: Lysine-specific demethylase (KDM) and Serine proteinase inhibitor (Serpins). Transient expression of CpBV-H4 into non parasitized P. xylostella was performed by microinjection of a recombinant expression vector, and showed the expression up to 70 h. Under this transient expression condition, we analyzed the effect of CpBV-H4 on the expression of target genes by RT-PCR at different time points. Interestingly, the CpBV-H4 significantly inhibited the expression of these target genes, while the truncated CpBV-H4 deleting the N-terminal tail did not show this inhibitory effect. This study also showed that the viral histone H4 suppresses expressions of lysine-specific demethylase and serine proteinase inhibitor (Serpin2) to inhibit host growth and development.

Like vertebrate insulins, insulin-like peptides (ILPs) play crucial roles in controlling immature growth, adult lifespan, and plasma sugar level in some insects. An ILP gene (SeILP1) was predicted from a transcription database of Spodoptera exigua. SeILP1 encodes 95 amino acid sequence, which shares sequence homologies (33~83%) with other insects ILPs. The predicted B and A chains possess six cysteine residences. SeILP1 was expressed in all developmental stages of S. exigua. However, its expression was detected in fat body, gut and epidermis, but not in hemocytes. Its expression increased with feeding activity. Plasma trehalose levels of fifth instar larvae maintained at relatively stable concentration of 2.31±0.62 mM. However, starvation induced a significant increase of plasma trehalose level by more than two fold in 48 h, at which SeILP1 expression kept at a low level. RNA interference of SeILP1 induced a significant increase of plasma trehalose level. Interestingly, a bovine insulin decreased plasma trehalose level in a dose-dependent manner. These results indicate mat SeILP1 plays a role in suppressing plasma trehalose level in S. exigua.

Upon freezing temperatures, most insects should avoid cellular freezing by migration to warm hibernating sites, by becoming cold-hardy or by undergoing diapause development. However, a highly threatened butterfly, Parnassius bremeri, terminates egg diapause at early winter season and grows during entire winter and spring. Thus, the cold hardiness of P. bremeri needs to be explored to understand its cold tolerance limit and physiological factors. Supercooling points (SCPs) of P. bremeri vary from -10℃ to -48℃ among season. Especially, the young larvae during Jan – Mar kept SCPs at below -20℃. Larval plasma contained high level of glycerol (39.7 mM) at March, but it decreased the level (2.4 mM) at May. Transcriptome analysis indicated high levels of gene expressions associated with glycerol synthesis. Temporal expression patterns of polyol synthesis genes supported the change of glycerol. This study suggests that glycerol is a major cryoprotectant of P. bremeri to be cold-handy against freezing temperatures during winter.

A novel oxidant fumigation (NOF) is a commercial bleaching and disinfection agent. Recent study indicates its insecticidal activity. However, its exact mode of action to kill insects is not known. This study sets up a hypothesis that reactive oxygen species released from NOF is a main factor to kill insects. Plodia interpunctella is a lepidopteran insect pest infesting various stored grains. Both larvae and adults were susceptive to NOF. To test the hypothesis, we needed to identify antioxidant genes in P. interpunctella. Superoxide dismutase (SOD) and thioredoxin-peroxidase (Trx) were identified from P. interpunctella EST library using ortholog sequences of Bombyx mori. Both SOD and Trx were expressed in larvae of P. interpunctella expecially against oxidative stress induced by bacterial challenge. The bacterial challenge also induced some heat shock protein (HSP) genes. Similarly, different doses of NOF significantly induced both SOD and Trx genes. There results suggest that NOF at sublethal doses releases reactive oxygen species, which may be detoxified by the antioxidant activities of SOD and Trx of P. interpunctella.

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.

A novel oxidant fumigation (NOF) has been considered as alternative fumigant to replace methyl bromide that is a serious ozone depleter. Its high oxidative activity has been used as a bleaching or sanitary agent. Though some reports an insecticidal activity of NOF, its insecticidal action is yet to be understood. This study was conducted with an observation of an insecticidal activity of NOF against Plodia interpunctella, which is a stored grain insect pest. Cytotoxicity test was performed by using MTT assay, NOF gave a significant cytotoxicity on both Sf9 cells and HiFive insect cell lines. Sf9 cells were higher susceptible (IC50 = 43.2+ 3.5 ppm) to chloride dioxide than HiFive cells (IC50 = 174.6 + 5.9 ppm). To understand its cytotoxic effect on P. interpunctella, the larval hemocytes were incubated in vitro with different doses of NOF for 40 min at room temperature. In a dose-dependent manner, NOF gave a significant toxicity to the hemocytes. When NOF was injected to larvae of P. interpunctella, it significantly reduced total hemocyte counts compared to control. These results indicate that NOF has cytotoxic effect against hemocytes of P. interpunctella. This hemolytic activity of NOF can be regarded as a lethal factor to the stored grain insect pest.

Phospholipase A2 (PLA2) catalyzes an ester hydrolysis at sn-2 position of phospholipids. Various PLA2 genes are classified into at least 15 groups. However, on the basis of physiological functions, PLA2 genes are classified into calcium dependent cellular PLA2 (cPLA2), calcium independent cellular PLA2 (iPLA2) and secretary PLA2 (sPLA2). In insects, several sPLA2 genes are known to be associated with venom or immune functions. However, no known cellular PLA2 genes are identified. This study reports an iPLA2 (SeiPLA2) encoded in Spodoptera exigua. SeiPLA2 has an open reading frame of 2448 bp encoding a sequence of 816 amino acid residues. Its predicted protein is 89.55 KDa and 6.15 pI. SeiPLA2 is expressed in egg, larva, pupa and adult stages. In larval stage, SeiPLA2 is expressed in hemocytes, fat body, epidermis, gut, malpighian tubules and salivary gland. To understand its physiological function, its RNA interference is under investigation.

Cotesia plutellae known as an endoparasitoid parasitizes larvae of the diamondback moth, Plutella xylostella which is a major pest in cruciferous crops. For the successful parasitization, maternal and embryonic factors of C. plutellae such as polydnavirus, ovarian proteins, teratocytes and venom are required. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae. cDNA of CRT was cloned from total RNA of the venom gland via PCR and encodes 403 amino acids harboring several structural motifs such as a signal peptide sequence, a repetitive sequence, a putative coiled-coil sequence encompassing, and endoplasmic reticulum-recognizing domain (-KDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes, indicating that the Cp-CRT belongs to the CRT family. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminal was constructed and expressed in E. coli. Recombinant Cp-CRT was successfully expressed via Western blot analysis and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT involves in suppression of cellular immune response in the host