배추좀나방(Plutella xylostella)은 시설재배지를 중심으로 국내 자연 상태에서 월동한다. 안동지역에서 이른 봄부터 배추좀나방 성페로몬트 랩을 이용하여 배추좀나방의 성충 발생 시기를 주기적으로 조사한 결과 연중 4 회의 성충 발생 피크를 보였다. 월동 집단을 대상으로 서로 다른 지 역 집단 간 생물적 특성을 조사한 결과 내한성, 약제감수성 및 발육속도에서 뚜렷한 집단 특성을 나타냈다. 분자마커로 집단변이를 분석한 결과 월동세대의 높은 집단변이는 계절이 진행됨에 따라 낮아지는 양상을 나타냈다. 이 결과는 배추좀나방의 국내 월동 집단 사이의 생물적 특성 차이 를 나타냈고, 이들의 높은 유전적 분화는 계절이 진행됨에 따라 감소하여 이들 집단 사이의 개체들의 이동에 따른 유전적 교환이 이뤄졌다는 것을 제시했다.

The pheromone biosynthesis in Plutella xylostella is stimulated a neuropeptide, pheromone biosynthesis activating neuropeptide which is produced in subesophageal ganglion. The pheromone production is more active in the scotophase than in the photophase, which suggests that there may be changes of gene expression in the pheromone glands. To analyze gene expression related to pheromone biosynthesis, we performed transcriptomes of pheromone glands which were isolated at every 4 h. Eleven pheromone biosynthesis-related genes, acetyl-CoA carboxylase, fatty acid synthase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, Δ9 desaturase, Δ11 desaturase, fatty acid reductase, alcohol oxidase, aldehyde oxidase, and aldehyde reductase were identified. Among these genes, the expression of aldehyde reductase and aldehyde oxidase were relatively higher in the scotophase than in photophase, which may affect increase of pheromone production in the scotophase. Expression of signal genes involving in pheromone biosynthesis such as acyl-CoA desaturase, FAR, PBAN receptor, fatty acid transporter and acyl-CoA binding protein did not exhibited any significant difference.

One of the endoparasitoid wasp, Cotesia plutellae (Braconidae), parasitizes young larvae of the diamondback moth, Plutella xylostella. For the successful parasitization, C. plutellae required suppression of immune response in P. xylostella. Maternal (polydnavirus, venom proteins and ovary proteins) and embryonic (teratocytes) factors have been involved in immune-suppression. In this study, we performed transcriptome analysis of venom of C. plutellae and identify neprilysin-1 (Cp-NEP1) as a potential immunosuppressive protein. Cp-NEP1 encoded 451 amino acids and largely belongs to the hymeopteran neprilysin family via phylogenetic analysis. It is of interest that Cp-NEP1 has no conserved motifs such as zinc-binding domain (HExxH), substate binding domain (NAYY/F) and protein folding and maturation domain (CxxW) generally identified in other neprilysin family. In order to examine the biochemical function of Cp-NEP1, the recombinant Cp-NEP1 tagged with N-terminally 6X His was constructed and expressed in Escherichia coli. Expression of Cp-NEP1 was confirmed with SDS-PAGE and peptide sequencing. Recombinant Cp-NEP1 significantly suppressed nodule formation when the co-injection with E. coli. These results suggest that Cp-NEP1 contributes to suppression of immune response in P. xylostella and that the conserved motifs reported from other neprilysin do not involve immunosupperssion.

Entomopathogenic fungi have been studied to develop for biological control agents as an alternative to chemical control agents in insect pest management. Two Lepidopteran insects, Spodoptera exigua and Plutella xylostella, are serious insect pests infest various crops, but not effectively controlled by commercial chemical pesticides due to its high insecticide resistance. A fungal isolate was isolated from S. exigua larvae collected from green onion field in Andong, Korea. To identify the fungal isolate, 18srRNA sequence for internal transcribed spacer (ITS) and β-tubulin regions were sequenced. The ITS and β-tubulin sequence were highly matched to Beauveria bassiana and morphological characteristics also was fit to known B. bassiana. Finally, isolated fungus has identified as B. bassiana and named B. bassiana ANU1. The result of bioassay, median lethal concentrations were 2.7×103 and 0.9×103 conidia/ml and medial lethal times were 65.6 and 60.8 h to S. exigua and P. xylostella, respectively. B. bassiana ANU1 showed high pathogenicity to two insect pests from 20℃ to 30℃ at 50% relative humidity (RH) and more than 40% RH at 25℃ with 107 conidia/ml of concentration.

The pheromone biosynthesis in Plutella xylostella is more active in the scotophase than in the photophase, which suggests that there may be changes of gene expression in the pheromone glands. To identify genes contributing to change in pheromone production, we analyzed transcriptomes from pheromone glands of both decapitated females in the photophase and normal ones in the scotophase. Comparative analysis were performed with transcriptomes of pheromone glands from non-decapitated (PG) females and decapitated ones for identification and expression of putative genes associated with pheromone biosynthesis pathway. Deep sequencing for mRNAs in the pheromone gland yielded approximately 7.5Gb and totally 17265 transcript were constructed under a homology cutoff of 10-6 Evalue. Genes putatively involved in pheromone biosynthesis were identified such as acetyl-CoA carboxylase, acetyl-CoAdehydrogenase, fatty acid synthase (FAS), desaturases (Δ9 and Δ 11) and fatty acid reductases (FAR) including pgFAR, alcohol oxidase, aldehyde oxidase and aldehyde reductase, etc. Expression of 6 signal genes involving in pheromone biosynthesis such as acyl-CoA desaturase, FAR, PBAN receptor, fatty acid transporter, acyl-CoA binding protein did not exhibited ant significant different in both transcriptomes. Quantitative RT-PCR revealed that expressions of FAS, Δ11 desaturase and pgFAR were higher in PG than that in ΔPG. Based on results, Δ11 desaturase and pgFAR may have a crucial role in sex pheromone biosynthesis of P. xylostella.

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.

잎브로콜리에 심각한 피해를 주는 배추좀나방의 적절한 방제시기 설정을 위한 기초자료로 이용하기 위하여 유충접종밀도와 잎브로콜리 피 해량의 관계를 분석하여 경제적피해허용수준과 요방제 수준을 설정한 결과는 다음과 같다. 배추좀나방 3~4령 유충을 주당 0, 0.5, 1.0, 1.5, 2.0 마리를 5월 24일에 접종하고, 6월 1일, 6월 9일, 6월 18일에 발생밀도와 엽수량을 조사한 결과, 초기 접종밀도가 증가할수록 배추좀나방의 발생 밀도는 증가하였고 시간이 경과할수록 엽수량은 감소하였다. 이를 토대로 배추좀나방의 접종밀도와 수량감소율을 회귀식을 이용하여 분석한 결 과 y=1636-394x (R 2 =0.79***)로 추정되었다. 잎브로콜리에서 5% 피해율을 경제적 피해한도로 볼 때 배추좀나방의 경제적피해허용수준은 10 주당 2~3마리이고, 요방제 수준은 10주당 1-2마리 수준으로 추정된다.

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.

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

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus. Our previous study indicated that overexpression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. This study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to analyze its inhibitory activity against cysteine protease and physiological role in the parasitism of an endoparsitoid wasp, Cotesia plutellae. The open reading frame (ORF) of CpBV-CST1 encodes a polypeptide of 138 amino acids (15 kDa). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was overexpressed by 0.5 mM IPTG for 4 h. In biological activity assay, partially purified GST-fused rCpBV-CST1 showed inhibitory activity against papain. It also inhibited larval development of P. xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 plays a role in retardation of larval development of P. xylostella during parasitism.

Insulin in vertebrates plays a crucial role in maintaining homeostasis of blood sugar level. Insulin-like peptide (ILP) has been identified in insects, such as Drosophila melanogaster and Aedes aegypti. Plasma sugars and polyols of the diamondback moth, Plutella xylostella were separated by a Bio-LC. Among seven peaks, trehalose was the most predominant blood sugar and maintained at approximately 3.5 mM in the larval plasma. However, the feeding activity affected the plasma trehalose level, in which starvation significantly up-regulated the trehalose level. Analysis of ILP expression upon feeding indicated that feeding stimulated the gene expression of ILP. Interestingly, an injection of a vertebrate insulin significantly suppressed the hypertrehalosemia induced by starvation. These results suggest that ILP is a endocrine signal to down-regulate the plasma trehalose level in P. xylostella.

Species-specific sex pheromones play an important role in mediating sexual behavior and pheromone biosynthesis activating neuropeptide (PBAN) stimulates the pheromone production. Previously, we identified PBAN and PBAN receptor gene and reported the functionality of these genes via heterologous expression and RNAi (BBA-general subjects, 2005 and IBMB, 2011). To find the differences in the transcriptional level between scotophase and photophase in the pheromone gland of P. xylostella, total RNA was extracted from the adults and transcriptomes of the pheromone glands were analyzed by RNA-seq. The genes related to the pheromone biosynthesis were identified and the putative pathway of pheromone biosynthesis was predicted. Compared to the expression level of pheromonebiosynthesis-related genes between scotophase and photophase, the expression of fatty acid reductase (FAR) exhibited the significant difference in the putative pheromone biosynthesis pathway, suggesting that FAR is the key enzyme regulating the pheromone biosynthesis.

Paenibacillus elgii HOA73는 신규 개발된 미생물 제제로 작물생장 및 병원균 방 제 등 다양한 효능에 대한 연구가 수행중이다. 본 연구는 P. elgii 배양액의 배추좀 나방 살충률 제고와 시판농자재 사용량 감소를 통한 방제비용 절감을 위하여 시판 친환경 농자재(1000배 ~ 10,000배)와 P. elgii 농도별(5 × 107 ~ 9 × 107) 혼합 살포 에 따른 살충효과를 분석하고자 하였다. 속효성과 약효성이 높은 세 종류의 시판 농 자재를 선발 한 후 이들의 희석 배율과 P. elgii의 배양액 농도별 혼합 실험 결과, 시 판 농자재는 권장 희석액의 두 배 희석농도(5 × 107 농도의 P. elgii 배양액)에서도 세 종류 모두 72시간내 96% 이상의 살충력을 나타내었다. 이들에 대한 복숭아혹진 딧물과 점박이응애에 대한 혼합 살충력 분석결과, 복숭아혹진딧물의 경우 권장 희 석액의 두 배 희석농도(5 × 107 농도의 P. elgii 배양액) 역시 80% ~ 100%의 살충력 을 그리고 점박이응애의 경우 모두 100%의 살비력을 나타내었다. 이로써 시판 친 환경농자재를 P. elgii 배양액과 혼합하여 사용할 경우 친환경농자재 단독 처리시 보다 최소 반의 가격 절감효과가 있으며 하나의 약재를 이용하여 저작성 배추좀나 방과 흡즙성의 복숭아혹진딧물과 점박이응애를 동시에 방제할 수 있을 것으로 판 단된다. 향후 소포장 검정과 확장된 실내검정을 통해 작물생장 및 병원균 방제에 효 능을 갖고 있는 P. elgii의 적용범위에 대한 추가 연구를 수행하고자 한다.

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.

The physiology of parasitic wasp control of their lepidopteran hosts' not only includes injecting their egg but also various factors such as symbiotic virus. This study was focused on the investigation of sophisticated interaction between parasitoid (Diadegma fenestrale) and their host (Plutella xylostella) in P. xylostella larva at transcriptome level, to check whether it is parasitized or not. Short-read deep sequencing method (Hiseq2000) was used for the transcriptome analysis. De novo assembly of cDNA sequence data generated 196,081 contigs between 201bp and 15,853bp in length. Some detoxification enzymes such as cytochrome P450 and Immune-related genes such as antimicrobial peptides were up-regulated after parasitism. Expression of symbiotic ichnovirus genes was detected in parasitized larvae with 55 contigs identified from five ichnovirus gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. This investigation provides a detailed information on differential expression of P. xylostella larval genes and symbiotic ichnovirus genes following parasitization.

국내 배추좀나방(Plutella xylostella) 집단은 피레스로이드 농약에 대해서 저항성을 보이며, 이는 이 살충제의 작용점인 소듐이온채널 유전자의 돌연변이에 기인된다. 더욱이 배추좀나방은 대부분 상용화된 살충제에 대해서 저항성을 발달시킬 수 있다. 본 연구는 배추좀나방을 효과적으로 방제하기 위해 내부기생성 천적인 프루텔고치벌(Cotesia plutellae)과 미생물농약인 Bacillus thuringiensis의 혼합처리 기술을 개발하기 위해 수행되었다. 프루텔고치벌이 감수성과 저항성 배추좀나방에 대한 기생 선호성에 차등이 있는 지 조사하기 위해 다섯 개 서로 다른 집단에 대해서 살충제 감수성과 프루텔고치벌 기생성 차이를 비교하였다. 이들 배추좀나방 집단들은 피레스로이드, 유기인계, 네오니코틴계 및 곤충성장조절제를 포함하는 세 종류의 상용 살충제에 대한 약제 감수성에서 뚜렷한 차이를 보였다. 그러나 이들 집단들은 프루텔고치벌에 의한 기생률에서는 차이를 보이지 않았다. 더욱이 기생된 배추좀나방은 B. thuringiensis에 대해서 감수성이 증가되었다. 프루텔고치벌이 갖는 면역억제인자 가운데 바이러스 유래 ankyrin 유전자(vankyrin)를 비기생된 배추좀나방에 발현시켰다. Vankyrin의 발현은 배추좀나방 3령충의 B. thuringiensis에 대한 감수성을 현격하게 증가시켰다. 즉, 프루텔고치벌에 의해 야기된 면역저하가 B. thuringiensis의 살충력을 증가시켰다. 이러한 결과들은 프루텔고치벌과 미생물농약인 B. thuringiensis의 혼합처리가 살충제 저항성 배추좀나방을 효과적으로 방제할 수 있다고 제시하고 있다.

Parasitism by an endoparasitoid wasp, Cotesia plutellae, results in significant immunosuppression of the diamondback moth, Plutella xylostella. Parasitized larvae significantly suffered higher susceptibility to a microbial biopesticide, Bacillus thuringiensis (Bt) than nonparasitized (NP) larvae. To find out an immunosuppressive agent causing the enhanced Bt efficacy, viral ankyrin (=vankyrin) genes encoded in C. plutellae bracovirus (CpBV) were analyzed by transient expression in NP larvae. CpBV segments containing different vankyrins were microinjected to NP larvae and expressed their encoded vankyrins. Expression of some vankyrins significantly inhibited immune response and enhanced Bt efficacy. This study suggests that expression of vankyrins suppress a cellular immune response and lose Bt tolerance of P. xylostella larvae.

An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella, with its parasitic factors of polydnavirus, venom, ovarian proteins, and teratocytes (TC). TCs are originated from embryonic serosal membrane at hatch of C. plutellae eggs. TCs, after released in hemocoel of parasitized larvae, increased their average cell size from 20.6 μm to 77 μm during whole developmental period of the parasitoid larvae, but did not increase their cell number by maintaining about 150 cells per larvae. TCs of C. plutellae, are considered to be involved to extend the host larval development period and to arrest larval-pupal metamorphosis, were cultured in an insect cell culture medium for 21 days. Like TCs in parasitized larvae, in vitro cultured TCs showed increase in cell size, but did not show increase of cell number. Microinjection of in vitro cultured TCs significantly inhibited larva-to-pupa metamorphosis of nonparasitized P. xylostella, in which pupated host also showed extended larval period. Larvae injected with TCs exhibited alteration in expression of ecdysone receptor (EcR) and insulin receptor (InR) as well as in parasitized larvae. Teratocyte-secretory factors in culture medium showed this antimetamorphic effect on P. xylostella, while heat treated TC culture medium lost the effect. However, a successful parasitization of C. plutellae required both TCs and polydnavirus to alter host physiology.

 ,  , The larvicidal activities of Piper nigrum fruit methanol extracts and its constituents against larvae of the diamondback moth, Plutella xylostella, were investigated using the leaf dipping method. Administering the P. nigrum methanol extract resulted in 100 and 97% mortality against diamondback moth larvae at 5.0 and 2.5 mg/mL concentrations, respectively. Larvicidal activities of the P. nigrum fruit-derived piperidine alkaloids, piperine, and N-isobutylamide, as well as pellitorine, guineensine, pipercide, and retrofractamide A against P. xylostella varied according to test compound. Based on the 48 h LC50 values, the most toxic compound to diamondback moth larvae was guineensine (0.013 mg/mL) followed by retrofractamide A (0.020mg/mL), pipercide (0.033mg/mL), and pellitorine (0.046 mg/mL). The LC50 value of piperine was >, 0.5 mg/mL.