배추나비고치벌(Cotesia glomerata L.)은 배추흰나비(Artogeia rapae L.)와 배추좀나방(Plutella xylostella L.) 유충을 공격하는 내부기생 천적으로, 여름배추를 주로 생산하는 고랭지 채소밭에서 배추흰나비와 배추좀나방을 동시에 생물적 방제하기 위한 천적자원으로서 활용 가능성이 높다. 배추나비고치벌에 대한 온도별 생육반응과 성비, 먹이에 따른 수명을 실험실내에서 조사한 결과, 배추나비고치벌의 알-유충 기간 및 번데기 기간은 20℃에서 각각 12.1 ± 2.1일, 6.4 ± 1.8일이었으며, 생육온도가 높아질수록 짧아지는 경향을 보였다. 이를 바탕으로 산출한 알-유충 및 번데기 시기의 발육영점온도는 각각 7.7℃, 8.5℃였다. 여러 상이한 온도에서 사육한 배추나비고치벌 우화성충의 암수를 조사한 결과, 15℃에 서 61.0 ± 4.5%, 20℃에서 44.2 ± 1.0%, 25℃에서 39.0 ± 2.3%의 성비를 보여 온도가 낮아질수록 암컷의 발생률이 높아지는 경향을 보였다. 배추나비고치벌 성충에 10% 설탕액을 급여한 결과 수명은 20.4 ± 0.2일이었으며, 아무것도 급여하지 않은 경우는 3.6 ± 0.1일이었다. 실내 대량 사육을 통해 확보한 배추나비고치벌 성충을 2007년부터 2018년까지 매년 8월 초에 고랭지 배추밭에 방사하고 노지 기생률을 조사하였다. 그 결과 햇수가 지날수록 노지 기생률이 증가하는 것을 확인하였다(Y=0.2696X+2.8633, R2=0.3994). 가장 높은 기생률을 보인 연도는 2013년의 7.6%이었고, 가장 최근인 2018년에는 6.5%의 기생률을 나타내었다.

When treating the 3rd instar larvae of the diamondback moth (DBM), Plutella xylostella, with sublethal doses (LC10) of chlorantraniliprole, indoxacarb and spinosad via leaf dipping, their tolerance to insecticides was significantly enhanced. By analyzing the differentially expressed genes (DEGs), we found a number of genes that respond commonly or specifically to the test insecticides. With the criteria of p value < 0.05 and Log2FC > 1/ < -1, a total of 476, 367 and 410 genes were determined to respond specifically to chlorantraniliprole, indoxacarb and spinosad, respectively. Gene Ontology (GO) analysis revealed that the cuticle reorganization is commonly associated in all treatments and the oxidative stress-related process is also shown in all insecticides except spinosad. Finally, the DEGs seemingly related with enhanced tolerance were chosen for further characterization, and reliability of the transcriptome data were confirmed by quantitative PCR. The functional categories of these DEGs included mostly detoxification related genes, cuticle proteins, energy metabolism and transcriptional regulation. While the commonly responding DEGs suggest that they are likely involved in defense against common intoxication process, the DEGs specifically responded to each insecticide suggests the presence of unique tolerance mechanisms to each insecticide depending on their different structure and mode of action. Their possible roles in the tolerance/resistance development were discussed.

Actinobacteria have been known to produce variety of bioactive metabolites that include antibiotics and plant growthfactor. Also, Insecticidal compounds such as avermectin and tetranectin have been reported from actinobacteria. In thisstudy, to identify novel insect growth regulator (IGR)-based insecticidal compounds, 1,283 actinobacteria isolates weretested for their juvenile hormone antagonist (JHAN) activity using the yeast-two hybrid β-galactosidase assay. Amongthem, 25 isolates showed high level of JHAN activity and insecticidal activity against 3rd larvae of diamondback moth,Plutella xylostella. These results suggested that actinobacteria could be unparalleled source of novel IGR-based compounds.

To identify genes that commonly respond to the treatment of different insecticides and are responsible for the toleranceenhancement, transcriptomic profiles of larvae treated with sublethal doses of the five insecticides were compared withthat of untreated control. A total of 117,181 transcripts with a mean length of 662 bp were generated by de novo assembly,of which 35,329 transcripts were annotated. Among them, 125, 143, 182, 215 and 149 transcripts were determined tobe up-regulated whereas 67, 45, 60, 60 and 38 genes were down-regulated following treatments with these five insecticides.The most notable examples of commonly responding over-transcribed genes were two cytochrome P450 genes and ninecuticular protein genes. In contrast, several genes composing the mitochondrial energy generation system were significantlydown-regulated in all treated larvae. Considering the distinct structure and mode of action of the five insecticides tested,the differentially expressed genes identified in this study appear to be involved in general chemical defense at the initialstage of intoxication. Their possible roles in the tolerance/resistance development were discussed.

To identify genes that commonly respond to the treatment of different insecticides, 3rd instar larvae of the diamondback moth, Plutella xylostella, were treated with sublethal doses (<LC10) of chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad via leaf dipping. Then, transcriptomic profiles of treated larvae were compared with that of untreated control. A total of 117,181 transcripts in average with a mean length of 662 bp were generated by de novo assembly, of which 35,329 transcripts were annotated. Among them, 207, 153, 336, 360, and 262 transcripts were determined to be up-regulated whereas 117, 47, 92, 115, and 81 genes were down-regulated following treatments with chlorantraniliprole, cypermethrin, dinotefuran, indoxacarb and spinosad, respectively. Finally, with the criteria of >10 X fold change (FC) and p < 0.05 or >4 X FC, p < 0.05 and q < 0.2, the genes commonly over-transcribed in all treated insects were selected and their over-transcription levels were confirmed by quantitative PCR. These commonly responding genes included three cytochrome P450 genes (Cyp303a1, Cyp6a20 and CYP9E2), three cuticle protein genes (LM-8, LM-19 and TM-A3A), lavesin-1, acyl-CoA D11 desaturase, glucose dehydrogenase, nose resistant to fluxetine protein 6, chorion peroxidase and protein yellow. As the five test insecticides have distinct structure and mode of action, the genes identified in this study were suggested to be involved in general chemical defense at the initial stage of intoxication. Their possible roles in tolerance and resistance development were further discussed.

The diamondback moth (DBM), Plutella xylostella, is a globally distributed and important economic pest. Chemical control is the primary approach to regulate populations of this pest. Chlorantraniliprole is the first commercial insecticide that belongs to the new chemical class of diamide insecticides. In this study, the resistant strain was observed 1578-fold resistance to chlorantraniliprole. Point mutation (G4946E) in ryanodine receptor (RyR) showed a high frequency. Enzyme assays indicated that glutathione S-transferase (GST) activity in the resistant strain was 2.4 times higher compared with the susceptible strain, whereas no difference was seen for P450 and esterase. In addition, the expression of two GSTs genes was up-regulated. These findings pave the way for the complete understanding of the mechanisms of diamide insecticides resistance in insects.

Cotesia plutellae, an endoparasitoid wasp, parasitizes larvae of Plutella xylostella, and disrupt immune response of the host through parasitic factors. These immune disruption factors are maternal (venom proteins, polydnavirus, and ovary proteins) and embryonic (teratocytes) factors. In this study, we performed transcriptome analysis of venom glands of C. plutellae and identified neprilysin-1 (NEP1) known to be potential immunosuppression gene. Cp-NEP1 encoded 451 amino acid and belongs to hymeopteran NEP1 via phylogenetic analysis. Based on the structural comparison Cp-NEP1 lacks in conserved motifs such as substrate binding (NAYY/F), zinc-binding site (HExxH), zinc-binding site, protein folding and maturation (CxxW). To investigate function of Cp-NEP1, we constructed a recombinant Cp-NEP1 harboring N-terminally fused 6X His tag. Peptide sequencing revealed successful expression of the recombinant Cp-NEP1 in Escherichia coli. Pre-heated E. coli as antigen induced spike of nodule formation whereas co-injection of the recombinant Cp-NEP1 and pre-heated E. coli exhibited suppression of nodule formation in the host. Quantitative real-time PCR revealed that expression of phenoloxidase related to nodule formation was suppressed under co-injection of the recombinant Cp-NEP1 and E. coli. These results suggest that Cp-NEP1 contributes to immunosuppression of P. xylostella via phenoloxidase suppression and conserved motifs of neprilysin family are not required for host immune suppression.

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.

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.

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.

Conservation biological control (CBC) is one of the pest management tactics based on protecting and maintaining natural enemies that already exist in the crop environment. Among many appropriate practices to control pests, CBC has been considered as a sustainable means and a less expensive alternatives to chemicals. It contains an introduction of non-crop plants as shelter habitat for providing natural enemies with food sources, overwintering or refuges shelters. As a small scale experiment of CBC, we tried to intercrop six kinds of floral plants (buckwheat, red clover, Agastache rugosa, Chrysanthemum indicum, Allium tuberosum, Lythrum anceps) with napa cabbage in order to attract some natural enemies, especially parasitic wasps that could be fed on them in the cabbage fields, which is a novel way for conservation of natural enemies, but none showed significant result. For the development of integrated pest management system by harmonizing biological and chemical control, we carried out evaluating toxicity of 30 pesticides to parasitoids and investigating effectiveness of traps. With leaf dipping method for adult and body dipping method for cocoons, 12 pesticides showed under 30% of toxicity being selected as safer by IOBC. Using sex-pheromone traps for attracting lepidopteran pests showed that DBM, CAW and CL occurred less than untreated fields; 67.5%, 70.6% and 44.0%, respectively. Also, yellow sticky traps could reduce some pests; 52% for flea beetle, 62% for cabbage sawfly and 41% for Phaedon brassicae. These results are expected to give basic information to develop conservation biological control of DBM with indigenous parasitoids in the cabbage fields on a large scale in the future. Before developing and distributing a promising pest control method, we should consider whether it can be compatible with other agricultural practices or various situations around fields.

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.

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.

Juvenile hormone is the most important hormone inside the insect, but its circulating titer should be under tight control. Then enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs) play critical roles in insect metamorphosis and reproduction. Within a set of 11 JHEs predicted in the diamondback moth (DBM) genome, Plutlla xylostella (Linn.) we identified one gene that contained the main functional motifs of insect JHEs. Its expression and transcript levels during egg, different instar of larvae, prepuae, pupae and adult stages were measured. Also, its expression in the epidermis, midgut and fatbody of the 4 th instar larvae was compared. The changes in enzyme activity in the 4th instar larvae were determined.

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.