Pheromone biosynthesis-activating neuropeptide (PBAN), produced in subesophageal ganglion, is known to stimulate pheromone biosynthesis in Plutella xylostella. 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 involving in pheromone biosynthesis for 24 hrs, we performed transcriptomes of pheromone glands which were isolated at every 4 h. Eleven pheromone biosynthesis-related genes, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, Δ9 desaturase, Δ11 desaturase, fatty acid reductase (FAR), alcohol oxidase, aldehyde oxidase, and aldehyde reductase were identified. Among these genes, ACC, FAS and chain shortening enzymes involving in early stage of pheromone biosynthesis exhibited their highest expression between AM9 and PM5. Desaturases, Δ9 and Δ11, showed the peak of expression at PM1 and AM5 or PM5, respectively. Interestingly, FAR expression was the highest at AM1, active reproductive period. These results suggest that genes involving in pheromone biosynthesis can be sequentially regulated for their biological roles.

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.

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.

A rapid cold hardening (RCH) and supercooling capacity usually play crucial roles in survival during the overwintering period in the tobacco budworm, Helicoverpa assulta, a freeze-susceptible species. Cryoprotectants such as polyol or sugar affect RCH and maintain fluid status of hemolymph. This study is performed to identify cryoprotectants as a RCH factor in H. assulta. Pre-exposure of H. assulta larvae to 4℃ significantly increased survival at -10℃ in all developmental stages from egg to adult. RCH was dependent on the duration of the pre-exposure period. RCH also significantly enhanced the supercooling capacity. Analysis of cryoprotectants from the hemolymph revealed that the pre-exposure treatment allowed the larvae to accumulate glycerol and trehalose among polyols examined. In addition, unknown materials from 2 peaks were also increased. TIC analysis revealed 3 predicted formulas for unknown materials, C26H24O20S or C3H4N6OS and C22H20O21. Glycerol-3-phosphate dehydrogenase (GPDH) and glycerol 3-phosphatase (G3P) that involving in glycerol biosynthesis were identified from the transcriptome of H. assulta 4th instar larvae. Based on the expression level of transcripts, the expressions of GPDH and G3P were relatively increased when compared to that of the control, suggesting that these genes contribute to overwintering and biosynthesis of glycerol.

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.

본 연구에서는 파프리카 수확기 개발의 일환으로 엔드 이펙터의 정확한 제어를 위하여 스테레오 영상으로 파프리카를 인식하고 인식된 파프리카의 공간 좌표를 획득하기 위하여 영상처리 알고리즘을 개발하고자 하였다. 먼저, 색상 정보를 이용하여 파프리카 영상을 추출하기 위하여 히스토그램 분석을 수행하였고 결과에 따른 임계값 을 설정하였다. 임계값에 의해 추출된 파프리카 영역에 대해 스테레오 대응을 수행하기 위해 실험에 사용된 스테레오 영상의 F 행렬을 구하였고 이를 이용하여 에피 폴라선을 구하여 대응을 수행하였다. 대응을 수행 할 때는 색상 영상을 이용하여 강조 마스크와 컨벌루션을 통해 중심 픽셀과 수직, 수평방향 이웃 픽셀에 가중치를 적용하여 강조한 후 최소 자승 오차를 갖는 점을 대응 점으로 추출하였다. 추출 된 대응 점간의 거리를 스테레오 영상의 기하학적인 관계를 이용하여 실제 거리를 계산하였고, 계산된 거리(Z)값을 이용하여 수평(X), 수직 (Y) 방향 공간 좌표를 획득하였다. 그 결과 수평 방향 오차 평균 5.3mm, 수직 방향 오차 평균 18.8mm, 거리 오차 평균 5.4mm로 나타났으며, 거리 400~450mm 구간과 영상의 모서리 부분의 왜곡이 발생하는 부분에서 오차가 다른 구간에 비해 크게 나타나는 것을 확인 할 수 있었다.

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.

The tobacco budworm, Helicoverpa assulta, is a freeze-susceptible species that overwinters in temperate zones with pupa diapause. A rapid cold hardening (RCH) and supercooling capacity usually play crucial roles in survival during the overwintering period. This study is performed to identify a cryoprotectant as a RCH factor in H. assulta. Pre-exposure of H. assulta larvae to 4°C significantly increased survival at -10°C in all developmental stages from egg to adult. RCH was dependent on the duration of the pre-exposure period. RCH also significantly enhanced the supercooling capacity. Cryoprotectant analysis using HPLC showed that the preexposure treatment allowed the larvae to accumulate glycerol in the hemolymph. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol 3-phosphatase (G3P), that involing in glycerol biosynthesis were identified from the transcriptome of H. assulta 4th instar larvae. From the result of transcriptome, the expressions of GPDH and G3P were relatively increased when compared to that of the control, suggesting that these genes contribute to overwintering and biosynthesis of cryoprotectant.

The bumblebee, Bombus terrestris, plays an important role as one of alternative pollinators since the outbreak of honeybee colony collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites affecting the life span and fecundity of their host have been discovered in B. terrestris. In this study, in order to detect viral infection in B. terrestris, we collected B. terrestris adults and isolated total RNA for diagnostic PCR. The PCR primers specific for pathogenic viruses were newly designed and applied to gene amplification for cloning and detection. Capsid protein gene of black queen cell virus (BQCV) among examined viral genes was only successfully amplified from collected bumble bee adults and sequenced. To optimize the detection of capsid protein gene of BQCV, 4 regions in the capsid protein gene were selected and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis revealed that capsid protein gene was directly detected with not more than 200 ng total RNA. This result suggests that an optimized detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terrestris.

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

After firstly identified sex pheromone components of Bombxy mori, those of many insect pests were synthesized by organic chemistry methodology. These synthesized components were used for monitoring, mass trapping, and mating disruption during five decades. For identification of pheromone biosynthesis mechanisms and control to many pests bring to serious damages also were proceeded. The transcriptome analysis from pheromone glands by Next Generation Sequence (NGS) showed many genes and pathway involved on sex pheromone biosynthesis.. The two main genes involved on production of acetate and alcohol, and aldehyde from fatty acid, fatty acid desaturase and fatty acid reductase (FAR) were identified and functional characterized via gene introduction to Brewer’s yeast Saccharomyces cerevisiae. This S. cerevisiae now used as a mediator as well as cell factory for sex pheromone producing. Recently, One group was published that the plant factory for producing via genetically modified plant (tobacco, Nicotiana benthamiana) as a step of semisynthetic preparation. These trials will be suggest that firstly, the possibility of yeast as a molecular toolbox to produce pheromone components and secondly, a novel and cost-effective way of producing moderate to large quantities of pheromones with high purity and a minimum of hazardous waste.

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.

본 연구에서는 과수의 형태, 거리에 따라 약대가 항상 적정 살포 거리를 유지하며 작업을 진행할 수 있도록 초음파 센서의 신호를 실시간으로 받아 약대를 제어하였고, 또한 약대의 보호를 위하여 진행 방향에 장애물이 존재할 경우 회피할 수 있도록 프로그래밍 하였다. 제작된 시스템으로 현장에서 비 자동제어 상태와 자동제어 상태로 실험을 수행하였다. 실험은 과수의 기둥과 잎 부위에 감수지를 일정간격으로 설치하고 시스템을 이용하여 분사한 후 감수지의 영상을 스캔하여 영상처리를 통해 분석하는 방법으로 이루어졌다. 상 방향 분사 실험에서는 시스템과 대상의 거리가 0.9m~1.1m로 설정해둔 적정거리를 벗어나지 않았기 때문에 비 자동제어와 자동제어 상태 모두 양호한 결과를 보였다. 하지만 측 방향 분 사 실험에서는 비 자동제어 시 우측 열은 98.09%의 분 사율을 보였으나 좌측 열은 69.25%로 낮게 나타났다. 이는 실험이 수행된 배 과원의 경우 과수의 좌측 열이 수평하게 식재되어 있지 않았기 때문으로 비자동제어 상태에서는 좌측열의 과수에 분사되는 양이 줄어들었으나 자동제어 상태에서는 좌, 우측열의 과수에 분사되는 양이 각각 92.66%, 94.64%로 균일하게 나타났다. 시스템 의 제어 속도를 측정하기 위하여 방향 별 약대의 속도를 측정하였고 각각의 속도는 수직방향 100mm/s, 수평 방향 100mm/s, 각 변화 3o/s로 측정되었다. 초기 목적했 던 바와 같이 과수의 형태, 거리에 따라 약대가 적정 거리를 유지하며 작업을 진행함으로 인해서 균일한 살포량을 유지 할 수 있음을 확인하였다.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema Spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema Spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA(SSU rRNA) gene of N. ceranae was successfully amplified and sequenced among examined genes, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR(qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentrations as low as 0.85 ng/μl genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators. Recently, pathogens and parasites affect the life span and fecundity of their host and been isolated from B. terristris. In order to detect viral infection in the field populations of B. terristris, we collected adults and isolated total RNA for reverse transcriptase-polymerase chain reaction (PCR). The PCR primers specific for several viruses such as deformed wing virus, Israel acute paralysis virus, Kashmir bee virus and black queen cell virus (BQCV) were newly designed and applied to gene amplification for cloning. Only BQCV was successfully amplified and sequenced, which suggests that BQCV may mainly infects the examined field population of B. terristris. To detect of capsid protein gene of BQCV, 4 selected regions were analyzed by primary PCR and 1 region was successfully amplified, which was further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that BQCV was detected at concentrations as low as 0.1ng/μl total RNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terristris.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors such as venom proteins, teratocytes and polydnavirus. In order to identify immune suppressive factors from teratocytes, we collected the supernatant from serum-free culture media of teratocytes. Concentration of secreted proteins from teratocytes was successfully performed with using centricons among tested methods such proteins precipitation and electrophoresed in sodium dodecyl sulfate polyacrylamide gel. The gel slices were directly digested with trypsin using in-gel digestion method and analyzed via LC-Ms-Ms. Molecular weight of peptide fragments were compared with protein database predicted by full-genome sequences of C. plutellae. We identified two immune responsive proteins, which are calreticulin, host cellular response-related gene and neprilysin 2, immune regulator. This result suggests that host immune response is suppressed or regulated by the immune suppressive factors of teratocytes.

Bombus terrestris has played an important role in the pollination in agricultural fields for the alternatives in colony collapsing in the honeybee. Recently, some pathogens or parasites such as viruses, bacteria, mites have been discovered in B. terristris, which affects its life span and fecundity. In order to detect a microsporidian, Nosema apis. in the field population, we collected honeybees and isolated genomic DNA. PCR primers specific for 16S ribosomal RNA (16S rRNA) were synthesized and applied to gene amplification for cloning and quantitative real-time PCR (qRT-PCR). The amplified gene was cloned and sequenced to confirm the 16S rRNA gene. qRT-PCR analysis showed the detection limit of 16S rRNA of Nosema apis was approximately 0.5 ng/μl genomic DNA. This result suggests that detection via qRT-PCR can be applied for the diagnosis of pathogen infection.

Cotesia plutellae has been known as a natural enemy against the Diamondback moth, Plutella xylostella via laying eggs into a larva. When the larva hatches from the egg, teratocytes also are released and expected to work as immune suppressor via secreting immune suppressive factors. In order to analyze the gene expression in teratocytes, total RNAs were isolated and genes expressed in the teratocyte were sequenced by Illumina HiSeq2000 RNASeq analysis. The information on RNA sequences was assembled by Trinity and contigs were annotated by Blast analysis. The levels of gene expression were calculated by FPKM. Approximately, 6.3 Gbs were obtained and 34,686 contigs were found and annotated. Forty two percent of contigs were homologous to previously reported genes and classified by gene ontologies: the highly abundant components are metabolic process, biological regulation and cellular process in biological function; binding, catalytic activity and transporter activity in molecular function; cell part, membrane part and organelle in cellular function, respectively. In addition, some teratocyte transcripts of C. plutellae are related to host regulation such as immunosuppression and nutrition: Ankyrin repeat proteins, Serpin, protease, lipase, chitinase and scavenger receptor.

Currently, honeybee colonies are not stable and suffer from the infection of pathogens, affecting the pollination. For the alternatives to this difficulty, Bombus terrestris has been imported and used for pollination in agricultural fields. Although imported insects for pollination are very useful, the potential risk exposing to novel pathogens has been raised. To assess the risk primarily, we designed and synthesized PCR primers for detection of pathogens and parasites in B. terrestris. The samples were obtained from companies importing B. terrestris or field collections and genomic DNAs not showing physical shearing were purified. PCR for detection of pathogen- or parasite-specific gene revealed several DNA fragments were amplified in expected molecular size including Kashmir Bee Virus, Varroa jacobsoni, V. rindereri, Acarapis woodi and Aspergillus flavus. These amplified DNA fragments are in the process of cloning for DNA sequencing to confirm the target gene amplification. We also have plans to optimize the PCR conditions for each amplified target gene and try to develop biomarkers for diagnosis.

Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. A cDNA isolated from female adult heads of Plutella xylostella encodes 193 amino acids including PBAN, designated as Plx-PBAN. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. The PBAN receptor (Plx-PBANr) gene was also cloned from the female pheromone gland and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. In addition, to assess molecular events occurring downstream of PBAN signaling, partial sequences of Δ9 and Δ11 fatty acid desaturases of P. xylostella. were cloned. Phylogenetic analysis indicated that these two desaturase genes were highly clustered with other desaturases associated with sex pheromone biosynthesis in other insects. RT-PCR analysis showed that Δ9 desaturase was dominantly expressed in adult females, whereas Δ11 desaturase was expressed in all developmental stages. When PBANr expression was suppressed by PBANr-RNAi, the treated females also showed significant suppression of expression of both desaturases. These results suggest that expressions of the two desaturases are controlled by PBAN and that the two desaturases may be involved as downstream components in sex pheromone biosynthesis of P. xylostella.