Identification of rapid cold hardening-related genes in the tobacco budworm, Helicoverpa assulta

차욱현,이대원 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.4

A rapid cold hardening (RCH) and supercooling capacity usually play crucial roles in the survival of the tobacco budworm, Helicoverpa assulta, which is a freeze-tolerant species during the overwintering period. Cryoprotectants such as polyols or sugars are known to affect RCH and maintain fluid status of hemolymph improving the survival rate. This study is performed to identify cryoprotectants 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 and also significantly enhanced the supercooling capacity. Analysis of cryoprotectants from the hemolymph revealed that the distinguished changes of polyolswere found in the peaks of glycerol and trehalose. As cold treatmentwas prolonged, the amount of trehalose was continuously increased. In contrast, glycerol showed the highest level at 4 h treatment, and was gradually declined. To understand the pathway of trehalose biosynthesis during the pre-exposure treatment, expression level of genes related to trehalose biosynthesis were analyzed from fat body transcriptome of the 4th instar larvae of H. assulta. Based on the expression level of transcripts, the expression of trehalose phosphate synthase (TPS) harboring TPS and trehalose phosphate phosphatase domain contributes to the accumulation of trehalose in the hemolymph, suggesting that trehalose is responsible for overcoming RCH in H. assulta.

온도변화에 따른 담배나방 유충 지방체의 유전자 발현 비교 분석

차욱현,김광호,이대원,Cha, Wook Hyun,Kim, Kwang Ho,Lee, Dae-Weon 한국응용곤충학회 2018 한국응용곤충학회지 Vol.57 No.3

곤충은 넓은 범위의 온도영역에 사는 것으로 알려져 있으나, $40^{\circ}C$가 넘는 고온이나 빙결온도 이하의 저온에서는 생존이 어렵다. 본 연구는 사육온도 조건이 다른 환경에서 대사중심 조직인 지방체의 유전자 발현을 분석하기 위해, 온도조건을 달리하여 담배나방을 저온 사육충 ($3{\sim}10^{\circ}C$), 고온 사육충 ($35^{\circ}C$)로 나누고 상온 사육충 ($25^{\circ}C$)을 대조구로 사용하여 전사체 분석을 수행하였다. 저온에서 특이적으로 높은 발현을 보인 유전자는 표피단백질, ${\Delta}9$ 불포화효소, 글리세롤 3-인산 탈수소효소이며, 저온에서 발현이 낮아진 유전자는 키틴 합성효소, catalase, UDP-당전이 효소이다. 고온에서 특이적으로 높은 발현을 보인 유전자는 과산화물제거효소, metallothionein 2, phosphenolpyruvate carboxykinase, 트레할로스 운반단백질이었다. 고온에서 높고 저온에서 낮은 대조적 발현을 보인 유전자는 열충격단백질, glutathione peroxidase이었다. 이들 온도 특이적이거나 대조적 발현을 보이는 유전자는 기후변화에 관련한 특이마커로 활용이 가능할 것으로 사료된다. Insects are known to live at wide range of temperature, but can not survive when they are exposed to over $40^{\circ}C$ or below supercooling point. The larvae of Helicoverpa assulta have been reared at high ($35^{\circ}C$), low (3 to $10^{\circ}C$), and room temperature ($25^{\circ}C$; control). To identify stress-related genes, the transcriptomes of fat body have been analyzed. Genes such as cuticular proteins, fatty acyl ${\Delta}9$ desaturase and glycerol 3 phosphate dehydrogenase were up-regulated whereas chitin synthase, catalase, and UDP-glycosyltransferase were down-regulated at low temperature. Superoxide dismutase, metallothionein 2, phosphoenolpyruvate carboxykinase and trehalose transporter have been up-regulated at high temperature. In addition, expressions of heat shock protein and glutathione peroxidase were increased at high temperature, but decreased at low temperature. These temperature-specific expressed genes can be available as markers for climate change of insect pests.

Identification of G protein-coupled receptors in the pheromone gland of Maruca vitrata by transcriptomic analysis

차욱현,정진교,이대원 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.4

G protein-coupled receptors (GPCRs) belong to the cell membrane protein family, which regulate various physiological process such as reproduction, behavior and immune etc. In order to identify GPCRs in pheromone gland of Maruca vitrata, we carried out transcriptome analysis from virgin female adults. Fifty GPCR genes were identified such as pheromone biosynthesis activating neuropeptide receptor, prostaglandin receptors, neuropeptide receptor, 5-hydroxytryptamine receptor, galanin receptor, calcitonin gene-related peptide receptor, diuretic hormone receptor, gonadotropin-releasing hormone receptor, frizzled and orphan receptor, etc. Expressions of the identified GPCRs were confirmed with semi-quantitative RT-PCR. Expressions of various GPCRs in the pheromone gland indicate the biological roles of pheromone gland may not be limited to the pheromone production.

RNA interference of trehalose phosphate synthase inhibits metamorphosis and decreases cold tolerance in the diamondback moth, Plutella xylostella (L.)

차욱현,이대원 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.3

Cryoprotectants such as polyols or sugars affect rapid cold hardening (RCH) and maintain fluid status of hemolymph, improving the survival rate. RCH in Plutella xylostella larvae significantly increased the survival rate. Analysis of cryoprotectants in the hemolymph revealed that glycerol is a crucial cyroprotectant which is closely related to trehalose metabolism. To understand the importance of trehalose biosynthesis during the pre-exposure treatment, trehalose phosphate synthase (TPS) responsible for trehalose biosynthesis was identified from fat body transcriptome of the 3rd instar larvae P. xylostella. RT-PCR revealed that PxTPS was expressed in all developmental stages and largely detected in fat body among examined tissues. The suppression of PxTPS via RNA interference inhibited metamorphosis and decreased survival rate under RCH.

Putative pheromone biosynthesis pathway in Maruca vitrata by transcriptomic analysis

차욱현,김우진,정진교,이대원 한국응용곤충학회 2017 Journal of Asia-Pacific Entomology Vol.20 No.1

Pheromone biosynthesis in the pheromone gland is stimulated by pheromone biosynthesis activating neuropeptide (PBAN) produced in the suboesophageal ganglion. PBAN binds its receptor and transduces biological signal into themolecules for the pheromone biosynthesis. To understand pheromone biosynthesis pathway in legume pod borer, Maruca vitrata, total RNA from the pheromone gland was isolated and transcriptome of the pheromone gland was obtained. Total read bases from the transcriptome were 12.03 billion bp and 62,604 contigs were identified. A total of 191 contigs involved in the pheromone biosynthesis such as PBAN receptor, PBAN, fatty acid transport proteins, fatty acid synthases, fatty acid desaturases (FADs), fatty acyl-CoA reductases (FARs), alcohol oxidase/dehydrogenases (AOXs) and β-oxidation enzymes, were identified. Putative pheromone biosynthetic pathways for sex pheromone components inM. vitrata were proposed through transcriptomic analysis. Biosynthesis pathway for E10,E12-16:Ald and E10,E12-16:OH may initiate fromC16:CoAwith FAD2-like enzyme found in Manduca sexta, which converts the substrate into E10,E12-16:CoA. This intermediate is subsequently reduced by pheromone gland-specific FAR (pgFAR) which modifies end moiety with alcohol or by AOX which convert end moiety into aldehyde. The production of E10-16:Ald can be initiated from C16:CoA by Δ10 desaturase or from C18:CoA by Δ12 desaturase followed by with β-oxidation, and the intermediate can be reduced by pgFAR.

C-terminal conserved motifs of Neprilysin1 in Cotesia plutellae are not required for immune suppression of the Diamondback moth, Plutella xylostella (L.)

차욱현,이대원 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.4

Cotesia plutellae, an endoparasitoid wasp, parasitizes Plutella xylostella larvae and disrupts host immune responses through parasitic factors. These immune disruptive factors are known as maternal factors (venom proteins, polydnavirus, and ovary proteins) and an embryonic factor (teratocytes). In this study, we identified neprilysin-1 (Cp-NEP1) known as a potential immunosuppressive gene from the transcriptome of venom glands in C. plutellae. Cp-NEP1 encoding 451 amino acids belonged to the hymenopteran NEP1 family through phylogenetic analysis. Based on the structural comparison, Cp-NEP1 has lacked in conserved motifs such as a substrate binding (NAYY/ F), zinc-binding (HExxH and ExxxD), and protein folding and maturation (CxxW). In order to investigate the function of Cp-NEP1 in host immune response, we constructed a recombinant Cp-NEP1 (rCp-NEP1) harboring Nterminally fused 6X His-tag. rCp-NEP1 was successfully expressed in Escherichia coli and purified. Pre-heated E. coli induced the spike of nodule formation whereas co-injection of rCp-NEP1 with pre-heated E. coli exhibited suppression of nodule formation. Quantitative real-time PCR showed that expression of phenoloxidase related to nodule formation was also suppressed when rCp-NEP1 was co-injected with preheated E. coli. These results suggest that Cp-NEP1 contributes to immunosuppression and that conserved motifs lacking in Cp-NEP1 are not necessary for immune suppression in the host.

Identification and pheromonotropic activity of pheromone biosynthesis activating neuropeptide in Maruca vitrata

차욱현,정진교,김용균,이대원 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.1

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the suboesophageal ganglion stimulatespheromone biosynthesis in the pheromone gland, mediating sexual behaviors. The nucleotide sequences ofPBAN and PBAN receptor (PBANr) in the legume pod borer, Maruca vitrata, were obtained from the transcriptomeof the head and the pheromone gland, respectively. PBAN was expressed in all developmental stages. In contrast, PBANr was only detected in all developmental stages except for male adult. To examine the pheromonotropicactivity of PBAN (Mvi-PBAN) in M. vitrata, Mvi-PBAN was chemically synthesized and injected intofemale adult. The pheromone production was increased in the dose-dependent manner and exhibited themaximal increase 2 h post-injection.

Comparative transcriptome analysis of pheromone biosynthesis-related gene expressions in Plutella xylostella (L.)

차욱현,정충렬,황윤정,이대원 한국응용곤충학회 2017 Journal of Asia-Pacific Entomology Vol.20 No.4

The pheromone biosynthesis in Plutella xylostella is more active in the scotophase than that of the photophase, indicating that there may be changes of gene expression in the pheromone gland according to the photoperiod. To identify genes contributing to change in pheromone production, we analyzed transcriptomes of pheromone glands from both decapitated females (PG-minus) in the photophase and normal ones (PG-plus) in the scotophase. Deep sequencing for mRNAs in the pheromone gland yielded approximately 7.5 Gb and 10,303 transcripts showing positive FPKM value were analyzed. Differentially expressed gene analysis revealed that up- and down-regulated transcripts were 310 and 326 in the PG-plus transcriptome, respectively. Genes putatively involved in the pheromone biosynthesis pathway were identified such as acetyl-CoA carboxylase, acetyl-CoA dehydrogenase, fatty acid synthase (FAS), desaturases (Δ9 and Δ 11) and fatty acid reductases of pheromone gland (pgFAR), alcohol oxidase, aldehyde oxidase and aldehyde reductase, etc. Quantitative RT-PCR revealed that expressions of FAS, Δ11 desaturase and pgFAR were significantly higher in PG-plus, suggesting that they may have crucial roles in sex pheromone biosynthesis of P. xylostella

Calreticulin in Cotesia plutellae suppresses immune response of Plutella xylostella (L.)

차욱현,김용균,이대원 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.1

An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella, which is amajor pest in cruciferous crops. Successful parasitization requires bothmaternal and embryonic factors of C. plutellae, such as polydnavirus, ovarian proteins, teratocytes and venomproteins. In this study, we identified calreticulin (Cp-CRT) gene from transcriptome data of the venom gland in C. plutellae which encodes 403 amino acids harboring several structural motifs such as CRT family motif I and II, repetitive sequence (DP(X)3KPEDW), and endoplasmic reticulum-recognizing domain (-HDEL). Phylogenetic analysis showed that the Cp-CRT gene formed a unique cluster with other hymenopteran CRT genes. To examine the physiological function of Cp-CRT, recombinant Cp-CRT, fused with 6X-His at N-terminus, was expressed in Escherichia coli. Recombinant Cp-CRT was successfully expressed and suppressed significant nodule formation when co-injected with E. coli as immune response inducer. These results suggest that the Cp-CRT contributes to suppression of cellular immune response in the host.

콩명나방의 성페로몬 생합성

차욱현,박정준,이대원 한국응용곤충학회 2022 한국응용곤충학회지 Vol.61 No.1

나방류의 페로몬 생합성은 식도아래 신경절에서 생산된 페로몬 생합성 촉진 신경펩타이드(pheromone biosynthesis activating neuropeptide, PBAN)의 분비로부터 시작된다. 분비된 PBAN은 페로몬샘의 상피세포막에 있는 PBAN 수용체와 결합하고, 신호전달과정을 거쳐 페로몬 생합성과정이 활성화되어, 페로몬이 외부로 방출된다. 본 종설은 콩명나방(Maruca vitrata)의 성페로몬, PBAN과 수용체, 성페로몬 생합성 경로를 소개한 Pheromone biosynthesis in the pheromone gland is triggered from release of pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion. PBAN binds to its receptor on the epithelial cell membrane and activates signal transduction pathways for the pheromone biosynthesis. This study reviews sex pheromone, PBAN and its receptor, and pheromone biosynthesis pathway of Maruca vitrata.