차세대 염기서열 분석(Next Generation Sequencing, NGS)은 대량의 병렬 데이터 생산으로 유전체의 염기서열 을 고속으로 분석하는 기술이며, 이 기술은 바이러스 유전체 분석에도 광범위하게 사용되고 있다. 하지만, 바이 러스의 전장 유전체가 100kb를 넘을 경우, 동일한 raw data라도 분석 방법 및 소프트웨어 그리고 매개변수 (parameter)에 따라 유전체의 크기와 구조가 다르게 결정된다. 따라서 유전체가 큰 바이러스 분석 시, 최적화된 NGS 분석 방법을 선택하는 것이 중요하다. 본 연구는 장수풍뎅이 누디바이러스(Oryctes rhinocerous nudivirus, 120kb) 유전체를 기반으로, 다양한 Assembly 소프트웨어(metaviralSPAdes, metaSPAdes, velvet, shovill, Geneious, megahit)를 사용하여, 최적화된 NGS 분석 방법을 고안하였다. Assembly 소프트웨어에 따라 바이러스 유전체 크기와 특징(Single Nucleotide Polymorphism, Insertion&Deletion, repetitive genomic variants)의 차이를 확인하였 다. Assembly 소프트웨어 간의 차이가 있는 염기서열은 Sanger sequencing을 통해 재확인하여, 참조 유전체 (reference sequence)를 구축하였다. 이 참조 유전체를 기반으로 가장 정확한 Assembly 소프트웨어와 parameter를 평가하였다. 본 연구는 분석 방법에 따라 달라지는 유전체의 특성을 이해하고, 바이러스 유전체를 정확하게 구축 하는 분석 파이프라인을 제공할 것으로 기대된다.

쌍별귀뚜라미는 국내에서 식용 및 파충류의 먹이로 사용되는 중요한 산업곤충으로 알려져있다. 2023년 국내 쌍별귀뚜라미 농가에서 쌍별귀뚜라미 덴소바이러스 감염이 확인되었으며 바이러스 억제제가 요구되는 실정 이다. RNA 간섭(RNAi)은 dsRNA를 이용해 목적 유전자의 발현을 억제할 수 있는 작용기작을 가지며, 바이러스 특이적 시퀀스를 이용한다면 효과적으로 타겟 바이러스의 증식을 저해할 수 있다. 본 연구에서는 덴소바이러스 에 감염된 귀뚜라미에 덴소바이러스 특이적 dsRNA를 제작하여 주입(injection)하고, 바이러스 발현 저해 효과를 관찰했다. 바이러스 감염 수준은 qPCR로 평가하였으며 dsRNA를 접종한 결과 체내 바이러스양이 감소함을 확인 했다. 이 연구는 향후 RNAi를 이용한 쌍별귀뚜라미 덴소바이러스 억제제 개발에 도움이 될 것으로 기대된다.

한국 귀뚜라미 산업은 최근 큰 성장을 보였으며, 수직농장을 이용한 대량생산과 기술발전으로 사육수와 판매 량이 늘어나는 추세다. 그러나 귀뚜라미 사육시스템은 농가 간의 교류 및 밀집 사육으로 인해 곤충병원체에 의한 전염병에 매우 취약한 구조를 가진다. 사육 곤충의 질병을 이해하는 것이 중요함에도 곤충병원성 미생물에 대한 연구가 부족한 실정이다. 본 연구는 국내 쌍별귀뚜라미 농가에서 발견된 볼보바이러스를 식별하였으며, Gryllus bimaculatus volvovirus (GbVVV-KR)의 게놈 특성을 분석하였다. GbVVV-KR의 전체 게놈 서열은 Sanger 시퀀싱 을 통해 얻어졌으며, 게놈 크기가 2,515개 뉴클레오티드인 원형 단일가닥 DNA 바이러스임을 밝혔다. 단일 핵산 염기 다형현상 분석(Single nucleotide polymorphism, SNP)으로 정지코돈 돌연변이로 인한 ORF3 영역이 다른 귀뚜 라미 volvovirus 들에 비해 큰 변화를 확인하였다. 또한, 전국 11개 농장에서 수집한 귀뚜라미를 대상으로 PCR/qPCR을 실시하여 GbVVV-KR의 감염 정도를 확인하였다. 이러한 결과는 GbVVV-KR의 게놈 구조와 유전 적 특성, 계통발생 및 유병률에 대한 귀중한 통찰력을 제공하고 귀뚜라미 바이러스에 대한 이해를 도울 것으로 기대한다.

귀뚜라미는 단백질 함량이 높고 필수아미노산이 풍부하여 세계적으로 식용과 사료용 곤충으로서 가치가 높 다. 국내에서 쌍별귀뚜라미(Gryllus bimaculatus)는 사육이 용이하다는 장점이 있고 주로 파충류, 물고기 등의 사료로 대량 소비되고 있기 때문에 매우 중요한 산업곤충으로 자리매김하고 있다. 그러나 최근 쌍별귀뚜라미의 활동성이 저하되고 다리가 마비되는 등 이상 증상이 발생하여 대량 폐사된 농가가 급증하였다. 이에 대한 원인을 규명하기 위해 질병 발생 농가로부터 귀뚜라미 시료를 수집하여 RNA를 추출하고 metatranscriptomic analysis 를 수행하였다. 그 결과, 신규한 덴소바이러스(Densovirus)가 대량 폐사의 원인임을 밝혀내었고, 이 바이러스를 Gryllus bimaculatus densovirus (GbDV)로 명명하였다. 또한, GbDV의 유전체 분석 결과, genome size는 5,643 bp로 5개의 ORFs(Open Reading Frames)을 포함하며, 왼쪽에는 세 개의 Non-structural proteins을 포함하는 유전자(NS1, NS2, NS3)가 존재했고 오른쪽에는 두 개의 Structural proteins을 포함하는 유전자(VP1, VP2)가 존재했다. 본 연구 는 알 수 없는 곤충 질병의 원인을 밝혀내어 해당 질병에 대한 모니터링을 가능케 함으로써 산업곤충 질병의 예찰하고 확산을 방지하는 데 기여할 것으로 사료된다.

전 세계적으로 RNA 간섭(RNA interference, RNAi)을 활용한 해충방제제 연구가 활발히 진행되고 있다. 대표적 으로 Monsanto의 서부 옥수수 뿌리벌레(Diabrotica virgifera virgifera) 특이적 방제용 dsRNA (DvSNF7)를 발현하 는 옥수수 종자가 상용화 되었고, 2016년 이 종자가 국내 사료 및 식품용으로 수입이 승인 되었다. 본 연구는 국내 에 서식하는 좁은가슴잎벌레(Phaedon brassicae)를 Non-target 곤충으로 사용하여, 옥수수 종자에 사용된 DvSNF7 dsRNA의 잠재적 위해성을 평가했다. P. brassicae의 SNF7 유전자와 DvSNF7 dsRNA 간 Sequence유사성 을 확인했다. 다음으로 P. brassicae가 DvSNF7 dsRNA를 섭식할 수 있는 환경을 조성하여, 치사 효과 실험과 Sequence특이적인 Knockdown효과를 확인하였다. 그 결과, DvSNF7 dsRNA는 P. brassicae생존에 영향을 미치지 않았으며, P. brassicae의 SNF7유전자를 Knockdown시키지 않음을 확인했다. 이번 연구를 통해 D. virgifera virgifera와 같은 과인 P. brassicae 는 SNF7유전자 간 Sequence유사성이 있지만, Sequence특이성이 부족하여 생존과 유전자 발현에 영향을 주지 않음으로써 위해성이 낮음을 확인했다.

Climate change has led to increased insect pests and pest distribution changes. Traditionally, chemical control using synthetic pesticides has been the main method for pest management, but the emergence of pesticide-resistant pests has become a problem. There is a need to develop new pest control agents to overcome these issues. Entomopathogenic fungi used in pest management have minimal environmental side effects and possess a mechanism of action distinct from that of synthetic pesticides. However, there is a need for the development of technologies to maximize the insecticidal effects of fungi against pests, and expressing and releasing dsRNA within the fungi can preemptively knock out the activation of the insect’s defense system, thereby enhancing the insecticidal effect. Controlling insect defense genes and using entomopathogenic fungi as bio-carriers forms a new pest management strategy. This approach, described as a “microbial insecticide agents development strategy of cassette concept, ” can versatilely modify genes and microbes. It is expected to overcome the limitations of synthetic pesticides.

장수풍뎅이(Trypoxylus dichotomus)는 오래 전부터 애완·학습용으로 활용되는 정서곤충으로 곤충산업의 씨 앗이 되는 중요한 곤충이다. 2012년 청원군에서 처음 장수풍뎅이 유충이 일제히 사망한 사례가 있고 2014년부터 비슷한 증상의 사례가 전국적으로 발생했다. 2015년에는 이러한 원인이 장수풍뎅이 누디바이러스(Oryctes rhinoceros nudivirus, OrNV)로 인한 것임이 밝혀졌고 2017년에 raw sequencing data가 NCBI에 공개되었지만 추가 분석은 이루어지지 않았다. 따라서, 우리는 국내 장수풍뎅이에서 분리한 누디바이러스(Trypoxylus dichotomus nudivirus, TdNV-KR)로 명명하고, NCBI로부터 수집한 Malaysia, Solomon Islands, Indonesia, Philippines, Palau strain의 OrNV sequence와 비교 분석하였다. 그 결과, TdNV-KR의 genome size는 126,408bp임을 확인하였고 다른 OrNV strain이 비해 가장 적은 open reading frames(ORF)를 가지고 있었으며, 3개의 ORF가 TdNV genome에서만 부재함을 확인했다. 또한, Nudivirus core genes의 아미노산 염기서열 비교에서는 Single-nucleotide polymorphism 과 indels(insertion/deletion)로 인해 highly conserved 한 OrNV strain의 sequence들에 비해 TdNV의 시퀀스는 많은 변이/차이를 보였다. 우리의 연구는 누디바이러스의 종간 전파에 대한 이해도를 높이고, 바이러스 간의 유연관계 를 확인함으로써 유입 경로를 추정하여 산업곤충의 질병을 효과적으로 예방하는 데 기여할 것이다.

Six plant essential oils, vanillin, and their mixtures were tested for repellent activities against a dengue virus vector mosquito, Aedes aegypti. Their repellency was verified with two conditions: different doses and an addition of vanillin. Among the plant essential oils, lemongrass, lemoneucalyptus and xanthoxylum oils were selected as the potential oils for tertiary mixtures. In bioassays using tertiary mixtures with two essential oils with vanillin, the composition of 1:3:1 (v/v/w) consisted of lemongrass oil, xanthoxylum oil and vanillin provided 270 min-CPT. As a practical application into spatial repellent, the mixture of 1:1:1 formulation containing lemongrass oil, xanthoxylum oil, and vanillin (v/v/w) was enclosed into the Viscopearl, porous cellulose beads that provide gradual release of volatile compounds. We composed this Viscopearl into a module and installed into the air conditioner. Apparently, repellent activity was shown that average repellency of 83.5% was observed for an hour through the chamber test. We confirmed via GC-chromatogram that linalool, geraniol, citral and vanillin were the main compounds of our oil mixture. Our study indicates that personal repellents were possibility applicable to spatial repellents.

Six plant essential oils, vanillin, and their mixtures were tested for repellent activities and olfactory responses in a dengue virus vector mosquito, Aedes aegypti. Among the plant essential oils, cassia oil showed complete protection time (CPT) of 75 minutes. CPTs of lemongrass, lemoneucalyptus, xanthoxylum oils, and vanillin were within 30 minutes at 5% (0.21 mg/cm2) tested level, although their CPTs were not comparable to same concentration of DEET that showed 127.5 minutes of CPT. However, their repellency effects were significantly improved in two conditions; improving concentration up to 15% level (0.63 mg/cm2) and addition of vanillin. In bioassays using binary or tertiary mixtures with one or two essential oils with or without vanillin, the composition of 1:3:1 (v/v/w) consisted of lemongrass oil, xanthoxylum oil and vanillin provided 270 min-CPT. As a practical application, the mixture of 1:1:1 formulation containing lemongrass oil, xanthoxylum oil, and vanillin (v/v/w) was enclosed into the Viscopearl, porous cellulose beads that provide gradual release of volatile compounds. Efficiently, more than 90% of repellency for 2 hours was observed in cage and semi-field chamber tests using the formulation. In addition to behavioral assays, we subsequently examined how mosquitoes sense the blends of oils with vanillin by using electroantennogram (EAG) recording. Binary mixture with one oil and vanillin, which extended CPTs, showed no significant patterns of EAG alternation, while tertiary mixtures of oils and vanillin decreased patterns of EAG responses as an increase of vanillin contents in the mixture, implying further potential roles of vanillin as a synergist in mosquito repellency. Based on behavioral and electrophysiological data, cassia, rosemary, lemongrass, xanthoxylum, and lemoneucalyptus oils could provide the high possibility for development of commercial products for useful management strategies to control mosquitoes.

Cinnamaldehyde as the main component of Cinnamomum plants is well known as mammalian transient receptor potential ankyrin 1 (TRPA1) agonist, also activated by low temperature stimuli and mechanosensation. The other TRP subfamily, transient receptor potential vanilloid-1 (TRPV1) sensitive to pungent compounds such as capsaicin and allicin mediates the feeling of warmth. Both TRPA1 and TRPV1 channels are abundantly distributed in sensory neurons. Thus, there is possibility that these channels modulate repellent behaviors of mosquitoes and Drosophila through olfactory receptor neurons (ORNs). In order to confirm this hypothesis, we carried out laboratory repellent tests with cinnamaldehyde to Aedes aegypti females using arm-in-cage test and to a wild type and two TRP channel mutants Drosophila lines using a choice assay. Cinnamaldehyde showed strong repellency against Ae. aegypti and Drosophila wild adults at tested concentrations. However, a mutant fly line did not discriminate or detect the existence of the repellent. These behavioral data suggest that cinnamaldehyde may directly target the TRP channel. More studies to elucidate neural correlates of repellency to ainnamaldehyde compound are as follows: 1) Identifying the ORNs mediating cinnamaldehyde detection using single-sensillum recording techniques, 2) Co-localization of TRP genes on olfactory organs of Ae. aegypti and Drosophila using in situ hybridization and 3) Whether the Aedes TRP homologs might function in cinnamaldehyde repellency using rescued TRP chennels of Drosophila.

Olfaction as an important sensory modality in insects is essential for identification of hosts, mates, oviposition sites, and food resources in nature. In the cockroach, both olfactory sensitivity in the antennae and the formation of shortand long-term olfactory memories exhibit daily fluctuations that are regulated by the circadian system. An important problem is to characterize the signalling systems and molecules that are involved in this regulation of olfactory reception and olfactory behaviour. Recent results suggest that insect olfactory systems are modulated by both biogenic amines and neuropeptides. However, it remains elusive how these molecules modulate olfactory system in the peripheral systems. In the present study, our aim was to characterize the structure and organization of these signalling systems in the peripheral olfactory system of the American cockroach, Periplaneta americana. This work illuminated that tachykinin and its receptors regulate olfactory sensitivity in the antennae of the cockroach. Injections of tachykinin peptides caused decreases in the amplitude of the electroantennoogram (EAG), cells that produce tachykinin were localized in the antennae, and olfactory receptor neurons expressed tachykinin receptors. Interestingly, the tachykinin expressing cells also express receptors for the biogenic amine, octopamine and injections of octopamine also cause reductions in EAG amplitude. These results suggest that both octopaminergic and tachykinin peptide signalling pathways are important regulators of olfactory reception in the cockroach. We propose the hypothesis that octopamine regulates the release of tachykinin from cells in the antennae that, in turn, modulate the sensitivity of olfactory receptor neurons.

Aedes aegypti is a primary vector that transmits dengue and yellow fever around the world. To prevent the spreading and elimination of mosquitoes, insecticides and repellents like DEET (N, N-diethyl-m-methyl benzamide) have been broadly used. Even though DEET is considered as highly effective and proven protection against mosquito, it causes skin irritants and rashes, melts some synthetic plastics, and unpleasant smells. Therefore, there is a trend finding alternative mosquito repellents instead of using DEET. We tested repellent effects with plant essential oils and synergistic effects of those plant essential oils with additional vanillin, comparing them to DEET itself. Some of prepared mixtures showed better repellency than DEET. In addition, we evaluated the differences in the peripheral olfactory responses of Ae.aegypti females using EAG tests (electroantennogram). The aim of this test is to determined how the vanillin within plant essential oils or DEET acts in mosquito’s olfactory organs in aspect of molecular mechanisms. Revealing the novel function and localization of these putative repellent receptors may provide new insight into development of repellent as well as behavioral control agents in the future and contribute to understand the mechanism of processing patterns of repellent receptors in mosquitoes.

An olfactory system is one of the complicatedly-equipped sensory facilities in the insect sensory systems, which is most essential for insect olfactory-driven behaviors relevant to survival such as finding hosts, mates, oviposition sites, and food resources. These behaviors are mostly controlled by circadian rhythm. The american cockroach, Periplaneta americana, has been an ideal model to extensively study olfactory system associated with complex behavioral repertoires and circadian controls of certain behaviors, respectively. Even though it is known that olfactory-related physiology in peripheral and central olfactory systems seems to be highly variable by circadian rhythms, little is known about how these are controlled at the neuronal and molecular levels. It has been reported that the plasticity in the olfactory system is modulated by a set of neuropeptides. However, it remains still elusive how these neuropeptides and neuroendocrine system interact in the peripheral systems to change olfactory responses in cockroaches. Here, current study focuses on the localization of neuropeptides and their receptors by using in situ hybridization and immunostaining methods. Also, expression level of these genes are evaulated by qRT-PCR methods. Circadian fluctuation of these genes seem to be important neurotransmission machineries in the periphery. Our current study suggests that microcircuits of neuronal systems in the peripheral olfactory organ play an important in olfactory modulation by circadian rhythm

Olfactory sense is an essential modality for insects to locate hosts, mates, oviposition sites, and food resources in nature. Based on many studies so far, insect olfactory systems in periphery as well as central nervous systems seem to be highly plastic with behavioral and physiological changes associated with learning and memory, sensory processes, and other developmental processes. Eventually, it is also evident that these plasticity and processes in olfactory systems are modulated by a various ensemble or sets of neuropeptides. The olfactory reception in the peripheral systems of the cockroach, Periplaneta americana, associated with learning behaviors, is fluctuated by the circadian rhythm, which is subsequently thought to control the titer of hormones and various neuropeptides concomitantly. However, it remains still elusive how neuropeptides and neuroendocrine systems modulate olfactory system in the peripheral systems in cockroaches as well as any other insects. Here, our aims to characterize the ultrastructure of these neuro-endocrine systems in the peripheral olfactory systems in American cockroach, Periplaneta americana. Using in situ hybridization methods, we found out that tachykinin and its receptors seem to be important neurotransmission machineries in the periphery to convey the arousal signals from the insect brain. Our findings also indicate complicated endocrine systems connected with central nervous systems may modulate the olfactory reception in the periphery.