Autophagy is an important self-eating process to eliminate damaged or unused organelles. We identified nine autophagy-related genes (Atg) including AaAtg-1, -3, -4b, -4d, -5, -6, -8, -12 and -13 from the Asian tiger mosquito, Aedes albopictus. Developmental expression patterns indicate that mRNA levels of AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 were highly expressed in egg, whereas expression of AaAtg8 was high in 1stand3rdinstarlarvalstages. TissuespecificexpressionofthesegenesindicatesthatAaAtg1 was highly expressed in thorax and midgut in blood-fed adult female mosquitoes (BF), and head and thorax in sugar fed adult female mosquitoes (SF). Transcript level of AaAtg3 was high in thorax in BF, but head, thorax and Malpighian tubules in SF. AaAtg4b, -4d mRNA levels were significantly high in Malpighian tubules in BF, and head in SF, respectively. AaAtg-5 and -6 transcripts were highly expressed in head in BF, and expression of AaAtg-8 was high in Malpighian tubules in BF. Levels of AaAtg-12 and -13 mRNAs were significantly high in head and midgut in BF. Induction patterns of AaAtg genes against pathogens showed that AaAtg-1, -3, -4b, -8, -12 and -13 were strongly induced at 6 h-post injection of S. aureus, and mRNA levels of AaAtg-1, -3 and -13 were significantly induced by E. coli challenge after 3 h-post injection in SF abdominal carcass. In SF midgut, AaAtg-1, -3, -4b, -4d, -5, -6, -12 and -13 transcripts were drastically induced at 9 h-injection of E. coli and S. aureus, while expression of AaAtg-8 was highly induced by S. aureus and C. albicans at 9 h-post injection. Each AaAtg gene was slightly induced by E. coli, S. aureus or C. albicans at different time points in abdominal carcass in BF. Interestingly, AaAtg-8 was not induced by microbial challenge. While eight other Atg genes except AaAtg-8 were highly influenced by S. aureus at 6 and 9 h-post injection, E. coli at 3 h-post-treatment, and 3, 6, and 9 h-post inoculation. In the future, we will characterize the functional roles of autophagy during mosquito-microbes interaction.

It has been well known that IKK-β, -ε and –γ play a pivotal role in IMD pathway. In this study, TmIKK-ε was identified and their functions in countering pathogenic infections were investigated. We identified TmIKK-ε gene which including 2,196 bp nucleotides (encoding 731 amino acid residues). Domain analysis of TmIKK-ε indicates that there is one Serine/Threonine protein kinases catalytic domain. TmIKK-ε gene was highly expressed in 2 day-old pupal stage and the expression was gradually decreased until 1 day-old adults. Then the expression was slightly increased until 4 day-old adult stage. Tissue specific expression of TmIKK-ε mRNA was high in the gut, integuments and hemocytes in last instar larvae, and fat body, Malpighian tubules and testis in 5-daysold adult. In hemocytes, TmIKK-ε was drastically induced by E. coli injection after 3 h and by S. aureus at 3 and 12 h-post injection. In gut, expression level of TmIKK-ε was high at 6 h-post injection of microbial injection. Expression of TmIKK-ε in fat body was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-ε, gene specific RNAi and mortality assay were performed. TmIKK-ε RNAi caused increased larval mortality against E. coli, not S. aureus and C. albicans. Finally, to investigate the induction patterns of Tenebrio fourteen AMP genes in response TmIKK-ε RNAi, three microorganisms were treated into TmIKK-ε-silenced T. molitor larvae. Nine out of fourteen AMP genes were not induced by microbial challenge in TmIKK-β dsRNA-injected group. Taken together, our results indicate that TmIKK-ε may regulates nine antimicrobial peptide genes in response to microbial challenge in T. molitor fat body.

Host defense against pathogen invasion highly relies on immune defense machinery that is controlled by the nuclear factor-κB (NF-κB) of transcription factors. The Toll pathway are well known as an insect innate immune mechanism to protect host itself from invaded pathogens. Basically, in the edible insect, Tenebrio molitor, the Toll pathway is primarily activated by polymeric Lys-type peptidoglycans (PGNs), and components of fungal cell walls, β-1,3-glucan. Based on the current studies, the tremendous study has been focused on recognition and subsequent activation of spätzle in haemolymph, hence, there is a grave gap for intracellular event. Herein, in order to understand intracellular event of Toll signaling pathway, the Dorsal gene were identified. Moreover, domain analyses of TmDorsal2 gene indicate that there are two major domains such as Rel homology domain (RHD), ig-like, plexins, and transcription factors (IPT) domains. Based on the achieved results, TmDorsal2 mRNA was highly expressed in 1-day old pupa. Furthermore, TmDorsal2 was highly expressed in Malpighian tubules and fat body in last instar larvae (LL), and likewise mainly expressed in Malpighian tubules during adult 5-day old period, also the lowest expression of TmDorsal2 was observed in gonads. Moreover, TmDorsal2 mRNA levels after infection with E. coli appreciably went up at 6 and 9h time points. To investigate the effects of TmDorsal2 RNAi on larval susceptibility against various pathogens namely E. coli, S. aureus or C.albicans, dsRNA of TmDorsal2 has been synthesized the larvae dissected after 24h. As a result, TmAttacin1a, 1b and 2, TmDefencine1 and 2, TmTenecin1, 2, 3 and 4, TmCecropin2, TmColeoptericin1 and 2, Thaumatin-like protein 1 and 2 markedly reduced in the gut after injecting all mentioned microbes. In contrast, TmTenecin 2, Thaumatin-like protein 1 and 2 strikingly increased after microbe injection in the fat body. Interestingly, the most AMPs gene expression in whole body experimental case were upregulated. On the horizon, we will investigate effects of TmDorsal1 RNAi on larval susceptibility against various pathogens. Taken together, our studies may aid to understand insect innate immunity.

본 연구는 지속가능한 유기농업의 실천을 위하여 국내 작부체계와 농업환경에 적합한 한국형 현장적용 기법을 구축하고자 수행하였다. 본 연구의 기본 개념은 ‘Natural Enemy in First (NE가 먼저)’로 해충발생시기의 예찰 없이 주 작물을 정식함과 동시에 천적과 보조식물을 혼합 적용해서 해충발생 이전에 천적을 포장에 먼저 정착시키는 생물적 방제기법이다. 시설재배 딸기에서 칠레이리응애, 사막이리응애, 콜레마니진디벌과 천적의 서식처 3종을 주 작물의 정식과 동시에 적용한 결과, 관행방제구에 비해 80%이상의 해충 밀도억제효과를, 천적 단독처리구에 비해 3배 이상의 높은 천적의 밀도를 확인할 수 있었다. 또한 포장 환경과 천적의 방사방법에 구애받지 않고 천적의 해충 방제효과를 극대화시킬 수 있는 라인을 적용한 결과, 약제와 천적 혼합처리구에서 라인을 적용하지 않은 처리구에 비해 해충의 밀도가 급격히 감소하고, 천적의 밀도는 2배 이상 높게 유지되는 것을 확인할 수 있었다.

To identify the venom components and their expression patterns of some Aculeata bees/wasps, venom gland-specific transcriptome analysis was conducted. FPKM values were normalized with the average of the transcription level of reference gene (a-tubulin). Common components in both solitary and social wasp venoms include hyaluronidase, phospholipase A2, metalloendopeptidase, etc. Although it has been expected that more diverse bioactive components with the functions of prey inactivation and physiology manipulation are present in solitary wasps, the information on venom compositions of solitary wasps obtained in this study was not sufficient to generalizae this notion. Nevertheless, some neurotoxic peptides (e.g., pompilidotoxin and dendrotoxin-like peptide) and proteins (e.g., insuline-like peptide binding protein) appear to be specific to solitary wasp venom. In contrast, several proteins, such as venom allergen 5 protein, venom acid phosphatase, and various phospholipases, appear to be relatively more abundant in social wasp venom. In the venom gland trancsriptome of bumblebees, major allergens or pain producing factors were barely identified, implying that bumblebee venoms are relatively less toxic than those of social or solitary wasps.

IKK-γ is an essential protein to form IKK complex which regulate NF-κB. We identified TmIKK-γ (or TmKenny) gene which has 1,521 bp of nucleotides encoding 506 amino acid residues. Domain analysis of TmIKK-γ shows that there are one NF-κB essential modulator (NEMO) domain and a leucine zipper domain. Expression of TmIKK-γ gene was gradually increased from egg to 2-day-old pupal stage, dramatically decreased until 7 day-old pupal stage, and then it was gradually increased. TmIKK-γ transcripts were highly expressed in fat body and hemocytes in late instar larvae and integuments, fat body and Malpighian tubules in 5 day-old adult. TmIKK-γ was drastically induced by E. coli after 3 h challenges and by S. aureus at 3 and 12 h-post injection in hemocytes. TmIKK-γ was not induced by C. albicans although it was significantly induced by E. coli (at 3, 6 and 24 h) and S. aureus (at 9 h) in gut. In fat body, expression of TmIKK-γ was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-γ, gene specific RNAi and mortality assay was performed. larval mortality against microbial challenge was dramatically increased by TmIKK-γ RNAi. Furthermore, we investigate the tissue specific induction patterns of fourteen AMP genes in response TmIKK-γ dsRNA-treatment. In fat body, ten AMP genes out of fourteen was not significantly induced by microbial challenge in TmIKK-γ dsRNA-treated group. Based on these results, TmIKK-γ might play an important role in antimicrobial innate immune responses in Tenebrio molitor.

Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae) is considered as a serious pest in pine trees. To develop an eco-friendly strategy to manage this forest insect, we collected entomopathogenic fungi from Korean soil and assessed their virulence against the adults of the insect in laboratory conditions. As a result, two isolates with conidial suspension (1.0×107conidia/ml), showed 87% and 90% mortality 12 days after fungal treatment, respectively. We assessed the potential of the fungi-derived destruxin and protease as additives to the fungal isolates, and they showed insecticidal activity via feeding and spraying treatments. Finally, we produced fungal conidia in massive solid cultures and formulated wettable powders, and now studying optimal conditions of oil-based formulation with two isolates. Based on these results, we are evaluating the control efficacy of the fungal agents against M. alternatus in field conditions.

The beneficial effect of silicon (Si) in increasing salt stress tolerance has been observed in many plants, including the cereal crops rice, wheat, and barley. In this experiment, we examined the effect of Si on the survival and growth of torenia (Torenia fournieri L inden ex F oum) ‘ Duchess Blue and White’ cultured in vitro in the presence and absence of salt stress. Previous reports had suggested that torenia exhibited low salt tolerance. Shoot buds isolated from 16-day-old seedlings were cultured on Murashige and Skoog (MS) medium containing 0, 50, or 100 mM NaCl alone or in combination with 1.8 or 3.6 mM Si supplied as K2SiO3. Plant survival rate was significantly reduced by NaCl supplementation compared with the control. The survival rate significantly increased to 100% when 1.8 or 3.6 mM Si was added to the MS medium containing 50 mM NaCl. However, only 31% of plantlets survived when 1.8 mM Si was added to the culture medium containing 100 mM NaCl. Shoot and root lengths significantly decreased with increasing NaCl concentration in the culture medium, whereas addition of NaCl to the MS medium also significantly reduced fresh and dry weights. However, Si supplementation significantly increased fresh and dry weights under 50 mM NaCl, compared with the control. The greatest fresh and dry weights were recorded when shoot buds were cultured on MS medium containing 50 mM NaCl and 3.6 mM Si. The activities of the antioxidant-scavenging enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), but not peroxidase (POD), were markedly higher in the presence of 50 mM NaCl than the activity of the control. When Si was added to the medium containing 50 mM NaCl, activities of SOD, POD, APX, and CAT decreased as compared with the 50 mM NaCl treatment. Thus, Si-mediated tolerance to NaCl stress was not due to increased activity of antioxidant enzymes. Although Si was not effective in increasing tolerance to high salt concentrations, such as 100 mM NaCl, the results suggested that Si supplementation could effectively enhance tolerance to 50 mM NaCl stress.

The objectives of the present study were to examine the antioxidant activity of fractions with different isoelectric points from salmon enzymatic hydrolysates and obtain peptide fractions of sufficient amounts with higher antioxidant peptide fraction, which could be applied to the food and animal model systems. The salmon enzymatic hydrolysates were fractionated on the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing without toxic solvents and reagents, which is termed autofocusing. Acidic and basic fractions showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than the other fractions. The basic fractions showed higher hydroxyl (OH) radical scavenging activity. The weak acidic and weak basic fractions showed higher oxygen radical absorbance capacity (ORAC) values than the acidic and basic fractions. The acidic fraction showed higher metal chelating activity than other fractions. The acidic fraction suppressed lipid oxidation in the cooked patties to a greater extent than other fractions. These results suggest that peptides fractions from salmon enzymatic hydrolysate are effective antioxidant, and that autofocusing could be useful to increase antioxidant activity for application to food and animal model systems.

벗초파리는 우리나라에서는 심각한 해충으로 인식되지 않아 발생 기간 및 패턴, 기주 범위 등의 생태적인 연구뿐만 아니라 모니터링을 위한 우수 유인제 및 트랩의 연구도 제한적으로 수행된 실정이다. 본 연구는 기존 연구 결과를 참조하여 사과식초 함유 유인제 2종과 화학적 루어 2종 그리고 트랩 2종에 대한 선발 시험을 수행하였다. 선발 시험은 사육중인 벗초파리를 이용한 choice test와 함께 수목원, 딸기 농장, 블루베리 농장에서의 6반복 유인력 검증 실험을 수행하였다. 유인력 비교 결과 ACV + wine의 포획량이 높았으며, 블루베리 농장 인근 결과값은 전체 조사 항목에서 통계적 유의성을 보였다 (P < 0.05). 트랩 선발 실험 결과 모든 실험 장소에서 Dreves 트랩이 높은 포획량을 보였다. 이상의 결과를 통해 ACV + wine과 Dreves 트랩 조합을 선발하였으며, 이를 이용하여 향후 지속적인 모니터링, 월동 발생 조사 등의 연구에 활용할 수 있을 것으로 생각된다.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this paper, we investigated the molecular structure, tissue distribution patterns, and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was detected by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of sequestration and hydrolysis of acetylcholine (ACh)/choline (Ch) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. Kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions as a sequestering enzyme specific to ACh (not to Ch) by having a very strong affinity to ACh but an extremely long turnover time.

To elucidate the effect of cellular phone electromagnetic wave (EMW) exposure on the developing cerebellar cortex of neonatal Sprague-Dawley rats, animals were exposed to cellular phone electromagnetic waves for 1 hr per day for 3 weeks. At the end of the experimental period, animals were sacrificed by cardiac perfusion, after which histological samples were prepared and observed microscopically. In the EMW exposure group, external granule cells were remained partially in the external granular layer without migrating into the internal granular layer. In addition, dark stained shrunken Purkinje cells with pyknotic nuclei increased and the outline of cells became irregular and showed degenerative signs, such as mitochondrial swelling and disrupted cristae. Moreover, the cisternae of rough endoplasmic reticula and Golgi complex were severely swollen. Bergmann glial cells adjacent to the dark stained Purkinje cells were swollen and cytoplasmic organelles were scant. Dark stained shrunken granule cells were also observed and the outline of cells was irregular. The results of the present study suggest that cellular phone EMW exposure to neonatal Sprague-Dawley rats leads to a partial delay of early migration of cerebellar cortical cells and degenerative changes in Purkinje cells, Bergmann glial cells and granule cells.

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.