The mosquito is a dangerous disease vector, which transfers various diseases like dengue virus, Zika virus and malaria.This study used an alternative way of mosquito repellent agents rather than chemical repellents such as DEET. We haveemployed a novel emitters of ultra-sonic sounds, which showed repellent effects to mosquito behaviors that avoid ultra-sonicsounds. Using NGS analysis after ultrasonic treatment, we found that some subsets of sensory and other related geneswere affected, indicating that ultrasonic sounds affects gene expression associated with host finding behaviors in Aedesaegypti. Additionally, we performed electrophysiological recording and behavior assay testing repellency in a chamberin Culex pipiens, which is the most common species in Korea.

One of the overlooked points in mosquito blood feeding research is a final step before blood feeding. We provide the anatomical and chemosensory evidence that a piercing structure of the mouthpart of the mosquitoes is an essential apparatus for the penultimate stage in blood feeding in mosquitoes. Indeed, the stylet possesses a number of sensory hairs located at the tip of the stylet. These hairs house olfactory receptor neurons that express two conventional olfactory receptors of Aedes aegypti (AaOrs), AaOr8 and AaOr49, together with the odorant co-receptor (AaOrco). In vivo calcium imaging using transfected cell lines demonstrated that AaOr8 and AaOr49 were activated by volatile compounds present in blood. Taken together, we identified olfactory receptor neurons in the stylet involved in mosquito blood feeding behaviors, which in turn indicates that olfactory perception in the stylet is necessary and sufficient for mosquitoes to find host blood in order to rapidly acquire blood meals from a host animal.

The Plant Extract Bank was established by 21 Century Frontier R&D Program. It has began selling a research plant extract samples to support many Korean scientists since 2001. The plant extracts were tested for insecticidal activities. A total of 19,100 ethanolic and methanolic extracts of differentplant species from 23 nations including Costa Rica, Philippines, India and South Africa were evaluated for their larvicidal activities against Aedes aegypti, the major vector of dangue, dangue hemorhagic fever and yellow fever. The larval mortalities were observed 24h after treating the larvae to the extracts. At 500 ppm, 754 extracts showed >80% larval mortality in the 24h exposure. Among the extracts tested, the highest larval mortality was observed in the methanol extracts of Piper guianense, P. nigrum, P. mocropodum, P. sem-immersum, P. magen and P. pubicatulm.

The toxicity of red pine needle hydrodistillate, its constituents, and 12 structurally related compounds to third instar larvae of insecticide-susceptible Aedes aegypti and wild Aedes albopictus was examined using a direct-contact bioassay. Results were compared with those of the currently used larvicides fenthion and temephos. The red pine needle hydrodistillate constituents were identified by GC-MS and co-elution of authenticated samples following co-injection. Based on 24 h LC50 values, 3-carene (8.60–9.33 mg/L), limonene (10.34–10.77 mg/L), and thymol (10.65–11.45 mg/L) were the most toxic constituents. The moderate toxicity was produced by (1S)-(–)-α-pinene, p-cymene, geranyl acetate, myrcene, and β -caryophyllene (LC50, 27.62–38.01 and 28.61–39.52 mg/L). Overall, these compounds were less effective than either fenthion (LC50, 0.012–0.022 mg/L) or temephos (LC50, 0.010–0.015 mg/L). In the light of global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment, red pine needle hydrodistillate and the compounds described merit further study as potential larvicides for the control of mosquito populations.

We evaluated the larvicidal activity of 20 plant essential oils and components from ajowan (Trachyspermum ammi) and Peru balsam (Myroxylon pereira) oils against Aedes aegypti. Of the 20 plant essential oils, ajowan and Peru balsam oils at 0.1 mg/mL exhibited 100% and 97.5% larval mortality, respectively. At this same concentration, the individual constituents, (+)-camphene, benzoic acid, thymol, carvacrol, benzyl benzonate and benzyl trans-cinnamate caused 100% mortality. The toxicity of blends of constituents identified in 2 active oils indicated that thymol and benzyl benzoate were major contributors to the larvicidal activity of the artificial blend. We also tested the acute toxicity of these 2 active oils and their major constituents against the water flea, Daphnia magna. Peru balsam oil and benzyl trans-cinnamate were the most toxic to D. magna. Two days after the treatment, residues of ajowan and Peru balsalm oils in water were 36.2% and 85.1%, respectively. Less than 50% of benzyl trans-cinnamate and thymol were detected in the water at 2 days after treatment. Our results show that the essential oils of ajowan and Peru balsam and some of their constituents have potential as botanical insecticides against Ae. aegypti mosquito larvae.

The yellow fever mosquito, Aedes aegypti, is a vector for transmitting dengue fever and yellow fever. An assessment was made of the histopathological and molecular effects of pellitorine, an isobutylamide alkaloid, on third instar Ae. aegypti larvae. At 5 mg/L concentration of pellitorine, whole body of the treated larvae became dark in color, particularly damaged thorax and abdominal regions. Pellitorine targeted mainly on midgut epithelium and anal gills, indicating variably dramatic degenerative responses of the midgut through a sequential epithelial disorganization. The anterior and posterior midgut was entirely necrosed, bearing only gut lumen residues inside the peritrophic membranes. Pellitorine caused comprehensive damage of anal gill cells and branches of tracheole and the debris was found in hemolymph of anal gills. RT-PCR analysis indicates that the compound inhibited gene expression encoding V-type H+-ATPase and aquaporine 4 after treatment with 2.21 mg/L pellitorine. The results provide a fact that pellitorine merits further study as a potential larvicide with a specific target site or a lead molecule for the control of mosquito populations.

Melanization is a unique defence mechanism in arthropods involved in wound healing and pathogen encapsulation. Phenoloxidases (PPOs) are key enzymes of melanization, which mediate the enzymatic conversion of tyrosine to eumelanin. A serine-protease (SP) cascade, similar to the blood-clotting cascade of vertebrates, proteolytically activates prophenoloxidases to phenoloxidases. This proteolytic activation is tightly controlled by serpins and other melanization inhibitors. Melanization has been regarded as one of key immune responses against malaria parasites in mosquitoes. The ookinete melanization of both the simian malaria parasite, Plasmodium cynomolgi, and of the rodent parasite, Plasmodium berghei, prevent parasite development in the human malaria vector, An, gambiae. However, the recent studies revealed a melanization response regulated by Serpin-2 and two C-type lectins (CTL4 and CTLMA-2) was shown to result in ookinete melanization but did not affect the development of the natural human parasite P. falciparum in the mosquito. Instead of melanization, TEP1/APL1/LRIM1 complement-like pathway has been identified as major immune response that regulate parasite loads in the natural association of An. gambiae and P. falciparum. The studies by me and my colleagues revealed another melanization response independent on Serpin-2. Genome analyses of mosquitoes revealed a large expansion of the PPO, SP, and serpin genes potentially involved in the melanization pathway. This expansion was devoted to existence of at least two distinct SP-Serpin regulation modules in controlling separate melanization responses, tissue and hemolymph melanization, in the mosquito, Aedes aegypti. Tissue melanization regulated by Serpin-2 has role in melanotic tumor formation, but not in ookinete melanization. Hemolymph melanization regulated by Serpin-1 and a couple of SPs was activated by the infection of various pathogens and is involved in anti-malarial defense against the avian malaria parasite, P. gallinaceum. A new type of regulator, CLSP2, negatively modulate this hemolymph melanization. Cross-talk between hemolymph melanization and complement-like pathway will be discussed.

One of the overlooked points in mosquito blood feeding research is a final step before blood feeding such as finding a best position of blood feeding site and blood vessels underneath. How mosquitoes detect blood vessels especially using chemosensory organs prior to a final stage of blood feeding is totally unknown. Here we provide the anatomical and chemosensory evidence that a piecing structure of the mouthpart of the mosquitoes is an essential apparatus for the penultimate stage in blood feeding in mosquitoes. Indeed, mosquito mouthparts possesses a set of olfactory receptor neurons in sensory hairs, which are sensitive to volatile compounds present in host blood. Furthermore, the inhibition of gene expression of these odorant receptors delayed blood feeding of the mosquito from host animals. Taken together, these results identify that chemosensory perception in mouthpart is involved in mosquito blood feeding behaviors, which in turn allows mosquito to locate a feeding site more precisely.

The toxicity of materials derived from the seed of Pongamia pinnata to third instars of insecticide-susceptible Culex pipiens pallens and Aedes aegypti and wild Aedes albopictus was examined using a direct contact bioassay. Results were compared with those of the currently used insecticides fenthion and temephos. The active principles of Pongamia pinnata were identified as the karanjin (1), karanjachromene (2), pongamol (3), pongarotene (4), oleic acid (5), and palmitic acid (6) by spectroscopic analysis. Based on 24 h LC50 values, karanjin (14.61 and 16.13 mg/L) was the most toxic compound, followed by oleic acid (18.07 and 18.45 mg/L) and karanjachromene (18.74 and 20.57 mg/L). These constituents were less toxic than either fenthion (LC50, 0.0031 and 0.0048 mg/L) or temephos (0.021 and 0.050 mg/L) against Ae. aegypti and Cx. p. pallens. Low toxicity was produced by pongamol (LC50, 23.95 and 25.76 mg/L), pongarotene (25.52 and 37.61 mg/L), and palmitic acid (34.50 and 42.96 mg/L). Against A. alpopictus instars, oleic acid (LC50, 18.79 mg/L) was most toxic. Low toxicity was observed with the other five constituents (LC50, 35.26- 85.61 mg/L). P. pinnata seed-derived active principles, particularly karanjin, karanjachromene, and oleic acid, merits further study as potential mosquito larvicides for the control of mosquito populations in light of global efforts to reduce the level of highly toxic synthetic larvicides in the aquatic environment.

인도, 베트남, 칠레, 중국, 필리핀, 케냐, 탄자니아, 페루, 인도네시아, 코스타리카, 그리고 라오스 외 총 23개국의 서로 다른 5000종 식물의 에탄올과 메탄올 추출물을 이용해서 뎅기, 뎅기열, 황열병의 주 매개체인 Aedes aegypti 에 대해 살충 활성을 검정 하였다. 유충의 사충률은 처리 후 24시간 후에 확인 하였다. 500ppm에서 사충률 > 90% 이상 되는 식물체는 49 종의 식물체로 확인 되었다. 49종의 식물체 추출물의 처리 농도를 희석 과정을 거쳐서 확인 후 최종적으로 3종의 식물체를 선별 하였다. 3종의 식물체는 인도에서 채집한 Araceae과의 Scindapsus officinalis (Roxb.) Schott, 케냐에서 채집한 Commelinaceae과의 Commelina africana L., 그리고 탄자니아에서 채집한 Fabaceae과의 Millettia oblata Dunn 이다. 이 3종의 식물체를 유기용매 4가지를 이용 하여 분획 과정을 거친 후 살충 활성 검정 후 높은 활성을 보이는 분획물의 LC50 값을 확인하였다. Scindapsus officinalis (Roxb.) Schott 는 클로로포름 분획물 (LC50-13.93), 부탄올 분획물 (LC50-15.65)에서 높은 활성을 보였고, Commelina africana L 는 헥산 분획물(LC50-1.85), 에틸아세테이트 (LC50-5.5)에서 높은 활성을 보였고, Millettia oblata Dunn는 헥산 분획물(LC50-8.77), 에틸아세테이트(LC50-9.73)에서 높은 활성을 보였다. 이 세 가지 식물은 Aedes aegypti 유충 방제를 위한 좋은 원료가 될 것으로 예상한다.

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.

A total of 5,000 ethanolic and methanolic extracts of different plant species from 23 nations including Costa Rica, Vietnam, Philippines, India, South Africa, Pakistan and Peru were evaluated for their larvicidal activities against Aedes aegypti, the major vector of dangue, dangue hemorhagic fever and yellow fever. The larval mortalities were observed 24h after treating the larvae to the extracts. At 500 ppm, 179 extracts showed >80% larval mortality in the 24h exposure. Among the extracts tested, the highest larval mortality was observed in the extracts of Piper guianense, Piper nigrum, Piper mocropodum, Piper sem-immersum, Piper magen and Piper pubicatulum. The LC50 value of extract P. guianense, P. nigrum, P. mocropodum, P. sem-immersum, P. magen and P. pubicatulum were 8.84, 11.48, 8.84, 13.86, 9.48 and 10.12 ppm against Ae. aegypti. It is suggested that P. guianense, P. nigrum, P. mocropodum, P. sem-immersum, P. magen and P. pubicatulum can be developed as potent larvicidal agents against Ae. aegypti.

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.

The repellency of 104 plant essential oils to female Aedes aegypti was examined using a cage-distribution assay. Results were compared with those of the conventional mosquito repellent N,N-diethyl-m-toluamide (DEET). Coriander, oreganum, pennyroyal, lemoneucalyptus, and spearmint exhibited high repellency effect (>60 minutes at 50 mg/filter paper), followed by sage, leavander, tarragon, bergamot, and neroli oils which showed moderate effective (< 40 minutes at 50 mg/filter paper (5 cm diameter)). In the light of global efforts to reduce the level of highly toxic synthetic repellents, essential oils described merit further study as potential repellents for the control of mosquito populations.

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.

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.

The toxicity of materials derived from seed of Pongamia pinnata Pierre toward to third instar larvae of Aedes aegypti and Culex pipiens pallens was examined using direct contact bioassay. Results were compared with those of the currently used insecticides: fenthion and temephos. The active principles of Pongamia pinnata were identified as the karanjin (1), pongamone (2), palmitic acid (3) and karanjachromene (4), by spectroscopic analysis. Based on 24h LC50 values, karanjin (14.61 and 16.13 ppm) was the most toxic compound but less effective than fenthion (0.0031 and 0.068 ppm) and temephos (0.016 and 0.056 ppm) against Ae. aegypti and Cx p. pallens. Moderate toxicity was produced by pongamone (34.50 and 39.53 ppm), palmitic acid (36.93 and 42.96 ppm), and karanjachromene (43.05 and 48.95 ppm). P. pinnata seed derived materials, particularly karanjin, merit further study as potential mosquito larvicides for the control of mosquito populations in light of global efforts to reduce the level of highly toxic synthetic larvicides in the aquatic environment

The larvicidal activities of 11 Myrtaceae essential oils and their components against Aedes aegypti were tested by the immersion method. We also tested the acute toxicity of 4 active oils and their components against the water flea, Daphnia magna. Further, the aqueous residues of these oils and their components were determined at 2 and 7 days after suspending in water. Among the 11 oils tested, 0.1 mg/mL of Melaleucalinariifolia, M. dissitiflora, M. quinquenervia, and Eucalyptus globulus oils showed strong larvicidal activity against A. aegypti. Among the test compounds, allyl isothiocyanate, γ-terpinene, p-cymene, (+)-limonene, (-)-limonene, γ-terpinene, and (E)-nerolidol showed strong larvicidal activity against A. aegypti. The acute toxicity test revealed M. linariifolia was the most toxic to D. magna. Among test compounds, allyl isothiocyanate was the most toxic to D. magna. Two days after treatment, the residues of M. dissitiflora, M. linariifolia, M. quinquenervia, and E. globulus oils in water were 55.4, 46.6, 32.4, and 14.8%, respectively. Low concentrations of allyl isothiocyanate, γ -terpinene, p-cymene, (-)-limonene, (+)-limonene, and γ-terpinene were detected in the water at 2 days after treatment. Therefore, Myrtaceae essential oils and their components could be developed as control agents against mosquito larvae.