生殖母体の文化のA.の調製 ： <ol><li>水浴中で37℃に新鮮なヒト血液および血清をもたらす。 </li><li>滅菌条件下、ピペットエッペンドルフチュー​​ブに暖かい血液1mL下換気フード、で。 2200 rpmで5分間、遠心分離で血液を回転させる。赤血球をペレットにする必要があります。明確な血清を除去し捨てる。 gametocytesのための全血を得るために新たな血清3倍等量のerythocytesを洗ってください。 </li><li> CO 2インキュベーターやキャンドルの瓶から生殖母体文化を取得。この文化は文化の16日目に蚊にRPMI培地と供給に維持されるべきである。 gametocytemiaの％を決定するために（文化の日16）一滴の血でスライドガラス上に薄層塗抹標本を作成し、Geimsa染色行います。寄生虫文化の分野における配偶子の数を決定します。それは2の範囲である - 4％。あなたが希望する最終的な％のgametocytemia（0.3 -1％）を達成するために必要な全血の量を計算する。 </li><li> （メディアは赤/ピンクの光になる間、寄生虫が黒として表示される）寄生虫を妨害したり吸うように注意しながら、ピペットを使用して、生殖母細胞培養液からのメディアのほとんどを削除します。 15ミリリットルのファルコンチューブにピペットでメディアや寄生虫の残りを。 2200 rpmで5分間、遠心分離でスピン。寄生虫と赤血球はペレット、削除、および妨害の寄生虫のない上清のメディアを破棄します。 </li><li>あなたの前の計算によれば、所望の最終gametocytemiaを与えるために準備した全血の量（ステップA2から）で寄生虫を組み合わせる。完全に混合するピペット。 （〜1分） </li></ol>これは、今、あなたの蚊に供給されるサンプルです。水浴で37℃で保管してください。 B.は、蚊を食べさせる 。 <ol><li>カップに付属のゴムバンドやボール紙のふたによって保護されたネッティングメッシュで覆われているワックスが並ぶ段ボールのコップにあなたのハマダラカ蚊（〜30から50）を置く。我々は、このステップのためにバッテリ駆動アスピレーターを使用してください。 </li><li>ゴム管にガラスフィーダの両側にノズルを挿入することにより、循環水浴を準備。あなたが直列に必要な数のフィーダとして接続します。一連の一端が水浴（フィーダーを通して水をプッシュする部分）のサーキュレータにチューブで接続する必要が、シリーズのもう一方の端はゴムチューブで構成する必要があります風呂に空の背面に循環水を可能にするために風呂に沈め。水が約37 ° Cの水と餌を循環し、戻ってお風呂にされるようになるまでお風呂に入れます。これは、血液を暖かく保つと寄生虫が生き続けるために不可欠です。 </li><li>フィーダの開口部にしっかりとシールするために圧力を使用して、ガラス膜のフィーダの周りに延伸して膜を（我々は、ストレッチパラフィルムの小さな四角を使用）準備。この膜は、皮膚を模倣します。 </li><li>場所ごとに蚊のカップとクランプまたはテープを使用して、安全なフィーダー上に1フィーダ膜面を下に。蚊は、カップの内側からそれにアクセスできるように膜がネットと同じ高さに座ってください。 </li><li>慎重にピペット寄生虫血の食事（ステップA5から）フィーダの首に、蚊が血にアクセスできるように膜を接触させ、血液が貯留中に首を介してすべての道を行くことを確認して。我々は、フィーダあたりの血液の約200μlを使用していますが、これは変わることができる。 </li><li>蚊が供給することができます。我々は彼らが≈30分間供給することができます（theexperimentに応じて、変数を、ほとんどの実験のために、時折何がすでに腹部の血液の含有量を調べることで食事を取った監視。） </li><li>授乳後は、蚊カップからフィーダを取り外します。水浴の電源を切り、チューブからフィーダを取り外してください。 </li><li>メンブレンを外し、除染と水で洗浄して10％の漂白剤でフィーダを浸す。大規模なバイオハザード容器内の小さなバイオハザードバッグや預金に汚染された物質（膜、紙タオル、手袋、等）を配置します。 </li><li>冷蔵庫や低温室でカップを置くことによって、蚊を麻酔。一度蚊が完全に麻酔ですが、氷のバケツに埋め込まれたペトリ皿に移す。ソートの蚊は、血液の食事のいずれかの符号のための腹部を注意深く見て、そして食べていないものを捨てる​​。他の人が摂取した血液の狭い帯域を表示しますが、一部の蚊は完全に充血され、すべては&quot;供給&quot;として受け入れられます。段ボールカップに供給蚊を元に戻します。 </li><li>蚊に砂糖の食事を（10％ショ糖に浸した綿の部分）を提供し、7日間、通常の昆虫館の条件下でインキュベートする。毎日、蚊は、死亡率を監視する必要があります。また、綿は、それらが食物を奪われないように、ショ糖とウェットなされるべきである。それは、蚊は湿った保つために、蒸留水に浸したペーパータオルを追加するには、まだ、良いです。これらの蚊は、P.ですfalcipraumが感染し、そのすべてのケアはない蚊がESCAPできないように注意する必要がありますカップからE。小さなcagefor二重保護にカップを置くことが最良です。 </li></ol> C.は、蚊midgutsを解剖し、オーシストの負荷を推定する。 <ol style="margin-top: 0in"><li style="tab-stops: list .5in">バッテリ駆動アスピレーターを使用して段ボールカップから吸引蚊（ステップBから）。寒い部屋に移動したり、少なくとも5分間、氷中に貯水池を埋めてたり、蚊が移動して停止するまで、取り外し可能な吸引器のタンクの内側にある麻酔蚊、。 </li><li style="tab-stops: list .5in">貯水池からの氷の中に埋め込まれたガラスのペトリ皿に麻酔蚊を転送する。単層に蚊を広げて、プラスチックシャーレの蓋でカバーしています。皿に対応できない蚊は寒い部屋や氷に戻される、タンク内に残ります。 </li><li style="tab-stops: list .5in">先の細いピンセットを使用して、解剖顕微鏡下にマウントスライドガラス上に含まれる100μlのPBSに皿から単一の蚊を転送する。チルドペトリ皿で​​覆われた他人のままにしておきます。 </li><li style="tab-stops: list .5in">先の細いピンセットを用いて、胸部と後腹部で蚊を開催。腸が露出するまで離れて身体を引っ張る。 gut.The腸からの過剰な組織を除去するために鉗子を使用すると白、袋状のボディのように見えます。 gut.Mountからマーキュロクロム0.2％の5〜10μlの12ウェルスライドガラスと浸すの1つのウェルで腸の過剰な組織を削除します。スカッシュ他の蚊の湿った紙towel.Continueで死体と破棄を12ウェルスライドが一杯になるまで。ガラスのカバースリップでマウントされている内臓をカバーしています。 </li><li style="tab-stops: list .5in">かすかな/クリアピンクの腸組織に対する明るいピンクに染色ラウンドオーシストを探して、光学顕微鏡下で内臓を調べる。 、オーシストを数えて記録し、次のサンプルに進んでください。すべての実験的な治療から、蚊のすべてのために継続する。 </li><li style="tab-stops: list .5in">終わったら、ペーパータオルできれいにスライドさせ拭いてください。小さなバイオハザードバッグに、ガッツとペーパータオル、と死骸とペーパータオルを置き、ゴミ箱に捨てます。手袋は、作業中は身に着けられている。ケアは、感染蚊のエスケープを聞かせしないように注意しています。 </li></ol>

<ol>
	<li>Avila, S. L., Ferreira, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+diagnosis:+a+review.">Malaria diagnosis: a review.</a> Braz J Med Biol Res. 29 (4), 431-443 (1996).</li><li>Bell, A. S., Ranford-Cartwright, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+PCR+assay+for+quantifying+Plasmodium+falciparum+infections+in+the+mosquito+vector.">A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.</a> Int J Parasitol. 34 (7), 795-802 (2004).</li><li>Collins, K. M., Hochberg, L. P., Ryan, P. R., Collins, W. E., Wirtz, R. A., Ryan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Plasmodium+malariae+infection+in+mosquito+vectors.">Quantification of Plasmodium malariae infection in mosquito vectors.</a> Ann Trop Med Parasitol. 98 (5), 469-472 (2004).</li><li>Czeher, C., Labbo, R., Djibrilla, A., Here, L., Arzika, I., Duchemin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+study+of+oocyst-sporozoite+transition+in+malaria+vectors.">ELISA study of oocyst-sporozoite transition in malaria vectors.</a> Parasite. 13 (3), 257-261 (2006).</li><li>Haghdoost, A. A., Mazhari, S., Bahadini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+results+of+light+microscopy+with+the+results+of+PCR+method+in+the+diagnosis+of+Plasmodium+vivax.">Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax.</a> J Vector Borne Dis. 43 (2), 53-57 (2006).</li><li>Patsoula, E., Spanakos, G., Sofianatou, D., Parara, M., Vakalis, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step,+PCR-based+method+for+the+detection+and+differentiation+of+Plasmodium+vivax.">A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax.</a> Ann Trop Med Parasitol. 97 (1), 15-21 (2003).</li><li>Osta, M. A., Christophides, G. K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mosquito+genes+on+Plasmodium+development.">Effects of mosquito genes on Plasmodium development.</a> Science. 303 (5666), 2030-2032 (2004).</li><li>Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Dimopoulos, G., Kafatos, F. C., Adams, J. H., Fujioka, H., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+and+Anopheles+transcriptomes+during+oocyst+differentiation.">Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation.</a> J Biol Chem. 279 (7), 5581-5587 (2004).</li></ol>

さまざまな手法に従うことがマラリアの感染症や感染症表現型の決定と異なるラボでのいくつかのバリエーションがある場合もあります。

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

遺伝子がマラリアのための潜在的に耐火として識別されると、それは蚊の中マラリア原虫の感染症の予防にその役割を評価する必要があります。このプロトコルは、蚊のマラリア原虫の感染の程度をアッセイする方法を示します。生殖母体の文化、膜の給餌蚊ヒト血液、及び蚊の中腸におけるウイルス力価を測定することを準備するための技術が実証されています。

Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

蚊と感染症の表現型決定における熱帯熱マラリア原虫の感染症のためのプロトコル

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

protocol-for-plasmodium-falciparum-infections-mosquitoes-infection

Nanyang Technological University

Research

JoVE Journal

Biology

17.8K ビュー.  Johns Hopkins University. 遺伝子がマラリアのための潜在的に耐火として識別されると、それは蚊の中マラリア原虫の感染症の予防にその役割を評価する必要があります。このプロトコルは、蚊のマラリア原虫の感染の程度をアッセイする方法を示します。生殖母体の文化、膜の給餌蚊ヒト血液、及び蚊の中腸におけるウイルス力価を測定することを準備するための技術が実証さ?...

ビデオ: Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

デング熱蚊の感染症（A.ネッタイシマカ）と感染症の表現型決定のためのプロトコル

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

生物学

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成虫（A. gambiae）におけるRNAiのアッセイのためのプロトコル

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

初代腸オルガノイド培養におけるレトロウイルス感染のビデオプロトコル

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

マラリア蚊。送信のためのマルチ検出分析

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

画像解析のためのクロックスキャンプロトコル：ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

研究室住宅のターコイズ メダカ (による furzeri) のためのプロトコル

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

口腔の細菌感染とキイロショウジョウバエの開口

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

蚊を殺虫化合物小説の毒性試験のプロトコル

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

蚊のベクトル能力を分析する方法として唾液の分泌を強制

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...