著者は、蚊飼育用ジョンフレイとベンピーターソンに感謝します。我々はまた、血球の顕微鏡写真のヘルプはクリスティンデイビスとゲイリーRadiceに感謝。この研究は、A.テーラングにAA Qayumと教員の助成金にリッチモンドアーツ＆サイエンスサマーフェローシップ大学によって資金を供給された。

1。血球コレクションの事前準備<ol><li> （は98 mMのNaOHを、186 mMのNaCl、1.7 mMのEDTA、41 mMクエン酸、pH4.5の抗凝固剤）60％シュナイダーの培地、10％ウシ胎児血清（FBS）、および30％クエン酸緩衝液で構成される体液の希釈溶液を調製11。 -20℃で保存することができます1.0mLのアリコートを作る（資料を参照してください）​​一度だけ各アリコートを使用してください。 </li><li>ガラスエッチングのツールを使用して、一端に直径1cmの円形の得点によって顕微鏡用スライドガラス（75 × 25 × 1 mm）を準備する。また、2つの事前エッチング円と顕微鏡のスライドは、（例えば、フィッシャーサイエンティフィックまたはEMSの源）で購入することができます。蚊ごとにスライドガラスが必要となります。 </li><li>ガラス針のセットを準備する（資料を参照）、以下の推奨設定（サッタインスツルメンツ、モデルP - 87）によるとプル。 <table border="1"><tbody><tr><td>ヒートランプ5 </td></tr><tr><td>プル：45 </td></tr><tr><td>ヴェル：75 </td></tr><tr><td>時間：175 </td></tr><tr><td>圧力580 </td></tr></tbody></table> このプーラーで提案された設定を使用して、我々は、約3mmのシャンクの長さと全体の長さは500 mm程度である針を構築する。事前に引っ張ら針はまた（例えばTritech研究）購入することができます。 </li></ol> 2。血球コレクションの事前に微量注入器の準備<ol><li>製造元の指示に従って、インジェクターのニードルホルダーに引っ張ら針を挿入します。埋め戻しは、インジェクター（資料を参照）チューブ、注射器とは、製造元の指示に従って、鉱物油とガラスの針を引っ張った。 </li></ol> 3。血球コレクションの事前にメスの蚊の準備<ol><li>氷と接触​​することができる小さな固体ケージの容器に15、成人女性を吸引除去する。深く十分なケージとすべての側面のその高さ全体が氷と接触しているので、氷の蚊と浸しケージ。これは、すべての蚊が低温にさらされるようになります。 約8分まで、または冷たい麻酔の蚊は、彼らはもはやモバイルではありません。 </li><li>うつ病のスライドにone蚊を置きます。このスライドは、注射のためのサイトとして機能します。 </li></ol> 4。血球コレクション<ol><li>プルガラス針に血リンパの希釈液の12μlを取り上げ、7位と8腹部セグメント間の蚊に溶液を注入するインジェクターを設定します。約3蚊が取り上げ合計12μlの体積の3から3.5μLを注入することができます。注入が行われる間、蚊を穏やかに鉗子で定位置に保持されるべきである。希釈剤の少量が注入されるから、鉱物油を防ぐために、針の先端に残るはず。注入後腹部には&quot;充填&quot;や肥大化になるはずです。 </li><li> 5分で回復するために氷上で別々の清潔な容器に注入された蚊を置きます。蚊内部の抗凝固剤希釈液のインキュベーションでは、内部組織に付着した血球を取り除くのに役立ちます。平均で3〜5新鮮な蚊は、各5分間の回復期間中に注入することができる。 </li><li>回復の5分後、足、翼、そしてマイクロはさみを使って一度に各蚊つの腹部の先端（第8セグメントで）オフスニップ。これは、氷上に置く回復カップで行う必要があります。脚と翼を切断すると、コレクションのスライドに転送されるスケールの可能性を減らすことができます。注：あまりにも胸の付着部位に近い足と羽をカットしたり、エスケープする血リンパための他の開口部を作成する危険はありません。血リンパは、腹部の先端に作成された開口部を通して収集する必要があります。 </li><li> 70％エタノールでのガラスエッチング顕微鏡スライドを洗浄し、キムワイプでそれを乾燥させます。位置1は、エッチングされた円の外側の端にスライド上に蚊を注入して回復した。新鮮な血リンパの希釈液の12μlをピックアップし、その中胸の横側に蚊にこのボリュームを注入するインジェクターを設定します。注入されるから、鉱物油を防ぐために - 希釈剤の全体量（10μlの約8）ではない、ほとんどの実現が。希釈剤の少量は、針の先端に残るはず。 </li><li>カット腹部の開口部が1cmの円形の端になるように蚊を配置します。注入後希釈血リンパはマークされた領域内で収集する必要があります。針がmesothoracic注射部位から削除されると、鉗子の2つのアームの間に蚊を保持し、静かにエッチングされた丸い部分の上に血リンパを指示するために、これらの鉗子で腹部を圧迫を再開する。 </li><li>希薄化後の血リンパは約10分間、またはそれが目に見えて乾燥するまでスライド上で乾燥することができます。 </li></ol> 5。血球固定と染色<ol><li>ディップS：HEMA3 7（資料を参照）で別々に各スライドを染色たびに1秒のための固定液に5回の李徳、それぞれ1秒間と、最後に、ソリューションIIのディップスライドが3回、私は各1秒に対して3倍の溶液中でDipslide。 DI水でスライドの後ろをすすぐ。スライドの前面を洗浄してはいけません。ステンド組織を含むエッチングされた円の外側のスライドの前面にしみ、乾燥させます。 </li><li>エッチングされた中心の周りに取り付け媒体（例えば、シュール/マウントまたはPolymount）を適用し、その上にカバースリップを配置。ようにカバースリップ（25 × 25 mm）の上を軽く押し下げてそのカバースリップ（ステンド組織を含むエッチング円を含む）でカバーエリア全体への封入剤が広がる。スライドは3時間または一晩の最低を乾かせてください。血球は、最適な可視化と画像の収集後2週間以内に表示する必要があります。 </li></ol> 6。代表的な結果 ネッタイシマカ大人の女性から採取した固定とHEMA3ステンド血球の例を図1A - Cに示されています。図1Aは、我々は細胞質比（550x倍率）〜11核のサイズが小さく、球形、回転楕円体の形状及び高に基づいてprohemocyteとして識別血球を示しています。図1Bは、回転楕円体の形状と核の比（550x倍率）の高い細胞質に基づいてoenocytoidとして分類血球を示しています。最後に、図1Cは、顆粒球型血球（550x倍率）を示しています。顆粒球は自然の中で、よりきめ細かなものであり、彼らはガラスの表面に付着するような形状と動作に、よりアメーバの傾向にある。血球タイピングおよび回復11の以前に発行された基準を使用して、私たちの収集方法は、総血球の同じような数を生み出し、我々は同様に顆粒球は総血球11（図2）の最大の割合を構成していることがわかった。 <img src="/files/ftp_upload/2772/2772fig1.jpg" alt="図1" /> 図1代表的なHEMA3固定、染色したprohemocyteの光学顕微鏡像（）、oenocytoid（B）、およびAEから顆粒球（C）。ネッタイシマカの大人の女性。 C =細胞質、N =核、G =顆粒。 <img src="/files/ftp_upload/2772/2772fig2.jpg" alt="図2"/> 図2。合計カウント±私たちの体液の希釈と収集法でメス蚊ごとに得られるすべての血球と顆粒球のSE。

<ol>
	<li>Enayati, A., Hemingway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+management:+past,+present,+and+future.">Malaria management: past, present, and future.</a> Annu. Rev. Entomol. 55, 569-569 (2010).</li><li>Medlock, J., Luz, P. M., Struchiner, C. J., Galvani, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+transgenic+mosquitoes+on+dengue+virulence+to+humans+and+mosquitoes.">The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.</a> The American Naturalist. 174, 565-565 (2009).</li><li>Baton, L. A., Garver, L., Xi, Z., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+studies+on+the+innate+immunity+of+disease+vectors.">Functional genomics studies on the innate immunity of disease vectors.</a> Insect Sci. 15, 15-15 (2008).</li><li>Thomas, R. E., Wu, W. K., Verleye, D., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midgut+basal+lamina+thickness+and+dengue-1+virus+dissemination+rates+in+laboratory+strains+of+Aedes+albopictus+(Diptera:+Culicidae).">Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).</a> J. Med. Entomol. 30, 326-326 (1993).</li><li>Bartholomay, 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=Profiling+infection+responses+in+the+haemocytes+of+the+mosquito,+Aedes+aegypti.">Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.</a> Insect Mol. Biol. 16, 761-761 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15, 1-1 (2008).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+mortality+in+immune+challenged+mosquitoes+(Aedes+aegypti)+correlates+with+a+decrease+in+haemocyte+numbers.">Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.</a> Cell. Microbiol. 7, 39-39 (2005).</li><li>Chen, C. C., Laurence, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+humoral+encapsulation+of+microfilariae:+establishment+of+technique+and+description+of+reaction.">In vitro study on humoral encapsulation of microfilariae: establishment of technique and description of reaction.</a> International Journal for Parasitology. 17, 781-781 (1987).</li><li>Beerntsen, B. T., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dirofilaria+immitis:+effects+on+hemolymph+polypeptide+synthesis+in+Aedes+aegypti+during+melanotic+encapsulation+reactions+against+Microfilariae.">Dirofilaria immitis: effects on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against Microfilariae.</a> Exp. Parasitol. 71, 406-406 (1990).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+innate+immune+response+by+the+mosquito+Aedes+aegypti+is+mediated+by+hemocytes+and+independent+of+Gram+type+and+pathogenicity.">The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.</a> Microb. Infect. 6, 448-448 (2004).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36, 891-891 (2006).</li></ol>

利害の衝突は宣言されません。

血球収集方法は、以前に発行されたメソッドから変更と1つのより少ない手順でヒトスジシマカ蚊から血球のクリーンな準備を分離することができますされ、ここで説明。私たちの特定の関心は、 ヒトスジシマカ蚊の血球集団を特徴付けるにある一方で、我々は最初の試験が適切な注入量を決定するために実施された後、この手法は他の蚊のグループに適用できると考えていま?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>シュナイダーのミディアム</td><td>シグマアルドリッチ</td><td> S0146 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>シグマアルドリッチ</td><td> F0643 </td><td></td></tr><tr><td> Hema3染色キット</td><td>フィッシャー</td><td> 123から869 </td><td></td></tr><tr><td>ガラス針 （フィラメントとホウケイ酸塩） </td><td>サターのインストゥルメント</td><td> BF100 - 78から10 </td><td> 1.0ミリメートルOD 0.78ミリメートルとID </td></tr><tr><td>ニードルプラー</td><td>サターインスツルメンツ</td><td>モデルP - 87 </td><td></td></tr><tr><td>微量注入器</td><td> Tritech研究</td><td> MINJ - PD </td><td></td></tr><tr><td>針のホルダー</td><td> Tritech研究</td><td> MINJ - 4 </td><td></td></tr></tbody></table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

蚊は、黄熱病ウイルス、マラリア原虫やフィラリアワームなどの病気を引き起こす病原体の数のベクトルです。研究所は、そのような病原体1,2に抵抗性遺伝子組換え蚊を発生させることを望んで病気のベクトルの種の自然免疫系の抗病原体成分を調査している。蚊の自然免疫系は防御3の複数の行で構成されています。上皮で裏打ちされた蚊中腸4で課された障壁を逃れるために管理する病原体は血リンパを入力し、循環血球、カプセル化する重要な細胞成分と巻き込むの病原体5,6が発生する 。研究者は、蚊に造血組織のための証拠を発見していないと、現在の証拠は、血球の数が羽化に固定され、数字が実際に蚊の7歳と低下する場合があることを示唆している。適切に収集し、医学的に重要な昆虫から血球を識別する能力は、細胞性免疫の研究に不可欠なステップです。しかし、蚊と血リンパの限られた容積の小さいサイズは、免疫細胞を収集するために挑戦をもたらす。 蚊血球を収集するための2つの確立された方法は、頭部と胸部の間に膜necklike領域（すなわち、子宮頸部）に注入され、灌流血リンパが収集される生理食塩水でカット口先8、およびボリュームの変位（灌流）、からの血リンパの除名を含む腹部9、10の遠位領域内の引き裂かれた開口部から。これらの技術は、しかし、それぞれ11、血球の低い回収と脂肪体細胞による汚染の可能性によって制限されている。スケールと内部の組織11を汚染のレベルを低減しつつ、より多くの最近の方法は、抗凝固剤のバッファを使用することによって免疫細胞の高い注入/回収の改善回復と呼びます。その方式で初代培養のための血球を収集し、維持する改良法を可能にする一方で、それは注射の数を伴いますし、下流の目標は、収集、修正および診断のための血球を染色する場合に必要ではないステップを収集。ここでは、 ネッタイシマカ蚊の血球のクリーンな準備を分離するために、高いインジェクション技術の精度で、生理食塩水の代わりに抗凝固剤のバッファを使用して、血流のシンプルさを組み合わせた蚊の血リンパの収集の私達の方法を示しています。

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

記述されている蚊の血球細胞を収集して染色する、まだ正確な方法を簡素化。私たちの方法は、ヒトスジシマカ蚊の血球のクリーンな準備を分離するために、高いインジェクション技術の精度で灌流のシンプルさを兼ね備えています。このメソッドは、血球とその豊かさの種類の知識を必要とする研究を促進する。

黄熱病の蚊から血球を収集し、染色のためのプロトコルネッタイシマカ</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

a-protocol-for-collecting-staining-hemocytes-from-yellow-fever

Research

JoVE Journal

Immunology and Infection

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

15.2K ビュー.  University of Richmond. 記述されている蚊の血球細胞を収集して染色する、まだ正確な方法を簡素化。私たちの方法は、ヒトスジシマカ蚊の血球のクリーンな準備を分離するために、高いインジェクション技術の精度で灌流のシンプルさを兼ね備えています。このメソッドは、血球とその豊かさの種類の知識を必要とする研究を促進する。

ビデオ: A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

げっ歯類のマラリア原虫の遺伝的交雑の生産のためのプロトコル

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

免疫・感染学

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

からのDNA抽出のためのシンプルなChelexプロトコル<em&gt;ハマダラカ</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C型肝炎ウイルスの複製を分析するためのプロトコル

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

細菌感染や解析のためのキイロショウジョウバエ幼虫血球の抽出

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

共同血管や脂肪組織における神経線維を染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

狂犬病ワクチンの効力を評価するインビトロELISA試験

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

ジカウイルスを用いた Aedes aegypti蚊の ベクターコンピテンス分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

アエデスアエジプティ蚊における微小胞体寄生虫・エダリア・アダイスの伝播

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

アエデスアエジプティ蚊の動物由来血液と人工食事の摂食と定量化

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...