Name: a bacterial oral feeding assay with antibiotic-treated mosquitoes
Uploaded: 2020-09-12T14:00:00
Description: Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

この研究は中国国立自然科学財団(グラント81902094、81600497)と湖南省の科学技術計画プロジェクト(2019RS1036)によって支援されました。

1. 中腸の解剖と分節性細菌の分離
<ol><li>解剖のために蚊を準備します。<ol>
<li>アスピレーターで出現から7~9日後に蚊を集める。採取した蚊を4°Cの温度に3〜5分間加え、蚊を氷冷ペトリ皿で解剖するまで麻酔します。</li>
	</ol></li>
	<li>実験室の器具および蚊の表面を殺菌する。<ol>
<li>環境からの細菌による汚染を避けるために75%エタノールを噴霧することによって実?...

<ol>
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Z., McClure, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenome+and+culture-based+methods+reveal+candidate+bacterial+mutualists+in+the+Southern+house+mosquito+(Diptera:+Culicidae).">Metagenome and culture-based methods reveal candidate bacterial mutualists in the Southern house mosquito (Diptera: Culicidae).</a> Journal of Medical Entomology. 55 (5), 1170-1181 (2018).</li><li>Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gut+microbiome+across+life+history+of+the+malaria+mosquito+Anopheles+gambiae+in+Kenya.">Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya.</a> PloS One. 6 (9), (2011).</li><li>Xiao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mesh-Duox+pathway+regulates+homeostasis+in+the+insect+gut.">A Mesh-Duox pathway regulates homeostasis in the insect gut.</a> Nature Microbiology. 2 (5), 17020 (2017).</li><li>Gu&#233;gan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+impacts+of+anthropogenic+stressors+on+Aedes+albopictus+mosquito+vector+microbiota.">Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota.</a> FEMS Microbiology Ecology. 94 (12), 188 (2018).</li><li>Valzania, L., Coon, K. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+tripartite+interactions+between+the+Aedes+aegypti+midgut+microbiota,+innate+immune+system+and+dengue+virus+influences+vector+competence.">Reciprocal tripartite interactions between the Aedes aegypti midgut microbiota, innate immune system and dengue virus influences vector competence.</a> PLoS Neglected Tropical Diseases. 6 (3), 1561 (2012).</li><li>Dong, Y., Morton, J. C., Ramirez, J. L., Souza-Neto, J. A., 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=The+entomopathogenic+fungus+Beauveria+bassiana+activate+toll+and+JAK-STAT+pathway-controlled+effector+genes+and+anti-dengue+activity+in+Aedes+aegypti.">The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti.</a> Insect Biochemistry and Molecular Biology. 42 (2), 126-132 (2012).</li><li>Anglero-Rodriguez, Y. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Aedes+aegypti-associated+fungus+increases+susceptibility+to+dengue+virus+by+modulating+gut+trypsin+activity.">An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity.</a> Elife. 6, 28844 (2017).</li><li>Wu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gut+commensal+bacterium+promotes+mosquito+permissiveness+to+arboviruses.">A gut commensal bacterium promotes mosquito permissiveness to arboviruses.</a> Cell Host &amp; Microbe. 25 (1), 101-112 (2019).</li><li>M&#246;hlmann, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+gut+bacteria+on+the+infection+and+transmission+of+pathogenic+arboviruses+by+biting+midges+and+mosquitoes.">Impact of gut bacteria on the infection and transmission of pathogenic arboviruses by biting midges and mosquitoes.</a> Microbial Ecology. , (2020).</li><li>Llorca, M., Gros, M., Rodr&#237;guez-Mozaz, S., Barcel&#243;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preservation+for+the+analysis+of+antibiotics+in+water.">Sample preservation for the analysis of antibiotics in water.</a> Journal of Chromatography. A. 1369, 43-51 (2014).</li><li>Berendsen, B., Elbers, I., Stolker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+stability+of+antibiotics+in+matrix+and+reference+solutions+using+a+straightforward+procedure+applying+mass+spectrometric+detection.">Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection.</a> Food Additives &amp; Contaminants: Part A, Chemistry, Analysis, Control, Exposure &amp; Risk Assessment. 28 (12), 1657-1666 (2011).</li><li>Hill, C. L., Sharma, A., Shouche, Y., Severson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+midgut+microflora+and+dengue+virus+impact+on+life+history+traits+in+Aedes+aegypti.">Dynamics of midgut microflora and dengue virus impact on life history traits in Aedes aegypti.</a> Acta Tropica. 140, 151-157 (2014).</li><li>Eng, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+functional+implications+of+an+endogenously+expressed+tRNA+fragment+in+the+vector+mosquito+Aedes+aegypti.">Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti.</a> PLoS Neglected Tropical Diseases. 12 (1), 0006186 (2018).</li><li>Kajla, M. K., Barrett-Wilt, G. A., Paskewitz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria:+A+novel+source+for+potent+mosquito+feeding-deterrents.">Bacteria: A novel source for potent mosquito feeding-deterrents.</a> Science Advances. 5 (1), 6141 (2019).</li><li>Gon&#231;alves, G. G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+MALDI-TOF+MS+to+identify+the+culturable+midgut+microbiota+of+laboratory+and+wild+mosquitoes.">Use of MALDI-TOF MS to identify the culturable midgut microbiota of laboratory and wild mosquitoes.</a> Acta Tropica. 200, 105174 (2019).</li><li>Kuss, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+microbiota+promote+enteric+virus+replication+and+systemic+pathogenesis.">Intestinal microbiota promote enteric virus replication and systemic pathogenesis.</a> Science. 334 (6053), 249-252 (2011).</li><li>Rani, A., Sharma, A., Rajagopal, R., Adak, T., Bhatnagar, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+diversity+analysis+of+larvae+and+adult+midgut+microflora+using+culture-dependent+and+culture-independent+methods+in+lab-reared+and+field-collected+Anopheles+stephensi-an+Asian+malarial+vector.">Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.</a> BMC Microbiology. 9 (1), (2009).</li><li>Apte-Deshpande, A., Paingankar, M., Gokhale, M. D., Deobagkar, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serratia+odorifera+a+midgut+inhabitant+of+Aedes+aegypti+mosquito+enhances+its+susceptibility+to+dengue-2+virus.">Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus.</a> PLoS One. 7 (7), 40401 (2012).</li><li>Behura, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+microbiota+and+metagenomics,+and+its+relevance+to+disease+transmission.">Mosquito microbiota and metagenomics, and its relevance to disease transmission.</a> Nature. 436, 257-260 (2013).</li><li>Dickson, L. 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宿主と微生物の相互作用に関する研究は、異なる腸内微生物が発散メカニズムを介して宿主生理学に影響を与えることを発見した。本稿では、蚊の中腸を解剖し、培養可能な腸内細菌を培養し、抗生物質処理、関心のある細菌を再導入するなど、蚊の腸内微生物のそれぞれの役割を調査する方法を紹介する。
抗生物質治療を成功させるためには、実験を行う際に以下の?...

蚊は、ジカウイルス、デング熱ウイルス、および マラリア 原虫1を含む百の病原体を介して伝染する、ヒト病原性疾患の最も重要なベクターであると考えられている。蚊が卵子の栄養を得るために血液を摂取すると、感染した宿主から消化管2を介して誤って病原体を摂取する可能性がある。重要なことに、血液食事の消化と病原体の入り口の両方で重要な役割を果たす蚊の中腸は、非常にダイナミックなマイクロバイオーム3を抱えています。
いくつかの研究は、培養依存的な方法または細菌シーケンシングアッセイ44、5、65のいずれかを使用して、実験室で飼育され、フィールドに集められた蚊の微生物叢を特徴付けている。パントア、セラチア、クレブシエラ、エリザベスキンキヤ、エンテロコッカスなどの種は、一般的に様々な研究55、7、8、97,8,9で蚊から分離されています。興味深いことに、蚊の腸内微生物叢は、開発段階、種、地理的起源、および摂食行動4の影響を受ける、コミュニティの多様性と細菌種の量の両方で動的に変動する。研究は、血液供給が劇的に腸内細菌科からの種の急速な拡大と全体的な多様性の減少と総細菌負荷を増加することを示しています10,,11.さらに、幼虫期の蚊の腸内微生物叢は、通常、昆虫が子犬や分化の間に変態を受けると根絶される。したがって、新たに出現した成虫蚊は、微生物叢4を再移植する必要がある。
腸内微生物叢は、栄養吸収、免疫、発達、生殖、およびベクター能力12を含む様々な側面で昆虫生理学を調節する。無菌蚊の幼虫は、細菌の経口供給が発達を救う間、最初のインスターを超えて発達することができない、蚊の腸内微生物が幼虫の発達に不可欠であることを示す13、14。13,また、腸内細菌の枯渇は、血液の食事の消化と栄養吸収を遅らせる、卵母細胞の成熟に影響を与え、そして、卵子15を減少させる。さらに、腸内微生物叢を有する蚊は、抗生物質処理された蚊と比較してより高い免疫応答を引き出し、他の病原体に対して絶えず上昇した抗菌ペプチド発現を16に感染させる。抗生物質は通常、これらの研究でパン腸内細菌を除去するために経口投与され、その後、無菌蚊と蚊の違いを合成微生物と比較する実験が行われる。しかし、蚊の中腸は微生物の多様なコミュニティを収容し、各細菌種は宿主生理学に対して明確な効果を発揮する可能性があります。
蚊の微生物叢は、発散効果を持つベクター能力を調節します。デング熱流行地域のフィールド由来の蚊から分離されたプロテウスによる植民地化は、デング熱ウイルス感染に対する上駆除された抗菌ペプチド発現および耐性を与える。病原性真菌のボーベリア・バシアナは、アルボウイルス感染に対するトールおよびJAK-STAT免疫経路を活性化する。対照的に、Aedes aegypti midgutから単離された真菌タラロマイセスは、腸トリプシン活性18を調節することによってデング熱ウイルス感染を促進する。さらに、セラチア・マルセセンスは、蚊19の腸上皮上のムチン層を消化するSmコンハクチンと呼ばれる分泌タンパク質を介したアルボウイルス感染を促進する。
この手順は、蚊の中腸の解剖、品種細菌コロニーの分離、細菌種の同定、および経口摂食による再導入のための体系的かつ直感的な方法を提供する。これは、蚊の卵巣の発達と卵位に対する生来細菌、 菊えおろく髄膜炎菌との血液供給の代表的な結果を提供する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td >Sigma</td>
			<td >A2383</td>
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate has been used to prepare adenosine triphosphate (ATP) standard solutions</td>
		</tr>
		<tr >
			<td >Aedes aegypti</td>
			<td></td>
			<td></td>
			<td>Female mosquitoes</td>
		</tr>
		<tr >
			<td >Anticoagulant tube</td>
			<td>BD Vacutainer</td>
			<td>363095</td>
			<td>Collect fresh blood</td>
		</tr>
		<tr >
			<td >Centrifuge tube</td>
			<td>Sangon Biotech</td>
			<td>F601620-0010</td>
			<td>1.5 ml, Natural, Graduated, Sterile</td>
		</tr>
		<tr >
			<td >Cotton balls</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposable Tissue Grinding Pestle</td>
			<td>Sangon Biotech</td>
			<td>F619072-0001</td>
			<td>70 mm Long, Conical, Blue, Sterile</td>
		</tr>
		<tr >
			<td >Ethanol absolute</td>
			<td>Paini</td>
			<td></td>
			<td>Dilute it to 75% ethanol</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td>RWD</td>
			<td>F11029</td>
			<td>Dissection</td>
		</tr>
		<tr >
			<td >Hemotek Membrane Feeding System</td>
			<td>Hemotek</td>
			<td></td>
			<td>Components of the feeding system, including&#160; Hemotek temperature controller, feeder-housing assembly, metal feeder assembled.</td>
		</tr>
		<tr >
			<td >Incubator shaker</td>
			<td></td>
			<td>ZQZY-78AN</td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculation Loops</td>
			<td>Sangon Biotech</td>
			<td>F619312-0001</td>
			<td>10 &#956;l, Yellow</td>
		</tr>
		<tr >
			<td >LB Agar Powder</td>
			<td>Sangon Biotech</td>
			<td>A507003</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g&#65307; Agar 15.0 g.</td>
		</tr>
		<tr >
			<td >LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g.</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Zeiss</td>
			<td>Stemi508</td>
			<td></td>
		</tr>
		<tr >
			<td >Paper cup</td>
			<td></td>
			<td></td>
			<td>Place mosquito</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td>Sangon Biotech</td>
			<td>F104002</td>
			<td>4 inx 125 ft</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Sangon Biotech</td>
			<td>F611203</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin G procaine salt hydrate</td>
			<td>Sangon Biotech</td>
			<td>A606248</td>
			<td>White powder. Soluble in water, soluble in methanol, slightly soluble in water, ethanol</td>
		</tr>
		<tr >
			<td >Single Channal Pipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Streptomycin sulfate</td>
			<td>Sangon Biotech</td>
			<td>A610494</td>
			<td>Streptomycin sulfate is a glucosamine antibiotic that interferes with the synthesis of prokaryotic proteins.</td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792</td>
			<td>Soluble in water, ethanol and methanol, slightly soluble in glycerol and pyridine.</td>
		</tr>
		<tr >
			<td >TIANamp Bacteria DNA Kit</td>
			<td>TIANGEN</td>
			<td>DP302</td>
			<td>Extract DNA&#160;</td>
		</tr>
		<tr >
			<td >Utility Fabric-Mosquito Netting White</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td>Scintic Industries</td>
			<td>S1-0246</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5ml EP tube</td>
			<td>Sangon Biotech</td>
			<td>F600620</td>
			<td></td>
		</tr>
		<tr >
			<td >10X PBS buffer</td>
			<td>Sangon Biotech</td>
			<td>E607016</td>
			<td>This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4.</td>
		</tr>
	</tbody>
</table>

a bacterial oral feeding assay with antibiotic-treated mosquitoes

蚊の中腸は宿主の代謝、生殖、適性およびベクトルの能力に影響を与える非常に動的なマイクロバイオームを収容する。腸内微生物全体の効果を調査するための研究が行われています。しかし、異なる微生物が宿主に対して明確な効果を発揮する可能性がある。本稿では、それぞれの特定の蚊の腸内微生物の効果と潜在的なメカニズムを研究するための方法論を提供する。
このプロトコルには 2 つの部分が含まれています。最初の部分では、蚊の中腸を解剖し、殺虫性細菌のコロニーを分離し、細菌種を同定する方法を紹介します。第2部は、抗生物質処理蚊を生成し、1つの特定の細菌種を再導入する手順を提供する。

抗生物質で治療された蚊の中腸と抗生物質なしでDNA抽出のために取り出され、qPCRは普遍的な細菌プライマーで行われた。 図1 は、対照群および抗生物質治療群における細菌16S rRNAの発現を示す。その結果、腸内細菌の約98%が除去され、ペニシリンとストレプトマイシンの腸管滅菌が成功した。
記載された方法により、細菌株を単離し、同定した。 C. 髄膜?...

Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

抗生物質処理された蚊による細菌性経口摂食アッセイ

本稿では、蚊中腸の品種微生物の単離と同定、蚊腸内細菌の抗生物質枯渇、特定の細菌種の再導入など、個々の蚊腸内細菌の影響を調査するためのプロトコルを提示する。

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

蚊の中腸は、宿主の代謝、生殖、フィットネス、獣医の能力に影響を与える微生物の複雑なコミュニティを抱えています。各腸内微生物と異なる効果です。このビデオでは、各細菌株または宿主生理学のそれぞれの役割を研究する手順を紹介します。腸内細菌コロニーの培養を取得するには、無菌状態で蚊の中腸を解剖する。解剖中腸、セロトガネートを接地し、寒天プレートの上に広がります。プレートから単一のコロニーを選び、16個のSRNA遺伝子配列を介して細菌種を同定する。腸内微生物叢を取り除くために、連続して3日間抗生物質を含むCODエンボスで蚊を養います。抗生物質治療の有効性は、その後の過激派の前にコロニー形成ユニットアッセイのための無菌蚊の中腸を解剖することによって確認することができる。その後、経口摂食アッセイを介して以前に蚊の腸から単離された特定の細菌種を再導入する。宿主生理学に対する細菌の影響は、未治療の蚊、抗生物質処理された蚊、および蚊が特定の拘束を再導入したのを比較することによって評価することができる。中腸の解剖と培養可能な細菌の分離.まず、アスピレーターでケージ内の蚊を収集し、収集ボックス内の蚊を細胞にします。蚊を麻酔し、3~5分間4度の温度を下します。蚊を氷の冷たいペトリ皿に入れて麻酔をかけ続けます。ベンチを消毒し、顕微鏡を解剖し、環境からの細菌による汚染を避けるために75%エタノールを噴霧して鉗子を除去する。表面は、テーピングし、3分間75%エタノールでそれらを振ることによって蚊を殺菌し、PBSバッファーで2回リンスします。PBSのあなたの滴を含む無菌ガラススライド上で別々に蚊を二分します。慎重に鉗子を使用して蚊の足、翼と頭を取り除きます。蚊の胸と腹部の最後の部分をクランプし、消化管を分離するために引き離しました。鉗子を使用して、消化管から作物とマルピギアンチューブを取り除きます。作物は、中腸のpnteriorに配置され、砂糖水を摂取した後に膨らみます。マルピギアンチューブは、中腸と後腸の接合部にある細い排泄管のグループです。解剖した化粧を200マイクロリットルの無菌PBSバッファーを備えたEPチューブに入れ、クリーンベンチで無菌研削牧師と一緒に粉砕します。シリアルは、LB寒天プレートと絶望的なプレートに各妄想の50マイクロリットルで3つのテンポ妄想にホモジネートをロードします。単一のコロニーが実現可能になるまで、1〜2日間プレートを37度としてインキュベートする。50ミリリットルlbブラジャー培地を含む150ミリリットルの円錐形フラスコに単一のコロニーを選びます。一晩で37度で細菌を振る。種の識別。細菌ゲノムDNA構築キットによりTOTO DNAを抽出し、PCRにより16SRDNAを増幅します。アンブローズゲル電気泳動と駆動回収キットによるPCR産物からのDNA断片の回収と精製。精製されたDNA断片にDNAシーケンシングを行い、達成された細菌鎖配列を有する。細菌および古細菌およびデータベースに対して細菌を同定する16個のSRNA遺伝子配列を問い合わせにブラスト検索を実行する。種レベルの同定は、最も近いチームバンクエントリと類似性99%16 SRDNA配列以上と定義した。この分離株を対応する、16 SRDNA配列である場合に属に割り当て、類似性は99%未満であり、95%以上の抗生物質治療および細菌再導入であった。スクロースペニシリンとストレプトマイシンの必要量を量って、ペニシリンの20単位とミリリットル当たりストレプトマイシンの20マイクログラムを試すことを含む10%スクロース溶液を調製する。抗生物質を含むスクロース溶液中の綿球に浸透し、蚊を含む紙コップの上に置きます。水辺が蒸発するのを防ぐために、綿のボールを10センチメートルのペトリ皿で覆います。綿のボールを2日2回交換し、3日間連続して蚊に餌を与えます。蚊は2つのグループに分かれています。最初のグループは何の治療もなく蚊のカップに入れられ、綿球の医師は10%だから十字架が毎日与えられた。2番目のグループは抗生物質で治療され、3日間務める。各群で、セルゲイ蚊を解剖した。このミカを全DNA抽出のために抽出し、QPCRを普遍的な細菌プライマーを用いて実施した、図1は、対照群および抗生物質処理群における16SRNAの発現を示す。結果は、ペニシリンおよびストレプトマイシンのコストまたは実現が成功したことを示す。細菌溶液を調製し、分光光度計でウイルス中の細菌溶液を測定し、EPチューブに1つのOT細菌懸濁液を加える。5,000 RCAで懸濁液を4度で5分間遠心分離して上清を捨て、細菌パレットを滅菌PBS緩衝液で洗浄する。200マイクロリットルの無菌PBSバッファーで細菌パレットを再中断600マイクロリットル、10%スクロース溶液、200マイクロリットルATP、200マイクロリットルの細菌懸濁液を新しいAPチューブに加え、よく混ぜます。あるいは、細菌は、あなたの血液を活性化する熱で再懸濁することができます。加熱活性化血液の調製。抗凝固管で新鮮な血液を凝固し、2〜3ミリリットルの新鮮な血液を摂取する。遠心分離機は、血漿および血液細胞を分離するために4度で10分間10分間、1000で。プラズマを新しいEPチューブに集め、ここで1時間56度で活性化活性化する。滅菌PBSバッファーであなたの時間を通してブラストハウスを洗浄します。加熱活性化プラズマとの懸濁爆風の家をすすい。膜および供給システムを組み立てる。フィルム病を最大限に引き出します。プラスチックリングと職場のパラフィルムを固定して、準備された細菌溶液をフィーダーユニットに最大財布にゆっくりと追加します。気泡が一方向から落ちるのを避けるために、フィーダーユニットをシールします。電源を接続します。温度が37度に達するまで待って、フィーダーに細菌溶液とフィーダーユニットを取り付けます。細菌の餌を与える前に蚊は抗生物質を代謝するために24時間停止する必要があります。蚊と紙コップのフィードユニットのために、その後、90分間の給餌を許可します。完全に奨励された蚊は、さらなる研究のために選ぶことができました。代表結果。図2は、蚊1人当たりの平均称賛を示しています。抗生物質のプロット給餌が蚊を治療した後、我々は髄膜炎菌を見る。図3は、Cメニンゴセプチカムを含む血液食後24時間の変異発症関連遺伝子の発現を示す。結果は、対照群および退色群の卵の生産に有意な変化がないことを示す。細菌や抗生物質処理蚊との摂食エッセイに興味を持ってくれてありがとう。

a-bacterial-oral-feeding-assay-with-antibiotic-treated-mosquitoes

Research

JoVE Journal

Biology

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

生物学

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

遺伝子組換え蚊を使用してマラリアやデング熱の拡大防止

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

バキュロウイルスによるチョウ目幼虫の経口感染するためのプロトコル

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAiによる遺伝子ノックダウンと<emインビボで&gt;</em大人の女性の&gt;利尿アッセイ<em&gt;ネッタイシマカ</em&gt;蚊

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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, ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

幼虫のゼブラフィッシュのためのパラダイムを摂食高脂肪：摂食、ライブイメージング、および食物摂取の定量化

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

ネッタイシマカ蚊ボルバキアに感染維持

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

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 ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.