Name: maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions
Uploaded: 2018-08-31T14:00:00.000+00:00
Duration: 7 min 20 s
Description: Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

写真についてミシェル月 (ヴァンダービルト大学) に感謝したいと思います。PA と SSLB にヴァンダービルト大学提供のサポートは、地区ガバナーエレクト ・ 1445197 によってサポートされます。

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	<li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum.:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum.: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> BioEssays. 28, 747-755 (2006).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aphid Ecology: An Optimization Approach. , (1985).</li><li>Consortium, T. I. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Sequence+of+the+Pea+Aphid+Acyrthosiphon+pisum.">Genome Sequence of the Pea Aphid Acyrthosiphon pisum.</a> PLoS Biology. , 1000313 (2010).</li><li>Srinivasan, D. G., Brisson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aphids:+A+Model+for+Polyphenism+and+Epigenetics.">Aphids: A Model for Polyphenism and Epigenetics.</a> Genetics Research International. 2012, 1-12 (2012).</li><li>Wenger, J. 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=Whole+genome+sequence+of+the+soybean+aphid,+Aphis+glycines.">Whole genome sequence of the soybean aphid, Aphis glycines.</a> Insect Biochememistry and Molecular Biology. , 1-10 (2017).</li><li>Li, Z. -. Q., 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=Ecological+adaption+analysis+of+the+cotton+aphid+(Aphis+gossypii)+in+different+phenotypes+by+transcriptome+comparison.">Ecological adaption analysis of the cotton aphid (Aphis gossypii) in different phenotypes by transcriptome comparison.</a> PLoS ONE. 8, 83180 (2013).</li><li>Wang, D., Liu, Q., Jones, H. D., Bruce, T., Xia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptomic+analyses+revealed+divergences+of+two+agriculturally+important+aphid+species.">Comparative transcriptomic analyses revealed divergences of two agriculturally important aphid species.</a> BMC Genomics. 15 (1), 1023-1024 (2014).</li><li>Wu, S., 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=De+novo+characterization+of+the+pine+aphid+Cinara+pinitabulaeformis+Zhang+et+Zhang+transcriptome+and+analysis+of+genes+relevant+to+pesticides.">De novo characterization of the pine aphid Cinara pinitabulaeformis Zhang et Zhang transcriptome and analysis of genes relevant to pesticides.</a> PLoS ONE. 12, 0178496-0178517 (2017).</li><li>Birnbaum, S. S. L., Rinker, D. C., Gerardo, N. M., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profile+and+differential+fitness+in+a+specialist+milkweed+insect+across+host+plants+varying+in+toxicity.">Transcriptional profile and differential fitness in a specialist milkweed insect across host plants varying in toxicity.</a> Molecular Ecology. , (2017).</li><li>Dixon, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Herbivore-Host Dynamics. , (2005).</li><li>Goggin, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-aphid+interactions:+molecular+and+ecological+perspectives.">Plant-aphid interactions: molecular and ecological perspectives.</a> Current Opinion in Plant Biology. 10, 399-408 (2007).</li><li>Will, T., Furch, A., Zimmermann, 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=How+phloem-feeding+insects+face+the+challenge+of+phloem-located+defenses.">How phloem-feeding insects face the challenge of phloem-located defenses.</a> Frontiers in Plant Science. 4, 1-12 (2013).</li><li>Webster, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+olfaction+in+aphid+host+location.">The role of olfaction in aphid host location.</a> Physiological Entomology. 37, 10-18 (2012).</li><li>Agrawal, A. A., Petschenka, G., Bingham, R. A., Weber, M. G., Rasmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+cardenolides:+chemical+ecology+and+coevolution+of+specialized+plant-herbivore+interactions.">Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions.</a> New Phytologist. 194, 28-45 (2012).</li><li>Dobler, S., Petschenka, G., Pankoke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coping+with+toxic+plant+compounds-+the+insect's+perspective+on+iridoid+glycosides+and+cardenolides.">Coping with toxic plant compounds- the insect's perspective on iridoid glycosides and cardenolides.</a> Phytochemistry. 72, 1593-1604 (2011).</li><li>Opitz, S. E. W., M&#252;ller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemistry+and+insect+sequestration.">Plant chemistry and insect sequestration.</a> Chemoecology. 19, 117-154 (2009).</li><li>Birnbaum, S. S. L., Abbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+adaptations+toward+plant+toxins+in+milkweed-herbivores+systems+-+a+review.">Insect adaptations toward plant toxins in milkweed-herbivores systems - a review.</a> Entomologia Experimentalis et Applicata. 58, 579-610 (2018).</li><li>Rothschild, M., von Euw, J., Reichstein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+glycosides+in+the+oleander+aphid,+Aphis+nerii.">Cardiac glycosides in the oleander aphid, Aphis nerii.</a> Journal of Insect Physiology. 16, 1141-1145 (1970).</li><li>Harrison, J. S., Mondor, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+invasive+aphid+'superclone':+extremely+low+genetic+diversity+in+oleander+aphid+(Aphis+nerii)+populations+in+the+southern+United+States.">Evidence for an invasive aphid 'superclone': extremely low genetic diversity in oleander aphid (Aphis nerii) populations in the southern United States.</a> PLoS ONE. 6, 17524 (2011).</li><li>Mooney, K. A., Halitschke, R., Kessler, A., Agrawal, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+trade-offs+in+plants+mediate+the+strength+of+trophic+cascades.">Evolutionary trade-offs in plants mediate the strength of trophic cascades.</a> Science. 327, 1642-1644 (2010).</li><li>Grabherr, M. G., 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=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nature Biotechnology. 29, 644-652 (2011).</li><li>Haas, B. J., 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=De+novo+transcript+sequence+reconstruction+from+RNA-seq+using+the+Trinity+platform+for+reference+generation+and+analysis.">De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.</a> Nature Protocols. 8, 1494-1512 (2013).</li><li>Finn, R. D., 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=The+Pfam+protein+families+database:+towards+a+more+sustainable+future.">The Pfam protein families database: towards a more sustainable future.</a> Nucleic Acids Research. 44, 279-285 (2016).</li><li>The UniProt Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniProt:+the+universal+protein+knowledgebase.">UniProt: the universal protein knowledgebase.</a> Nucleic Acids Research. 45, 158-169 (2017).</li><li>Johnson, 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=NCBI+BLAST:+a+better+web+interface.">NCBI BLAST: a better web interface.</a> Nucleic Acids Research. 1 (36), 5-9 (2008).</li><li>Fu, L., Niu, B., Zhu, Z., Wu, S., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD-HIT:+accelerated+for+clustering+the+next+generation+sequencing+data.">CD-HIT: accelerated for clustering the next generation sequencing data.</a> Bioinformatics. 28 (23), 3150-3152 (2012).</li><li>Sim&#227;o, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., Zdobnov, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BUSCO:+assessing+genome+assembly+and+annotation+completeness+with+single-copy+orthologs.">BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs.</a> Bioinformatics. 31, 3210-3212 (2015).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30, 2114-2120 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9, 357-359 (2012).</li><li>Li, H., 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=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2.">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biology. 15, 31 (2014).</li><li>Rieu, I., Powers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+RT-PCR:+design,+calculations,+and+statistics.">Real-time quantitative RT-PCR: design, calculations, and statistics.</a> Plant Cell. 21, 1031-1033 (2009).</li><li>Malcolm, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+defence+in+chewing+and+sucking+insect+herbivores:+plant-derived+cardenolides+in+the+monarch+butterfly+and+oleander+aphid.">Chemical defence in chewing and sucking insect herbivores: plant-derived cardenolides in the monarch butterfly and oleander aphid.</a> Chemoecology. 1, 12-21 (1990).</li><li>Agrawal, A. A., Underwood, N., Stinchcombe, 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=Intraspecific+variation+in+the+strength+of+density+dependence+in+aphid+populations.">Intraspecific variation in the strength of density dependence in aphid populations.</a> Ecological Entomology. 29, 521-526 (2004).</li><li>Zehnder, C. B., Hunter, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+maternal+effects+and+current+environment+on+vital+rates+of+Aphis+nerii,+the+milkweed-oleander+aphid.">A comparison of maternal effects and current environment on vital rates of Aphis nerii, the milkweed-oleander aphid.</a> Ecological Entomology. 32, 172-180 (2007).</li><li>Hartbauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+defense+of+Aphis+nerii+and+Uroleucon+hypochoeridis+(Homoptera,+Aphididae)+against+natural+enemies.">Collective defense of Aphis nerii and Uroleucon hypochoeridis (Homoptera, Aphididae) against natural enemies.</a> PLoS ONE. 5, 10417 (2010).</li></ol>

著者が明らかに何もありません。

長いこと、ディアボルスA. neriiパターンと植物の防御、特に化学隔離18,37への抵抗のメカニズムに洞察力を提供できるが認識されています。ゲノム リソースの数は、 A. nerii10, A. neriiを使用して、モデルとして生態学的機能のゲノム研究の新しい機会を提供するため最近浮上しています。アブラムシ、植物文化のこの種の今後の作業がられるゲノムおよび機能の生態学的アプローチを活用した研究を伴う可能性が高いことを前提に、分子/ゲノム技術の基本的なプロトコルの概要を説明します。カルデノライド解毒とA. neriiで隔離の意義とメカニズムについて多くの未解決の問題が残っています。RNAi などの方法によってノックダウン式または遺伝子編集アプローチをこの点で貴重な証明します。
アブラムシを培養する際の課題の 1 つは繁殖と分散戦略のための驚異的な能力です。同質遺伝子系統が実験のために必要な場合、これらの特徴は、なぜ彼らが深刻な作物害虫のアブラムシの文化として、ほぼ毎日注意が必要ことを意味に直接関係するよくとして細心の注意です。遺伝子発現の分析のためのデータを生成するためのそれらを含む上記で説明したテクニック、アブラムシの飼育と分子解析のための一般的なプロトコルと同様に、提供十分な生物を生成する特定のステップ バイ ステップ ガイド分子および生態学的なアプリケーションの多様なセットのa. neriiの材料。
このためには、機能または生態学的なゲノム研究はA. neriiのための地平線上にある場合、完全に彼らを提供する実験の機会を活用する生活文化と結合されるこれら必要があります。草食昆虫が彼らのホスト植物複雑なコミュニティに住んでいるし、種間相互作用40と同様、両方の種内38,39形宿主植物にA. neriiの最終的な応答.A. neriiホスト植物に特化、発散の生活史戦略15、21、カップリングと純粋な genomic または生理学的アプローチの重要性を強調を表す植物の多様なセットを表すA. neriiコミュニティの自然発生する変化の実験操作。ここで説明する方法は、 A. neriiと有毒な宿主植物との相互作用に関する機能と生態学的なゲノムの観点のためのポイントを始めています。

アブラムシは、世界中の多様な植物家族の植民地 hemimetabolous、小さな昆虫です。最も特にから不足している栄養素を供給する酵母や細菌の共生とその偏栄養共生と離散 polyphenisms 循環単為生殖を含む複雑なライフ サイクルのいくつかの機能の独特であります。sap の1工場の彼らの食事療法。ほとんどアブラムシはホスト植物の専門家、いくつか種がどちらか直接にある作物で、かなりの経済ダメージを与える重要な作物害虫や病原体やウイルスを介して彼らはベクトル2。最初アブラムシにおけるゲノム学 2010 年エンドウ アブラムシエンドウヒゲナガアブラムシ3、パブリケーション アブラムシの生物学の研究で重要なマイルス トーンをマークは、昆虫のアドレス指定についての遺伝子資源を提供されているため良いコントロールの戦略4につながる可能性のあるものを含む、草食性のライフ スタイルに適応します。それ以来、追加ゲノム リソース大豆アブラムシワタアブラムシ グリシン5注釈ゲノムと別の 3 アブラムシ種 (モモアカアブラムシの公に利用可能な全ゲノム リソースのパブリケーションと蓄積してきたcerasi (ブラック チェリー アブラムシ)、モモアカアブラムシ(桃ジャガイモ アブラムシ)した padi (鳥チェリー エンバク アブラムシ)6。貴重なデ novoトランスクリプトーム リソースが他のアブラムシの数も利用可能 (e.g.,Aphis gossypii (綿アブラムシ)7, Sitobion 褐(穀物アブラムシ)8, Cinarapinitabulaeformis (松アブラムシ)9、ワタアブラムシ nerii (トウワタ キョウチクトウ アブラムシ)10)。
アブラムシは植物11植物昆虫相互作用と生命の生態学の私達の理解に永続的な貢献をしたも。アブラムシが特に重要な貢献をした 1 つの領域は、宿主植物の化学生態学の私達の理解にあります。共同植物の防御、自分の選ぶも克服する植物の防御、およびいくつかの草食性昆虫表現多様な適応は利益12,13,14です。たとえば、トウワタ キョウチクトウ モモアカアブラムシ、ワタアブラムシ neriiは、明るい黄色、侵襲的なアブラムシ世界中の温帯、熱帯地域は、トウワタ家族 (キョウチクトウ科) 植物の定着です。キョウチクトウ科の植物は、乳状のラテックスとカチオン キャリア Na, K-atpase をバインドし、ジェネラ リスト草食動物15,に効果的な抑止力は、cardenolides として知られている強心配糖体などを含む、多様な化学防御を進化しています。16. トウワタ専門家エクスプレス、cardenolides に対する抵抗性のさまざまなモード、選択的または受動的蓄積や捕食を阻止するための手段としてまたは他の利点の17のための組織の cardenolides を変更あり。A. neriiはこの方法で cardenolides を隔離するメカニズムと機能上の利点のまま不明10,18です。
手で、ゲノム リソース、 A. neriiを提供します優れた実験モデル有毒な宿主植物との専門家の化学生態学的相互作用に関与する分子および遺伝のメカニズムの調査のため草食動物。A. nerii cardenolides19, その時以来の隔離に焦点を当てたの初期の研究のいくつか、 a. neriiの研究が広範な進化と生態学的な質問に洞察力を提供している注目に値する侵襲的な昆虫20と草食動物密度21のボトムアップとトップダウンの規則間の相互作用の遺伝的構造を含みます。A. neriiはこうして特に広範な昆虫植物相互作用の研究のための実験モデルとしての良い候補です。重要なA. neriiと任意の研究の成功に注意カルチャですアブラムシの植物のアブラムシが依存の文化を含む集団として高品質弔データの効率的な生成。私たちの目標は、両方を介してリーダーを指導することです。世代やメンテナンス、温室効果と研究所、DNA と RNA の抽出、マイクロ サテライト解析、 de novoトランスクリプトーム アセンブリおよび注釈、トランスクリプトームで植物とアブラムシの文化のための方法を以下に発現解析と qPCR 性遺伝子が発現。一方、これらのメソッドは、 A. neriiの書かれている、培養、抽出、および分析の一般的な方法をさまざまな種のアブラムシに拡張できます。

<table><tbody><tr><td>Sun Gro Fafard Germination Mix</td><td>Hummert International</td><td>10-0952-2</td><td></td></tr><tr><td>Sun Gro Fafard 3B/ Metro Mix</td><td>Hummert International</td><td>10-0951-2</td><td></td></tr><tr><td>2&quot; x 4&quot; Round Standard Pot</td><td>Anderson Pots</td><td>1503</td><td></td></tr><tr><td>DreamTaq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>EP0701</td><td></td></tr><tr><td>Trizol</td><td>ThermoFisher Scientific</td><td>15596026</td><td></td></tr><tr><td>SuperScript&amp;reg; III First-Strand Synthesis kit</td><td>ThermoFisher Scientific</td><td>18080051</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix</td><td>ThermoFisher Scientific</td><td>4367659</td><td></td></tr></tbody></table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

アブラムシは宿主植物と昆虫の相互作用をめぐる質問に共生の進化と polyphenisms の開発に至る生物の質問の様々 な優れた実験的モデルです。アブラムシ数種の遺伝子資源があり、非モデル生物のゲノムを欠いているに延長されるトランスクリプトーム研究次世代シーケンシングの進化により、です。さらに、アブラムシの文化はフィールドから収集し、生態学的な遺伝学の間のギャップを埋めるための生物・分子の実験で使用する実験室で飼育できます。最後に、多くのアブラムシは無性再現遺伝子型の比較を可能にする永久的な単為のライフ サイクルでの優先ホスト植物研究所で維持できます。ワタアブラムシ nerii、トウワタ キョウチクトウ アブラムシは、生物分子の実験を使用して有毒な植物と昆虫の相互作用を研究するようなの 1 つのモデルを提供します。世代と温室効果と研究所、DNA および RNA の抽出、マイクロ サテライト解析、 de novoトランスクリプトーム アセンブリおよび注釈、トランスクリプトーム発現の植物とアブラムシの文化の維持の方法分析、および特異的発現遺伝子の qPCR 検証は概説し、ここで説明します。

植物の文化:種子は季節に応じて約 2 〜 4 週間の時間がかかります、大きく十分に鉢植え (図 1A)。再鉢植え苗は、アブラムシの文化 (図 1B) の最適なサイズに成長する別の 2 〜 4 週間かかります。
アブラムシ文化:大人A. neriiいくつかの暗い馬尾によって区別され、翼のあるかもしれない (無翅、図 3A、 B) または翼 (翼、図 3C, D)。ニンフ (図 3E, F) の 3 齢に達するとき、発展途上の翼パッドが表示されます。株式文化ベスト 1 ~ 3 の中間齢と 1 つ大人高齢者 unwinged アブラムシ; 転送によって維持されます。これにより、健康年齢人口を混合します。実験に使用する集団は、(2.4) を上記のように unwinged アブラムシを使用して培養する必要があります。A. nerii大人 1 人産むことができます 3-10 日あたり、宿主植物とアブラムシ10の年齢によって異なります。
DNA および RNA の抽出:シングル、大人A. neriiを行えば、およそ 100-200 ng/μ L の DNA (80 μ L の溶出。図 4A) および 150-300 ng/μ L の RNA (30 μ L の溶出。図 4B). 代表マイクロ サテライト ピークは、図 5に示します。3 つの条件 (制御、治療 1、処理 2) 下候補遺伝子の代表的な相対式は表 2に計算され、図 6に示します。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58044/58044fig1.jpg"> 図 1: アブラムシ文化の代表的な植物。彼らは真の葉の最初の完全なセットを開発した後 (A) 苗は鉢植え再することができます。(B) 植物は、彼らは真の葉の 3-4 セットを開発しているとき、アブラムシ文化の使用できます。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58044/58044fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58044/58044fig2.jpg"> 図 2: アブラムシを培養するために使用するツールの例です。(A) 口のピペットは、3/16"x 1/4" 外径チューブ、1,000 μ L ピペット チップ 200 μ L ピペット チップ ID を使用して作成できます。(B、C)カップ ケージ (上部を切断し、細かいメッシュで保護と透明なプラスチック カップ) を使用して、アブラムシ文化用 4 インチの鍋の上に合わせて、確実に。これは十分なライトおよび換気のためアブラムシおよび植物に適した環境を作成することができます、含まれているアブラムシを続けています。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58044/58044fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58044/58044fig3.jpg"> 図 3: 代表的な大人とワタアブラムシ neriiニンフ。(A, B)(Unwinged) 大人の無翅A. neriiは、後部端に暗い馬尾によって識別されます。(C, D)翼 (翼) 大人が十分に発達した翼によって識別され、暗く、後方で馬尾。(E, F)発展途上のA. neriiニンフが 4 齢段階を経るし、開発翼パッドは 3 齢段階で明らかになります。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58044/58044fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58044/58044fig4.jpg"> 図 4: 代表的なゲル。(A) DNA 抽出 (はしご 1 kb)。7 A. nerii DNA の抽出に 3-9 のレーンで視覚化されます。否定的な制御は車線 10 で。(B) RNA の抽出を行11 A. nerii RNA の抽出に 3-13 のレーンで視覚化されます。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58044/58044fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58044/58044fig5.jpg"> 図 5: マイクロ サテライト代表ピーク。タグ付き FAM の 6 ピークに青で視覚化されます。リズ 500 はしごがオレンジ色で表示されます。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58044/58044fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58044/58044fig6.jpg"> 図 6: 特異的発現遺伝子の qPCR 検証します。代表的な相対的な量 (RQ) 発現 (表 2ΔΔCt メソッドを使用して計算される) 3 つの条件の下で興味の候補遺伝子の表示: 制御、処理 1、処理 2。グラフでは、1 と 2 のコントロール (表 2) に比較して治療の下候補遺伝子の発現の低下を示しています。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58044/58044fig6large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>プライマー名</td>
			<td>方向</td>
			<td>シーケンス (5' 3')</td>
		</tr>
<tr>
<td>Ago24_F</td>
			<td>楽しみにして</td>
			<td>TTTTCCCGGCACACCGAGT</td>
		</tr>
<tr>
<td>Ago24_R</td>
			<td>リバース</td>
			<td>GCCAAACTTTACACCCCGC</td>
		</tr>
<tr>
<td>前 53_F</td>
			<td>楽しみにして</td>
			<td>TGACGAACGTGGTTAGTCGT</td>
		</tr>
<tr>
<td>前 53_R</td>
			<td>リバース</td>
			<td>GGCATAACGTCCTAGTCACA</td>
		</tr>
<tr>
<td>前 59_F</td>
			<td>楽しみにして</td>
			<td>GCGAGTGGTATTCGCTTAGT</td>
		</tr>
<tr>
<td>前 59_R</td>
			<td>リバース</td>
			<td>GTTACCCTCGACGATTGCGT</td>
		</tr>
<tr>
<td>前 66_F</td>
			<td>楽しみにして</td>
			<td>TCGGTTTGGCAACGTCGGGC</td>
		</tr>
<tr>
<td>前 66_R</td>
			<td>リバース</td>
			<td>GACTAGGGAGATGCCGGCGA</td>
		</tr>
<tr>
<td>前 69_F</td>
			<td>楽しみにして</td>
			<td>CGACTCAGCCCCGAGATTT</td>
		</tr>
<tr>
<td>前 69_R</td>
			<td>リバース</td>
			<td>ATACAAGCAAACATAGACGGAA</td>
		</tr>
<tr>
<td>前 84_F</td>
			<td>楽しみにして</td>
			<td>GACAGTGGTGAGGTTTCAA</td>
		</tr>
<tr>
<td>前 84_R</td>
			<td>リバース</td>
			<td>ACTGGCGTTACCTTGTCTA</td>
		</tr>
<tr>
<td>前 89_F</td>
			<td>楽しみにして</td>
			<td>GAACAGTGCTCGCAGTCTAT</td>
		</tr>
<tr>
<td>前 89_R</td>
			<td>リバース</td>
			<td>GACAGCGTAAACATCGCGGT</td>
		</tr>
<tr>
<td>前 126_F</td>
			<td>楽しみにして</td>
			<td>GGTACATTCGTGTCGATTT</td>
		</tr>
<tr>
<td>前 126_R</td>
			<td>リバース</td>
			<td>TAAACGAAAAAACCACGTAC</td>
		</tr>
</tbody></table>表 1: 遺伝子型に使用されるマイクロ サテライト プライマー シーケンスワタアブラムシ nerii20.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>ターゲット</td>
			<td>サンプル</td>
			<td>Ct の意味</td>
			<td>Ct 標準開発</td>
			<td>ΔCt</td>
			<td>平均 ΔCt</td>
			<td>ΔΔCt</td>
			<td>RQ=2^(-ΔΔCt)</td>
			<td>RQ SEM</td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.1</td>
			<td>22.59</td>
			<td>0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.2</td>
			<td>20.31</td>
			<td>0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.3</td>
			<td>20.36</td>
			<td>0.226</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.4</td>
			<td>20.27</td>
			<td>0.036</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.5</td>
			<td>20.55</td>
			<td>0.003</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.6</td>
			<td>20.52</td>
			<td>0.245</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.1</td>
			<td>20.49</td>
			<td>0.082</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.2</td>
			<td>19.86</td>
			<td>0.033 お</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.3</td>
			<td>20.19</td>
			<td>0.037</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.4</td>
			<td>19.67</td>
			<td>0.058</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.5</td>
			<td>20.25</td>
			<td>内径 0.188</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.6</td>
			<td>18.16</td>
			<td>0.089</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.1</td>
			<td>20.93</td>
			<td>0.157</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.2</td>
			<td>20.22</td>
			<td>0.003</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.3</td>
			<td>20.44</td>
			<td>0.039</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.4</td>
			<td>20.91</td>
			<td>0.559</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.5</td>
			<td>20.63</td>
			<td>0.017</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.6</td>
			<td>20.3</td>
			<td>0.135</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.1</td>
			<td>24.6</td>
			<td>0.173</td>
			<td>2.01</td>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.2</td>
			<td>24.25</td>
			<td>0.019</td>
			<td>3.94</td>
			<td>2.975</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.3</td>
			<td>24.79</td>
			<td>0.04</td>
			<td>4.43</td>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.4</td>
			<td>25.23</td>
			<td>0.285</td>
			<td>4.96</td>
			<td>4.695</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.5</td>
			<td>24.6</td>
			<td>0.103</td>
			<td>4.05</td>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.6</td>
			<td>25.08</td>
			<td>0.033 お</td>
			<td>4.56</td>
			<td>4.305</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.1</td>
			<td>27.52</td>
			<td>0.155</td>
			<td>7.03</td>
			<td></td>
			<td>5.019033762</td>
			<td>0.03084042</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.2</td>
			<td>27.23</td>
			<td>0.061</td>
			<td>7.37</td>
			<td>7.2</td>
			<td>3.428355679</td>
			<td>0.092888533</td>
			<td>0.031024057</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.3</td>
			<td>27.18</td>
			<td>0.058</td>
			<td>6.99</td>
			<td></td>
			<td>2.56158174</td>
			<td>0.169389724</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.4</td>
			<td>27.45</td>
			<td>0</td>
			<td>7.78</td>
			<td>7.385</td>
			<td>2.820764967</td>
			<td>0.141535419</td>
			<td>0.013927153</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.5</td>
			<td>27.44</td>
			<td>0.032</td>
			<td>7.19</td>
			<td></td>
			<td>3.138956897</td>
			<td>0.113521944</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.6</td>
			<td>28</td>
			<td>0</td>
			<td>9.84</td>
			<td>8.515</td>
			<td>5.284272079</td>
			<td>0.025661119</td>
			<td>0.043930413</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.1</td>
			<td>27.23</td>
			<td>0.143</td>
			<td>6.3</td>
			<td></td>
			<td>4.292437371</td>
			<td>0.051032588</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.2</td>
			<td>27.05</td>
			<td>0.088</td>
			<td>6.83</td>
			<td>6.565</td>
			<td>2.891234282</td>
			<td>0.134788164</td>
			<td>0.041877788</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.3</td>
			<td>27.45</td>
			<td>0.109</td>
			<td>7.01</td>
			<td></td>
			<td>2.578145722</td>
			<td>0.167456035</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.4</td>
			<td>27.58</td>
			<td>0.019</td>
			<td>6.67</td>
			<td>6.84</td>
			<td>1.709038085</td>
			<td>0.305863936</td>
			<td>0.069203951</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.5</td>
			<td>27.06</td>
			<td>0.067 倍</td>
			<td>6.43</td>
			<td></td>
			<td>2.384498984</td>
			<td>0.191511246</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.6</td>
			<td>27.36</td>
			<td>0</td>
			<td>7.06</td>
			<td>6.745</td>
			<td>2.513723938</td>
			<td>0.175103043</td>
			<td>0.008204101</td>
		</tr>
</tbody></table>表 2: qPCR ΔΔC の計算t候補遺伝子の検証します。候補遺伝子発現 ef1a を基準にして計算されます (図 6)。1.1 1.6 表す 6 生物複製処理; 下のサンプルします。2.1 2.6 表す 6 生物複製処理 1; 下のサンプルします。3.1 3.6 表す 6 生物学的治療法 2 下で複製をサンプリングします。Ct標準の開発は、3 つの技術的な複製から計算されます。

Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

アブラムシワタアブラムシ nerii付いているバシクルモン家族 (Apocyanaceae) で強く擁護した植物の定着植物昆虫の相互作用を研究する多くの機会を提供しています。ここでは、我々 の文化と生成分子の解析植物とアブラムシの保守のためのプロトコルのシリーズの紹介とA. nerii用プロファイルー データ。

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

maintaining-biological-cultures-measuring-gene-expression-aphis-nerii

Nanyang Technological University

Research

JoVE Journal

Genetics

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

7.5K ビュー.  Vanderbilt University. アブラムシワタアブラムシ nerii付いているバシクルモン家族 (Apocyanaceae) で強く擁護した植物の定着植物昆虫の相互作用を研究する多くの機会を提供しています。ここでは、我々 の文化と生成分子の解析植物とアブラムシの保守のためのプロトコルのシリーズの紹介とA. nerii用プロファイルー データ。

ビデオ: Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

世界的な農業害虫昆虫における胚のマイクロインジェクションを介して、核酸の送達、<em&gt;チチュウカイミバエ</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

遺伝学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

エンドウアブラムシ胚における免疫染色のための重要な条件の同定：組織透過性を増大し、バックグラウンド染色を減少

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

発生生物学

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

の飼育とインジェクション<em&gt;たばこスズメガ</em細菌毒性を評価するための&gt;幼虫

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

免疫・感染学

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

トルトリシダ科害虫の大量飼育と分子研究

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

環境学

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

ミツバチにおけるRNAiによる二重遺伝子ノックダウンと味覚知覚測定（<em&gt;ミツバチ</em&gt;）

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

神経科学

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

昆虫病原性線虫の培養と遺伝子操作

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

培養昆虫細胞における一過性発現および組換えタンパク質の細胞内局在

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

生物学

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, ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

ナソニアビトリペニスにおけるRNAiおよびCRISPR遺伝子編集のためのプパルおよび成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

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世界的な農業害虫昆虫における胚のマイクロインジェクションを介して、核酸の送達、

隠すビートルにノックダウン飼育し、二本鎖RNA媒介遺伝子、