Name: systemic bacterial infection and immune defense phenotypes in drosophila melanogaster
Uploaded: 2015-05-13T13:00:00.000+00:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster at JoVE.com

We would like to thank the entire Lazzaro lab, and especially Susan Rottschaefer, for their help in both reviewing and testing these protocols. This is a product of their cumulative expertise. Work in the Lazzaro lab is supported by grants R01 AI083932 and R01 AI064950 from the US National Institutes of Health.

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None of the authors have competing interests or conflicting interests.

ここで説明する手順は、 キイロショウジョウバエの厳格な、高品質の感染をもたらします。図示の例は、主にプロビデンシアrettgeriとEでの感染に焦点を当て大腸菌が、プロトコルは、高度に適応可能であり、ホストの飼育および維持条件の範囲に亘って多様な細菌の感染に適用することができます。 最適な実験的アプローチの詳細については、感染のために使用される細菌は、宿主の遺伝子型、および全体的な実験条件に依存するであろう。それは強く、新しい実験条件より野心的なプロジェクトを開始する前に、パイロットテストに推奨されます。出発点は、100倍の範囲にわたって3感染用量を試験することです。フライあたり100細菌細胞 - 高病原性の病原体は、多くの場合、最善の10程度で、非常に低い感染量で導入されます。より穏やかな病原体は、FLあたり約1,000の細菌の高用量で注入することができますyおよび非病原体フライ10,000細菌などの高用量で注射することができます。これは、縦方向の時系列の上に病原体負荷、ホストの死亡率、および免疫系の活動を追跡することによって、新たな感染症の動態を定義することがしばしば有益です。病原体負荷および宿主遺伝子発現の測定は、破壊的なアッセイであるので、測定されるすべての時点について、実験の開始時に異なるハエに感染することが必要です。 針を刺しまたはマイクロキャピラリーベースの注入を使用するかどうかを決定する際には、各アプローチには利点と制限があります注意することが重要です。キャピラリー注入は両方が適度膨圧を増加させ、担体中に懸濁または溶解された塩またはその他の分子を導入するハエへの液体の量を導入します。キャピラリー注射も注入設備や必要な機器の購入にアクセスする必要があります。浄化槽の針を刺しは、特別な機器や紹介を必要としません無視できる程度のフライにメディア、一般的にハエを​​大量に感染させるための、より効率的です。しかし、針で刺す感染症は、毛細管注入で達成することができる感染量を正確に制御することはできません。本プロトコルは、機械的に注入量を調節する噴射装置に焦点を当てたが、加圧された空気の離散パルスに基づいて噴射システムも存在する。20,21、これらは一般的に装置は、ここで紹介さよりも高価であり、それぞれの空気パルスのキャリブレーションを必要とします一貫性の注入量を確保するために、針。 ハエは、野生での細菌と全身感染する方法にかなりの議論が、非常に少ないデータがあります。一部の研究者は、 ショウジョウバエの病原性細菌を摂取するとき、自然感染の大部分が発生し、細菌が、その後全身感染を確立するために、腸を脱出することができることを断定します。しかし、非常にFEがありますD.の腸を横断できることが知られている細菌のw ショウジョウバエ 、およびこの能力を持っているものは、ハエ22,23に対して非常に致死的です。代替理論は、定期的に失敗した捕食試行または外部寄生虫ダニによる攻撃からの脱出を介してクチクラ負傷飛ぶことです。この仮説は、野生Dの頻繁なコレクションでサポートされていますメラノ治癒創傷（未発表の観察）の指標であるメラニンのスポットを有します。ダニは、 ショウジョウバエ 24で細菌感染を送信することが示されており、ダニが残した傷は二ミツバチ25中の細菌に感染することができます。しかし、Dのダニ·ドリブンまたはその他の日和見感染症の自然の中で、周波数キューティクルの侵害を介してメラノは知られていません。ここで説明するプロトコルは、直接、任意の上皮障壁または行動IMMUをバイパスし、定量注入による血リンパへの細菌の導入を可能にします無限大。 Dに病原性細菌を供給するための方法ショウジョウバエは Vodovar ら 22及びNehme ら 23に記載されています。 多くの昆虫病原性細菌は、研究者が快適に彼らと一緒に作業できるように、ほとんど、あるいは全く人間の健康上のリスクをもたらします。また、非常に少数の細菌が実験介入なしに接触したショウジョウバエに感染する能力、その汚染された表面を介して実験室を介して細菌感染の「流行」の広がりの危険性を持っているか、一般的に非常に低いハエを脱出しました。それにもかかわらず、適切な封じ込め対策が逃げるの感染ハエを防ぐために、所定の位置に、任意のハエを免れ奪還するためのものであることを保証することをお勧めします。研究室では、使用されている病原体のそれに見合った生物学的に安全なレベルで装備されるべきであり、微生物学における標準的なベストプラクティスを採用すべきです。 実験infectiここに記載の方法に任意の細菌のいずれかの用量でキイロショウジョウバエの感染を可能にします。感染が確立されると、それは、細菌の増殖またはクリアランスの速度を測定するために宿主の死亡率を追跡するため、および宿主免疫系の誘導をアッセイすることは簡単です。感染したハエは、容易に形作るか、または感染によって成形することが生理学的機能の試験を含む他の表現型アッセイにかけることができます。記載された手順は、安価で比較的少ない特殊な装置を必要とし、容易に研究と教育ラボの幅全体で多様なプロジェクトでの使用に適して作り、学習されます。

フルーツフライ、キイロショウジョウバエは、免疫防御の機能と進化を研究するための最高のモデル生物の一つである。 ショウジョウバエ 、安価で、背面に簡単で、実験操作に非常に適している、と幅広いを開発した大規模な科学コミュニティが付いています研究ツールの配列。 Toll様受容体によって媒介されるシグナル伝達およびNF-kBのファミリー転写因子、JAK / STATシグナル、およびJNK経路の応答を含む、昆虫、哺乳類間で保存されている自然免疫の多くの側面。1,2これらの遺伝子および経路の機能することができますDに照会しますショウジョウバエの変異を使用して、またはRNAiは、その増減経路活動をノックダウン3 - 。さらに6、 ショウジョウバエは、進化の生活史理論の文脈を含め、感染および疾患の生理学的結果を研究するために使用することができます7。- 9すべてのこのような研究は、しかし、確実に定義された処理条件の下で実験的なハエに感染する能力に依存します。ここで説明する手順は、 キイロショウジョウバエに堅牢かつ反復細菌感染を配信し、その後、感染の重症度を測定し、宿主免疫応答を定量化するための方法論的枠組みを提示します。 ショウジョウバエは、天然及び実験細菌、真菌、ウイルス、線虫および寄生蜂の寄生生物および病原体を含む多種多様なにより感染され得ます。現在のプロトコルは、全身細菌感染を提供することに焦点を当てています。多くの異なる細菌は、ハエに感染させるために使用することができ、実験者の選択が求められている正確な科学的質問に基づくべきです。例えば、ヒトの臨床分離株は、細菌の病原性機構10を研究するために使用され得る、または生態学的に関連する分離株はpreferreであってもよいです進化の研究のためのD。11一部の細菌は、Dの有能な病原体でありますショウジョウバエ、感染時に増殖し、ホスト病気や死を引き起こします。他の細菌は、効果的に宿主の免疫系によって管理され、数日以内にクリアされます。このデモでは、 プロビデンシアrettgeriは、ホストの死亡率の原因となり、ホストの存続に固執することができる増殖性病原体として使用されます。 大腸菌は、宿主免疫系によってクリアされた非病原体として使用されます。 感染は直接ハエの体腔内への細菌の導入によって確立されます。このアプローチは関係なく、変速機の固有モードの全身感染の調査を可能にする、上皮障壁や防御行動をバイパスします。実験的に全身感染を確立するための2つの主要な方法があります。最初にナノインジェクタと引っ張らガラスキャピラリ針の正確な数を注入するために使用されハエへの細菌。この方法は、感染用量の大きなダイナミックレンジを可能にし、定量的に高度に反復可能であるという利点を有します。第2のアプローチは、敗血症性針を刺したの感染を提供することです。この手法は、迅速であり、かつ特別な装置を必要としないという利点を有します。感染が確立されると、それは全身の病原体負荷、ホスト死亡率、および誘導性免疫系の活性を測定することが可能となります。もちろん、追加の表現型の任意の数の考えられる感染D.で測定することができ感染後の産卵数12、学習能力13、代謝状態14、または想像することができ、実質的に任意の他の形質を含むメラノ 。

<table><tbody><tr><td>Incubator</td><td>Powers Scientific, Inc</td><td>DROS52SD</td><td></td></tr><tr><td>Paintbrush</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Flypads</td><td>FlyStuff</td><td>59-114</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td>CD FG50</td><td></td></tr><tr><td>Drosophila rearing mix&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>6 oz Square Bottom Bottles, polypropylene</td><td>Genesee Scientific</td><td>32-130</td><td></td></tr><tr><td>Nosterile Extra Large Cotton Balls</td><td>Fisher brand</td><td>22-456-882</td><td></td></tr><tr><td>Microscope</td><td>Olympus Corporation</td><td>SZ51</td><td></td></tr><tr><td>Drosophila Vials polystyrene</td><td>VWR international</td><td>89092-720</td><td></td></tr><tr><td>Nosterile Large Cotton Balls</td><td>Fisher brand</td><td>22-456-883</td><td></td></tr><tr><td>2 L flask</td><td>VWR international</td><td>89000-370</td><td></td></tr><tr><td>Petri Dishes with Clear Lids, Raised Ridge; 100 x 15 mm;</td><td>VWR international</td><td>25384-302</td><td></td></tr><tr><td>LB Agar, Miller</td><td>Difco</td><td>244520</td><td></td></tr><tr><td>Innoculing Loop</td><td>VWR international</td><td>80094-488</td><td></td></tr><tr><td>Rainin Clasic Pipettes in various sizes&lt;br/&gt; 0.1 &amp;micro;l to 2 &amp;micro;l,&lt;br/&gt; 2 &amp;micro;l to 20 &amp;micro;l,&lt;br/&gt; 20 &amp;micro;l to 200 &amp;micro;l,&lt;br/&gt; 100 &amp;micro;l to 1,000 &amp;micro;l</td><td>Rainin</td><td>PR-2&lt;br/&gt; PR-20&lt;br/&gt; PR-200&lt;br/&gt; PR-1000</td><td></td></tr><tr><td>Micropipette tips (assorted sizes)</td><td>VWR international</td><td>30128-376&lt;br/&gt; 53503-810&lt;br/&gt; 16466-008</td><td></td></tr><tr><td>Luria Broth Base, Miller</td><td>Difco</td><td>241420</td><td></td></tr><tr><td>Disposable Culture Tubes Borosilicate Glass</td><td>VWR international</td><td>47729-576</td><td></td></tr><tr><td>S-500 Orbital Shaker</td><td>VWR international</td><td>14005-830</td><td></td></tr><tr><td>Centrifuge</td><td>VWR international</td><td>37001-300</td><td></td></tr><tr><td>PBS pH 7.4 10X</td><td>Invitrogen</td><td>70011044</td><td></td></tr><tr><td>SmartSpec 3000 Spectrophotometer</td><td>Bio-Rad</td><td>170-2501</td><td></td></tr><tr><td>Semimicrovolume Cuvettes</td><td>Bio-Rad</td><td>223-9955</td><td></td></tr><tr><td>Vertical Capillary Puller</td><td>Kopf Needle Pipette Puller</td><td></td><td></td></tr><tr><td>3.5&#039;&#039; Replacement glass Capillaries for Nanojet II</td><td>Drummond Sientific Company</td><td>3-000-203-G/X</td><td></td></tr><tr><td>Nanoject II</td><td>Drummond Sientific Company</td><td>3-000-204</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Mineral Oil, White, light</td><td>Macron Fine Chemicals</td><td>6358-10</td><td></td></tr><tr><td>Minutein pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>1.5 ml&amp;nbsp; Microcentrifuge tubes; Seal Rite</td><td>USA Scientific Inc.</td><td>1615-5500</td><td></td></tr><tr><td>Motorized Pestle; Talboys Laboratory Stirrer</td><td>Troemner</td><td>103</td><td></td></tr><tr><td>Talboys High Throughput Homogenizer</td><td>OPS Diagnostics</td><td>930145</td><td></td></tr><tr><td>5/32&#039;&#039; Grinding Balls</td><td>OPS Diagnostics</td><td>GBSS 156-5000-01</td><td></td></tr><tr><td>Vortex Genie</td><td>Scientific indurstries inc.</td><td>G560</td><td></td></tr><tr><td>Multichannel Pipettor (10 &amp;mu;l - 300 &amp;mu;l)</td><td>Sartorius</td><td>730360</td><td></td></tr><tr><td>WASP2 Whitley Automated Spiral Plater</td><td>Microbiology International</td><td></td><td></td></tr><tr><td>ProtoCOL automated colony counter / plate counter/ plate reader&amp;nbsp;</td><td>Microbiology International</td><td></td><td></td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596-026</td><td></td></tr><tr><td>qPCR tubes; Low-Profile 0.2 ml 8-Tube Strips</td><td>Bio-Rad</td><td>TLS0801</td><td></td></tr><tr><td>qPCR caps; Optical Flat 8-Cap Strips</td><td>Bio-Rad</td><td>TCS0803</td><td></td></tr><tr><td>RQ1 RNase-Free DNase</td><td>Promega</td><td>m610a</td><td></td></tr><tr><td>M-MLV Reverse Transcriptase</td><td>Promega</td><td>m170b</td><td></td></tr><tr><td>dNTPs</td><td>Promega</td><td>U1240</td><td></td></tr><tr><td>Oligo-dT</td><td>IDT</td><td></td><td></td></tr><tr><td>&amp;nbsp;SsoAdvanced SYBR Green Supermix</td><td>Bio-Rad</td><td>172-5260</td><td></td></tr><tr><td>CFX Connect Real-Time PCR Detection System</td><td>Bio-Rad</td><td>185-5200</td><td></td></tr><tr><td>RNasin Ribonuclease Inhibitor</td><td>Promega</td><td>N2115</td><td></td></tr></tbody></table>

systemic bacterial infection and immune defense phenotypes in drosophila melanogaster

果実はキイロショウジョウバエは、免疫防御の機能と進化を研究するための最高のモデル生物の一つで飛びます。自然免疫の多くの側面は、昆虫および哺乳動物の間で保存され、 ショウジョウバエを容易に遺伝的および実験的に操作することができるので、それらは免疫系の機能と疾患の生理学的影響を研究するための強力です。ここで実証された手順は、上皮障壁と防衛のより受動的な形態をバイパスし、全身感染にフォーカスを可能にする、直接体腔内に細菌を導入することによってハエの感染を可能にします。手順は、ホストの死亡率、全身病原体負荷、および宿主免疫系の誘導の程度の測定速度のためのプロトコルを含みます。この感染手順は、安価で堅牢で再現性がある定量的、および機能的遺伝学、進化の生活史、および生理学の研究に使用することができます。

このセクションでは、1感染用量を注入するために使用される細菌懸濁液の光学濃度に応じて変化することを示す図 。キイロショウジョウバエの細菌感染後に得られる結果を示しており、確実に均質化し、めっきすることによって推定することができる送達用量は、注射直後に飛ぶこと。 図2に示すように、異なる病原体は、宿主の死亡率（ 図2A）の異なるレベルを引き起こすことができ、宿主の死亡率は、用量依存的（ 図2B）とすることができます。重要なのは、このプロトコルは、達成すべき感染症の様々なタイプを可能にする： プロビデンシアrettgeriは 20日以上（ 図3A）持続する慢性、亜致死感染を引き起こす可能性があります。しかし、Escerichiaコリのような他の細菌は、ほとんどが6時間後に感染（ 図3B）内のホストフライによってクリアされます。免疫SYSの誘導TEMは、抗菌性ペプチド転写物（ 図4AおよびB）のRNAの単離およびその後の定量RT-PCRによって推定することができます。同様あまり定量的、免疫系（ 図4C）の誘導を可視化するために使用することができる抗菌ペプチド遺伝子プロモーターの制御下でGFPを発現するハエ。 <img alt="図1" src="/files/ftp_upload/52613/52613fig1.jpg" /> 。図1：感染量を決定するハエは、光学密度（0.0001から0.05）の範囲をカバーする細菌懸濁液の50 NLを注射しました。ハエは、すぐに注射によって導入細菌の数を決定するために、均質化し、プレーティングしました。初期の細菌負荷が強く、初期ODが注入さと相関した（r 2 = 0.96） <img alt="図2" src="/files/ftp_upload/52613/52613fig2.jpg" /> <strong&gt;図2：生存細菌性病原体の注射後には5〜7日齢の雄は、細菌懸濁液または滅菌媒体のいずれかの50 NLを注射し、生存をモニターしました。 （A）野生型のハエは、3つの異なる種のいずれかからの約5,000の細菌を注射しました。ホストの死亡率は、感染のために使用細菌種に大きく依存します。 50、500、およびフライ5,000細菌- （B）野生型のハエは、3つの異なるEnterococcousフェカリスの投与量を注射しました。免疫系の変異体およびその野生型同質遺伝子の制御線は約500 セパシア菌を注射した高い感染量（C）に感染したときにハエがより早く死にます。ペアごとのログランク比較は、野生型のハエは、変異体よりも有意に良好感染（χ2 = 59.02、DF = 1、P &lt;0.0001）が生き残っていることを示しています。 図3：注入後の細菌負荷ハエは約（A）5,000 P.注射しましたrettgeri（B）3,400 E.大腸菌 。細菌負荷は、注射直後及びその後の様々な時点で測定しました。各データ点は、単一のハエの細菌負荷を表す。E. P.ながらコリを速やかに挑戦ホストからクリアされますrettgeriは、ホストの人生の残りのために持続します、。 <img alt="図4" src="/files/ftp_upload/52613/52613fig4.jpg" /> 図4：注射後の免疫遺伝子発現の誘導 。 （A）ハエはP. 50 NLを注射しました腹部や胸部のいずれかでrettgeriまたは滅菌PBS、 またはCOは別に操作されていない残っていました<s&gt; 2麻酔UB。六時間の感染後、ハエ遺伝子は定量RT-PCRを用いて決定したDiptericinのRNAの単離および発現のために収集しました。 （A）の発現レベルは、Diptericinの比としてrp49転写物に転写物をグラフ化し、N（CO 2制御をバーは、各条件の比率の平均値と標準誤差を表す1の発現レベルを有するとして定義されるようにスケーリングされ= 4）。 （B）発現レベルは、標準的な非誘導条件としてCO 2 aesthesia制御ハエからの平均C T値との2ΔΔCTとしてグラフ化されています。バーは、各条件からΔΔCTの平均値と標準誤差を表す（N = 4）。による注射部位への転写誘導に差がないが、パネルAおよびBの比較は、ΔΔCT法は、潜在的に誘導レベルを過大評価する方法を示しています。 （Cは）Diptericin Aプロモーターの制御下でGFPを発現しているハエは、Pの50 NLを注射しましたその後rettgeri（OD = 0.1）および7日後に画像化しました。 GFPパネルはDiptericinの活性化細菌に感染したハエ中のプロモーターではなく、メディアを注射のコントロールを示し、感染したハエの腹部脂肪体におけるGFPの発現を示します。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 遺伝子 </td><td> フォワード </td><td> リバース </td></tr><tr><td> rp49（もrpL32呼ばれます） </td><td> 5 &#39;AGGCCCAAGATCGTGAAGAA 3&#39; </td><td> 5 &#39;GACGCACTCTGTTGTCGATACC 3&#39; </td></tr><tr><td> Diptericin A </td><td> 5 &#39;GCGGCGATGGTTTTGG 3&#39; </td><td> 5 &#39;CGCTGGTCCACACCTTCTG 3&#39; </td></tr><tr><td> Drosomycin </td><td> 5 &#39;CTGCCTGTCCGGAAGATACAA 3 &#39; </td><td> 5 &#39;TCCCTCCTCCTTGCACACA 3&#39; </td></tr><tr><td> デフェンシン </td><td> 5 &#39;GAGGATCATGTCCTGGTGCAT 3&#39; </td><td> 5 &#39;TCGCTTCTGGCGGCTATG 3&#39; </td></tr><tr><td> Attacin A </td><td> 5 &#39;CGTTTGGATCTGACCAAGG 3&#39; </td><td> 5 &#39;AAAGTTCCGCCAGGTGTGAC 3&#39; </td></tr><tr><td> Metchnikowan </td><td> 5 &#39;AACTTAATCTTGGAGCGATTTTTCTG 3&#39; </td><td> 5 &#39;ACGGCCTCGTATCGAAAATG 3&#39; </td></tr></tbody></table> 表1：定量RT-PCRのためのプライマー。

Watch this Scientific Journal Video about Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster at JoVE.com

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身細菌感染や免疫防御表現型<em&gt;キイロショウジョウバエ</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

systemic-bacterial-infection-immune-defense-phenotypes-drosophila

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

24.5K ビュー.  Cornell University. Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

ビデオ: Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

免疫・感染学

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

フルーツフライを飼育<em&gt;キイロショウジョウバエ</em&gt;無菌およびノトバイオート条件の下で

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

発生生物学

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

免疫応答やスリープ中の定量的測定<em&gt;ショウジョウバエ</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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 ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

ミバエショウジョウバエVivo で貪食能アッセイを使用しての細胞性免疫能を評価します。

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

キイロショウジョウバエ 幼虫注入プロトコル

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

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 ...

生物学

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

インビボファゴサイトーシスアッセイを用いた成人ショウジョウバエのメラノガステル血球の年齢特異的食細胞能力の評価

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Infecting Flies with Bacterial Suspension Using a Nanoinjector

Source: Khalil, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52613">Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.</a>&#160;J. Vis. Exp.&#160; (2015)
This video demonstrates the nano-injection of bacterial suspension into the fly body cavity, resulting in bacterial multiplication and infection. The fly&#39;s immune response clears some bacteria, while others escape immune clearance and survive within the host causing an infection.

ナノインジェクターを用いたハエへの細菌懸濁液の感染

Source: Khalil, S. et al., Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.&#160;J. Vis. Exp.&#160; (2015)
This ...

全身細菌感染や免疫防御表現型

要約

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この動画の章

の寄生蜂の紹介

フルーツフライを飼育

免疫応答やスリープ中の定量的測定

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

ウイルス感染とキイロショウジョウバエのホストウイルスの相互作用の解析

ミバエショウジョウバエVivo で貪食能アッセイを使用しての細胞性免疫能を評価します。

キイロショウジョウバエ幼虫注入プロトコル

口腔の細菌感染とキイロショウジョウバエの開口

インビボファゴサイトーシスアッセイを用いた成人ショウジョウバエのメラノガステル血球の年齢特異的食細胞能力の評価

ナノインジェクターを用いたハエへの細菌懸濁液の感染

全身細菌感染や免疫防御表現型

要約

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この動画の章

の寄生蜂の紹介

フルーツフライを飼育

免疫応答やスリープ中の定量的測定

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

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

ミバエショウジョウバエVivo で貪食能アッセイを使用しての細胞性免疫能を評価します。

キイロショウジョウバエ 幼虫注入プロトコル

口腔の細菌感染とキイロショウジョウバエの開口

インビボファゴサイトーシスアッセイを用いた成人ショウジョウバエのメラノガステル血球の年齢特異的食細胞能力の評価

ナノインジェクターを用いたハエへの細菌懸濁液の感染

ウイルス感染とキイロショウジョウバエのホストウイルスの相互作用の解析

キイロショウジョウバエ幼虫注入プロトコル