この作業は、LCグリフィスに授与健康グラントR01 GM54408の国立研究所によってサポートされていました。私たちは、正方形のチャンバーを設計し、当社のチャンバーを構築し、そして分析の問題に関する有益な会話のためのDanバレンテとティムLebestkyのためのブランダイス大学の機械工場弊社のアッセイ、フランクメロのセットアップのすべての彼の助けのためにフレッドウルフに感謝します。

パート1：ハエの餌と管理<ol><li>ハエは、酵母のない標準のメディアを含むボトルで増殖させてください。あなたは、ハエを大量に必要になりますので、十字架は、それに応じて設定する必要があります。 25℃で暗サイクル：ハエは、12時間の光で栽培する必要がある</li><li>ハエはすぐに羽化（1-3日）後に収集する必要があります。ハエは、二酸化炭素のディフューザーを使用して、この段階で働いたことができ、オートクレーブまたは酵母培地を含む試験管にソートしておく必要があります。我々は、試験管にのみ、保存されている男性10を使用してください。 </li><li>行動実験を実行する前に、二酸化炭素の使用後に少なくとも一日か二日を許可する。私たちは行動アッセイ3-5日以下の羽化にハエを実行する。 </li><li>アッセイは、常に概日リズムの問​​題を回避するために、毎日同じ2〜3時間の時間ウィンドウ内で実行する必要があります。 </li></ol>パート2：管理された環境での追跡システムのセットアップ<ol><li>全ての実験は、70％の湿度と25℃の一定温度を維持する環境の部屋で行われます。 </li><li> （カメラが下向き）に記録される領域の上にデジタルビデオカメラを一時停止します。私たちのトラッキングソフトウェアは、コントラストをベースにしているので、我々は、ライトボックス上のカメラを一時停止する。カメラは、FireWire接続を介して直接のDellコンピュータにビデオを記録します。我々は、ビデオの取り込みのためのWindows映画制作者（Vistaのバージョン）でのDellコンピュータにフックアップする、シャープデジタルビデオカメラを使用してください。 </li><li>空気パルスがこのプロトコルで投与される、空気の流れのソースは、客室内に存在しなければならないので。ゴムチューブを使用して、空気ろ過用活性炭フィルターに空気源を接続してください。 </li><li>より多くのゴ​​ムチューブを使用して、中空のゴム栓を経由して三角フラスコに活性炭フィルターの反対側を接続し、そして水の半インチ程度とフラスコを埋める。これは、空気を加湿します。 </li><li>フラスコには、ゴムチューブを経由してY字型バルブに接続するサイドアームを、持つ必要があります。 </li><li> Y -バルブの一枝は、流量計に接続する必要があります。他の支店は、正方形の運動室に空気を配布する必要があります。 </li></ol>パート3：運動室にハエを配信<ol><li>正方形の運動室は、Wolf らの設計に基づいています。2002年1は 、チャンバ内に飛んでからハエを防ぐための小さなドロップトレイを追加して。チャンバーを分解する必要があり、すべてのコンポーネントが完全に70％エタノールでダウン拭いて、使用前に乾燥する必要があります。 </li><li>すぐに行動試験を行う前に麻酔は、潜在的にパフォーマンスが損なわれる可能性があります。これを避けるために、ハエが優しく小さなアウトレットで漏斗を用いてチャンバーにノックされています。正しいサイズのアウトレットを作成するには、漏斗の端に青色のピペットの先端を（P1000用）を取り付けます。通過するフライ用に十分な開口部が大きいために、ピペットチップの非常に端を切断するはさみを使用してください。 </li><li>私たちの部屋の上部は、エッジの周りのねじで固定プレキシガラスの小片です。ネジが削除されたときに、穴がチャンバー内にハエを提供するために使用することができます。ネジ穴は、ネジが正常に座っている場所の代わりに、内部の部屋の上に直接座っているように、プレキシガラスの上に置きます。ハエを含む試験管を取るし、ネジ穴に配置する必要が漏斗、上の逆さまに置きます。 </li><li>これは、ハエを傷つけたり、破損の原因と、漏斗を移動しないでください。すべてのハエがチャンバー内になるまで、その代わりに、穏やかに、全体のチャンバー、ファンネルとすべてをたたく。このノッキングは、このバンギングの衝撃を吸収するためにマウスパッド上で行ってください。ハエが内側にある場合はネジのベッド上のプレキシガラスの上を、漏斗を削除し、再位置。所定の位置にして再度ネジプレキシガラスのトップ。 </li></ol>パート4：運動アッセイを実行する<ol><li>ハエが正しくロー​​ドされたら、それらを30分間チャンバーに順化てみましょう。この順化期間が制御された環境（25℃、湿度70％）内で起こるべきであり、チャンバーは、（これがオンしてください）​​ライトボックスの上に配置する必要があります。他の部屋の照明をオフにしてください。 </li><li>順応期間の後、空気のみを流量計に流入するように空気の流れのシステムでY -バルブを切り替える。空気の電源をオンにして、希望の速度（4.0〜6.0 L /分）に空気を増加させる。我々は一般的に5.0〜5.5 L / minを使用しますが、その範囲内の任意の速度で動作します。 </li><li>希望の対気速度が達成されると、空気が移動チャンバーに流れるようにY -バルブを切り替える。時間は、この空気は、15秒間パルスし、突然空気をオフにしてください。あなたが空気を切るように、モードを記録するカメラを切り替えます。 （この部分は一度にすべてを行うには練習が必要）。 </li><li>希望の時間後に録画が停止して保存。我々は、毎秒10フレーム（遺伝子型ごとに少なくとも8試験）で30秒の試行を記録する。 </li></ol>パート5：動画の分析<ol><li>我々は、使用動的画像解析システム（DIAS）2モーショントラッキングするためのソフトウェア3.2。このソフトウェアを使用するためには、我々は最初のQuickTime 7.5.5で、AVIファイルフォーマットに私たちの動画を変換。 </li><li>コントラストに基づいて、ハエを追跡するために、我々は、関数&quot;しきい値で自動トレース&quot;を使用してください。私たちは、その後、デジタル化への&quot;トレースからパスを作成&quot;機能は、これらのトレースを使用してください。 </li><li>各フライの瞬時速度を出力するには、我々は（DIAS固有）データベースファイルを生成するために&quot;計算パラメータ&quot;機能を使用してください。この機能を使用して、我々はまた、&quot;5,15,60,15,5&quot;Tukeyの平滑化ウィンドウを使用してデータを平滑化。 </li><li>この情報はデータベースファイルとして出力されると、それはMicrosoft Excelで開くことができます。我々は、Excel内の瞬時速度をコンパイルし、運動のダイナミクスと試合の構造を理解するために必要な追加パラメータを計算するためにMatlabのスクリプトを使用してください。 </li></ol>代表的な結果： 図1は、野生型カントンS生物（図1、左パネル）から代表的なトレースを示しており、二つの大きなオーバーラップ失（DF（3R）X307とDF（3R）x313）（図1を交配することによって生成されたdCASK遺伝子にnullを飛ぶ、）パネル右側。dCASKヌルハエは、以前にビュリダンのパラダイム3を使用して運動に問題があることが示されている、と私たちのパラダイムでは、カントンSと比較して、彼らは大幅に減少運動を示す。我々は常に条件と動作が特定の日の正常範囲内であることを確認する最初の野生型の微生物を実行します。私たちは、試験日の5％未満で、標準の応答からの偏差を観測した。これらの違いは、通常、私たちの制御された環境のパラメータを持つ問題に起因することができます。 <img src="/files/ftp_upload/1096/figure_1.png" alt="" width="600" height="298" /> 図1。DIAS -生成カントンS野生型のハエ（上記、左）とオーバーラップ欠失（上記、右）から生成された運動欠損dCASKのヌルハエの両方のトレースを。トレースが記録されたビデオで見ていることを象徴している。 80から10ハエから両方の画像contrainトレースは、空気のパルスは、次のビデオトラッキングアッセイで30秒間実行する。 <img src="/files/ftp_upload/1096/figure_2.png" alt="" width="600" height="438" /> 図2野生型のハエは、空気のパルスに続く歩行分析（上）（紫のバー）にして空気パルス（青色）なしで実行されました。分析プログラムによって計算されたすべてのパラメータでは、2つの条件（両側スチューデントt -テストで決定）の間に有意差はなかった。これは空気パルス後、我々のセットアップでは正常に動き回る飛ぶことを示している。

<ol>
	<li>Wolf, F. W., Rodan, A. R., Tsai, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Analysis+of+Ethanol-Induced+Locomotor+Stimulation+in+Drosophila.">High-Resolution Analysis of Ethanol-Induced Locomotor Stimulation in Drosophila.</a> J Neurosci. 22 (24), 11035-11044 (2002).</li><li>Soll, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+computers+in+understanding+how+animal+cells+crawl.">The use of computers in understanding how animal cells crawl.</a> International review of cytology. 163, 43-104 (1995).</li><li>Martin, J. R., Ollo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+Drosophila+Ca2+/calmodulin-dependent+protein+kinase+(Caki)+is+localized+in+the+central+nervous+system+and+implicated+in+walking+speed.">A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed.</a> The EMBO journal. 15 (8), 1865-1876 (1996).</li><li>Levine, J. D., Funes, P., Dowse, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+the+Circadian+Clock+by+Social+Experience+in+Drosophila+melanogaster.">Resetting the Circadian Clock by Social Experience in Drosophila melanogaster.</a> Science. 298, 2010-2012 (2002).</li></ol>

我々のセットアップでは、やや強い空気のパルスは、一時的にハエの動きを停止します。空気のパルスが終了すると、ハエは、図2に示すように、この定常状態と正常に動き回るから解放されています。この空気のパルスが効果的に人口の移動を同期化されるため、我々はまた勢い破りの刺激に続く運動の発症を学ぶことができるように試験を開始するために使用。開始後自発運動のパラメー...

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<td>Square Chamber</td>
<td>Tool</td>
<td>Machine Shop</td>
<td>N/A</td>
<td>Design from Wolf et al. 20021</td>
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<td>Digital Camera</td>
<td>Camera</td>
<td>Sharp</td>
<td>ViewcamZ VL-23</td>
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<td>Flowmeter</td>
<td>Tool</td>
<td>Cole Parmer</td>
<td>SY-32003-12</td>
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<td>Light Box</td>
<td>Tool</td>
<td>DNASTAR, Inc.</td>
<td>Seq-Easy</td>
<td>&nbsp;</td>
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<td>Charcoal Filter</td>
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high-resolution video tracking of locomotion in adult drosophila melanogaster

ハエは、この分野の研究者に利用可能な遺伝的なツールの過多に起因する複雑な挙動を研究するための重要なモデルを提供しています。における運動行動を研究して<em&gt;キイロショウジョウバエ</em&gt;中または特定のタスクに対応して動きの変化を定量化できるようにする能力に依存している。そのため、このような我々が本稿で説明する1つのような高解像度のビデオトラッキングシステムは、リアルタイムでの移動を測定するための貴重なツールです。我々のプロトコルは、正方形のチャンバー内の第三十二撮影期間が続くハエの勢いを、破るために初期の空気パルスの使用を含む。追跡プログラムは、10ミリ秒単位でチャンバー内の各フライの瞬時速度を計算するために使用されます。解析ソフトウェアは、このデータをコンパイルし、麻酔または固定化せずにハエを処理し、このプロトコルは、行動的なタスクのためにハエの適切な給餌と管理について議論するような平均速度、最高速度、運動で消費された時間、加速度、などのさまざまなパラメータを出力します、制御された環境をセットアップし、最初から最後までアッセイを実行する。

Watch this Scientific Journal Video about High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster at JoVE.com

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

の複雑な運動挙動の研究<em&gt;キイロショウジョウバエ</em&gt;与えられたハエの動きの変化を定量化する能力に依存しています。この記事では、高分解能のトラッキングシステムを使用してこれを行う方法を示します。

アダルトキイロショウジョウバエのロコモーションの高解像度のビデオトラッキング

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

high-resolution-video-tracking-locomotion-adult-drosophila

Research

JoVE Journal

Biology

13.4K ビュー.  Brandeis. の複雑な運動挙動の研究キイロショウジョウバエ与えられたハエの動きの変化を定量化する能力に依存しています。この記事では、高分解能のトラッキングシステムを使用してこれを行う方法を示します。

ビデオ: High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

ショウジョウバエのフライトの間に視覚的に仲介臭気の追跡

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

大人からOenocytesの解剖キイロショウジョウバエ</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

の分離キイロショウジョウバエ精巣

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

インスリン注射と成人のインスリン感受性を測定する血リンパの抽出キイロショウジョウバエ</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

における寿命の測定<em&gt;キイロショウジョウバエ</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

のメンテナンス<em&gt;キイロショウジョウバエ</em&gt;人口ケージ

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

キイロショウジョウバエ成虫の間接飛翔筋の光学断面筋決定

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

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 ...