Name: probing the limits of egg recognition using egg rejection experiments along phenotypic gradients
Uploaded: 2018-08-22T13:00:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients at JoVE.com

まあまあは、イリノイ大学アーバナ ・ シャンペーン校で HJ ・ ヴァン ・ クリーブ教授によって賄われていた。さらに、資金調達のため我々 は (M.E.H. および T.G.) に人間のフロンティア科学プログラムと欧州社会基金、チェコ共和国の国家予算を感謝プロジェクトの no。(T.G.) に CZ.1.07/2.3.00/30.0041。私たちは、出版費用をカバーするためオーシャンオプティクスを感謝します。

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卵除去実験がひなの寄生虫-ホストの共進化の66を勉強する最も一般的な方法、材料、技術、またはプロトコルを標準化する努力を欠いています。これはメタ分析のため特に問題となります。ないホストの卵は今のところ拒絶の私たちの知識に、メタ分析研究67,68, 擬態または非擬態と考えられるものを含むの中で方法論的不一致の?...

ひな寄生虫は、子育てし、親の介護1,2,3に関連付けられている費用を支払う可能性があります他の種の巣に卵を産みます。ホスト側の寄生虫を検出する寄生虫と追跡側ホストの裏をかく欺瞞のこの行為は、両方の俳優の強力な選択圧を提供します。鳥類育児寄生者のいくつかのケースで異種寄生卵のホストの認識は、ホストと寄生虫の4間の進化的軍拡競走を生成するホストの卵を模した寄生虫の選択します。托卵の勉強は共進化のダイナミクスを調べるためのモデル システムだから重要と野生の5の意思決定です。卵除去実験は、鳥の托卵と種間相互作用6を調査して、生態学者の重要なツールの分野での勉強に使用する最も一般的な方法の 1 つです。
卵除去実験の過程で、研究者通常紹介自然卵をモデルや標準化された期間にわたってこれらの実験卵のホストの応答を評価します。そのような実験は、巣の7日間 (つまりの外観が変わる) 本物の卵を交換または染色を含むことができるまたはその卵の本当の卵 (必要に応じて追加パターン) と、元の巣に8、それらを返すまたはモデルの生成の面を塗装色9、10、サイズ11、および/または図形12をスポッティング スコープなどの特徴を操作しています。外観の異なる卵に対する宿主の反応は卵拒絶決定13と14の応答を引き出すためにする必要がありますを卵だけでどのように異なる到達している情報コンテンツに関する貴重な洞察を提供できます。最適な受容閾値理論15状態のホストが誤って寄生卵 (受け入れエラー) を受け入れるか、自分の卵との違いを調べることで自分の卵 (拒否エラー) を誤って削除のリスクをバランスする必要があります (または、これらの卵の内部のテンプレート) と寄生の卵。など、これを超えるホスト決定を容認する刺激が違いますも受容閾値が存在しています。寄生リスクが低い、受け入れエラーのリスクは寄生の危険性が高い; 場合よりも低いしたがって、決定は特定のコンテキスト、知覚リスク変更14,16,17として適切にシフトされます。
最適な受容閾値理論は、ホストと寄生虫の表現型の連続的な変化の時に意思決定をホストにベースを前提としています。したがって、さまざまな寄生虫の表現型へのホスト応答の測定はどのように耐性 (独自の表現型変異) を持つホスト人口は、寄生の表現型の範囲を確立する必要。ただし、実質的にすべての先行研究は、カテゴリカル卵の色や斑紋の発現の治療 (例えば、擬態/非擬態) に頼ってきました。ホスト卵殻形質変化しない、生物学的に現実的な期待ではない場合にのみ、すべての応答は (擬態の度合い) に関係なく、直接に匹敵します。そうでなければ、「擬態」卵モデルは、ホストの卵内および18の調査結果を比較するときの混乱を招く可能性の人口間に同様の方法で異なります。理論では、寄生の卵と自分14、必ずしも特定の寄生虫の卵の色の違いに基づいて決定をホストするを示唆しています。したがって、1 つの卵モデル タイプを使用してホスト決定しきい値または弁別能力に関する仮説をテストするための理想的なアプローチが限りは卵モデルの種類と個々 のホストの卵の色と丁度可知差異 (以下 JND)、興味の変数です。これは自然の色19の範囲にホストの応答をテストする自然卵を追加または交換する実験的研究にも適用されます。ただし、これらの研究を行うホストおよび寄生虫の表現型の変化のことが、特に同種卵7を使用する場合の特徴6の自然な変動によって制限されます。
対照的に、いろいろな色の人工卵を作る研究者、自然変動の制約から解放 (例えば、superstimuli20レスポンスを調べることができます)、ホストの認識6の限界をプローブするように。最近の研究では、絵画実験卵一致し、自然の卵殻9およびスポット カラー21の変動の範囲を凌駕するのにように設計によって表現型の範囲にわたってホスト応答を測定するのに手法を使用います。受け入れしきい値15キノコアリ擬態4などの理論的な予測は、連続的な違いに基づいているので根本的な認知プロセスを発見することがグラデーションで色卵を宿主反応を勉強特徴です。たとえば、Dainsonら、このアプローチを使用して21は、卵殻の地面の着色とスポット着色有彩色コントラストが高い、アメリカのロビンつぐみ migratorius卵をより強く拒否する傾向が設立。この発見は、このホストを処理する方法については、この場合スポッティングを寄生虫の卵を削除するかどうかを決定するために貴重な洞察力を提供します。塗料混合物をカスタマイズすると、研究者正確に操作できる実験の卵の色とホストの卵の色の類似性スポッティング パターン10、卵サイズ22卵など他の交絡要因を標準化しながら図形23。
さらにレプリケーションおよび metareplication24の古典的なと最近卵除去作業を奨励し、科学者が系統 (別のホスト種)7,22, に対して標準化された方法論を使用することが重要です。(別のホスト集団)7,22,25,26を空間し、時間 (異なった繁殖シーズン)7,22,25,26 ,27、稀だけ行われていた。方法論を標準化された28ではなかった後 artefactual 結果29,30にリードする示されていた。このタイプの連続的な変化への応答を調べ、重要な方法論的概念の数を強調する卵除去実験を複製しようとすると、研究者のためのガイドラインのセットとして本稿: コントロール巣の重要性先験的な仮説、metareplication、pseudoreplication、および色およびスペクトル解析。鳥ホスト寄生虫共進化の分野を支配、卵除去の実験にもかかわらず包括的なプロトコルが存在しません。したがって、これらのガイドライン間- とラボ内再現性 metareplication、すなわちである任意の仮説の真のテストとして、系統にわたって全体の研究を繰り返してスペースを高め、24時間に貴重なリソースとなりますが一貫した方法29,30,31を使用するときにすることができますだけ意味深長行われます。

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probing the limits of egg recognition using egg rejection experiments along phenotypic gradients

ひな寄生虫は、孵化し、若いリア ホスト両親を残し他のメスの巣に卵を産みます。勉強方法ひな寄生虫は、子供を育てるにホストを操作し、ホストが寄生を検出する方法は、共進化生物学の分野で重要な洞察を提供します。彼ら自身の若い子育ての費用を支払う必要はありませんので、カッコウなど、ウシドリのひな寄生虫は進化の利点を得る。しかし、これらのコストは卵、彼ら若者や大人を含め、寄生虫のすべての発達段階に対するホスト防御の選択します。卵除去実験は、ホストの防御を研究するために使用する最も一般的な方法です。これらの実験の間に、研究者はホストの巣に実験卵を配置し、ホストの応答を監視します。色は操作多くの場合とは卵の差別とホストと実験卵間の非類似度の可能性に積極的に関連していることを期待。本稿はそのような実験の結果を分析する一貫性のある卵の色を作成する方法を説明したから卵除去実験を実施するためのガイドとして機能します。特別な注意は、ホストの認識における色バイアスを探る可能性がある色のグラデーションに沿った一意に着色された卵を含む新しいメソッドに与えられます。標準化を行わなければ、それは意味のある方法で研究の調査結果を比較することはさらに実験のますます正確なと同等の結果を得るのためこのフィールド内の標準的なプロトコルになります。

カラフルな卵モデルの生成
カスタム ペイントの混合物と自然卵の反射スペクトルは図 1A-Dに表示されます。托卵研究で使用される塗料の混合は必要があります密接にスペクトル形状 (色) と大きさ (明るさ) の面で自然な反射率計測に対応します。達成される場合実験卵の色?...

Watch this Scientific Journal Video about Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients at JoVE.com

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

表現型の勾配に沿って卵除去実験を用いた卵認識の限界を探る

このプロトコルは、卵除去実験を実行するためのガイドラインを提供します: アウトライン化実験的絵画卵自然鳥の卵、フィールドワークを実施し、収集したデータを分析の色をエミュレートするモデル。このプロトコルは、匹敵する卵除去実験を行うための統一された方法を提供します。

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

probing-limits-egg-recognition-using-egg-rejection-experiments-along

Research

JoVE Journal

Behavior

8.2K ビュー.  Long Island University-Post. このプロトコルは、卵除去実験を実行するためのガイドラインを提供します: アウトライン化実験的絵画卵自然鳥の卵、フィールドワークを実施し、収集したデータを分析の色をエミュレートするモデル。このプロトコルは、匹敵する卵除去実験を行うための統一された方法を提供します。

ビデオ: Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

のアップライトイメージング<em&gt;ショウジョウバエ</em&gt;エッグチェンバース

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

発生生物学

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

用いた機能クローニング<em&gt;アフリカツメガエル</em&gt;卵母細胞発現システム

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

発達研究のためのウィンドウ鶏卵

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

生物学

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 ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

グリルス・ビマキュラトゥス・エッグを注入する

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

ファクトブラトサーモビア家電におけるゲノム編集のための卵マイクロインジェクションと効率的な交配

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

個々の産卵環境を調べるために、ハイスループットアッセイ<em&gt;キイロショウジョウバエ</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

神経科学

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

ショウジョウバエ幼虫の温度設定を決定する温度勾配試験

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

カエノハブディティス・エレガンスを使用して組織特異的シャペロン相互作用をスクリーニングする

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

産卵アッセイ:C.エレガンスの産卵行動を定量化する方法

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

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