私たちは、プライマーの開発とテストを支援したアルヴィ・ワドゥドとトルディ・フロストに感謝します。本研究で報告されたアッセイ設計のための資金は、国防総省戦略環境研究開発プログラム(RC19-1156)によって提供されました。貿易、製品、または会社名の使用は記述目的のものであり、米国政府による承認を意味するものではありません。この調査中に生成されたデータは、USGS データ・リリース https://doi.org/10.5066/P9BIGOS5 として入手できます。

<ol>
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著者らは利益相反を宣言しない。資金提供者は、研究の設計に何の役割も持っていなかった。データの収集、分析、または解釈に含まれる。原稿の執筆に。または結果を公開する決定に含まれます。

他の研究と同様に、対処する質問を定義することは最初のステップであり、eDNAアッセイの設計は研究の範囲に依存する26.例えば、研究または調査の目的が1種または数種を検出することである場合、標的プローブベースのアッセイが最善である。しかし、より大きな組み合えまたは種の集合体を評価することが目標である場合、高スループットシーケンシングメタバーコーディングアッセイの方が適しています。どのアプローチを取る必要が決定されたら、検定の設計、テスト、最適化を含むパイロットスタディを推奨します。24.アッセイの設計は、次の説明に従って種のリストから始まります。 図 1.このリストは、アッセイが特異性と適用される可能性のある地理的範囲の観点から、アッセイがどの程度優れたパフォーマンスを発揮するかを理解するための基礎となります。6,10.特定の地理的領域に対するアッセイを設計し、設計者がその地域の他の種に対する交差反応性のアッセイをより良くテストすることを可能にし、標的種が発生する可能性のある他の地域にアッセイを拡張することに関する限界を認識することが奨励される24.リストが完成すると、パブリック遺伝データベースからシーケンスをダウンロードできます。これらのデータベースは不完全であるため27、アッセイ設計で使用される配列のローカル参照データベースを完了するために、一つは、家の中でリスト上のできるだけ多くの種をシーケンスする必要があります。これらは増幅する最も可能性の高い非標的であるとして、密接に関連する種の共生を優先する。ターゲット種と同じ属または家族内のすべての種に焦点を当てることは、開始する良い場所です。密接に関連する種との比較は、標的種に特有の配列領域を同定するのに役立つ。これは、アッセイが他のシステムまたは場所でどのように実行されるかを知らせるのに役立ちます。ミトコンドリア領域は、より多様な種からのより多くの配列情報が、バーコード・オブ・ライフ・プロジェクトで使用されてきたミトコンドリア遺伝子で入手可能であり、ミトコンドリアDNAが核DNAよりもコピー/細胞にはるかに高い濃度で存在するため、アッセイ開発のための通常の選択である24,28,29.配列カバレッジは、遺伝的リポジトリデータベース内の分類によって異なるため、複数の遺伝子領域をさらなるアッセイ開発について評価する必要があります。この参照シーケンスのローカルデータベースが作成された後、配列配列データとコンピュータソフトウェアプログラムの手動可視化を組み合わせてプライマー/プローブアッセイを設計します。どのアッセイをテストするかを決定するために、ソフトウェアに厳密に依存すべきではありません。プライマーとプローブがターゲットと非ターゲットに座っている位置合わせを視覚的に検証して、PCRでどのように作用するかをよりよく理解することが重要です。最後にアッセイスクリーニングと最適化は、3つのレベル(インシリコ、インビトロおよびinitu)を含む6,7,24,25.シリコの設計とテストは、成功の可能性が高いアッセイの短いリストを生成するために重要ですが、経験的な(インビトロ)テストは、最高の実際のパフォーマンスでアッセイを選択するために重要です。インビトロでの最適化とアッセイのテストには、反応効率の測定とアッセイの感度と特異性の定義が含まれます。検出と定量の限界は、アッセイの開発において見落とされがちであるが、データ解釈にとって重要な2つのパラメータである。アッセイ用の標準曲線の複数の反復を実行することにより、LODとLOQを簡単に測定することができます1,5,30.アッセイのLODまたはLOQに関する結果について議論する研究はほとんどありませんが、Senguptaら(2019)は、アッセイのLODとLOQをデータ解釈とグラフィックスに組み込み、結果を明確に理解します。31.内部の肯定的な制御は、同様に設計されたアッセイに多重化されるべきである。サンプル内の PCR 阻害をテストしないと、偽陰性が発生する可能性があります。24,32.PCR阻害試験の最も簡単な方法として、標的アッセイによる多重化IPCアッセイの使用を提案する23.最後に、現場および実験室から採取したサンプルからのアッセイの現場試験では、環境試料中でターゲット増幅が確実に起こる必要がある24.
eDNAサンプルを用いた種特異的なプローブベースのqPCRアッセイの使用には制限があります。例えば、テスト用の複数のアッセイの設計は、配列の可用性によって制限され、アッセイ性能の面で妥協が必要な場合があります。これらの選択は、研究の目標によって導かれ、結果26と報告されなければならない。例えば、希少種の検出が目標であり、陽性が少ない場合、すべての検出がシーケンシングによって検証されるならば、不完全な特異性(すなわち、非標的種の増幅)を伴うアッセイを使用することができる。目標が種の地理的範囲を監視する場合、eDNA濃度データが不要な場合、不完全な効率を持つアッセイを使用し、データはパーセント検出としてのみ報告されます。さらに、可能な限り不可能な実験室ですべての潜在的な特異性がテストされない限り、アッセイの真の特異性を絶対的な確実性をもって知ることはできません。例えば、アッセイはクリンチ川のいくつかの淡水ムール貝種に対して設計され、テストされました。このアッセイを別の河川システムで使用するには、新しい場所の種のスイートに対してテストする必要があります。アッセイの発生中に試験されない種または集団内の遺伝的変異も特異性に影響を及ぼす可能性がある。最後に、アッセイが高い技術的性能を有することが確認された場合でも。フィールドで作業すると、条件が変化します。水流、pH、動物の挙動などの非アッセイ関連条件は、異なるeDNA採取および抽出プロトコルを使用できるようにeDNA検出可能性を変化させる可能性があります。最適化され、よく説明されたアッセイを使用すると、そのようなパラメータがeDNA検出に及ぼす影響を理解しやすくなります。
eDNAの分野は、探索的分析の段階を超えて、方法や技術の標準化を高める段階で成熟しています。これらの開発により、eDNAの技術、能力、および制限に関する理解が深まりつつあります。上記の最適化プロセスにより、アッセイの感度、特異性、再現性が向上します。eDNA法の改良と標準化の最終目標は、eDNAデータに基づいて推論を行う研究者の能力を向上させるとともに、結果に対するエンドユーザーとステークホルダーの信頼を高めることです。

研究者や管理者は、種検出のための環境DNAアッセイの使用にますます関心を持つようになっています。30年にわたり、核酸1,2の配列特異的検出および定量化のために、定量的またはリアルタイムPCR(qPCR/rtPCR)が多数の分野で使用されてきました。eDNA研究の比較的新しい分野では、eDNAサンプルの体積または重量当たりの標的DNAのコピーを定量化するための標準曲線を用いたアッセイの使用が、今や日常的な実践となっている。ミトコンドリアのDNA配列は、ミトコンドリアゲノムが細胞当たり数千部に存在するため、一般にeDNAアッセイで標的とされているが、核DNAまたはRNA配列のアッセイも可能である。eDNAサンプルに対して公開されたアッセイは、必ずしも性能が等しいとは限らないということを理解することが重要です。標的種のDNA(特異性)と低量の標的DNA(すなわち、感度)の検出におけるアッセイの信頼性は、アッセイの設計、選択、最適化、試験方法の違いにより大きく異なる可能性があります。アッセイ性能の定量的尺度報告は以前はほとんど見落とされてきたが、最近ではアッセイ開発の透明性を向上させる基準が3、4、5、6、7、8に浮上している。
eDNA調査結果の設計と解釈の研究に役立つアッセイ性能の最適化と報告。非標的種DNAとの交差反応は偽陽性検出につながる可能性があるが、感度の低いアッセイは、サンプル中に存在する場合でも標的種DNAを検出できない可能性がある(偽陰性)。アッセイ感度と選択性を理解することは、希少種の検出に必要なサンプリング作業を知らせるのに役立ちます。eDNAには多くの自然な変動源があるため、eDNAアッセイ3を完全に最適化し、特性を付け込むなど、制御可能な変動源を可能な限り制限する必要があります。
アッセイの特異性や感度に直接影響する条件は、アッセイの性能を変えます。これは、異なる実験室の条件下で発生する可能性があります (すなわち、異なる試薬、ユーザー、機械など)。したがって、このプロトコルは、新しい条件下でアッセイを適用する際に再検討する必要があります。文献に良く特徴付けられるアッセイでさえ、新しい実験室で採用されたとき、または異なる試薬(例えば、マスターミックス溶液)5,9を使用する場合に、試験および最適化されるべきである。アッセイ特異性は、異なる地理的領域に適用されると変化する可能性があり、アッセイが検査されていない非標的種を含む新しい生物学的コミュニティからのサンプルにアッセイが適用されているため、標的種の遺伝的変異が生じる可能性がある。再び、アッセイは新しい場所で使用する場合に再評価されるべきである。フィールド PCR 阻害剤はサンプルに存在する可能性が高いため、フィールド条件は実験室の条件とは異なります。PCR阻害剤は、増幅反応に直接影響を与え、アッセイ性能に影響を与えます。このため、eDNAアッセイを開発する際には、内部の正のコントロールが必要です。
最後に、現場の環境条件は、標的種のDNA分子に影響を与え、DNA分解、輸送および保持を通じて捕捉する可能性があります。さらに、DNAの収集と抽出のための異なるプロトコルは、その効率とDNAを保持する能力が異なります。しかし、これらのプロセスはeDNAの検出可能性に影響を与えますが、分子アッセイの性能には影響しないことに注意することが重要です。したがって、フィールドサンプル中の標的種からのDNAの検出可能性は、qPCRアッセイの技術的性能と、フィールド条件および収集、保存、抽出プロトコルの両方の機能である。よく特徴付け、高度に実行アッセイを使用する場合、ユーザーはアッセイの能力に自信を持つことができます。研究者は、eDNA検出に影響を与える外部アッセイ因子(すなわち、環境変数、捕獲または抽出プロトコルの違い)の理解に集中できるようになりました。
ここでは、厳密な設計と最適化によるアッセイ技術性能に特に焦点を当てています。米国クリンチ川でサンプリングした水から淡水ムール貝(アクチナガイアス・靭帯)を検出するために開発されたプローブベースのアッセイを用いてプロトコルを実証します。最近、Thalingerら(2020)は、標的eDNAアッセイの検証のためのガイドラインを提示した。私たちのプロトコルに従ったアッセイ設計は、Thalingerらのレベル4にアッセイに加えて、レベル5 6に向けた追加のステップをもたらすでしょう。この時点で、アッセイの技術的性能が最適化され、実験室および現場でのアプリケーションでの定期的な使用に備える。実験室でのアッセイのさらなる使用、メソコスム、およびフィールド実験は、次に、eDNA検出および検出性に影響を与える因子に関する質問に対処することができる、レベル5検証6のための最終ステップ。

<table>
	<tbody>
		<tr>
			<td>96 Place Reversible Racks with Covers</td>
			<td>Globe Scientific</td>
			<td>456355AST</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean gloves (ie. latex, nitrile, etc.)</td>
			<td>Kimberly-Clark</td>
			<td>43431, 55090</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855196</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>5408129</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 2.0mL</td>
			<td>Fisher Scientific</td>
			<td>2681332</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear</td>
			<td>Bio-Rad</td>
			<td>#HSP9601</td>
			<td></td>
		</tr>
		<tr>
			<td>IPC forward and reverse primers</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>IPC PrimeTime qPCR Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>IPC Ultramer DNA Oligo synthetic template</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>Labnet MPS 1000 Compact Mini Plate Spinner Centrifuge for PCR Plates</td>
			<td>Labnet</td>
			<td>C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge machine</td>
			<td>Various</td>
			<td>-</td>
			<td>Any microcentrifuge machine that hold 1.5mL and 2.0mL tubes is typically okay.</td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; PCR Plate Sealing Film, adhesive, optical</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (not DEPC-Treated)</td>
			<td>Invitrogen</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 1000 &#181;L F 768A/8</td>
			<td>Rainin</td>
			<td>30389272</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 20 &#181;L F 960A/10</td>
			<td>Rainin</td>
			<td>30389274</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 200 &#181;L F 960A/10</td>
			<td>Rainin</td>
			<td>30389276</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Rainin</td>
			<td>Various</td>
			<td>Depending on lab preference, manual or electronic pipettes can be used at various maximum volumes.</td>
		</tr>
		<tr>
			<td>TaqMan Environmental Master Mix 2.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>4396838</td>
			<td></td>
		</tr>
		<tr>
			<td>Target forward and reverse primers</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>Target PrimeTime qPCR Probes</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product</td>
		</tr>
		<tr>
			<td>Target synthetic gBlock gene fragment</td>
			<td>Integrated DNA Technologies, Inc.</td>
			<td>none</td>
			<td>custom product. used for qPCR standard dilution series</td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Invitrogen</td>
			<td>AM9849</td>
			<td></td>
		</tr>
		<tr>
			<td>VORTEX-GENIE 2 VORTEX MIXER</td>
			<td>Fisher Scientific</td>
			<td>50728002</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and testing of species-specific quantitative pcr assays for environmental dna applications

漁業や野生動物保護管理を支援するために、種の存在を検出し、監視するための新しい非侵襲的な方法が開発されています。マクロビオタを検出するための環境DNA(eDNA)サンプルの使用は、急速に普及し、国家管理プログラムで実施されている方法の一つです。ここでは、プローブベースの定量PCR(qPCR)アプリケーションに対する種特異的標的アッセイの開発に焦点を当てています。プローブベースのqPCRを使用すると、プライマー単独で可能な限り特異性が高くなります。さらに、サンプル中のDNAの量を定量化する能力は、eDNAの生態と、現場におけるeDNA検出パターンの解釈を理解するのに役立ちます。環境試料から標的種を検出する感度と特異性を確保するために、これらのアッセイの開発と試験には慎重に検討が必要です。このプロトコルでは、標的種の検出のためのプローブベースのアッセイを設計およびテストするために必要な手順を説明します。シーケンスデータベースの作成、アッセイ設計、アッセイの選択と最適化、テストアッセイ性能、フィールド検証など。これらの手順に従うと、効率的で、敏感で、かつ、信頼できる特定のアッセイを実現するのに役立ちます。このプロセスは、米国クリンチ川で見られる淡水ムール貝種であるマケット(アクチナガイアス・シネラシナ)の集団のために設計されたアッセイで実証しています。

Watch this Scientific Journal Video about Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications at JoVE.com

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

環境DNAアッセイでは、現場データの収集を開始する前に、厳格な設計、テスト、最適化、検証が必要です。ここでは、環境試料から標的種DNAの検出と定量を行うための、種特異的なプローブベースのqPCRアッセイを設計する各ステップを通じてユーザーを取るプロトコルを提示する。

環境DNA応用に関する種特異的定量PCRアッセイの開発と試験

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

development-testing-species-specific-quantitative-pcr-assays-for

Research

JoVE Journal

Environment

12.6K ビュー.  U.S. Geological Survey. 環境DNAアッセイでは、現場データの収集を開始する前に、厳格な設計、テスト、最適化、検証が必要です。ここでは、環境試料から標的種DNAの検出と定量を行うための、種特異的なプローブベースのqPCRアッセイを設計する各ステップを通じてユーザーを取るプロトコルを提示する。

ビデオ: Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

別の雑草種の堅牢な除草剤抵抗性試験のためのプロトコル

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

環境学

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

毒性のための飲料水試料の迅速な評価のためのRTgill-W1細胞とのインピーダンスベースの流体バイオチップの製造および試験

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

ろ過水のサンプルおよび環境DNAの抽出のためのフィルターカートリッジの使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

水生生物に浮遊堆積物の影響を評価するためのフロースルー露光装置

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

個々のコロニーレベルにミツバチ開発に関する環境化学物質の影響を評価します

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

水溶液中におけるナノ材料の分散: プロトコルの最適化に向けて

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

DMMTAV DMDTAV環境用 DMAVを使用しての作製: 合成、精製、および確認

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

休眠復活オオミジンコ: プロトコルおよびアプリケーション

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

急性および慢性毒性試験ターコイズ メダカによる furzeriのためのプロトコル

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

若い淡水パール ムール貝原露出メソッドを使用して Bioindication ストリーム環境適合性のテスト

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Methods Article

環境DNA応用に関する種特異的定量PCRアッセイの開発と試験