飼育第1の給電ゼブラフィッシュの完全および更新された「ワムシPolyculture方法」
Summary
Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This “polyculture” strategy promotes high growth and survival with minimal labor and disturbance to the larvae.
Abstract
The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an “on-demand”, nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.
Introduction
5 –ゼブラフィッシュ（ ゼブラフィッシュ）は、発生遺伝学、毒物学、行動、水産養殖、再生生物学、多くのヒトの疾患1のモデル化を含むがこれらに限定されない科学分野の増加、で利用卓越した実験動物です。種は実験室で維持することは比較的容易であるが、彼 らの文化6に関連する管理上の課題がいくつかあります。これらの中で最も顕著なのは魚が第一ガス膀胱インフレ7以降のフィードを開始する場合は特に、幼虫の飼育です。成長の5日7特に重要である-通常の、制御された条件の下では、この発達イベントには、次の3で、受精後（DPF）〜5日後に発生します。フィード項目は、diges適切なサイズでなければなりません – この段階で中心的な技術的な難しさは十分に第1の給電幼虫の栄養要求を満たすためにあります培養タンクに過度の廃棄物を作成せずに、ほぼ連続的に魅力的、および使用可能なtible、。歴史的にこのルーチン水交換8,9とともに、タンクで魚に餌の多数の少量を提供することで、一般的に達成されました。これらの方法はある程度成功しているが、それらは、非効率的で高い労働投入を必要とし、唯一の変数と成長と生存10の制限されたレートを返します。
自然界では、ゼブラフィッシュの幼虫は、おそらく水柱11に豊富に小さな動物プランクトンの存在を餌に。このため、このようなゾウリムシ 、ワムシ、およびアルテミアなどのライブフィードを組み込むlarvicultureプロトコルは、通常、7最も効率的です。 2010年、最もよく、共著者は、外因性の摂食12の最初の5日間塩水ワムシと一緒に静的な、汽水水に幼虫のゼブラフィッシュを成長させることが可能であることを実証しました。このアプローチは、どのハーネスエスワムシ培養の自然な高生産性が低い労働投入12,13と幼虫の成長および生存の収率が非常に高い料金、水を汚染することなく、十分な、栄養価の高い獲物を提供します。近年では、世界中の研究室の数は増加し、このプロトコルのバリエーションを採用しており、その多くが今保育園のシステム14をサポートするために、連続的にワムシを培養しています。
過去数年にわたり、ワムシ/ゼブラフィッシュpolycultureとワムシ生産の両方のための方法は、洗練され、より標準化されたと容易に拡張可能になるように改善されました。この記事では、1のためのステップバイステップの手順）は、連続かつ堅牢なワムシの生産と2）外因性の給餌の最初の5日間魚の力強い成長をサポートするために使用ワムシ/ゼブラフィッシュpolycultureシステムの確立を提供します。
Protocol
Representative Results
Discussion
魚を養うために、連続ワムシ培養系の確立と維持、及び同タンク内のワムシと一緒に最初に送りゼブラフィッシュの幼虫を培養：早期幼虫のゼブラフィッシュを供給するためのワムシpolyculture方法の実装を成功させるには、二つのタスクのための有効なプロトコルが必要です。
最初のローレンスおよび共著者14で説明したゼブラフィッシュの研究室用の連続塩水?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
このプロトコールに記載の代表的な結果を得るために生成魚のケアと使用はボストン小児病院、プロトコル番号の14-05-2673Rで機関動物実験委員会によって定められたガイドラインに完全に従って行きました。
Materials
| Rotifer Culture Infrastructure | |||
| 100 Liter Culture Vessel | Aquaneering | Custom | Polycarbonate culture vessel, conical bottomed, with drain valve |
| 5 Gallon Culture Bucket Kit | Reed Mariculture | CCS Starter Kit | Small volume culture vessel for small facilities |
| Rigid Clear Tubing 1/2" O.D., 36” | Pentair Aquatic Ecosystems | 16025 | Rigid clear tubing for air delivery |
| Mesh tube | Pentair Aquatic Ecosystems | RT444X | Mesh tube support for floss filter |
| Rotifer Floss | Reed Mariculture | Rotifer floss 12” x 42” | Particulate waste trap |
| Peristaltic Metering Timer Pump, 5 GPD | Grainger | 38M003 | Metering pump with timer for dosing feed to rotifers |
| Peristaltic Metering Timer Pump, 1-100 mL/h (for smaller-scale culture) | Coral Vue | SKU: IC-LQD-DSR | Metering pump with timer for dosing feed to rotifers |
| Silicone Tubing | Cole Parmer | Tubing for algae delivery to rotifer vessel | |
| Rigid Clear Tubing " O.D.,36” | Pentair Aquatic Ecosystems | 16025 | Rigid clear tubing for air delivery to algae paste |
| Rigid Clear Tubing O.D., 36” | Pentair Aquatic Ecosystems | 16025 | Rigid clear tubing for algae delivery |
| Rotifers | |||
| Live Rotifers Brachionus plicatilis Type L | Reed Mariculture | Type L 5 million | Rotifer stock culture for system startup |
| Rotifer Feed | |||
| Sodium hydroxymethylsulfonate | Reed Mariculture | ClorAm-X® 1lb tub | Ammonia reducer for algae feed mix |
| Sodium Bicarbonate | Fisher Scientific | S25533B | pH buffer for algae feed mix |
| Microalgae concentrate | Reed Mariculture | Rotigrow Plus® 1 liter bag | Nutritionally optimized rotifer feed |
| Water Preparation | |||
| Reef Crystals Reef Salt | That Fish Place | 198210 | Salt for making culture water (NOTE: this item is an example only; any contaminant free salt formulations may be used). |
| Refractometer | Pentair Aquatic Ecosystems | SR6 | measuring salinity |
| Rotifer Culture Equipment | |||
| Plankton Collectors 12" Dia, 53 microns | Pentair Aquatic Ecosystems | BBPC20 | Mesh screen for collecting rotifers |
| Scrub Pads | Pentair Aquatic Ecosystems | SCR-58 | Scrub pad for cleaning inside of culturing vessels |
| Scrub Brush | |||
| Bucket | Grainger Supply | 43Y530 | Graduated bucket for mixing culture water |
| Hatching Jar | Pentair Aquatic Ecosystems | J30 | Storage of algae feed mix |
| Lugol’s Solution, Dilute | Fisher Scientific | S99481 | Agent used to immobilize live rotifers for counting |
| Sedgewick-Rafter plankton counting slide with grid | Pentair Aquatic Eco-Systems | M415 | Counting rotifers |
| Miscelleneous | |||
| Tea Strainer | Kitchenworks | 971972 | Used for collecting zebrafish embryos after spawning |
Referenzen
- Ribas, L., & Piferrer, F. The zebrafish (Danio rerio) as a model organism, with emphasis on applications for finfish aquaculture research. Reviews in Aquaculture. 6, 209-240 (2014).
- Poss, K. D. Advances in understanding tissue regenerative capacity and mechanisms in animals. Nature reviews. Genetics. 11, 710-722 (2010).
- Gemberling, M., Bailey, T. J., Hyde, D. R., & Poss, K. D. The zebrafish as a model for complex tissue regeneration. Trends in genetics : TIG. 29, 611-20 (2013).
- Santoriello, C., & Zon, L. I. Hooked! modeling human disease in zebrafish. Journal of Clinical Investigation. 122, 2337-2343 (2012).
- Selderslaghs, I. W. T., Blust, R., & Witters, H. E. Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reproductive Toxicology. 33 (2), 142-154 (2012).
- Lawrence, C. The husbandry of zebrafish (Danio rerio): A review. Aquaculture. 269, 1-20 (2007).
- Harper, C., & Lawrence, C. The Laboratory Zebrafish (Laboratory Animal Pocket Reference). CRC Press (2010).
- Nusslein-Volhard, C., & Dahm, R. Zebrafish, A Practical Approach. Oxford University Press (2002).
- Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish. University of Oregon Press (2007).
- Carvalho, P., & Arau, L. Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance. Aquaculture Research. 37, 1107-1111 (2006).
- Spence, R., Gerlach, G., Lawrence, C., & Smith, C. The behaviour and ecology of the zebrafish, Danio rerio. Biol Rev Camb Philos Soc. 83 (1), 13-34 (2008).
- Best, J., Adatto, I., Cockington, J., James, A., & Lawrence, C. A novel method for rearing first-feeding larval zebrafish: polyculture with Type L saltwater rotifers (Brachionus plicatilis). Zebrafish. 7 (3) 289-295 (2010).
- Lawrence, C. Advances in zebrafish husbandry and management. Methods in Cell Biology. 104, 429-451 (2011).
- Lawrence, C., Sanders, E., & Henry, E. Methods for culturing saltwater rotifers (Brachionus plicatilis) for rearing larval zebrafish. Zebrafish. 9, 140-6 (2012).
- Tucker, C. S., & Hargreaves, J. A. Biology and Culture of Channel Catfish. 34, 634-657 (2004).
