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THE ZEBRAFISH BOOKA Guide for the Laboratory Use of Zebrafish Danio (Brachydanio) rerioby Monte Westerfield, Institute of Neuroscience, University of Oregon248CHAPTER 11GENERAL METHODS FOR ZEBRAFISH CARE1Availability and simple care1Food1Water1Fish Diseases3Quarantine Room Procedures6Cleaning Tanks7Shipping Fish8Keeping Track of Stocks15Chapter 2 - Breeding20Simple Method For Steady, Low- Level Embryo Production20Method For Maximal Embryo Production20Embryo Collection21Simple Schedule For Breeding Zebrafish21A Zebrafish Breeding Schedule For Maximal Embryo Production22Detailed Methods for Breeding Over Marbles22Pair-Wise Breeding of Individual or Natural Cross Fish24Embryo Production By In Vitro Fertilization26CHAPTER 3 - EMBRYONIC AND LARVAL CULTURE33A Simple Method for Raising Babies33Raising Larvae in a Nursery33Simple, Quick Methods For Raising Paramecia38A Semi-Sterile Method For Raising Paramecia39Growing Paramecia Under Sterile Conditions41Paramecia Culture Medium42Purification of Paramecia43Microworms as a Food Source45A Simple Method for Raising Larvae46CHAPTER 4 - MICROSCOPIC OBSERVATIONS136REMOVING EMBRYOS FROM THEIR CHORIONS136VIEWING CHAMBERS136AGAR MOUNTING137METHYL CELLULOSE MOUNTING138CHAPTER 5 - CELLULAR METHODS140A Device to Hold Zebrafish Embryos During Microinjection140Blastomere Lineage Analysis142DiI Labeling147Labeling Single Cells With Lineage Tracers148Laser-ablation of Single Cells151Overview of Single Cell Transplantation152Detailed Procedure for Transplanting Single Cells154An Inexpensive and Easy Microinjection Embryo-Tray157CHAPTER 6 - DISSOCIATED CELL CULTURE159Preparing Embryos for Cell Culture1590 Ca2+ - Mechanical Dissociation159Protease Dissociation159Dissociation of Embryos by Dissection160Using Coated Glass Coverslips for Cell Culture160Recipes:160CHAPTER 7 - GENETIC METHODS162Conventions for Naming Zebrafish Genes162Suggested Guidelines for Maintaining Mutant stocks164Overview of Methods for Parthenogenesis166Production of Haploid Embryos169Production of Homozygous diploid embryos170DELAYED IN VITRO FERTILIZATION USING COHO SALMON OVARIAN FLUID175PRODUCTION OF ANDROGENETIC HAPLOIDS AND DIPLOIDS178Freezing Sperm182Thawing and Using Frozen Sperm for In Vitro Fertilization183Fin Amputations184Gamma Ray Mutagenesis184Chemical Mutagenesis185Testosterone Treatment to Produce Males187ZEBRAFISH STRAINS188CHAPTER 8 - HISTOLOGICAL METHODS189Preparing subbed slides for sections189OCT Embedding for Cryostat Sectioning of Embryos or Larvae189Agar Embedding for Cryostat Sectioning of Embryos or Larvae190Gelatin Embedding for Vibratome Sectioning of Embryos or Larvae191Staining Sections with Antibodies Using PAP192Staining Whole-Mount Embryos with PAP193Solutions for Antibody Staining Protocols195Whole-Mount Staining of Biotin-Dextran Injected Zebrafish Embryos197Photoconversion of Fluorescently Labeled Profiles for EM198Chromosome Spreads200Gentle Fixation by Freeze Substitution201The following is a list of available antibodies at ZIRC:203Chapter 9 - Molecular Methods209Extraction of Proteins from Zebrafish Embryos for SDS-Gel Analysis209Western Blots of Zebrafish Embryos209Preparation of Genomic DNA213Preparation of Genomic DNA for Southern Analysis215Rapid Isolation of RNA from Zebrafish216Extraction and Purification of RNA from Zebrafish Embryos216Preparation of High Quality RNA from Zebrafish Embryos217Whole-Mount in situ Hybridization218High Resolution whole-mount in situ hybridization222TWO COLOR WHOLE-MOUNT RNA IN SITU HYBRIDIZATION227Sections of Whole Mount in situ Hybridization Preparations233Total Nucleic Acid Extraction Procedure for Zebrafish Embryos234A Simplified Ribonuclease Protection Assay for Embryos235CHAPTER 10 - RECIPES237CHAPTER 1GENERAL METHODS FOR ZEBRAFISH CAREAvailability and simple careZebrafish are available at pet stores throughout the world. They can be most easily maintained in 10 gallon (45 liter) aquaria heated to 28.5C (above 31C and below 25C, zebrafish probably wont breed and development will be abnormal) with 25 fish per tank. If you replace 1/3 of the water each day by siphoning up debris from the bottom of the tank, a separate tank filtering system will not be necessary. Otherwise use a filter and replace about half the water at least once a week. Tap water, aged a day or more in an open (heated) tank to release chlorine, is adequate although more consistent conditions may be obtained by adding commercial sea salts to deionized or distilled water (60 mg of Instant Ocean per liter of water, for example). Adults should be fed 1-2 times per day with a variety of food (see below). It is a good idea to clean the tank by siphoning after the second feeding.Food(Source: M. Westerfield) To keep fish in good breeding condition it is best to feed them a variety of foods. Feed manually ground dry or moist trout pellets (Ranger 1/4 inch brood food or Oregon wet pellets) as well as dry flake food like Tetra brand (available at most pet stores). Add enough food to each tank so that all the fish get some and all the food is eaten within 5 minutes. Feed adults at least twice a day although multiple light feedings allow the fish better opportunity to utilize the food. The best possible food for breeding adults is live adult brine shrimp. Brine shrimp.i.Brine shrimp; eggs can be purchased at most pet shops. To make baby shrimp, add 10 ml of shrimp eggs to 2000 ml of salt water (or Instant.i.Instant Ocean; Ocean, Aquarium Systems Inc.). Aerate vigorously. After 48 hr at 28.5ree;C, filter the shrimp through a cloth, wash with fresh water and dilute into dH2O at a ratio of 1 volume shrimp to 3 volumes water. Feed 1 pasteur pipette of diluted shrimp per 8 adult fish. Other possible foods are daphnia, Drosophila and Drosophila larvae. Beware of tubifex worms which may carry diseases (also, see Raising Larvae in a Nursery). Water(Source: M. Westerfield) Several different types of water are used for procedures described in the following sections. These solutions have been adapted to balance ease and cost of production with the changing needs of zebrafish during their life cycle. In general, adults can be maintained in tap water, conditioned by letting it set. This depends critically on the quality of the local water source and on the demands for embryo production. Poor water quality will adversely affect the health of the fish, their susceptibility to disease, and their breeding potential. If there is any question about water quality, deionized or distilled water should be used to which a small amount of salts and minerals are added. If an adequate supply of deionized water is unavailable, a closed-system which recirculates the water after purification can be used. Embryos and young larvae have stricter requirements and should be raised in egg water. Embryos removed from their chorions require additional calcium and should be maintained in embryo medium. System (or tank) water: water out of the facilitys water system. This water is dripped into clean tanks and is used for setting up pairwise crosses. Egg water: Used for in vitro fertilization and raising young embryos. Stock salts: 40 g Instant Ocean Sea Salts added to 1 L distilled water Egg water = 1.5 ml stock salts added to 1 L distilled water = 60 g/ml final concentration. Embryo medium: Dont confuse with egg water above. Used in handling dechorionated embryos and storing young embryos in dishes. This is basically 10% Hanks with full strength calcium and magnesium. Embryo medium 1.0 ml Hanks Stock #1 0.1 ml Hanks Stock #2 1.0 ml Hanks Stock #4 95.9 ml dd H2O 1.0 ml Hanks Stock #5 1.0 ml fresh Hanks Stock #6 Use about 10 drops 1 M NaOH to Ph 7.2 Hanks solutions: Hanks solutions can be made from stock solutions (kept refrigerated, they will last for several months). A premix of the salts can be stored in the refrigerator for several weeks. Sodium bicarbonate does not store well, so it is made up fresh each time Hanks solution is made. Full Strength Hanks 0.137 M NaCl 5.4 mM KCl 0.25 mM Na2H PO4 0.44 mM KH2 PO4 1.3 mM CaCl2 1.0 mM Mg SO4 4.2 mM NaH CO3 Hanks Stock Solutions Stock #1 8.0 g NaCl 0.4 g KCl in 100 ml ddH2O Stock #2 0.358 g Na2HPO4 Anhydrous 0.60 g KH2PO4 in 100 ml ddH2O Stock #4 0.72 g CaCl2 in 50 ml H2O Stock #5 1.23 g MgSO4-7H2O in 50 ml ddH2O Stock #6 0.35 g NaHCo3 10.0 mls dd H2Oz Hanks Premix - Combine the following in order: 10.0 ml Solution #1 1.0 ml Solution #2 1.0 ml Solution #4 86.0 ml ddH2O 1.0 ml Solution #5 Store Hanks Premix in the refrigerator along with the Hanks solutions. Final Hanks 9.9 ml Hanks Premix 0.1 ml fresh Stock #6 Fish Diseases(Source: C. Walker) For any large colony of fish, precautions should be taken to prevent the spread of epidemic disease. The easiest strategy for combatting disease is prevention by minimizing contact between fish and water in different tanks. Avoid mixing fish from different tanks as much as possible. Sterilize all equipment that comes in contact with the fish or tanks. For example, use fish nets, siphons, and cleaning sponges on only one tank at a time and autoclave them before using them in a different tank. Sterilize the water (i.e. with a flow-through ultra-violet sterilizing lamp) before adding it to the tanks. Remove sick fish from tanks as quickly as possible. Quarantine fish from pet stores before adding them to the colony (see Quarantine Room Procedures, page 1). Wash hands and arms thoroughly if they come into contact with tank water. The two most common diseases that affect zebrafish are velvet disease and fish tuberculosis (mycobacteriosis). Velvet diseaseZebrafish are highly susceptible to velvet disease, Oodinium pillularis, a parasitic dinoflagellate algae. This oval-shaped parasite attaches to the fish near the fins, especially the dorsal fin, and around the gills. You can see it under a dissecting microscope. When the parasite is mature, it drops off the fish and multiplies 60 times on the bottom of the tank. These new parasites then reinfect the fish. Fish with velvet disease have the characteristic behavior of rubbing their sides and flipping around in the corners of the tank. As the disease progresses, fish become lethargic, the fins (particularly the dorsal fin) are held close to the body, and the fish stop producing eggs. Most fish from pet stores or dealers carry velvet disease. Symptoms of velvet disease: rubbing behavior lethargy fins held close to the body parasites near fins and gills Although velvet disease is extremely contagious, it can be cured with minimal damage to the fish using a 3-day treatment with Atabrine (Quinacrine hydrochloride). Treatment for velvet disease: Day 1 Turn off incoming water. Slowly drip 2 liters of sea salts into an infected 10-gallon tank. Add 3.3 ml of the Atabrine stock solution. Day 2 Add 3.3 ml Atabrine stock. Day 3 Add another 3.3 ml Atabrine stock for a total of 9.9 ml. At the end of the 3-day period, clean the bottom of the tank thoroughly and slowly dilute out the salt and the Atabrine with fresh water. Continue cleaning the bottom of the tank daily for several days. Solutions: Atabrine Stock:10 mg/ml dH2O. Store in light tight bottle. Salt Stock: 20 tablespoons (280 g) Instant Ocean Sea Salts (Aquarium Systems, Inc.) dissolved in 2 liters distilled water MycobacteriosisFish tuberculosis or mycobacteriosis is also a common disease. The agent, Mycobacterium marinum, is a rod-shaped, gram positive, non-motile, non-spore-forming bacterium1. Fish are infected by ingesting the bacteria in unpasteurized food or through abrasions in the skin. Snails often harbor the bacteria. Symptoms of Mycobacteriosis: o listlessness; the fish may isolate itself from other fish and refuse to eat o emaciation; hence, the name skinny o skin ulcers o spinal curvature o pigment change o grey-white nodules in internal organs Definitive diagnosis should be made by a pathology lab. There is no known effective antibiotic to treat mycobacteriosis. Some level of disease control can be obtained by eliminating sick fish, by sterilizing tanks routinely and all equipment that comes in contact with the fish or the tank water, and by reducing stress caused by moving fish between tanks or by changes in temperature, water flow, or feeding regimen. A word of caution regarding fish tuberculosis: Some cases of transfer of fish tuberculosis to humans has been documented. Precautions should be taken by wearing disposable gloves and washing hands with betadine (providone iodine) when handling sick fish. Persons with immuno-deficiency problems should not handle fish with TB. Other diseases that occur less frequently include raised scales, tumorous eyes, and hemorrhages. In these instances, remove the diseased fish as soon as they are seen and clean the tank. Reference: Post, G. (1987) Textbook of Fish Health, T.F.H. Publications, Neptune City, NJ, pp. 228. Intestinal Capillariasis(Source: M. Pack, J. Belak, C. Boggs, M. Fishman and W. Driever) Capillarids are thin and transparent worms that can reach one centimeter length. The eggs have a characteristic oval shaped appearance with a plug-like structure at either end and are visible in the adult worm, and the gut or feces of infected fish. Photographs and a good description of Capillarids at different developmental stages can be found in the Handbook of Fish Diseases, Dieter Untergasser, Editor (TFH publications, 1989, p. 104-105). Worms in the intestinal bulb of adult zebrafish are motile and can be easily seen when the dissected gut, in egg water (0.03% Instant Ocean), is viewed with transmitted light using a high power (50X) dissecting microscope. Outside the fish gut Capillarids are no longer motile. A combination of two drugs, Trichlorfon and Mebendazole (Fluke-tabs; Aquarium Products, 180-L Penrod Court, Glen Burnie, M.D. 21061) has been reported to be extremely efficacious for removing monogenetic trematodes from fresh water fish. Trichlorfon is an insecticide with anti-cholinergic activity that is considered toxic to humans. Mebendazole, a common anti-helminthic used to treat human intestinal infections, inhibits glucose uptake and is cidal for adult helminths and embryos. Use the dosage recommended by the supplier; one tablet per 38 liters once trials to assess toxicity are completed. Add the drugs as a slurry (100 tablets per one liter water-let stir 10 minutes) because they are poorly soluble in water. Repeat the treatment after 24 and 48 hours with a 10% water change every day thereby increasing the effective concentration. Remove carbon filters during treatment. Wear plastic gloves to avoid contact with water. Reinstall carbon filters at 72 hours to remove the drugs from the system. Repeat the treatment protocol at 10 days because the cidal effect of these drugs on freshly fertilized eggs or dauerlarvae is not reported. Continue testing fish at regular intervals and retreat if necessary. References: Goven, B.A. and Amend D.F. (1982) Mebendazole/trichlorfon combinations: A new anti-helminthic for removing monogenetic trematodes from fish. J. Fish Biol. 20:373-378. Quarantine Room Procedures(Source: S. Russell) Fish brought in from the outside world (i.e. not born in the facility) must be isolated in a quarantine room to reduce the risk of contaminating the existing stocks with infectious diseases. The following procedures are designed to prevent the accidental spread of diseases from the quarantine area. 1. Only pre-authorized people are allowed in the quarantine room. 2. All incoming fish are examined and, if necessary, treated for velvet disease. 3. All new fish are to be checked daily for two weeks for signs of illness. Any potential sickies are removed. 4. Dead fish.i.Dead fish; are transported in a plastic bag and flushed down the sink in the quarantine area with lots of water. 5. No live adult fish are to be moved from the quarantine room unless they leave the building entirely. 6. Embryos obtained in the quarantine room (after the initial 2 week period) must be treated in bleach before leaving the room. To bleach eggs, prepare two beakers of bleach solution, containing 0.1 ml of 5% sodium hypochlorite in 170 ml of system water. Mix thoroughly. Place the eggs in the first beaker, and allow them to stand for 5 min. Pour off the bleach solution, and rinse the eggs with system water. Allow the eggs to stand in system water for 5 min. Place the eggs in the second beaker of bleach solution for 5 min. Rinse the eggs with system water. Place the eggs into a small disposable petri dish. Eggs that have been properly bleached can be removed safely from the quarantine room. 7. No equipment can leave the quarantine area without being bleached or autoclaved. Equipment is transported, cleaned and stored in containers that are kept separate from the central facility containers and are labeled Q-room. No equipment is to be moved in or out of the Q-room without prior approval. 8. Always wash hands (and arms) carefully in the local sink using antiseptic soap after working in the quarantine room. 9. When working in the quarantine area, assume that everything (including yourself) is contaminated. Think about what youre doing! Cleaning Tanks(Source: T. Montgomery) Siphon (cleaning by vacuuming) the bottoms of all tanks at least once per week. Use siphon tips with a length that corresponds to the size of the tank: tips for 5-gallon tanks are 12 long; the 1