Fish Flashcards
Metals to avoid in facility materials?
Copper and lead
Which metal is the most corrosive resistant?
Titanium
Which disinfectant to avoid?
Phenols (One-Stroke Environ, Pheno-Tek, Tek-Trol, Lysol)
Which disinfectants can be used?
- 2% bleach (Na-hypochlorite)
- 1% virkon aquatic (potassium peroxymonosultate + NaCl) - a peroxygen compound (oxidizing)
- 70% ethanol for tanks
Preferred method of ventilation of aquatic environments
Regenerative blower better than air compressor (latter is better for high pressure needs not constant, low pressure)
Types of life support systems
- Closed System - no additional water or air provided from external sources after the initial set-up
- Recirculating System - water is filtered and reused within the system
- Flow-through System
- water is removed at a certain % and replaced with new water
Preferred mechanical filter
Uniform medium with effective particle size of 0.3 mm which will remove 95% of particles down to 6 um
Common order of filtration steps in recirculating system?
Mechanical –> biological –> chemical
Acceptable levels of ammonia, nitrite, and nitrate?
NH3 = 0
NO2 < 0.1-0.5 mg/L (ppm)
NO3 < 200 mg/L (ppm)
What converts ammonia to nitrite (NO2)?
- Ammonia oxidizing Bacteria (AOB) – Nitrosomonas, Nitrospira, Nitrosococcus
- Ammonia oxidizing archaea (AOA)
What converts nitrite to nitrate (NO3)?
Nitrogen oxidizing bacteria (NOB) - Nitrobacter and Nitrospira
Types of chemical filters
- activated carbon/charcoal (nonspecific) = the most common; binds cations and anions
- foam fractionation/protein skimmers – protein contaminants removed with rising bubbles
- UV filtration
What is zebrafish maintenance temperature? What should temperature fluctuations be limited to?
75-82F
24-28C
limit temp change to +/- 1.5C per day
What temperatures determine male vs female?
Male = 22C
Female = 31C
Which water quality issue causes the most morbidity/mortality?
Decreased oxygen saturation
Will see surface breathing behavior
What is ideal pH? What is caused by low or high pH?
Ideal = 7-8
High pH results in increased unionized ammonia (NH3)
Low pH 1) inhibits nitrifying bacteria causing increased ammonia
2) Zinc, copper, Fe, aluminum, etc are more soluble in acidic water causing greater chance of toxicity
What is recommended conductivity?
125 - 2,500 uS (microsiemens)
What is recommended water hardness for zebrafish?
Zebrafish are hardwater species 80-200 ppm (Calcium & Magnesium)
What is recommended light level for zebrafish?
5-30 ft-candles or 54-354 lux at water surface
How to remove Chlorine/ chloramines
- 24-hr period of aeration/“aging” (***but does not remove chloramine)
- Activated charcoal
- Commercial dechlorinator (sodium thiosulfate)
- Near-boiling
How much water volume to change daily in recirculating system?
1-10% of total water volume daily
What types of algae are good vs bad?
Green = good
Red = bad
How long should quarantine be?
2-3 wk (minimum)
4-6 wk (for Mycobacterium)
What are refined vs traditional quarantine strategies?
Refined = import surface-disinfected embryos (bleach-import-spawn-move)
Traditional = spawn within quarantine facility and then bleach (import-spawn-bleach-move)
Which organisms may not be effectively removed by bleaching embryos?
Vertically transmitted pathogens e.g. P. neurophilia
What is a common live feed for adult zebrafish?
Drosophila larvae
How do n6:n3 fatty acid requirements differ for zebrafish?
zebrafish have a higher demand for n6 than most coldwater fish
How to store fish feed?
Refrigerate and use within ONE MONTH
What is stocking density of zebrafish per the Guide?
5 adults per liter
What specialized structure in the heart ensures continuous supply of blood to the gills?
Bulbus arteriosus
How many chambers in heart for fish?
2 (1 atrium, 1 ventricle)
Which mutation in zebrafish selectively perturbs assembly of the artery (the aorta), causing a syndrome similar to coarctation of the aorta in humans?
Gridlock (grl) mutation
What are two distinct regions of fish kidney?
- Head kidney: the main hematopoietic organ; also site of maturation for lymphocytes; endocrine (interrenal and chromaffin cells analagous to adrenal)
- Trunk kidney (nephrons, hematopoietic, and lymphoid)
What cell type is important for lateral line system?
neuromast cells (mechanosensory hairs)
Which gland secretes calcitonin to regulate calcium? Where is it located?
Ultimobranchial gland
Located ventral to the esophagus, separating heart from abdominal cavity
Which gland secretes hypocalcin, which acts with calcitonin to regulate Ca metabolism? Where is it located?
Corpuscles of Stannius
eosinophilic granular cells located on the ventral surface of the kidneys
Which structure in the stomach of salmonids looks like worms and secretes digestive enzymes?
Pyloric ceca
What anatomical features are fish lacking?
Lymph nodes, urinary bladder, prostate, sex chromosomes
At what density do you see more aggression?
Low density
Examples of abnormal/maladaptive behavior in zebrafish
tight/cohesive shoaling, frequent aggression, erratic activity bursts, freezing, bottom dwelling
Examples of normal behavior in zebrafish
Loose shoaling, infrequent aggression, moderate activity, occupying entire water column; Live in upper strata in wild
How long after fertilization does first cleavage occur?
45 minutes
When is midblastula transition (MBT) and what occurs at this time?
3 hpf (hours post-fertilization)
Activation of zygotic transcription
When is gastrulation?
5.5-10 hours post fertilization
3 germ layers, endo-, meso-, ectoderm, take final positions
When is first heart beat?
24 hours
When do embryos hatch from chorions?
day 3 or 4
When do zebrafish begin feeding (no longer rely on yolk sac)?
day 4 or 5
How many chromosomes do zebrafish have (diploid)?
50
What are the other names for MS-222?
Ethyl 3-aminobenzoate methanesulfonic acid
Tricaine methanesulfonate
With MS-222 combined with ____ at 65 ppm, zebrafish enter surgical plane of anesthesia in 90 sec, remain there longer, and recover more quickly than MS222 alone
Isoflurane
What is NOT appropriate as sole agent for surgical anesthesia and must be combined with MS-222?
Isoflurane
What are the pros and cons with eugenol (4-allyl-2-methoxyphenol)?
- Active ingredient in clove oil
Pros: cheap, wide margin of safety, quick induction, rapid metabolism
Cons: isn’t water soluble (commonly mixed with ethanol), long recovery, not FDA approved
What are the pros and cons for metomidate?
Pros: safe for fish
Cons: illegal for food fish, increase fish pigmentation (not Zf), lower cort and glucose, no analgesia, not good GA
What temp is appropriate for gradual cooling? When is this method appropriate?
10-12C
Can be used for quick, nonpainful procedures but not for invasive procedures
What are planes of anesthesia for zebrafish?
- Stage I, plane 2=deeply sedated, stop voluntary swimming, reduced opercular movement
- Stage II, plane 1=light narcosis
- Stage II, plane 2=deep narcosis (loss of equilibrium) –> external sampling
- Stage III, plane 1=Dec. resp and near loss of muscle tone –> minor Sx
Stage III, plane 2= bradycardia, low RR, loss of reactivity to manipulation –> Sx
Stage IV=medullary collapse
What spinal pathway is the most important for pain transmission?
Spinothalamic tract (STT)
What is allowed volume for survival blood collection?
Maximum single collection = 2% body weight; maximum repeated collection = ≤1% body weight every 2 weeks
Where to collect blood survival?
Withdraw blood from dorsal aorta or posterior cardinal v.
When does spawning occur?
Spawn in the morning – initial light after darkness; Females scatter clutches
How many embryos can fish produce every week?
Can produce 200 embryos at weekly intervals
When is sexual maturity in zebrafish?
10-12 weeks
How to tell male and female zebrafish apart?
females are larger, more silvery, slightly rounded; males are more streamlined and more brightly colored
Zebrafish melanoma model has mutation in which gene?
Braf
Which Mycobacterium is most prevalent? Which is most zoonotic? What are clinical signs for both.
M. chelonae = most prevalent –> diffuse granulomatous
M. marinum = most zoonotic —> skin ulceration
Culture media for Mycobacteria
Tryptic Soy Agar (TSA), Middlebrook 7H10, or Lowenstein-Jensen slants culture media
What to do about Mycobacteria?
NO TREATMENT – CULL & CLEAN
Clean w/ dilute bleach (50ppm)
What causes “hole in the head” lesions? How to detect this organism?
Edwardsiella ictaluri with chronic infection and organism entering CNS
acute more common though with high mortality
PCR kidney
Which bacteria causes DEEP hemorrhage (muscle)?
Aeromonas
What pathogen causes haystack formation on wet mount?
Flavobacterium (due to filamentous nature of bacteria)
What pathogen causes red gills and haystack formation on wet mount?
Flavobacterium branchiophilum (bacterial gill disease)
Which pathogen causes “saddleback lesion” (loss of pigment and scales around dorsal fins)?
Flavobacterium columnare (cotton wool disease)
What clinical sign is associated with swim bladder infection (bacterial aerocystitis)?
Erythema of THE BASE of ventral and dorsal fins
What is most common zebrafish disease? What are two clinical signs?
Pseudoloma neurophilia
- Emaciation
- Scoliosis/lordosis
How to differentiate Pseudoloma neurophilia from Pleistophora hyphessobryconis?
P. neurophilia spore looks like bedroom slipper
P. hyphessobryconis only found in muscle not CNS; spore has prominent posterior vacuole
What causes gold dust disease/velvet disease?
Piscinoodinium, a protozoa
What causes white spot disease?
Ichthyophthirius multifillis
Life cycle of Ich
Trophont feeds within the epithelium fin, skin, and/or gills –> falls off host and forms a reproductive cyst (tomont) –> Tomont secretes a sticky capsule that sticks to substrate. Binary fission occurs up to 10 times w/in the tomont to produce tomites –> Tomites break through the nodule wall and differentiate into motile, infective theronts.
What is pathognomonic for Ich?
ID trophont within host’s epithelium on a wet mount or microscopic exam is pathognomonic for Ich; trophont also has characterisitic horeshoe shaped maronucleus “rolling motion”
How to treat Ich?
Theronts (infectious stage in water) can be killed by UV light, raise temp
Which protozoa causes excess mucus production and can be identified by circular movement?
Ichthyobodo necatrix
Ddx for Ich that looks more patchy (vs Ich more defined white spots)?
Epistylis (Heteropolaria colisarum)
What is the most common fish pathogen? What are clinical signs and tx?
Saprolegniaceae aka water mold
“cottony” masses on skin and gills
Tx: 1. salt water at >3ppm
2. malachite green (but it’s a mutagen/carcinogen/teratogen!)
What is the common zebrafish roundworm? What are clinical signs and prevention?
Pseudocapillaria tomentosa
Models of cancer, inflammation, or GI dz may be impacted by clinical presentation and P. tomentosa associated intestinal neoplasia
Prevention – avoid sources such as oligochaete worms (Tubifex tubifex - sludge worm) feed sources, which can act as paratenic hosts
Name other nematodes that affect fish. What is common clinical sign?
- larvae of Eustrongylus spp
- adults of Capillaria sp & Camallanus sp.
Large red cysts
What are two types of fish flukes and how to tell apart?
Dactylogyrus
- Gill fluke
- Oviparous
- Four-pointed anterior end
- See flashing (pruritus)
Gyrodactylus
- Mongolian flatworm (affects skin rather than gills)
- Viviparous
What causes “cheesy bile” and particularly affects the kidney of zebrafish?
Myxospores
Name a naturally occurring viral pathogen of zebrafish.
NONE
Which virus causes cream-colored to pink/grey nodules (wart-like growths) on body surface and internal organs (causes fibroblast hypertrophy) in laboratory fishes?
Lymphocystis disease (Iridovirus)
ICIB (intracytoplasmic inclusion bodies)
Which virus causes whirling in laboratory fishes? Which type of fish has high mortality?
Infectious pancreatitis necrosis (IPN) virus
Salmonids
Also see anemia
ICIBs
What is gas bubble disease and what is the cause?
too much dissolved oxygen or nitrogen
usually due to air becoming trapped in plumbing or pumping water from well w/o gas stripping, and injection of air from filters at water change
What is dx and tx for gas bubble disease?
dx: presence of gas emboli is pathognomonic
tx: aeration of water in the system to drive excess dissolved gas out of solution
Which form of ammonia is more toxic? What is effect of pH?
Unionized ammonia (NH3) is highly toxic to fish, whereas the ionized ammonium (NH4) is less toxic
Unionized ammonia is dependent on pH and temp of water; More toxic at HIGH pH
What causes methemoglobinemia (fish have pale tan/brown gills) in zebrafish?
Nitrite toxicity
Increased levels associated with inappropriate levels of nitrite oxidizing bacteria (NOB) (Nitrobacter and Nitrospira) that convert nitrite (NO2) to nitrate (NO3)
What causes cherry-red, swollen gills in zebrafish?
Chlorine/Chloramine toxicity
Tx: Remove chlorine from water by allowing a 24hr aeration period or adding dechlorinator; Chloramine can’t be removed by aeration, but must be filtered through activated carbon or tx with chemical like sodium thiosulfate; Near boiling water can also remove chloramines
What causes nephrocalcinosis in zebrafish?
Associated with high levels of CO2 in water or unbalanced levels of diet calcium or magnesium
What are the effects of copper toxicity?
- RESP: increased RR and O2 consumption, gill lesions
- IMMUNE: reduced Ab production, increased susceptibility to infections
- NEURO: lethargy, incoorindation, lateral line damage
- REPRO: reduced sexual development, reduced egg production, poor survival rates, teratogenic
- LIVER: liver damage
Fish with swelling cranial ventral aspect?
Pericardial effusion caused by heart failure
What is the most common neoplasia of zebrafish?
Seminoma
What causes distended coelomic cavity and focal ulceration?
Egg-associated inflammation
What is zebrafish family?
Cyprinidae
Name some wild-type zebrafish strains.
o AB
o TU - Tuebingen
o TL - Tupfel long fin
o WIK
o Ekkwill (EK)
o HK
o SJD
many others
Which generation to look at for mutagenesis screens?
F3
What does N-ethyl-N-nitrosourea (ENU) cause?
Random point mutations
Zebrafish model of T-cell acute lymphoblastic leukemia has mutation in which gene?
C-Myc
What are genetics of zebrafish model of acute myelogenous leukemia (AML)?
Translocation leading to AML1 gene (imp for hematopoiesis) fused to ETO gene (imp for gene reg)
What is effect of radiation on DNA?
Results in large deletion and inversion genetic mutations bc causes DNA strand breaks –> repair
How to screen for phenotypes affecting early embryonic development?
Haploid method - eggs from F1 female are collected + fertilized in vitro with ultraviolet (UV)- irradiated sperm that cannot contribute any genetic material
uses parthenogenesis which can be induced in zebrafish
In zebrafish research, what is the result of directly microinjecting a pseudotyped retrovirus into blastula-stage embryos?
The viral genome is randomly integrated, disrupting gene function and creating mosaic animals.
What are the differences in direct microinjection of DNA vs mRNA?
RNA construct is inherited more evenly when injected into a zebrafish embryo - doesn’t need to integrate like DNA and will be in cytoplasm and spread more evenly during rapid cell divisions. This also helpful if gene in question is important very early in development bc RNA does not need to integrate into genome to be active.
What is the name of the modified transposon allows intro of a specific gene of interest?
Tol2 transposon
What causes direct knockdown of a particular gene by disrupting protein translation of transcriptional splicing?
Morpholino antisense oligonucleotides
Which bacteria is Cas9 endonuclease from?
Strep pyogenes
What is use of zebrafish in research?
Cancer
-epidermal papilloma after ENU exposure
-acute myelogenous leukemia
-melanoma
Muscular dystrophy (Duchenne)
Tissue regeneration
Embryonic development
Gene function analysis
Mutagenesis
Toxicology
What are Cut-throat trout
(Salmo clarki) and Brown trout (Salmo trutta) models of?
Hepatocellular carcinoma
Which fish is a model of wasting dz caused by excess oxidation of UFA (unsaturated fatty acids) from eating silkworm pupae? What is prevention?
Carp (Cyprinus carpio)
Prev: add a-tocopherol (vit E)
Which fish develops Sekoke disease (spontaneous diabetes)?
Carp (Cyprinus carpio)
A model of juvenile diabetes (T1DM)
Which fish is a model for radiation?
Japanese Medaka
(Oryzias latipes)
Which fish is a model for malignant melanoma?
Swordtail
(Xiphophorus helleri)
Platyfish
(Xiphophorus maculatus)
Albino swordtail X F1 melanotic hybrid –> 50% offspring with amelanotic melanoma
Macromelanophere factors with specific pigment:
Xh – if pigment transplanted then will develop melanosis due to no modifier gene
Xm – sex linked co-dominant
Which fish is model of Copper storage disease (Wilson’s disease)
(along with Bedlington terrier, Long-Evans Cinnamon rat, sheep)?
White perch
(Merone americana)
Which fish is a model for Neurofibromatosis (von Recklinghausen’s Dz)
(also induced in Syrian hamsters with transplacental ENU)?
Bicolor Damselfish
(Pomacentrus partitus)
Also can see Schwannoma
Which fish is a model of renal regeneration?
Goldfish
(Carassius auratus)
Which fish is used for EPA toxicity testing?
Fathead minnow
(Pimephales promelas)
Which fish gets dermal sarcomas?
Walleye
(Stizostedion vitreum)
Caused by Walleye Dermal Sarcoma Virus
What tumors are most commonly induced by N-Nitroso-N-ethylurea (ENU) in zebrafish?
Epidermal papillomas
Time for euthanasia by rapid cooling with 4C ice water in fry and adults?
Adults: 10 min
Fry (4-7 dpf): 20 min
What is appropriate treatment for gyrodactylids (flukes)?
Praziquantel
Do not use organophosphates - associated with resistance
What does MS-222 cause if not buffered appropriately?
Acidosis and reduced Na/K in CNS
How long to perform immersion method of euthanasia for finfish?
At least 30 minutes after cessation of opercular movement
What is acceptable for euthanasia of finfish by immersion?
- Benzocaine
- 95% Ethanol
- Iso/sevoflurane
- Quinaldine
- Tricaine
- 2-phenoxyethanol
- Lidocaine
- Clove oil/eugenol
What is acceptable with conditions for immersion euthanasia of finfish?
- CO2 (may have hyperreactivity)
- Eugenol, isoeugenol, clove oil (at least 10 min after opercular movement stops)
When is rapid chilling acceptable vs acceptable with conditions?
A: zebrafish, Australian river gizzard shad (N. erebri) aka bony bream
AC: Other small-bodied tropical/subtropical finfish
NOT in coldwater fish!
Acceptable with conditions finfish
Physical
1. Rapid chilling in tropical/subtropical
2. Maceration (when instant death)
3. Captive bolt (large fish)
Immersion
1. MS-222 + adjunctive method in hypoxia tolerant spp
Injection
- pentobarbital or ketamine, ketamine/medetomidine, propofol then pentobarbital
Physical
- decapitation + pithing, blunt force trauma then pithing or exsanguination, rapid chilling then Na or Ca hypochlorite for zebrafish embryos)
What are adjunctive euthanasia methods for finfish (2nd step after rendering unconscious)?
Decapitation, pithing, exsanguination, freezing
Dilute Na or Ca hypochlorite solution (500 mg/L) after rapid chilling for embryos
Unacceptable euthanasia methods for finfish
Flushing, slow chilling, freezing unanesthetized, anoxia, caustic chemicals, 70% ethanol (onset too slow)
Which mutation of zebrafish is the ortholog of the mouse brachyury (T) gene which provided evidence that homologous genes perform common functions and could be translated to other vertebrate model systems including mammals?
Ntl (no tail)
How to treat gas bubble disease?
Aeration, increase temp
Survival blood collection technique in zebrafish
dorsal aorta or posterior cardinal v.
Zebrafish fry with beards and high mortality
Lecythophora mutabilis (fungus)
Occludes mouth/gills –> starve/asphyxiate
