BB Ch19 Zebrafish Flashcards
What is the genus and species of the zebrafish?
Danio rerio
How is this species used in biomedical research?
model of vertebrate embryonic development, gene function analysis, and mutagenesis.
T/F Zebrafish are sexually dimorphic.
T; Females are slightly larger, more silvery , and slightly rounded. Males are more streamlined and more brightly colored than females.
How is the female zebrafish induced to spawn?
After a period of darkness, the initial appearance of light, and persistent rubbing of the female by the male induces her to spawn.
In the laboratory, how do you prevent the adults from consuming the spawned eggs prior to collection?
Egg collection devices or rows of glass beads or marbles placed at the bottom of the tank
Name three attributes of the zebrafish that contribute to its popularity as a research model.
Small size, low maintenance, large litter size, easily manipulated embryos (develop outside of the mother), easily observed embryos (transparent ova), rapid development of embryos
What is the standard temperature for zebrafish maintenance?
28.5°C (83.3°F)
T/F The initial stages of embryo development involve a series of cleavages that occurs over several hours to days.
F: The initial series of cleavages takes place over a period of a few hours. The entire process from fertilization to hatching takes 3-4 days.
What does MBT stand for in regards to development of the zebrafish embryo and what is it’s significance?
MBT= midblastula transition & occurs 3 hrs post-fertilization; MBT represents the activation of zygotic transcription - all processes up to MBT are regulated by maternal factors; important because DNA constructs injected prior to MBT are NOT expressed until after MBT, RNA constructs ARE active prior to MBT
Define gastrulation and when does it occur?
Gastrulation, the process whereby the three germ layers take up their final positions in the embryo, begins about 5.5 hpf and is complete at 10 hpf.
Define “epiboly” and when does it occur?.
epiboly = When the embryonic cells begin to spread down over the yolk after sitting in a ball of cells on top of the yolk. This occurs about 4.5 hpf.
At what point does the embryo become responsive to touch?
24 hpf; This signals the presence of neuromuscular connections.
When do embryos hatch from their chorions? When do they begin to feed?
Hatch on day 3-4 Begin to feed on day 4-5
T/F The zebrafish is currently the only model organism for which large-scale genetic screens can be performed rapidly.
False: these studies can be done using C. elegans or Drosophila, but zebrafish ARE the only VERTEBRATE species in which these studies can be conducted
Describe the basic two-generation mutagenic screening process commonly employed using zebrafish.
Males are mutagenized and outcrossed to wild-type females. The offspring are inbred, and the F3 generation is scored for mutations.
ENU primarily generates ___________ mutations while radiation leads to ___________________. What is a drawback to these methods?
ENU primarily generates POINT mutations while radiation leads to LARGER DEFECTS (inversions, deletions). Drawback: rapid methods of mutagenesis, but veery labor intensive to ID the mutated genes
In addition to chemical mutagenesis and radiation, what is another means of inducing mutations in zebrafish embryos? What is an advantage of this method?
Viral infection to integrate a piece of DNA a known sequence into the genome. Advantage is that PCR can be used to rapidly ID the mutated gene(s)
What advantage does the zebrafish have over Xenopus and the mouse in regards to embryological studies?
Allows the unique combination of embryological and genetic tools. Xenopus genetics is not as well characterized or manipulable as the Zebrafish. Zebrafish embryos can be observed through development while mouse embryos develop in the uterus and cannot be observed as closely.
Define the terms “fate” and “committed” in the context of embryologic development.
FATE = the final differentiation state of particular cells. Cells are said to be COMMITTED if, following transplantation or explantation, the cells differentiate along the same pathway as dictated by the fate map. In other words, committment relates to the influence of environmental conditions on the fate of cells.
How is analysis of gene function performed in zebrafish?
By transient or stable gene expression accomplished by pressure-driven microinjection of DNA or RNA into immobilized embryos viewed under a stereomicroscope
Injected DNA constructs are not expressed until ________________, while injected RNA is translated __________________________.
Injected DNA constructs are not expressed until MIDBLASTULA TRANSITION (MBT), while injected RNA is translated IMMEDIATELY UPON INJECTION.
________________ions function as a “universal second messenger” carrying information across cells, tissues, organs, and organisms.
Calcium
What is aequorin and what is it used for?
A calcium-sensitive bioluminescent protein that emits light when it comes into contact with calcium ions and is used to study the role played by calcium signaling in biological systems.
Why is the monitoring of calcium signaling in the zebrafish embryo important to toxicologic studies?
One of the first responses of a cell to toxicant-induced injury is a loss of its ability to regulate intracellular calcium levels.
As a general rule, temperature changes should be limited to ____°C/day to avoid an internal shock reaction.
+/-1.5
What is the optimum temperature range for zebrafish?
75-82°F (24-28°C) Higher temperatures are recommended to stimulate egg laying/reproductive behavior and to facilitate development of embryos
Larvae are usually more/less tolerant to temperature changes than adults.
less
What is the cause of gas bubble disease (GBD)?
Too much dissolved oxygen or other compressed gases. This results in air emboli that disrupt the flow of blood across the gill or in some instances can rupture the vessels. The phenomenon is similar to the “bends” in humans.
Fish gathering at the surface of the tank may be an indication of __________?
Low dissolved oxygen levels within the water
What is the preferred pH range for zebrafish?
6.8-7.2 w/ pH 7.0 being optimal
High/low pH results in higher concentrations of un-ionized ammonia (NH3)?
Higher (>8)
In a closed, recirculating system, the pH will gradually ___________ due to the production of _____________ during the nitrification process as the bacteria within the biofilter convert ammonia to nitrate.
In a closed, recirculating system, the pH will gradually DECREASE due to the production of ACIDS during the nitrification process as the bacteria within the biofilter convert ammonia to nitrate.
The pH will ___________ in poorly aerated systems due to accumulation of carbon dioxide.
Decrease
Conductivity is an imprecise method to measure ________________.
salinity
What is the preferred conductivity range for optimal growth and breeding of zebrafish?
3-500ms
Total water hardness is a measure of what ?
calcium and magnesium salts in the water.
T/F Zebrafish are generally considered to be a “hard” water species.
True (optimum calcium and magnesium levels = 80-200ppm)
Commercial test kits usually measure hardness in terms of ____________ content in the water.
Amount of CaCO3 - calcium carbonate
What are the primary sources of nitrogen (in the form of ammonia) within an aquatic system?
Uneaten, decaying food and excretion from fish
Ammonia is converted to nitrite by ___________, and the nitrite is then converted to nitrate by ________________.
Ammonia is converted to nitrite by Nitrosomonas spp., and the nitrite is then converted to nitrate by Nitrobacter spp.
How are nitrates removed from the system?
Periodic water changes and plant metabolism
Approximately _________% of the total water in a recirculating system should be drained off and replaced daily.
5-10%
T/F As with rodent colonies, a key component to maintaining a healthy zebrafish colony is disease surveillance of sentinel animals within each water system.
True
In most cases, municipal tap water must be treated to move compounds such as _________, ____________, and ______________that are toxic to fish.
chlorine, copper, and chloramines
What must be done to distilled or reverse osmosis water to make it acceptable for housing fish?
It must be conditioned by the addition of mineral compounds.
Name two disadvantages to using fresh water snails to control algae in a laboratory aquatic system.
Snails rapidly multiply and may climb into pipes and obstruct water flow. They also act as the intermediate host or vector for the larval stages of a number of parasites (e.g. digenetic trematodes).
What are signs of vitamin C deficiency in fish?
Reduced growth, reduced egg viability, scoliosis, lordosis, fin/tail erosions, and mortality
Due to their small size, zebrafish up to 10-14 days old are often fed an exclusive diet of __________________.
Paramecium spp. or brine shrimp
Live prey items that are often used to supplement the diet of zebrafish include__________.
Brine shrimp nauplii, Drosophila larvae, and rotifers
Adult zebrafish should be fed _________ times daily.
1-2 times daily
Fish obtained from local pet shops should be considered high risk for harboring pathogens and quarantined for a minimum of ________________ if they are to be introduced into a research facility.
30-45 days: acquisition of zebrafish from pet stores is strongly discouraged
Name three species of Mycobacterium that may affect zebrafish.
M. marinum, M. fortuitum, M. chelonae
What clinical signs are associated with Mycobacteriosis in zebrafish?
Highly variable, but include poor growth rate, chronic wasting, emaciation, decrease in reproductive rates, and slightly increased mortality within a colony for chronically infected fish. Acutely diseased animals often demonstrate “dropsy syndrome” which consists of abdominal distension and scale edema.
This the wet mount prep from the gut of a zebrafish. What is the organism?
Pseudocapillaria tomentosa
Multiple granulomas are observed in the liver, spleen, kidney and reproductive organs of a zebrafish. What is the most likely diagnosis.
Mycobacteriosis
Tissue from zebrafish stained with Ziehl-Neelsen. What is the diagnosis?
Mycobacterium spp. in imprints stained with Ziehl-Neelsen.
A. Acid-fast (red) bacilli.
B. Imprint showing Mycobacterium bacilli (arrows) in pigment-containing macrophages (M = melanin granule).
What is the diagnosis?
Tail Rot.
In addition to Bacterial Gill Disease, gliding/filamentous bacteria (i.e., Flavobacterium, Flexibacter or Cytophaga spp.) can cause skin and fin infections, resulting in diseases such as fin or tail rot. Note frayed tail fin and whitish appearance of the tail region due to loss of the epithelium.
Zebrafish are reported with lethargy and hanging near the surface of the water column exhibiting labored breathing. Some of them have a greyish to rusty color sheen on the surface. Below is a histopath section of the gill.
What is the diagnosis?
Velvet Disease: caused by Piscinoodinium spp.: yellowish, parasitic dinoflagellate in which one stage infects the skin and gills of fish.
Histological section of gills section heavy infected with Piscinoodinium (X).
Arrow = early, small stages.
These fish are observed in your colony. Although these clinical signs are not pathognomonic for any disease, what disease is at the top of your differential diagnosis?
Pseudoloma neurophilia
Name the disease and etiologic agent.
White Spot Disease - Ichthyophthirius multifiliis
Name the disease.
Gas Bubble Disease
Zebrafish with gas bubbles around eye due to GBD
Fish are reported exhibiting labored breathing and accumulating at the surface. Samples shown below are collected at necropsy. What is the diagnosis?
Bacterial Gill Disease (BGD): caused by Flavobacterium brachiophilum.
A. Wet mount of gills showing severe, diffuse epithelial hyperplasia. Proliferation of epithelial cells has fused primary lamellae (P).
B. Histological section of gill with BGD. Arrow = masses of bacteria.
C. Wet mount preparation, phase contrast showing numerous filamentous bacteria associated with gill.
