Here we demonstrate how to breed the Mexican Tetra Astyanax Mexicanus, raise the larvae, and perform whole-mount immunohistochemistry on post larval fish to compare the phenotypes of river and cave adapted fish. We raise the fish at the same density on a nutrient rich diet of rotifers. This technique ensures consistent growth and provides advantages in cost, biosecurity, and water quality.
To begin, place a plastic mesh in the bottom of a five gallon tank. Then fill the tank with fish ready water. Affix a water heater to the side of the tank.
Add one female and two male Astyanax Mexicanus fish that are more than one year old to the tank. Set the temperature of the water heater to 24 degree Celsius. After 24 hours, increase the temperature by one degree Celsius.
Shine a flashlight into the bottom of the tank everyday to check for eggs. Once the eggs are identified in the breeding tank, remove the adult fish and the plastic mesh. Use a beaker to reduce the water depth to 10 cm.
To estimate the time of fertilization, use a transfer pipette to place several eggs into a petri dish and view them with a stereo microscope to determine their stage. Remove opaque eggs or feces. Then fill the tank with fish ready water and add seven drops of methylene blue to the water.
After this, add a heater and aquarium bubbler to the tank. Set the heater to 24 degrees Celsius and adjust the airstream regulator to produce a gentle stream of bubbles. Then cover the tank to maintain water temperature.
Add 20 grams of salt to eight liters of fish ready water and stir it until it is dissolved. Then add one liter of the prepared water to each nursery container. Next use a transfer pipette to move 20 of the hatched larvae to each nursery container.
Agitate the water in the larger tank to ensure that all larvae have been removed. To prepare the fish food, add three ml of algae mixture to one liter of harvested rotifers. Five days post fertilization add three ml of the fish food to each nursery container.
The rotifers should be visible in dense groups at the corner of the containers unless visible near the center. Check the container for rotifers everyday and add more food if the concentration is depleted. Remove any dead larvae.
First, to clear the gastrointestinal tract of food contents, pour the nursery container with the fish of the desired age through a nylon mesh strainer. Place the strainer into a container with clean fish ready water and withhold food for 24 hours. After euthanizing the fish, according to institutional guidelines, use a transfer pipette to transfer the fish to a conical tube.
Replace the euthanasia solution with fixative. And incubate the tube with rocking overnight at 4 degrees Celsius. After this, remove the fixative and replace it with PBST.
Incubate the tube for another 15 minutes with rocking. And repeat the wash process one more time. Next, use a transfer pipette to move the fish to a four ml glass vial with a screw top cap.
Remove the PBST from the vial and add three ml of blocking solution. Incubate the vial at room temperature for one hour with rocking. After this, use a transfer pipette to remove the blocking solution and replace it with primary antibody diluted in blocking solution.
Incubate the vial overnight at room temperature with agitation. Next, wash the fish with PBST three times, as described previously. Replace the PBST with secondary antibody diluted in blocking solution.
And incubate the vial overnight at room temperature with agitation. Finally, wash the fish three times for 15 minutes with PBST. Using this protocol, surface fish Tinaja, Molino and Pachon cave fish were bred in static breeding tanks.
The surface and Pachon spawns, with fertilized embryos, always produced hatched larvae. While the Molino and Tinaja were unsuccessful some of the time. In general, surface fish produced the greatest number of larvae per spawn, followed by Pachon, Tinaja, and Molino respectively.
The whole-mount immunostaining technique was successfully used to label neurons and pancreatic cells at stages up to 12.5 days post fertilization. It's important to remove any dead fish as you raise the larvae and record the number of surviving fish. If many fish die it may be due to bacterial or fungal contamination.
Tanks should be sterilized with 70%ethanol prior to use. Immunostaining technique we described has been successfully used for the antibodies listed in the materials table. After immunostaining, fish can be whole mounted or sectioned to view any tissue of interest.
In combination, the breeding, hatching and immunostaining procedure provide a robust method for assessing gene activity NC2 in a manner comparable between laboratories.
