A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

Laboratory housing of turquoise killifish can be scaled up to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

The overall goal of this protocol is to successfully raise a laboratory colony of turquoise killifish to study aging and age-related diseases in a rapid and high throughput manner. This method can help answer key questions in the aging field such as whether single gene mutations or environmental interventions can modulate the aging process in vertebrate species. The main advantage of this technique is that by following our basic protocols, several laboratories can now perform experiments using turquoise killifish.

Generally, individuals new to this method will struggle as killifish husbandry has some unique features. For example, embryos are raised on a dry substrate prior to hatching which is very different from canonical fish models such as zebrafish. To begin, set up 9.5 liter breeding tanks each with a five-week old male and two six to seven-week-old females.

Then fill a plastic container with autoclaved sand approximately two to three centimeters deep and place the sandbox in the center of the breeding tank. Allow the turquoise killifish to breed continuously and harvest embryos once a week for embryo incubation. To harvest one cell stage embryos to use for injection and generation of transgenic lines, set up a breeding tank with one male and two female fish as just demonstrated.

Two days prior to embryo collection, transfer the male into an individual tank and keep it in visual contact with the adult females. On the day of collection, add the male and a sandbox to the breeding tank and let the fish spawn for two hours. After spawning and removing the sandbox from the tank, empty the sandbox into a strainer and use system water to rinse it.

Then partially submerge the strainer in system water and gently swirl it so that the embryos group together in the center. Using a two milliliter Pasteur pipette, collect the embryos and transfer them to a 90 millimeter Petri dish with approximately 40 milliliters of system water. Under a light microscope, inspect the embryos in the Petri dish and remove those presenting a ruptured chorion and signs of damage.

After bleaching the embryos according to the text protocol, inspect and remove any dead embryos that would be stained blue. Once the embryos have been allowed to incubate in fresh methylene blue solution, check that the developed embryos have visible black eyes. Then using a disposable Pasteur pipette or fine curved tweezers, transfer embryos onto a filter paper plate.

Retain any undeveloped embryos in methylene blue and monitor them daily. Once black eyes have developed, transfer them to solid substrate medium. With forceps, spread the embryos approximately five millimeters apart up to 100 embryos per 90 millimeter plate.

Then use paraffin to seal the dish and incubate the embryos at 28 degrees Celsius for two to three weeks until they have fully developed golden irises and are ready for hatching. For long-term storage up to one year, following incubation in methylene blue solution for three days, transfer embryos onto solid substrate medium plates as just demonstrated and incubate them at 17 degrees Celsius. To hatch the fish, use fine curved tweezers to carefully transfer 50 to 100 developed embryos into the hatching box and fully immerse them in humic acid solution no deeper than two centimeters at four degrees Celsius.

A critical step in this protocol is hatching killifish embryos which is achieved by transferring embryos incubated on a dry substrate to a hatching solution containing humic acid provided with sufficient aeration. Cover the hatching box with a lid and incubate it at 28 degrees Celsius in a hatching incubator. To supply sufficient aeration, connect the box to an air supply with tubing.

To maintain adequate water quality in the hatching box, once per day from the day after hatching, replenish the box with a one to one ratio of autoclaved system water maintaining a final depth of two centimeters. Transfer any unhatched embryos back to the solid substrate and attempt hatching a week later. To raise juvenile and adult fish at five days post hatching, move juveniles to the water recirculation system by using disposable plastic pipettes or a plastic spoon to carefully transfer five juveniles per 0.8 liter tank equipped with a 400 micrometer fry screen.

At 14 days of age, transfer juvenile fish to a 2.8 liter tank equipped with an 850 micrometer fry screen. From this point onwards, label each tank with a fish ID.For survival assays, individually house each fish in a single tank. Fish raised for survival assays are also individually housed in a 2.8 liter tank.

For the following seven days, feed juveniles twice a day with five milliliters of brine shrimp supplemented with one to three live blood worms. At four weeks of age, feed each fish twice per day with brine shrimp and one milliliter of blood worms. At this stage, ensure that the fish reaches complete sexual maturation by checking for the presence of large dorsal, anal, and caudal fins with signs of coloration in males and round abdomens full of eggs in females.

Shown here is a representative survival curve for 70 male turquoise killifish. Proper husbandry results in median survival ranging from 12 to 28 weeks in the GRZ strain. Variations in median survival depend on diet, feeding frequency, and housing temperature conditions.

The survival curve for fish raised under poor husbandry reveals an increase in early mortality and repetitive sudden drops in survival throughout time as compared to fish raised under optimal husbandry conditions. While following this protocol, it's important to closely monitor water condition and excess food from freshly hatched fry as it's essential that these are maintained in a clean environment. After its development, this technique paved the way for researchers to explore experimental manipulations in turquoise killifish including transgenesis, genome editing, and dietary manipulations.

After watching this video, you should have a good understanding of how to successfully raise turquoise killifish in the laboratory and use this model to explore the biology of aging in the shortest-lived vertebrate that can be raised in captivity.