The African turquoise killifish (Nothobranchius furzeri) has emerged as a promising new model organism in biology, particularly in the fields of aging and ecology. Given their short life span and rapid life cycle, turquoise killifish are rapidly growing as a promising new model organism in biology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test - in a short time - experimental interventions that can lead to alterations of the aging rate and life expectancy.
This species is characterized by a unique life cycle for a teleost, consisting of embryonic diapause, rapid sexual maturation, and an extended post-reproductive life stage. Turquoise killifish live in seasonal fresh water bodies that form during the rainy season in the African savannah in Zimbabwe and Mozambique. Recent work has contributed to elucidating the biology of this species both in captivity and in the wild.
The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span - ad hoc husbandry practices are in urgent demand. Although a laboratory protocol has already been published for this species, in the present protocol we develop a comprehensive list of experimental laboratory guidelines that are specifically aimed at studies that investigate aging and survival.
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 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. The present protocol enables researchers already familiar with zebrafish and medaka husbandry to become versed in turquoise killifish husbandry by adopting a minimum number of key adjustments.
A male African Turquoise Killifish.
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Setting Up Your Killifish Tank
Three different tank sizes are recommended: 0.8 L, 2.8 L, and 9.5 L. Prepare humic acid (hatching) solution by dissolving 1 g/L humic acid in system water. Prepare methylene blue solution by dissolving 100 µL/L of methylene blue stock solution in previously autoclaved system water. Since methylene blue is light-sensitive, keep the solution in dark bottles, or cover with foil.
Prepare coconut fiber as a solid substrate for embryo incubation. Alternatively, use a filter paper. Presoak coconut fiber with distilled water. Compact coconut fiber to a height of 1 cm, with sterile tissue. Remove most of the moisture from the coconut fiber by pressing a paper towel on top of the plate, letting the paper to absorb the excess water. Heat a metal spoon over the flame, and press down on the entire surface of coconut fiber. Prepare the filter paper as a solid substrate for embryo incubation. On the day of embryos transfer, place 3 layers of filter paper disks that fit the 90-mm Petri dish.
Breeding Turquoise Killifish
Following this protocol, sexual maturity is reached at ~4 weeks post-hatching and fecundity peaks between 7 - 9 weeks. Setup a 9.5 L breeding tank. As male African turquoise killifish display dominance during mating, which might lead to harassment of females, choose a male with a slightly smaller body size than the female to reduce mating stress and increase reproductive output. Let turquoise killifish breed continuously and harvest embryos once a week for embryo incubation.
Embryos to use for injections need to be synchronized at the one-cell-stage, and this requires that they are collected immediately following fertilization.
Embryo Collection
Embryo collection is performed by sieving and harvesting embryos from the sand box. Under normal conditions, each sand box should contain from 30 to 200 embryos. On the day of collection, remove sand box from the breeding tank. Empty the sand box into a strainer (~0.9 mm strain size) and rinse with system water. Proceed directly to Embryo bleaching.
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Embryo Bleaching
Embryo bleaching prevents microorganisms present in fish tanks from contaminating the incubation media. Remove H2O2 solution with disposable Pasteur pipette and wash embryos three times for 5 min with 50 mL of methylene blue solution. Incubate embryos at 28 °C to increase synchronous embryos development, at a maximum density of 100 embryos per 90 mm Petri dish in 40 mL of methylene blue solution.
Do not extend embryo incubation in the bleaching solution. This may cause damage to the egg chorion and increase embryo mortality.
Embryo Incubation in Methylene Blue
Liquid incubation in methylene blue solution prevents parasite growth and enables detection of dead embryos and unfertilized eggs. Return Petri dish to 28 °C incubator (Figure 1A). Within 7 - 10 days, ensure that the developed embryos show visible black eyes. Repeat steps 3.3.1-3.3.3 daily until embryos have visible black eyes.
Embryo Transfer to Filter Paper
Turquoise killifish embryos can develop on a dry substrate, recapitulating natural conditions. Additionally, dry embryo incubation enables researchers to synchronize embryos and hatch them on the same day. Incubate embryos at 28 °C for 2 - 3 weeks, until they have fully developed golden irises and are ready for hatching (Figure 1C).
Embryo Transfer to Coconut Fiber
Autoclaved, sterile coconut fiber (or organic peat moss) can be used as a valid alternative medium for solid substrate incubation. Incubate embryos at 28 °C for 2 - 3 weeks, until they have fully developed golden irises (e.g. in Figure 1C).
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For long-term storage (up to one year), transfer embryos at 3-days post collection from methylene blue solutions to a solid-substrate plate at 17 °C.
Figure 1: Representative embryonic developmental stages with respective incubation substrate.
Hatching Turquoise Killifish
Using fine curved tweezers, transfer carefully 50 - 100 developed embryos into the hatching box filled with the humic acid solution at 4 °C. The humic acid solution consists of 1 g/L humic acid in system water. Autoclave and store at 4 °C for up to 10 weeks. Make sure that all embryos are completely immersed. The humic acid solution must be shallow, not deeper than 2 cm. Place the hatching box into the 28 °C hatching incubator. Cover the hatching box with the lid.
To supply sufficient aeration, connect hatching box by tubing with air supply. Transfer unhatched embryos back to the solid substrate, and attempt hatching a week later.
Upon hatching, turquoise killifish are readily capable to uptake and consume live food. For optimal growth, feed fry twice per day with excess freshly hatched brine shrimp (Artemia salina). The sign of full satiation is the orange-colored abdomens of fry after 10 - 15 min of each feeding.
Raising Juveniles
At five days post-hatching, move juveniles to the water re-circulation system. Using disposable plastic pipettes (or a plastic spoon), carefully transfer five juveniles per 0.8 L tank equipped with 400 µm fry screen. It is possible that a portion of juvenile killifish will not have filled the gas bladder, resulting in a typical "belly-slider" phenotype, characterized by fish not reaching proper buoyancy, forcing them to continuously swim, causing severe malformations in adult fish.
Feed juveniles twice per day with freshly hatched brine shrimp in excess until 14 days post-hatching. At 14 days of age, transfer juvenile fish to 2.8 L tank equipped with an 850 µm fry screen. From this point onwards, label each tank with fish ID, indicating hatch date, strain information, fish gender and fish identification number (Figure 3).
For the following 7 days, feed juveniles twice per day with ~2 mL of brine shrimp per fish. At this stage fish can be supplemented with 1 - 3 live blood worms (in case the blood worm larvae are too large for the fish, chop them into smaller pieces with a razor blade). After 3 weeks from hatching, remove fry screen from the back of the tank and start to feed each fish twice per day ~2mL of brine shrimp and 0.5 mL of blood worm. At 4 weeks of age, feed each fish twice per day with ~2mL of brine shrimp and 1 mL of blood worm. At this stage ensure that fish reach complete sexual maturation.
Check for the presence of large dorsal, anal and caudal fins with signs of coloration in males and round abdomens full of eggs in females. Raising adult fish in individual tanks for survival cohort studies may negatively affect fish behavior and health.
Feeding
Laboratory turquoise killifish can be fed a combination of baby brine shrimp (Artemia salina nauplii) and blood worm (Chironomus spp. larvae). Turquoise killifish fry are fed exclusively baby brine shrimp. Juvenile and adult fish are fed twice a day both brine shrimp and blood worm (Figure 2). Add 20 g of brine shrimp cysts into the hatching solution. Inspect that brine shrimp cysts do not float on the surface of the water and ensure proper oxygenation and circulation of the culture.
Harvesting Hatched Brine Shrimp
After ~36 h from the starting culture, brine shrimp are ready for harvesting (instar II phase). After 10 min, remove brine shrimp shells (brown color) from the top of the 5 L container and filter the hatched brine shrimp (orange color) through a mesh. Transfer brine shrimp from the 2 L container into squeeze bottles for feeding. Culturing brine shrimp is fairly robust and reliable.
After 18 - 24 h, supply the culture once more with 500 µL of HUFA. Immediately prior to feeding, filter an appropriate amount of blood worm through a strainer using RO water. With a plastic Pasteur pipette (narrow tip removed), take up the blood worm mixture for feeding.
Feeding laboratory killifish colonies with live food from un-controlled sources adds a risk for external contaminations from parasites and potentially pathogenic microbial communities.
Why an Aquaculture Feeding System?
Water Parameters and System Maintenance
Husbandry of organisms whose intended use is adult phenotyping requires highly stable husbandry conditions throughout the life span of the target species. Therefore, culturing water organisms, such as turquoise killifish, necessitates strict control of water parameters. Water recirculation, with additional four-steps water filtration, ensures a robust basis to attain control over water parameters, providing all the tanks with the same water conditions over time.
Water recirculation system scheme: First, waste-water from fish tanks flows through solid particles metal filter that captures all un-eaten food debris and larger particles. Metal filters are rinsed three times a week; Second, following the first mechanical filtration, water is conveyed in large sumps and then pumped to a biofilter, where bacteria convert ammonia to nitrites and nitrates; Third, from the biofilter, water is pumped to 25-µm filter sleeves, which trap finer size particles. Finally, water flows through ultraviolet (UV) lamps that sterilize water from bacteria and viruses.
Although killifish tolerate wide range of salinity, to avoid oodinosis, maintain conductivity within the range 650 - 710 micro-Siemens. A year-long 12 h light/dark cycle ensures colony health and productivity. Turquoise killifish proper husbandry results in median survival ranging between 12 - 18 weeks in the GRZ strain (e.g. Figure 4A). Variations of median survival depend on diet, feeding frequency, and housing temperature conditions.
Figure 4: Representative survival curve for 70 male turquoise killifish.
Critical steps within the protocol include shipping embryos within 8 - 30 °C temperature range. In case of breeding, fecundity depends on feeding frequency and food quality; therefore, we recommend at least two feedings a day per breeding tank to raise embryos yield. During embryo bleaching, do not extend embryo incubation in the bleaching solution. This may cause damage to the egg chorion and increased embryo mortality. When incubating embryos with methylene blue, do not prolong incubation of ready-to-hatch embryos for longer than 2 weeks as their viability will be dramatically reduced. For hatching turquoise killifish, low temperature of humic acid solution improves hatching and complete immersion of the embryos in the solution allows synchronized hatching.
Constant exposure of embryos to methylene blue may induce long-term changes in adult fish physiology. However, group housing for survival cohort studies adds significant confounding factors due to the establishment of social dominance and male territories, leading to strict social hierarchies. Therefore, we judge that isolation of male fish for survival studies is a reasonable compromise.
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.
Housing Density and Lifespan
To investigate the effect of housing density on juvenile growth, newly hatched fish were reared at densities of 1, 2, 4, 10, and 40 fish per S-tank (15 × 9 × 10 cm, 0.65 L water). At three weeks posthatching (wph), body weight was investigated, and it was found that lower housing densities resulted in faster growth.
The average weight in single housing (one fish per tank) was 5.4 times greater than the average weight in group housing at the highest density (40 fish per tank). The average weight in single housing was also 1.7 times greater than the average weight in group housing at the second-lowest density (two fish per tank). In addition at three wph, single-housed juveniles reached the onset of sexual maturity stage, where male fin coloration was observed. On the other hand, group-housed fish at the highest density (40 fish per tank) had just reached the middle stage of juvenile growth.
Regardless of sex, it took 22 days for group-housed fish and 12 days for single-housed fish to grow from stage 2 to stage 6, indicating that the growth rate of single-housed juveniles in stages 2-6 was 1.8 times faster than that of group-housed juveniles. However, regardless of sex, it took 14 days for group-housed fish and 10 days for single-housed fish to grow from stage 4 to stage 6, indicating that the growth rate of single-housed juveniles even in the stages 4-6 was 1.4 times faster than that of group-housed juveniles.
Unexpectedly, the mean adult lifespan in single-housed fish was longer than that in group-housed fish in both sexes (Figure 3A, male, 32%; female, 17%; male and female, 24%). Both fish housed at a density of one or two fish per tank exhibited a longer mean adult lifespan than fish housed at a density of 10 fish per tank in both sexes.
The cumulative number of live embryos in single-housed fish was less than that in group-housed fish. In group-housed fish, the number of eggs laid was high for the first two weeks, medium for the subsequent five weeks and low thereafter, whereas, in single-housed fish, it was medium for the first nine weeks and low thereafter.
Automated Feeding System
Controlling feeding automatically is a critical component for the development and scalability of a model organism. To address these challenges and develop the scalability of the killifish as a model system, an automated feeding system for killifish feeding has been created. The automated feeder that was designed and built is placed on top of each animal’s 2.8 L tank (Figure 1A) and drops dry food (e.g., Otohime fish diet) from a small feed hopper (Figure 1B) directly into the tank (which houses one individual fish). The feeder is powered by an attached battery, and the food pellets are automatically segregated from the hopper by a rotating acrylic disc, with the resulting pieces of food dropping onto the water through a 3-mm diameter opening cut out in the supporting acrylic plate below (Figure 1C, Figure 1-figure supplement 1A, Figure 1-video 1).
The 3-mm opening rotates from under the feed hopper, collects food, and travels to the drop site above the tank opening. The automated feeder can also feed during the night, if desired, which would not be practical for manual feeding. The automated feeder can deliver up to 5 mg per unit every 10 min, or 720 mg per 12 hr period, representing a potential 20× increase over the baseline dietary regime for the day (Table 1).
The automated feeders also allow us to design a novel associative learning assay to test cognitive function in the African killifish. Manual feeding requires individual attention to each tank on a daily basis, multiple times per day, whereas automated feeding requires an initial setup and biweekly checks to replace batteries and add food to the devices.
In killifish, dietary restriction has been done by every other day feeding because it is difficult to do otherwise with manual feeding. The automated feeder can also feed during the night, if desired, which would not be practical for manual feeding.
Conclusion
The African turquoise killifish is a valuable model organism for aging research due to its short lifespan and genetic tractability. Proper care and husbandry are essential for maintaining healthy colonies and conducting accurate experiments. By following the guidelines outlined in this protocol, researchers can successfully raise and study these fascinating fish.
