Annual Killifish: Peat Incubation and Hatching of Diapause Eggs

How Nothobranchius and Austrolebias eggs survive the dry season in three diapause stages, and how breeders dry peat, store it for months, wet it to hatch fry and deal with belly sliders.

Annual killifish and their temporary pools

Annual killifish live their whole life in water bodies that exist for only part of the year. According to Wikipedia, the African genus Nothobranchius ranges mainly across East Africa from Sudan to northern South Africa, with the greatest diversity in Tanzania, and inhabits ephemeral pools that fill only in the rainy season. The South American genus Austrolebias, with 46 recognised species, lives in periodically drying ponds of the Río de la Plata, Patos–Mirim and Mamoré basins; most species stay under 10 cm, a few reach 15–22 cm.

When the pool dries the adults die and only the eggs remain. Wikipedia describes Nothobranchius eggs as having a very hard chorion that resists desiccation and hypoxia, and notes that annual killifish eggs would not survive more than a few weeks if kept permanently under water. A review of Nothobranchius furzeri in PubMed Central (PMC) gives the scale of the problem: the dry season lasts 5–11 months, many wild pools persist only 3–10 weeks, and water temperature swings between 20 and 36 °C within one day. The fish answer with speed: per Wikipedia, N. furzeri reaches sexual maturity 14 days after hatching. Seriously Fish gives aquarium lifespans of 6–12 months for N. rachovii and 6–16 months for N. eggersi, with maturity at around three weeks.

Diapause: three developmental pauses

The embryo, not the adult, is the survival stage, and two open-access papers in PMC describe three points at which it can stop developing.

• Diapause I follows epiboly, during a "dispersed cell" phase unique to annual killifish. It is facultative: laboratory embryos usually skip it, while low temperature or lack of oxygen can induce it.
• Diapause II occurs midway through development, at about 38 pairs of somites, when rudimentary organs are visible but the circulatory system is inactive. It is the stage most resistant to temperature extremes, drying and oxygen deprivation; egg banks in dry sediment consist exclusively of diapause II embryos, and the pause can last well over a year.
• Diapause III occurs when the embryo is fully developed and able to hatch but waits, at a reduced metabolic rate, for a hatching cue, probably heavy rain and inundation of the pool.

Not every egg follows the same path. Direct-developing "escape" embryos skip diapause II and are hatch-ready 24–26 days after fertilisation, whereas diapausing embryos may need far longer. The trajectory responds to maternal effects, temperature and light and has a random component, which the PMC review reads as bet-hedging against false rains. Increased moisture combined with a drop in oxygen is thought to move embryos from diapause II to III.

Spawning substrate and collecting the eggs

Seriously Fish notes that N. rachovii and N. eggersi do not require peat, although peat moss is the usual spawning medium. Tropical Fish Hobbyist (TFH) uses fertiliser-free, horticultural-grade sphagnum peat, soaked or boiled so that it sinks; Practical Fishkeeping (PFK) lists peat moss, peat fibre or coconut coir. South American "diving" species submerge in the substrate to spawn, so TFH advises a peat layer at least 50 percent deeper than the length of the largest fish. A female releases her eggs within about eight hours, 24 at most, so TFH leaves a pair in the spawning container for 10–24 hours without feeding and then rests the fish for a week. PFK breeds N. rachovii with one male and at least two females at around 22 °C.

Drying the peat: the moisture test

The critical step is bringing the peat to a moisture level that mimics drying mud.

• TFH: squeeze the netted peat until little water remains, spread it between layers of newspaper for 24 hours and pack it when only slightly damp; for South American species the test is that squeezing produces no water.
• PFK: let the peat dry slowly in a container until the surface turns lighter brown and crusty, which locks enough moisture underneath for the eggs.

TFH packs the peat in a plastic bag that is tied or folded rather than heat-sealed and marks the date. Seriously Fish stresses that captive strains carry collection codes such as "Beira 1998" to prevent hybridisation, so species and population belong on the label too.

Incubation times and temperatures

Incubation length is species-specific and temperature dependent. Seriously Fish stores N. rachovii eggs at 21.1–25 °C for 5–7 months before wetting and N. eggersi eggs at the same temperature for 3–4 months. PMC laboratory work with N. furzeri develops embryos at 27–28 °C, keeps them at 17 °C for storage of up to a year, and finds that a lower incubation temperature increases the share of embryos entering diapause. Species detail is covered in the existing guides nothobranchius-furzeri-breeding-guide and nothobranchius-guentheri-breeding-guide.

Wetting and hatching

1. Check readiness. The PMC husbandry protocol treats visible black eyes and later golden irises as signs of a developed embryo. TFH does a test hatch with about a quarter of the peat: if the eggs are ready, fry appear within 24 hours.
2. Wet the peat in shallow water. PFK uses a few centimetres; TFH stirs the peat into a small tank of water from an established aquarium. Most developed fry hatch within two days (TFH).
3. Use cool water. PMC protocols hatch N. furzeri in a chilled humic-acid solution (4 °C) that is then moved to 28 °C; low temperature improves hatching and full immersion synchronises it.
4. Re-dry what did not hatch. TFH dries the peat again and stores it for another month or two; PFK confirms that wetting can be repeated several times.

Belly sliders

"Belly sliders" are fry whose gas bladder never inflates, so they slide along the bottom or swim continuously. The PMC housing protocol for N. furzeri names insufficient aeration during incubation as the cause and excludes such fish from breeding. PFK reports an angelfish study (Sanabria and colleagues, 2009) in which egg disinfectants such as methylene blue and hydrogen peroxide increased gas-bladder non-inflation, while rearing at 28 °C instead of 26 °C reduced belly sliders. The species differs, but the finding explains why breeders keep dyes out of the hatching water and aerate the hatching container.

Raising the fry

TFH states that most annual killifish fry take small live foods immediately, while the smallest species need microscopic food for a few days; the progression is paramecia or infusoria, then newly hatched brine shrimp, then microworms and finely ground prepared food. TFH changes water daily in the first week and at least doubles the water volume weekly during the first month. Fish are sexually mature in about three weeks (Seriously Fish) to four weeks (PMC), and the cycle can begin again.