Nothobranchius Killifish Breeding

[Schuyler]

This journal is meant to hopefully be a guide to breeding and raising African annual Killifish (genus Nothobranchius).

I will add the caveat the I've only been doing this for a few months at this point and this will be the first batch of eggs from my own fish that I will attend to hatch. I have raised another batch but those eggs were given to me by someone else.

I'm not a grizzled, veteran breeder so what I share will be flawed. If you're more experienced and you see an issue please comment so I can correct it. That said, my main goals, in order, are:

1) Create a single place that walks through the process from start to finish as a starting point

2) Consolidate references to good resources and people who do know what they are talking about

3) Breed Nothobranchius Eggersi

4) Earn points for the local clubs BAP

5) Have fish worth bringing to the GSAS auction in April

Edited February 2, 2023 by Schuyler

[lewk]

Cool, I'm looking forward to reading this. I would guess these would be a long way down the line before I would consider this myself, but it's a super interesting project for some really beautiful fish.

[Schuyler]

TLDR Sales Pitch

Killifish are beautiful, unique fish that are full of color, personality, and color. Annuals are the leasing a sports car of the fish world, if you like a species you can breed them and have more or if you don't then they are only about a year commitment.

Want something that fits the Betta niche but doesn't need a heater, doesn't have as many health issues, and is less mainstream? Try Nothobranchius Killifish 

Overview

If you've read about, watched, or listened to anything about Killifish they tend to all guy the same points so I'll paraphrase.

Killifish Basics

"Killi" comes from a Dutch word meaning ditch. They're called that because these types of fish tend to live in small bodies of water like pools, ditches, and creaks.

There are a few classifications of Killifish:

Non-annual - Live in water that didn't dry up (eg Florida Flagfish)

Semi-Annual - (aka Switch spawning) come from streams that dry out occasionally but not every year. They tend to have a more elongated and streamlined body. They can lay their eggs on plants or in the soil depending on how they are feeling (which is why they are called switch) their eggs can survive drying out like in this video about Gardner's Killifish hatching in an elephant's footprint

Annual - Live in pools of water that go through a regular dry season every year and dry out completely. They lay their eggs in the soil where they develop and then sit in suspended animation until rain comes back

Annuals are divided into two main groups: African and South American. African annuals have a stocky body that they use to bury their eggs in the top layer of soil. South American annuals take that a step further, they are even stockier because they tend to actually dive into the soil to lay their eggs.

African Annuals

Nothobranchius falls under the African annual category. Because of their breeding habit of isolated populations, they take evolution to the next level. There are dozens of species and each species could have dozens of slightly different patterns based on where they were collected. That's why you'll often see them listed with something after the species name. Like Nothobranchius Rachovii Birra 98, Birra describes the location and 98 is when they were collected. Take for instance the two Eggersi examples on the first line of this chart, same species yet dramatically different look:

Because they don't know how long their pool will last everything they do everything with intensity and speed. If they think there's a chance that things are better somewhere else they'll just jump out and look for another pool. They mature as fast as possible so that they can make before the pool is gone. One species, N. furzeri can be sexually mature within 14 days. 

Their quick lifespan and suspended animation have made them the subject for a ton of scientific papers. That mixed with the fact that they are a fairly niche in the hobby leads to some interesting search results like a website with a species profile saying that an annual lives for about three years (maybe one did once) followed by a scientific paper delving deep into the adaptation sex ratios to environmental conditions.

Here is a scientific article about nothobranchius with pictures of their habitat, what they look like as they age, and how they are used in research.

There's even someone on this forum who works in a university and does research involving Killifish.

Resources

These are some good resources I've found for getting information about Killifish

killi.co.uk - The webpage is a bit dated but they do have lots of information about different species including other breeders experiences with the fish

American Killifish Association (AKA) affiliated clubs - a list of local Killifish clubs in the US and Canada to see if there is are other breeders near you that you can with with. The AKA is also a good resource if you're serious about killis. Joining includes a free pair of a good beginner species.

AKA Beginner's Guide - A fairly in-depth guide to Killifish of an kinds

The Aquarist Podcast episode

There are a few threads on here from others keeping or breeding killis.

@Fish Folk's thread about killis with lots of great pictures (probably a much better sales pitch than what I gave)

My other batch of killis I hatched and raised

Other people journals about raising nothobranchius 

 

 

 

Edited February 3, 2023 by Schuyler

[Schuyler]

Tank Setup

Any time you hear someone talk about tank setups, inevitability they will say "the real extreme breeders keep them in little shoe boxes but I like to give them a little more space" it's like they're trained to say that in the AKA.

Most places recommend doing a 5 gallon tank for a pair or trio.

It's also recommend to keep a ratio of 2 females for every male to reduce stress on the females. Some keep them separated and then put them together for in another tank for a few days. This allows the female to recover and get conditioned. This article with procedures for raising them in a lab setting recommends putting a younger male worth larger females so he can't push them around as much which is an interesting idea.

That article also recommends feeding food high in protein and fat 2-3 times a day if possible to condition the females. Serious breeders well feed live black worms or white worms but frozen bloodworms and high quality prepared foods will be ok.

Example of my fat and happy female when she was at her prime:

Spawning Media

In your tank you will need to have a place for the fish to lay eggs that you can collect from regularly. You can use a small bowl or cheap Tupperware.

This will be filled with about an inch of some kind of media. Examples I've seen used:

Coconut coir/fiber - neutral in the water but tends to have more little bits that spread around the tank

Peat moss - adds acidity. Can be purchased in 20 lb bales at garden/hardware stores just be sure it's pure and doesn't have fertilizer. Some even use the seed starting pellets (jiffy7) because they are already pre-portioned size.

Less common things that require moving eggs to something else to incubate

Sand - that academic article recommends using sand so that you can rinse and strain the eggs out. I've never seen this anywhere else

Spawning Mop - A few guys in a Facebook group swore their annuals would use a mop in a spawning area. I'm testing this but my mop isn't sinking yet...

For coco coir or peat moss here is how you prepare it:

1) Break off a piece and boil it for a few minute to kill off bacteria or fungus. It will expand a lot so you don't need to get much.

2) Let it cool

3) Rinse it in a net to try and get as many of the little bits out. With rinsing vs without:

4) Place it in a bowl and pack it in evenly (if you're using plastic it may help up put a rock in first to weigh it down)

5) Fill the container with tank water all the way to the brim

6) Give it a few hours to settle (I do this overnight)

7) Scoop off any coir still floating

😎 Slowly lower it in the tank

With any luck they'll find it and put it to use fairly quickly like this:

 

Edited February 3, 2023 by Schuyler

[Schuyler]

Here I'm going to use that classic cooking show trick of having a cake in baking already in the oven while I demonstrate how the batter is made. Here I'm going to say "coir" for coconut coir but you can replace that with peat moss in most of the places.

Collecting and Incubating Eggs

After a week or two of spawning it's time to collect the eggs and store them until they are ready to hatch.

To do this:

1. Carefully remove the container so that the coir doesn't go everywhere
2. Strain the coir through a fine net and give a squeeze to get most of the water out. Don't worry about crushing the eggs, they are very sturdy.
3. Spread the coir out on a piece of paper towel
   
4. Pick out any plant debris, snails, uneaten food, or anything else you see that could rot while being stored
5. Check for eggs. This isn't totally needed but it helps give you an idea of how many eggs you started with and what they initially look like. I also like to pick out a few and place them on the side of the bag where I can hopefully find them again easily.
   1. Notes: 
      1. Know that for every one you see the are probably five you missed.
      2. Coco coir will have tiny crystals that can occasionally look like an egg because it is translucent but when you see an egg it will be obvious because of how perfectly round and smooth they are.
      3. My method is to use a magnifying glass while slowly moving bits of the coir from one side to the other. I've found that a light on the table facing me at a 45° angle seems to make the eggs easier to see. I've asked around online and no one really has a magic way of making this easier but if you figure one out please tell me!
6. Dry the eggs. You want the coir to be moist but not wet. Coco coir doesn't hold as much water as peat moss so it may not need to be dried any more.
7. Bag the eggs. Prep a ziplock bag by writing the species name (with collection site if you have it), the date you collected the eggs, and the date you need to check on it. Here is a page from the AKA with a list of estimated incubation periods. The timeframe varies based on the temperature, moisture of the eggs, and other factors. Be sure to check the paper and you hands in case any stayed stuck.
   
8. Store the eggs. Place the bag in a warm dark place that has fairly consistent temperatures. The optimum temp is about 75F or 24C (Source) but there is a decent amount of wiggle room. Cooler temps will mean a longer incubation period. If the eggs get too cold (<50F) or too hot (>100F) they may die and dissolve. I use the aquarium stand next to the canister filter. Lots of people use a linen closet of bathroom cabinet.
   
9.  Check on the eggs. Once the expected date has come it's time to check the eggs to see if they have "eyed up". If you look closely at the egg you'll see the little gold iris looking back at you. This is the indicator that the fry are developed and ready to hatch. It can be hard to see though even with a magnifying glass. Sometimes you don't see it specifically but you can tell that something is in there. Here is an example of an egg that is ready to hatch:
   
   Ignore that white circle that's the reflection of my magnifying glass' light but at the bottom of the circle if you zoom in you'll see a little eye.
   Here is an example of an egg that isn't ready and one that has fungused up (possibly wasn't fully fertilized):
   
   
   Note: Those incubation times are minimum times. Many people online have stories about pulling out a year old bag of eggs and being surprised by the eggs still being viable.

 

Edited February 3, 2023 by Schuyler
Adding pictures and cleaning up autocorrect

[Schuyler]

Last night I did some research on how people hatch/raise their notho fry. There were a lot of results from scholarly articles. That's when the thought came to me, maybe I can ask them what their process is for breeding.

So I sent an email to an author of one of the more recent papers. Then I googled the local university plus "nothobranchius" and turns out there was an assistant professor who mainly works with Nothobranchius as a model species.

I don't really expect any responses but it would be cool to hear about their process.

[Schuyler]

One of the researchers responded to me with a link to this "A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)"

It looks like this is the same paper I shared before but from a different site.

This podcast also had some interesting info. He said that the first males to mature are often the first to die and if you always use them your line will become shorter lived. That's part of what they did with the N Fuzeri stain used in aging research.

Edited March 13, 2023 by Schuyler

[Schuyler]

I got another response from a professor about an open source book that will be coming out in about a month with a chapter about care and breeding.

I also found this care sheet that specifically names the type of sand and the type of strainer they use.

Soon I'm going to try experimenting with using sand like they do in the lab and using aqua soil as media and letting the eggs fall through the strainer. But I still need to get strainer with holes big enough for the eggs to fall through. I'm thinking a cheap sand toy should work.

[Schuyler]

On 2/8/2023 at 11:13 AM, Simon Buys said:

If anyone is interested in some general information about Nothobranchius, here is a very good paper that covers a lot of info. 

It is written from a research point of view but still very interesting for everyone who loves annual killifish i think.

https://scholar.google.be/scholar?hl=nl&as_sdt=0%2C5&q=from+the+bush+to+the+bench+cellerino&oq=#d=gs_qabs&t=1675883508784&u=%23p%3DiuuzEfvlCwIJ

That's interesting that in the wild they normally saw a bias towards females. The article they cite for that actually says that the different species overlap and share pools often. I wonder if the fish can tell the difference. Otherwise how are they still distinct in the same pool?

Nice find!

I got distracted pretty early on when reading that because I looked up Pronothobranchius kiyawensis it's like a Notho version of a Gardner's killi

[Schuyler]

On 2/9/2023 at 10:43 AM, Simon Buys said:

Do you know which university this care sheet came from?

I had to search for it again. It looks like it may University of Idaho but it's probably not the university's official care sheet.

This is the base website: https://bernadettebiology.weebly.com/

[Schuyler]

At 3:40 in this interview he shows a way of finding the eggs that feels like magic. He uses a special rag to rub the coir and then the eggs just kinda appear

 

Edited February 12, 2023 by Schuyler

[Schuyler]

On 2/12/2023 at 12:06 AM, Schuyler said:

At 3:40 in this interview he shows a way of finding the eggs that feels like magic. He uses a special rag to rub the coir and then the eggs just kinda appear

 

That guy clearly has a lot of experience with this. He makes it look a lot easier than it is. All I did was make a mess:

Here is another interesting article that breaks down the phases of diapause and explains that the eggs are designed to bind to the substrate:

https://thenode.biologists.com/a-day-in-the-life-of-a-killifish-lab/lablife/

[Schuyler]

Today I learned about the three stages of diapause from a portion of this article

Diapause I Once the eggs are fertilized they will develope a little then enter diapause I. From what I've read they will stay in this stage until they are removed from water. This means that even if eggs were laid over a period of time they are basically at the same point when pulled from the water.

Diapause II once the basic organs have started forming the egg may go into diapause II. In this stage it can handle more extreme conditions better (cold, lack of O2, etc) and it doesn't use as much energy to sustain itself.

Diapause III after the egg leaves diapause II it will grow into it has the fully developed fry ready to hatch. This is the "eyed up" egg in one of the previous posts. In this form is ready to go whenever the water comes but it also uses up energy faster. If it develops too soon it may have issues like being a belly slider.

Each egg will gamble on how long it should wait to leave diapause II. If it develops too soon it may be waiting in III for too long and have negative impacts or it may hatch during an early rainfall and die off before the real rainy season comes. If it waits too long the other eggs will have a head start and may eat the smaller fry.

[Schuyler]

My dad let me borrow his cheap USB microscope to get some egg pictures.

Some of these are eggs harvested yesterday. Some are from mid December. The only difference I saw was a slight browning in the shell. Now looking at them again I don't remember which was which...

My dad let me borrow his cheap USB microscope to get some egg pictures.

Some of these are eggs harvested yesterday. Some are from mid December. The only difference I saw was a slight browning in the shell. Now looking at them again I don't remember which was which...

[Schuyler]

First ever Aquabid purchases

[Schuyler]

I found a new place to incubate the eggs: under my cat's heated house. It seems to keep the eggs at roughly 80°F. I put them in a box that should protect them from getting crushed to much.

Interesting thing from the book. The being killifish book suggests giving your killis as much variety as possible to make sure that they get everything they need. He even said that he would give them meat, fish, and scrambled eggs at times. But more realistically he recommends culturing wingless fruit flies.

[Schuyler]

The eggs still haven't eyed up yet. At this point with scheduling I probably won't be hatching them until April.

I have found some more interesting information from scholarly articles. Most of these were from citations in this article: "Nothobranchius as a model for aging studies"

It's more of an overview but has quite a few interesting facts like:

1) "the elimination of males from a population results in larger female fish that produce fewer eggs"

2) "the number of eggs laid each time and their viability decrease once the fish stops growing"

3) The age that males reach sexual maturity is a strong indicator for how long they will survive. Males that mature later have longer lifespans.

Edited March 13, 2023 by Schuyler
Late bloomers live longer rather than shorter

[Schuyler]

Here is a paper "Adaptation in a variable environment: Phenotypic plasticity and bet-hedging during egg diapause and hatching in an annual killifish" that has information about environmental impacts on incubation. Unfortunately I can only get the abstract and the graphs but just that is really useful.

It explained that things like how in nature the eggs use temperature as an indication of the season. That has an impact on wether or not the eggs enter diapause II. Basically if it's cold the eggs think that the rainy season is further away so they go into diapause II. If it's warm the eggs are more likely to skip diapause II and just develop directly.

At 20° C essentially all of the eggs went into diapause II and the mean incubation time was ~160 days. But if that was bumped up to 30° C almost all skipped diapause II and the incubation time went down to ~70 days. This is N Fuzeri which have shorter but more erratic incubating times from what I've read elsewhere.

Still, that much of a different is worth knowing and makes you want to be sure they are warm enough.

[Schuyler]

Today I met someone who used to be the librarian for a killifish club for a few decades. He had old a bunch of periodicals that he had collected in that time and he let me have then.

There's a ton to go through here and it's all at least 40 years old but I'm are there's plenty that's still applicable.

He also shared some wingless fruit flies 

[Schuyler]

That paper in the previous post is really interesting.

He says that if you pull the eggs, remove them from the peat, incubate them in 80° F water for three weeks, move them back to peat, and incubate for another 3-6 weeks (depending on the species) they would develop much faster. He managed to hatch Rachovii 8 weeks rather than six months. He mentioned three other species working.

This definitely seems like it's worth experimenting with.

[Schuyler]

On 3/11/2023 at 7:53 PM, Schuyler said:

This definitely seems like it's worth experimenting with.

Fun fact, in the ~60 years since this article was written this has been experimented with.

The professor I emailed a while back guy back to be with his recently published lab protocols. He sent them via email because online they require a subscription so I don't think I can share them. But I will share that they use this method for getting eggs to bypass diapause II. They even have specific temp ranges. A temp of 27-27C can be used to have more eggs develop directly.

They cite "Vertebrate diapause preserves organisms long term through Polycomb complex members" for that temperate. Something else interesting I find in the was that eggs from older females were more likely to enter diapause II.

[Schuyler]

If I'm reading this correctly (at least the abstract that's freely available) this study suggests that fry from younger parents develop reach sexual maturity faster than from older parents. It also reiterates what was said in the article from the last post that eggs from older females develop more slowly.

"Breeders Age Affects Reproductive Success in Nothobranchius furzeri"

[Schuyler]

On 3/3/2023 at 1:37 PM, Schuyler said:

3) The age that males reach sexual maturity is a strong indicator for how long they will survive. Males that mature later have longer lifespans.

I asked the professor about this because it wasn't mentioned in his lab protocol which was specifically tailored towards longevity studies.

He said the study "wasn't followed up rigorously" and that there is a hypothesis that faster maturation means shorter life but it's difficult to "prove rigorously"

[Schuyler]

Looks like it's almost time to hatch. I just checked some of the eggs and it looks like eyes have started developing in some of the eggs.

This one looks like it is still early in is growth

This one looks like it's close to reaching diapause III. The eyes are big but there isn't that reflection of the iris:

[Schuyler]

We pulled out the microscope today to get some better pictures of the embryos developing. Luckily the eggs that we picked had a few different levels of development.

Here's one that looks fully developed and another that looks like it's just a day or two away. While watching them they were moving around occasionally in their eggs.

A closer view of the fully developed embryo:

Then the less developed one:

You can see the veins branching out to the yolk sac which is much large than the fully developed embryo's. If you look closely you can see that the veins lead back to a red splotch behind the tail, that is (or will become) the heart. If this were a video you would be able to see the blood pumping through the veins.

There was also an even less developed embryo. The eyes are clearly less developed and the yolk takes up the majority of the egg.

Next time I'll see about posting a video.