Over the last few years there has been an increase in the number of odd recessive mutations that are popping up on axolotl fan sites.  Most of this is due to rampant inbreeding, rather than any new mutations.  

Just to be clear;  Inbreeding DOES NOT produce new mutations.  It just increases the probability that babies will inherit two copies of a recessive gene from their closely-related parents.  When relatively rare recessive genes start increasing in frequency it is a good indicator that increased  inbreeding is taking place - particularly when the recessive gene reduces or prevents breeding of affected animals.

One of the recessive mutations that is increasingly common over the last four years is the “eyeless” gene. I had read a great deal regarding physiological studies of the eyeless gene in ambystoma, but having never had any eyeless animals. I was curious as to whether the mutation is actually harmful, or just another oddity. When I was told that another batch was out there, I decided to take the time this year to find out.

Thanks very much to CL for acquiring these animals for me (Don’t want people hitting her up for more, or thinking she produced them. She got them from an acquaintance).

First, a little background:
The eyeless mutant axolotl was discovered by Rufus Humphrey back in 1969 [1].  Humphrey, like many early axolotl researchers, looked for recessive genes by the simple method of repeatedly inbreeding axolotls (in this case leucistic animals held at the Indiana University colony) and looking for anything unusual in the offspring.  Please note that inbreeding like this does not produce new mutations; It simply increases the probability that offspring will inherit two copies of recessive genes.  Since most mutations are recessive (they are basically broken or missing versions of the dominant genes) they are only expressed in individuals that get a copy of the gene from both parents.  After just 2-4 generations of successive inbreeding about all recessive genes that have phenotypic (observable) effects can be identified in a dozen (or so) animals.  No new axolotls have been introduced to our domestic stocks for MANY generations now - so don’t start thinking this is a good way for you, personally, to find new genes!  

Humphrey noted that some of the homozygous babies actually had some eye tissue, but that this tissue failed to develop into functioning eyes.  Most have no eye tissue at all.  All are totally blind, of course.  He also found that those that survived to become mature adults were unable to reproduce.  Many of the eyeless animals had unusual pigmentation - basically, they looked like very dirty leucistics - with dark patches on the head and face, and down the back.

Many of the animals he produced in the lab died before maturity, but due to the methods generally used for rearing axolotl larvae at the time (crowded conditions and relatively low-nutrition food sources) this was common even for axolotls that didn’t have unusual recessive genes.

A bit more detail (if you’re not a Genetics or Embryology geek you might want to skip the next three paragraphs):

We now know that the eyeless phenotype is caused by a mutation in the RAX (retina and anterior fold homeobox) gene.  This gene encodes a transcription factor that controls the development of several structures in the brain and eyes early in development.  Please note that humans (and most other vertebrates) have a nearly identical RAX gene, and similar mutations have been identified in humans, mice, frogs, and many other organisms.

The eyeless mutation is actually a deletion of five DNA base-pairs from near the front end of the gene.  This results in a “frame-shift” error, which turns the rest of the instructions on the gene into gibberish. It would be like cutting the first five letters from this sentence off, and moving all of the rest of the sentence five letters to the left, resulting in the sentence “ld belik ec utti ngthefir stfiv…” you get the idea.  The gene no longer makes any sense, so the cells can’t produce the transcription factor they need to produce eyes, pineal glands, parts of the pituitary gland, and some important structures in the brain.  

Eyeless axolotls are sterile because they don’t have a functioning anterior pituitary gland. Without the anterior pituitary there is no production of gonadotropin-releasing hormone (GnRH), which results in no production of luteinizing hormone or follicle-stimulating hormone, so the testes and ovaries fail to mature.  The lack of GnRH also affects their behaviour, making them permanent juveniles in terms of their response to other axolotls.  The males don’t dance for females, for example.  

I received ten juvenile eyeless axolotls and ten of their eyed siblings in March of 2022.  On arrival, they were 2-3 inches long, with the eyed sibs generally a bit larger than the eyeless.  All were badly bitten, with missing toes or limbs.

All were separated into individual tubs and treated with nitrofurazone to reduce risk of infection from their injuries.  Two of the eyed sibs died within the first week of unknown causes.

All were fed blackworms until they reached four inches.  The eyeless animals grew more slowly than their eyed siblings, but most were aggressive eaters.  They were then transitioned to floating pellets as they reached four inches in length.  

As you might expect, the eyed animals were much more successful at transitioning to floating pellets than the eyeless ones.  It was (and still is) necessary to present the food directly to the eyeless animals.  If not, they searched all over more-or-less at random, snapping at the surface until they found a pellet to eat.

By the end of April there were a few obvious differences in the eyed and eyeless siblings.  The eyed siblings were growing quickly, having gained about an inch in length since March, while the eyeless animals had grown less than half as much.  Some of the eyeless animals seemed listless and relatively inactive, and were eating far less than I felt they should be.  Others were hyperactive, being easily startled by vibrations and loud noises.

The difference in growth rates became more pronounced as the animals have grown.  A few of the eyeless animals became so lethargic that they had to be hand-fed every day just to keep weight on.  By June two of the eyeless died.  

Just to be clear, here;  Axolotls of this age don’t just randomly die.  I have raised many hundreds of axolotls, and have lost only a handful of juveniles over four months old over the course of MANY years, and none at all over the last two years. For two to die without obvious signs of disease or injury means something is terribly wrong.  

Also note that these eyeless animals snap at anything that comes near them. While most axolotls can be housed with others once they reach sexual maturity, these eyeless animals must be kept apart forever.  They will never stop biting each other.

The eyed siblings continued to grow at a normal rate, and by September all had matured to the point that they were clearly identifiable as male or female.  The eyeless animals, however, were still quite small and sexually immature.  See pic to the right (eyeless on the right, eyed on the left) By late September two more of the eyeless animals had died for no obvious reason.  They seemed fine one day and were gone the next.

As of now I have six of the eyeless animals left.  Of these, two (a melanoid copper and a leucistic)  appear reasonably healthy and are still growing reasonably well.  The others are far too small for their age.  The smaller ones are still very lethargic compared to their eyed siblings.  I suspect that they will not survive much longer even with hand feeding.

Interestingly, the eyeless axolotls are, in fact, sensitive to light - especially in the UV range, and become agitated when exposed to either bright full-spectrum light or bright UV.  This is due to the presence of photosensitive cells in the skin - a common feature of many fish and amphibians.  The ability to detect light intensity (and even color) with the skin is useful for species that have the ability to change color or shading in order to match their surroundings.  The melanophores can even respond directly to light exposure by expanding or contracting to become darker or lighter under different lighting conditions, which is why you may notice that axolotls tend to get pale at night and darker in the daylight.

You may be wondering how eyeless animals ended up in our pets after Humphrey found them in the Wistar line at IU back in the ‘60s…  Well, it seems that the IU colony didn’t have much of a security system back then.  Many animals from the IU colony “escaped” over the years to end up in home tanks.  Every eyeless animal and carrier of the gene is descended from a couple of animals smuggled out of the IU lab by grad students in the 1970s.

Given my experience with these eyeless animals I do not think there is any ethical justification for deliberately producing more eyeless axolotls. I feel that the eyeless gene should be eliminated from our pets. The gene is maintained in genetic stocks for laboratory studies, so there is no good reason to keep the gene circulating in our stocks.

That means culling the siblings of eyeless babies (since ⅔  of them are carriers of the gene)  and either culling or never breeding parents of eyeless babies (since they are carriers of the gene).

The gene is not at all common, so there is no excuse for repeatedly producing clutches of eyeless babies. One clutch is enough to identify the problem and eliminate it from breeding stocks. Please note that animals that are not siblings, first cousins or parent-offspring pairings should only very rarely produce eyeless offspring.  A breeder who repeatedly produces eyeless animals IS INBREEDING THEIR AXOLOTLS.  

With careful record keeping and diligent effort, we should be able to eliminate the eyeless gene from domestic stocks in a few generations.