The Lightning Project

The ongoing saga of the PNG Lightning Maroon Clownfish Breeding Project

Browsing Posts tagged dominant

As promised, I finally got time to look at the offspring that Mike Doty had managed to rear from my F1 Lightning X Lighting Maroon Clownfish pair.  While Mike had initially stated he felt the split was 50/50, today’s visit showed something different.

All told, I only managed to count 3 white stripe offspring in the BRT. Meanwhile, there appeared to be 6 distinctive Lightning Maroon-type offspring. What I didn’t see were any fish that, at this point, looked atypical from either known phenotype.

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With only 9 (approximate) juveniles, the sample is far from conclusive. The 3/6 split could be representative of anything from a 50/50 split to a 25/75 split. A new phenotype could be missing because none simply survived in this first successful run.

That said, the 3/6 split, if a valid sample, would represent something we perhaps don’t want to  see.  It would imply that Lightning is straight dominant, but it would also imply that a double-dose of Lightning is in fact fatal to the offspring and they fail to develop.  This is one of the current working hypotheses for the Snowflake gene in Ocellaris, and anecdotal reports continue to bolster that line of thinking (reports are anecdotal in so much as a breeder saying “It’s about a 60-70% snowflake result” is not the same as a breeder saying “I got 140 Snowflakes and 65 wild types in this clutch”).

It is fair to say that there is still hope for these fish – they are QUITE YOUNG and all we can truly discern at this point is that they outwardly either are white stripes or aberrant.  My tune could change as these babies develop, or if subsequent results are different. It is not surprising to me though, to see similarities emerge in various mutation types.  We already have Picasso/Platinum and the sister mutation of DaVinci/Wyoming White. Much as I wonder if these twin mutations could represent the same gene in different species (or simply a similar genetic mutation in sibling species), would we come to find out that Lightning is in fact not unlike Snowflake? After all, genetic analysis has revealed that the Maroon Clownfish are in fact very closely related to the Percula/Ocellaris complex, so it would not be surprising to find similar or the same genes present in all these species based on their common shared ancestor.

It is also an interesting footnote to observe that these tiny juvenile clownfishes DO represent a the first F2 generation of Lightning Maroons.

Here’s a quick rundown.

The 5th spawn of the F1 Lightning X Lightning Maroon Clownfish pairing was collected and hatched by Mike Doty while I was away.  In short, he scraped off the eggs, hatched them in a 1 gallon jar in a water bath with simple aeration, 75% clean new water.  Come November 28th, Mike relayed that settlement had started.  The moment we’ve been waiting for was here – is there something new?

Well, Mike’s first words were “About 90% sure we have some normal striped fish.”

This, of course, does the following:

  • likely rules out Lightning as a simple recessive gene. If it WAS recessive, then both parents would be “double dose” aka. homozygous, represented as l/l, which means that each parent could only contribute a recessive lightning gene, and thus, each offspring would also get one copy each, one from mom, one from dad, and thus, could only be l/l as well. For the moment, while another couple test matings will bolster the data, the fact that there are white-stripe offspring pretty much precludes this being a standard single allele, single locus, recessive trait.
  • does not rule out straight dominance. If it was straight dominance, and each parent is “single dose” aka. heterozygous, represented as L/+, then 25% of the offspring would not get a gene from either parent, and thus, 25% would be white stripe maroon clownfish.
  • nor does it rule out partial dominance. This of course, would work the same way as dominance, except that 25% of the offspring would get a lightning gene from EACH parent, and would be homozygous for Lightning, represented as L/L. This is the scenario that most people are hoping for, because with the new homozygous state, there comes the potential for a new phenotype that could be different from the Lightning that we know.

As of today, 12-8-2014, I spoke with Mike briefly and have to relay this news – while he doesn’t have many babies left, he believes that the phenotype split is roughly 50/50.  That is to say, half white stripes, half lightnings.  So far, he also has not seen anything unique or new in this F2 generation.  I have yet to see the babies for myself, and have yet to take pictures or do a headcount, but these cursory, informal results, mirror another clownfish mutation that seems to not fit the mold as we’d expect – SNOWFLAKE in Ocellaris.  It’s my hope to get over to Mike’s today yet to see for myself.

In other news, the 6th spawn of the F1 Lightning X Lightning pair was put down on 11/29/2014. It appears I finally won the battle of the tiles:

LxL Spawn #6

LxL Spawn #6

Meanwhile, I brought some new clownfish into the fishroom earlier in November (the 19th and 22nd) and was trying out the new Ick-Shield food from New Life Spectrum.  This is basically a Chloroquin-laced pellet food that is meant primarily to prevent disease such as Crytopcaryon, Amyloodium, Brooklynella etc…pretty much the things which are sensitive to the active ingredient. I decided to not simply feed this fish to the new arrivals, but also to feed it to one of my holding systems AND the wild Lightning Maroon and her mate as preventative medication, just in case.

Well, it turns out that there is an unfortunate side effect to this feed; it seems to shut down breeding activity.  All my routine pairs stopped spawning. The Lightning and her mate did finally put down a spawn on December 1st, 2014, #46.

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Unfortunately, it appears as though the spawn was not fertilized…the eggs didn’t develop, and after 48 hours they were gone. A few days after that, I read, anecdotally, that Chloroquin can cause male sterility??? Not permanent according to the rumor, but certainly a potential setback. As far as the efficacy of the food, my jury is out. The larger fish which were feeding well on it by and large remained disease free, but not all did.  I still had a Brooklynella outbreak, although not in the fish I would have necessarily expected. Once that outbreak started, it then affected other fish as well despite their feeding on the pellets.  I’m also seeing either Cryptocaryon or Amyloodium on fish which were visually “clean” upon arrival, which were in dedicated QT systems, feeding on this food from day one.  So the question here is were they simply getting ENOUGH feed as they were small fish which cannot readily eat the small pellet size.

So of course, one is left with questions, not answers.  There is no way to say the food didn’t work, nor is there any way to prove that it does work. Absence of disease is not proof of prevention, that much I know for certain. Lack of a cure, or lack of prevention, which IS documented, only raises questions about why it didn’t work as suggested and certainly requires investigation (eg. would a smaller pellet size be better accepted…could these failures stem from simply lack of feeding, or lack of sufficient feeding, thus insufficient dose to the fish?).

Circling back to LxL Spawn #6, as the week progressed an interesting change in behavior occurred starting around December 4th, 5 days post spawn.  The larger female F1 Lightning became belligerent towards the male, and over the day drove him from nest tending duties.  December 5th, a Friday, would have been 6 days post spawn, and the night of the first hatching.  I was simply swamped with preparations for sending our dog to live with my brother, and failed to pull the tile.  By morning, Saturday, December 6th, 2014, it appeared that I had not missed much…most if not all the eggs were still there. The pair remained at odds.  We left for the weekend to ship our family dog, and upon Sunday, December 7th, there were still a few dozen eggs remaining, although they appeared potentially dead and disappeared throughout the day.  By nightfall, the pair was starting to be less antagonistic, but I am still keeping a close eye on them.  Hopefully, we’ll get another spawn soon – this was the only mature pair in the house that didn’t receive Chloroquin-laced foods (as they don’t reside in the fishroom with the rest of the fish).

Since the PNG Lightning Maroon hit the online world, there’s been over 100+ threads on it.  I’m not about to link to them all here, but I found the conversations  I had with a few of my fellow TCMAS (Twin Cities Marine Aquarium Society) members very interesting.  The original thread is here ( http://www.tcmas.org/forums/showthread.php?t=25958 ), but I’ve summarized my side of things here for easier consumption :)

Clint (who helped me fund this project by purchasing some of my fish) posed some really interesting questions.

#1.  Why did I start out with a larger female to pair the Lightning Maroon with vs. a smaller fish (to make the Lightning Maroon become female)?

Several people have posed this question and it’s a very valid one.  Afterall, pairing the Lightning Maroon with SMALLER fish would minimize the pairing risk, minimizing the chances of death.  Still, I had (and still have) several reasons for thinking keeping the Lighting Maroon male (if it is male) is the way to go:

Simple answer – Blue Zoo Aquatics was already sold out of all the smaller PNG juveniles before they even had a chance to set some aside for me, so the only way to go when the Lightning Maroon was shipped was to send me a big female.

More complicated answer – If I could keep the “Lightning Maroon” as a male, there was/is a greater chance of a quicker spawn by trying to pair it with a female vs. waiting for a sex change to occur. Additionally, since Maroons, most notably LARGE FEMALES, tend to lose their stripes as they age, better to keep this guy as a male so the awesome lightning bolts stay!

Really complicated answer – If SEASMART were to find a second Lightning Maroon, and IF I were to obtain it, I would have much greater chances of pairing “Lightning” with  “Lightning” if the first Lightning Maroon remains male.  If the Lightning Maroon is allowed to become female, and a future discovered Lightning Maroon is also female, I won’t be able to pair them.  So, pairing possibility drops by at least 50% if I allow the first Lightning Maroon Clownfish to become a female.

Hearsay answer – there has been talk that “male” clownfish have more influence on “patterning”, whereas females have more influence on “coloration”. I think that’s bunk, but at the same chance, I’m not going to dismiss it outright.

#2.  Is there any evidence to show that the pattern is a dominant or ressesive gene carried by the male?

My initial answer is that  genetically we can speculate anything we want. We are even speculating that the Lightning morph has a genetic basis in the first place. It may not be a genetic trait. We may never see another Lightning Maroon Clownfish even if I do everything perfectly.

#3.  To paraphrase Clint’s third question, he asked if there was any difference in the likelihood that Male Lightning X Female Regular = more Lightnings vs. the flipside, Male Regular X Female Lighthing = more Lightnings.

My Response: There are a number of things to remember about clowns, starting with the fact that they’re all born male, and the dominant one turns female. Any male can later become a female.

A rough genetic picture might be to look at a straight up recessive trait like albinism for a comparison. LL = Lightning. LN or NN = Normal fish. Thus, the Lighting Clown being LL, mated with a normally colored NN fish, will produce only LN offspring. No lightnings in the first generation (F1). Mating those offspring together would produce LL, LN and NN fish, at a 25%, 50%, 25% rate. So 25% Lightnings, 75% normally colored, with 2/3 of the fish carrying the recessive L.

However, a LL fish, mated with an LN fish, will produce 50% LL and 50% LN right off the bat. Given that there have been other “Lightning Maroons” seen in PNG, including the other one collected in 2008, the chance of this being a “possibility” is partially why it is so critical to select mates from PNG, ideally the same reef. It increases the chances that the normally colored mate may in fact be LN. Just as easily could be NN, and probably more likely is, but you cannot tell if the trait is recessive.

I think if the trait was dominant, we’d see many more Lightning Maroons out there.

Now, here’s the reality. Even if this genetic, it may not be a simple straight up recessive trait. It could be something like Platinums and Picassos, where there may be more going on.   Based on the breeding outcomes of Picasso Percula offspring as reported by David Durr and Tal Sweet, the “Platinum” and “Picasso” genetic mix is starting to look like the following:

Platinums = PP. Picassos may be PN. Normal Percs might be NN.

Again, that is PURELY SPECULATION. It could actually turn out that Platinums = PPP, Picassos are PPN or PNN (A and B Grade anyone) and Normals are NNN. Or something different. We don’t know enough yet. Good observation and careful records, along with SHARING OF DATA, will be what reveals the truth.

#4.  TCMAS member “lr9788″ then asked, “How does breeding continue down the line? Since this is a one of a kind doesn’t it present genetic issues? Or can a lighting be paired with any maroon (in theory)?”

This question inspired a very long response!  So here it is, largely unedited, and I’ll let that wrap up this first post on “Genetics”, elaborating a bit on the thought process behind how I’m approaching the breeding of this fish.

Well, truthfully there are not any concerns about line breeding, generally in fish, until about the F6 generation. The parents here (Lightning PNG X Reg PNG) are F0. Their offspring are F1. I assume F1 will all be normal. Pairings of the F1 Offspring will produce F2, and it is there that we might first see Lightnings (of course, I could be wrong and we could see them in F1, but heck, we don’t even know if this is genetic yet!).

So, lets say you get F2′s that are Lightnings. It is here that we could first see “Lighting X Lightning” crosses. All are still PNG maroons as well at that point, having descended from the original PNG-collected normal & lightning pair. It is possible/likely/probably that the matings of F2 X F2 lightings would yield a much higher number of Lightning Offspring in that F3 generation.

Now, since Clownfish can be productive spawners for 10-20 years easily, we need to realize that we could work with F2 Lighting X Lightning crosses to produce all the Lightning Maroons needed for a long time. Why?

Well, let’s say we get this pair spawning in a year or two. So 2012-ish. Their F1 offspring could be spawning as early as 2014 perhaps? Which means the first F2 offspring were we likely see Lightning Maroons in quantity (if it all works) would be maybe in 2015. Any pairing that starts throwing lightnings could continue to do so for the next 10-20 years (so let’s say 2035 is when they start dying off). However, we could realistically continue to create new F1 pairs that *might* throw F2 lightnings for another 5-10 years with the initial offspring of the wild parents. So that means, time wise, we could have F1 generation fish still producing F2 offspring in 2040.

If we take another 2 years to get those first Lightning X Lightning crosses spawning, that puts us at maybe 2017 for their F3 generation. Again, conservatively we could go 10-20 years from the point this generation STARTS, so 2027 to 2037 easily. But again, same math for the F1′s that throw out Lightnings as F2. Based on that RANGE, knowing we could still get F2 Lightnings at 2040, means realistically we could still be producing line bred F3 Lightnings at 2050 or even 2060.

And we’re only at the F3 generation in line breeding which yes, is inbreeding. But remember, the general rule is that we can go to around F6 before we start seeing genetic problems as a result of this inbreeding in fish. So realistically, given the LONG reproductive lifespan of Clownfish, we could easily be into 2100 before we really have to worry about hitting that F6 generation.

The reality is that there are several other items at play. There may be more WC lightnings brought in…we certainly see that happen with Picasso Percs. That would open up all sorts of outcross possibilities.

Even if another Lightning never makes it into captivity, the reality is that we know these are PNG Maroons. Knowing the methodology that fixes the Lightning Strain, we could easily then take and outcross to other PNG Maroons to infuse new genetics and then go through the selective process again. So, long term, with either one crazy dedicated breeder, or the cooperative work of several, we could find ourselves with only semi-related Lighting Maroons. Pile on the simple fact that we are talking about mounds of sexual reproduction (and the inherent genetic variation that can occur), and the reality is that fairly quickly we could have a solid captive population that has descended from a tiny handful of seed stock from the wild.

Beyond THAT, it is also quite likely that the breeding of Lightning Maroons might leave the confines of only Maroons from Papua New Guinea (PNG). I think this is a big mistake, but I already know it will happen. People will take Lightning Maroons and cross them with Gold Stripe Maroons in the hopes of making Gold Striped Lightning Maroons. They’ll mate them with other abberant Maroon Clownfish varieties that show up (i.e. there are gray-barred Maroons..which honestly I kinda like). In the long run, this is where the guppification happens and we get into captive-produced varieties. This is what I don’t like, but know will happen. And yes, I will have played a part in it.

But for me, it’s about keeping the PNG location intact. Even if these fish never turn out another Lightning, we know that they were PNG collected and represent the genetics (and whatever distinct minute difference there may be) of Maroon Clownfish from PNG. To me, this is important. This is why I own “Sumatran” Fire Clowns and “Vanuatu” Pink Skunks (which also happen to carry that nice Orange “sunkist” color variation). I.e. on the Skunks, the color variant is a nice wild coloration, but who knows if it carries on to the offspring. Thankfully, the value for me comes first in knowing that they’re Vanuatu Pink Skunks vs. Fiji Pink Skunks…the fact that they have the more orange-yellow coloration is an interesting side benefit.

And finally, I’d like to simply take the question of Genetics and cite the recent article in CORAL magazine by Ret Talbot.

Ret put the question of genetics to two of the Marine Fish Breeding community’s premier experts, Martin Moe and Matthew Wittenrich.  Bottom line, you should read the whole article (starts on page 8 of the May/June 2010 issue of CORAL).  But I’ll tell you, it’s a 50/50 split as to whether the Lightning Maroon represents a genetic variation or not.  I’ll let you go find out which person held which stance!

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