The Lightning Project

The ongoing saga of the PNG Lightning Maroon Clownfish Breeding Project

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Surprised to hear that tonight, Mike Doty’s pair of Lightning Maroon Clownfish had thrown down eggs. This is one of the freely distributed “genetic repository” pairs I placed locally, just in case anything ever happened here at home.

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The female Lightning Maroon, MD1, and male Morse Code Maroon, MD2, represent the first F1 sibling pair that I am aware of which should replicate the pairing of their parents. We already know of the results Soren Hansen had when pairing an F1 Lightning Maroon Clownfish with a wild White Stripe Maroon; I expect that Mike will see a 50/50 White Stripe/Lightning spread in the F2 generation from Mike’s parents.

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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:

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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).

It’s the 7th night for Spawn #43 which was spawned on 10-22; we’re on the night of 10-29 going into 10-30 now; there wasn’t any noticeable hatch on the tile over the 6th night (I left it with the parents yesterady)….it will get pulled.  This is what Spawn #43 looked like when it was laid fresh on 10-22…I should also point out this is the first time I’ve taken pictures of the F0 original wild pair of Lightning Maroon X White Stripe PNG Maroon Clownfish.

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The larvae from spawn #42 that are still alive have largely settled out…it was a small group, but I’ll find a way to work with them.  More importantly, it looks like LM X LM Spawn #2 might be over…I have not found any larvae in the BRT, so one more look, I repurposed  it for Spawn #43.  The eggs got the usual dip in H2O2, but started hatching during it, so they were moved straight into the BRT with 8 gallons of broodstock water and a few ML of RotiGrow Plus (only thing I had thawed…it’ll be fine for the first 24 hours and by that point I’ll have RotiGreen Omega up and running).

The most frustrating news is arguably LM X LM Spawn #3.  So..to recap.  Here was the first spawn:

F1 (Lightning X Lightning) first spawn for the Lightning Maroon Clownfish

 

So…I took some proactive steps and tiled the back wall of the tank.

This was LM X LM (or LxL if you prefer) Spawn #2 on the first attempted hatch night:

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Yeah..that gray patch on the side..those are the eggs…

So that didn’t turn out obviously…so I tiled the side wall as well.

Here is Spawn #3, laid on 10/27/2014:

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The internet shorthand FML seems somehow appropriate. Clearly the pair is flipping me the middle fin.

 

Spawn #27 came back to my fishroom on 6/20/2014, as Mike Doty has opted to do another run of Lightnings with spawn #36 (update on that as of 6-30, there were only about 10 that made it from Spawn #35 – Mike had some hatching issues this time ’round).

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I took the opportunity to do a headcount on Spawn #27 as I released the group into the BRT – provided I didn’t make any mistakes, the rough split was 28 Lightning Maroons, and 24 White Stripe Maroons.  Out of those 24 White Stripes, I maybe only noticed 3 that had extra markings and would fall into the “Morse Code” notion.

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I should point out that Mike and I discussed his rearing methodologies at great length; you’ll recall that part of the reason for allowing him to rear batches was to see if he could do any better, and my own effort to learn from him and work in collaboration. Say what you will, but the bottom line is that Mike reared spawn #27 with what amounts to a 50% water change weekly.  I can guarantee I was nowhere near that on some runs.  Of course, that’s not the only difference involved here…his methodology was to drain the standalone BRT half way each week, and gradually fill it back up over the course of the week, and then repeat.

It seemed like a very manageable system, and frankly it should be obvious that good base maintenance will get you good results.  There are still some deformed fish, but if I have to be honest, I think he reared a superior batch than the first big one. I’m going to look into segregating them out, as well as stepping up the water changes overall, as that can only help produce superior fish. It’s interesting to note that the sizes do vary immensely within the group, whereas the group of 10 I had going from around the same time is more homogeneous in size and has more patterning, and is comparable in size to these.  It will take a lot of really specialized research to hone in on some of the variables in play, but I look forward to doing that if I’m able.

Mike’s routine is hardly surprising – my breeding of Angelfish relied strongly on 50% weekly water changes, and based on published recommendations, while I took 3-4 months to hit market size doing 50% weekly water changes on growout, if I had stepped it up to 50% daily water changes, I would have cut my growout time down to 6 week!  Think about that.

On 5/21/2014, initially the hatch didn’t appear so good, but by afternoon, it was clear I had a solid hatch with hundreds in the BRT. I checked the tile, looked like 50% had hatched perhaps, so I let it go in the BRT overnight again, with only ambient light.  THAT might have been a mistake, because this morning, there was no additional hatch, but many of the larvae had perished. Seems like I have a pretty reliable hatching protocol with H2O2 dip and broodstock water yielding reliable results on the first night. Moving the batch for a second night hatch might just be the ticket.  The OTHER interesting thing – I do have to wonder if we have hatches going on during the day. I’ve long since wondered if that could be happening…

On the other front, Mike sent me an update video of Spawn #27.

Looks like I’m gonna owe him a Gold Nugget Maroon from ORA.

I decided to try something different this time…I’d been putting it off but figured “might as well”.  I decided to try snagging a hatch with the Vossen Aquatics Larval Snagger (aka. Larval Trap).

Thursday night, the 24th, was officially the 6th night post spawn and assuredly I’d get a hatch.  I reconfigured the broodstock tank slightly to allow me to pull all the cords for water current devices in one shot.  I thought about simply using a light timer to turn pumps off before lights out, and then on two hours later, but with the Lightning Maroons, I wasn’t going to risk it. I’d rather stay up late and BE SURE I got everything back up and running.

I set up the larval snagger in the broodstock tank and was sure to get the placement correct – the top of the snagger must reside above the water’s surface, as shown here.

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After that, I made sure to get the air flowing, which runs water through the trap gently, without harming the larvae.

 

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Then, set up the suction cup light, which serves to attract the larvae to the intake of the trap.  Turn off all the lights in the room, and walk away for a couple hours…

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I checked around 4:30 AM and saw that I had snagged a few larvae, but not many.  The nest still looked rather full, and I scanned the tank for wayward larvae, using my cell phone’s flashlight feature.  I didn’t see any, so this was a very weak / small hatch.  It wasn’t enough to really try rearing, so I just figured I’d leave them in the trap and take some photos come morning.  This is what I found when I checked.

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It didn’t seem like nearly enough to bother a run with, so I left the snagger running all day and figured I’d simply try again on Friday night.

Unfortuantely, I might have screwed up on Friday night.  I can’t be sure, but here’s what happened.  The nest looked full most of the day, but I had shifted the light timing just a little bit.  So the lights on the system went off at 2 AM vs. 2:30 AM, but I didn’t make it down into the basement to turn off the filtration until 2:30.  The nest appeared to have hatched significantly before I had a chance to set things up.

I checked at 4:30 AM, and sadly I found one wayward larvae in the tank, and it appeared that the trap had maybe only captured a few more.  Did I “miss” the hatch window Friday night?  Possibly.  I set up the larvae I had captured in a clean BRT with 8 gallons of broodstock water, rotifers, and some RotiGreen Omega, and that was it.  I was gone Saturday night, so wasn’t able to see if I’d get any more on the 8th night.

Overall, based on the number of larvae caught vs. the number of larvae found in the tank, it’s fair to say that the snagger probably did perform well.  I’ll probably try it again on batch 31, and maybe this next time I’ll TEST a timer and trust it to do my bidding.  Maybe a little more automation will allow me to “score big” on a hatch in the broodstock tank, avoiding some of the problems I’ve encountered when artificially hatching.

It’s the morning of January 13th, 2014; time to play a little catch-up.

Back on January 9th, I split the larvae from Spawn #20 from one 10 gallon tank into the 10, plus a 15 gallon BRT on my larviculture racks. When I did the split, I took the opportunity to take a photograph of the babies I moved to the BRT (Black Round Tub). I finally took a moment to do a headcount this morning. First, the unedited shot:

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Now, since I scooped all this water out by hand into a clean 5 gallon bucket, in theory EVERY little smudge / smear / blurry little dark spot *should* be a baby clownfish. Remember, you have to keep in mind that the depth of field was pretty narrow on this shot – babies at the bottom of the bucket, or even just a few inches deep, were not captured in-focus. So the headcount is an estimate at best, and I’m going to say it’s probably high, and if it is, it’s high by 20 or so fish, ballparked.

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So my rough headcount puts it at 140 babies moving into the BRT. What I don’t know is how many were left behind in the 10 gallon tank!

On Friday, January 10th, the larvae got their first small taste of TDO A. This has been added to the diet daily since then, and they’ve taken to it well. By Sunday evening, 1/12/2014, I noticed the first little whitish-blue spots on the tops of the heads of a few babies, which means only one thing. Metamorphosis has started.

I’ve been sticking with the water changes…they might not get done every day, but they get done at least every other day.

That’s the story of Spawn #20 for now. It could be a very productive run if things go well.

I have photos of this spawn stashed away in the camera, but I figured I better get this on the books before I completely forget. Spawn #19 was put down on 12-14.  With tons of stuff going on with the holidays, I did a boneheaded thing and pulled the nest on the night of the 19th going into the 20th (because I looked at my phone after Midnight, didn’t realize it was that late, saw the 20th, and said “shoot I gotta pull this tonight!”).

Spawn #19 during H2O2 sanitizing dip.

Spawn #19 during H2O2 sanitizing dip.

So the eggs went into a clean tank with all new water after a 15 minute bath on hydrogen peroxide.  It was at least 24 hours early, and yet despite that, I had a handful of offspring the next morning…that’s technically 5 days after spawning!  A few more offspring hatched out the subsequent night (which would have been 6 days later), and yet after that, the nest died.  There has been plenty of RotiGreen Omega and rotifers in the 10 gallon blacked out tank, but by the time I departed for our holidays away, there were maybe only 3 babies I could see alive.  Upon returning the day after Xmas (12-26), I could find only one baby in the tank, and by the 27th I could no longer find any larvae.  Pulling my hair out.

There is one thing I’m looking at right now – I haven’t had access to wooden airstones for incubation in ages, and Joe Lichtenbert swore by them for direct egg aeration during hatching.  I’ve been having all sorts of nest deaths in the Lightning Maroons when putting coarse air directly on them, so I’ve generally tried to get good ambient flow, which works better but not consistently.  Well, if it’s my damn airstones I’m going to be really ticked, but my plans are to have wooden airstones on hand very soon, going out of my way to obtain them since most online suppliers have long since dropped them from their offerings.

And yet, I’ll get another shot at it still.  I discovered the latest nest, Spawn #20, when checking out my tanks when I returned home from the holidays.  I cannot say for certain, but given that the Lightning Maroon’s ovipositor was still down, I believe that Spawn #20 was laid on 12-26-2013.  Based on that timing, the nest was pulled on the evening of 1-1-2014, going into the morning of 1/2/2014.  Once again the 10 gallon tank was drained, cleaned, rinsed with freshwater, and set up. This time I used 50% new water, 40% broodstock water, and 5% RODI water to bring the specific gravity down slightly.  I did not sanitize the eggs prior to pulling.  There was very little orange left in the larvae, which I believe bodes well for my pull occuring at the right time to maximize whatever hatch I get.  A small LED flashlight was set up at the far end of the tank to draw hatched larvae away from the heavy aeration – wooden airstones have yet to arrive, so once again I’m pummeling the eggs with coarser bubbles from a glass airstone.

You may recall the “other” pair of wild (F0) White Stripe Maroons I’ve had set up from PNG.  I’ve had the pair a long time..they were paired before the Lightning Maroon ever got around to it.  By my records, they put down their first spawn on 12-20-2013.  Upon returning home today, I checked their nest; just a few unhatched eggs remained, which means that they hatched even FASTER than the Lightning Maroon.  I would have had to pull them the late evening of 12-25….just over 5 days post spawn.  The other possibility is that the parents ate most of the eggs prematurely, so I’ll have to watch the pair another few times through before I see what their pattern is.  But seriously…as little as 120 hours from spawn to hatch?!

I will absolutely raise a batch from this other pair this year, and the reasoning is simple.  First, we’ll want to see if by some odd chance they throw out Lightning ( I don’t believe for a second they will).  Next, we’ll want to see how many show up with horns and spots and other aberrant, but not “Lightning”, markings. Ultimately, regardless of what we get, this new and completely unrelated line of F1 PNG maroons will be the IDEAL outcross for the F1 Lightning offspring.  Instead of the F1 generation of PNG Maroon Clownfish stemming from only 2 foundation fish (which is really not enough), we’ll double the foundation population to 4 fish.  According to FAO documentation on conservation breeding of fishes in captivity, this is still too small of a genetic bottleneck, but since I am aware of a couple other breeders who have Lightning Maroons and claim to have unrelated F0 White Stripes to mate them with, we could realistically be on very solid footing for the long term captive viability of the PNG provenance lineage of Maroon Clownfish even if we have no further access to PNG fish for months, years, decades….

It’s delightful to see that the Lightning Maroon pair has finally settled into a spawning routine.  No sooner had I botched spawn #11, and they go put down spawn #12.  On an interesting side note, I discovered what apparently happened with Spawn #10 – about a week after the larvae had all vanished, I took a look at the black round tub that was sitting there, now empty.  It was absolutely OVERRUN with hyrdoid medusas.  I’m not sure how hydroids got into the mix; the other BRT, which contains a single now post-settlement juvenile Lightning Maroon baby (so cute) has not had any hydroid issues crop up.  I’ll have to drain and sterilize the BRTs before using them for Spawn #12.

Spawn #12 was laid on September 15th, 2013.  This means, based on our current “144 hours” to hatch rate, means I need to pull the eggs as early as this coming Saturday night, September 21st.  This is spawn number #12, photographed 24 hours after being laid.

Lightning Maroon Clownfish Spawn #12

Lightning Maroon Clownfish Spawn #12

Lightning Maroon Clownfish Spawn #12

Lightning Maroon Clownfish Spawn #12

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