The Lightning Project

The ongoing saga of the PNG Lightning Maroon Clownfish Breeding Project

Browsing Posts tagged Breeding

Last week was a flurry of clownfish sex.  March 4th, at 7 AM, I got a text image of the Lightning Maroon clownfish pair on display at the Great Lakes Aquarium; a small next was on the tank wall (I’m guessing laid on March 3rd?). This wasn’t the first spawn out of the pair; another had been seen, and evidence of prior spawns was also observable when the first spawn was discovered.

Mike Doty’s pair ALSO spawned; he shot me a text just a few hours later (10:30 AM) on March 4th as well.  I’m guessing they too spawned on the 3rd.  On the evening of March 5th, I found that the Nebula percula pair in my basement had spawned, and Friday, March 6th, the recently reunited Lightning X Lightning pair had thrown down their first nest as well; it’s a tiny nest, but it is viable.

It’s interesting how much of this breeding activity centered around the full moon.

Spawn #36 missed

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Not really sure what happened on this one.  By all schedules, the evening of July 2nd, going in the morning of July 3rd, was the first “hatch night” for Spawn #36.  Somewhere shortly before midnight on the 2nd, we had a power outage.  It maybe lasted an hour, and when I got down into the basement, there was not a single egg on the tile, and not a single larvae swimming around in the tank. If I’m honest, it appears as though they may have hatched far earlier in the day, or perhaps were even eaten by the parents – normally there would have been SOME eggs left, but this time, nothing.  Did the fish freak out and eat the eggs during the power outage?  Frank, I don’t know. I don’t recall checking the nest at all today, so I really can’t say if it was even there in the AM.

And so ends spawn #36…

 

So 8 days definitely would have been the right call.  I’m going to heed this warning to myself on the next spawn.

On 7-26-2013, there was definitely a hatch of offspring.  Somewhere between 25 and 50 larvae had hatched out, but the REST OF THE NEST remained unhatched.

By the morning of 7-27-2013, what I *should* have seen was more fish hatch out, but sadly, instead what I found were a bunch of dead eggs scattered all over the bottom of the black round tub. All of these were siphoned out. So despite the addition of Methelyne Blue, which if anything should have helped the eggs remain viable until their hatch, the vast majority of the nest, easily 500-700 eggs, died instead of hatching.

But the real kicker, by the late evening of 7-28, all those larvae that HAD hatched out?  Gone.  Vanished.  Nowhere to be found and no BODIES seemed to swirl up either.  There was ONE swimming around that I could find, and by the morning of 7-29, that one was gone too.

Not to worry though, late in the afternoon of 7-29-2013, the next spawn occurred. By my online records this should be spawn #8. So I get another shot at this.  It will be very hard to resist, but I will NOT pull the nest out for hatching in a larval tank until the night of August 6th, 2013.  What I *might* do is run a larval snagger on the night of the 5th, just in case the babies get all uppity and decide to all hatch on the “first night” of hatching.

It figures just as I’m starting to contemplate moving the Lightning Maroon Clownfish and her mate out of the basement and back upstairs into a display tank, she goes and throws down a whopping big nest of eggs back on 3-16-2013.  I sat on that news a bit mainly because I’ve been fundamentally too busy to share it; among other things, this has been a month on edge as we are expecting our second child.  At this point, we had labor over a month ago…so every little pain or contraction has me (and my wife) wondering if “this is it”.  For a month.  To say I’m frazzled is an understatement.

But sitting on this news did make for an excellent double-gotcha on this year’s “traditional” Reef Builders Lightning Maroon Clownfish April Fools for 2013.  No, I did not partner with Monsanto to clone the Lightning Maroon.  But those with real keen eyes did notice something they hadn’t seen before – Lightning Maroon eggs in a flowerpot ;)  Curtis Kramer, Brandon Mehlhoff, and “Ted” – kudos to you for your mad powers of observation.

Here’s the spawn from 3-16-2013.

So this “third spawn” from 3-16-2013 hatched out on 3/24.  Here’s a photograph of the next the night before hatching, 3/23/2013.

By 3/27, it appeared that most babies were gone, but “no worries” I thought.  Why?  Because that evening, Lightning and her mate threw down another big nest that night, 3/27/2013.  That is the spawn I included pictures of on our April 1st, 2013, prank.

I pulled spawn #4 on the evening of 4/3 (technically wee hours of 4/4/2013 – my fishroom lights go off at 2:30 AM these days); the eggs went into a black round tub with 5 gallons of the water from the broodstock aquarium. Of course, it appears I jumped the gun as they didn’t hatch overnight and were still intact on 4/4/2013.  The bulk of the hatch occurred on the night of 4/5/13 leading into the next morning, although many babies and eggs were found dead yet upon the evening of 4-6, there were still apparently very viable unhatched eggs.  So a hatch potentially spread out over 3 nights?

As of today, 4/10/2013, there were maybe 5 larvae swimming around in the BRT.  If this was any other clownfish, I’d probably skip trying to rear them and just wait for my next run.

Admittedly, I am frustrated by this – my Onyx Percula pair has always had split hatches, and it seems that now the Lightning Maroon is as well and even my newly spawning Fire Clown pair (Amphiprion ephippium) is doing so as well.  The mortality following hatch is drastically high right now; it could be how I’m hatching them in the BRT.  There must be something about my husbandry or technique causing this, but I’m not sure what it is.  Off to ask the experts…

And sadly, the result of this is that there are STILL only a handful of Lightning Maroon offspring that will be put up for sale – even if I got a massive run at this point, it wouldn’t be until fall or winter that they’d be market size.  But there is hope.  As of 10:30 PM on 4/10/2013…yet another nest laid (Spawn #5).  I’ll be blunt; given that we could have a newborn baby in the house any day now, the odds are good that I’ll totally botch or not even get to spawn #5.  So for now, scant few, if any additional lightnings on the horizon beyond what I’ve already raised!

This does raise one burning question – do I move the pair back upstairs and risk putting them off their stride, simply to have them back on battery backups and such, or do I place a pairing of their offspring upstairs.  No easy call there…

A special thanks goes out to Shane & Len at Advanced Aquarist for co-publishing this blog entry there - it seemed like the perfect contribution given the style of material they like to cover.

The Lightning Project

Just to bring you up to speed, it’s been over 2 years since the “Lightning Maroon” from PNG made it’s way to the US and ultimately into my home tank.  It was a long time coming but in the late spring and early summer of 2012, we finally got a glimmer of success with the first and second spawning between the “Lightning” Maroon, and a normally patterned “wild-type” Maroon clownfish.  The pairing below are the two parents responsible for everything we’re about to cover.

The Lightning Maroon Clownfish and her wild-type mate.

The Lightning Maroon Clownfish and her wild-type mate.

The Results Are In

So not only did we get a spawn that made it, but we got roughly 50 juveniles post-settlement in the very first rearing attempt.  Now, more than 2 weeks in, it seems ever more likely that we have a roughly 50% rate of “Lightning” babies in our group of offspring – note I have yet to do an actual headcount, this is just a ballpark guesstimate on the numbers.  Initially the babies showed up with blue “caps”; thicker headstripes that were readily discernible.  As they progressed, they looked more and more like Picasso Percula babies.  While still possibly premature to say conclusively we have “Lightnings”, we’re definitely starting to see signs that the Lightning trait will come through with defining characteristics that will clearly match up with those that the two original wild Lightning Maroons shared.

17 day old Offspring from the Lightning Maroon & a wild type Maroon from the same island in Papua New Guinae

17 day old Offspring from the Lightning Maroon & a wild type Maroon from the same island in Papua New Guinae

Dismissing the Hybrid Hypothesis

Before going into the genetics discussion, I’m going to address one “possibility” that some creative thinkers might propose, either though just being “creative”, or through having read, misread, or misunderstood what someone has posted on some forum somewhere.  The hypothesis is this; the Lightning Maroon is a different species than a normal white stripe maroon.  And thus, are these offspring “hybrids”?

Categorically I firmly believe no, the Lightning Maroon does not in any way represent a species other than Premnas biaculeatus.  In most hybrid scenarios between two species, the initial primary hybridization generally yields a predictable intermediate form between the two parental species – I am sure there are examples of a primary hybrid where the offspring “range” from one parent to the other, but that is far more common in the second generation if it’s going to happen.  Since we have no intermediate forms in the offspring of this pairing, I believe we can safely rule out the “hybrid” hypothesis without further delay (the same cannot necessarily be said if we look at the “White Stripe” vs. “Gold Stripe” Maroon…the more I read and learn and see…leads me to believe these may in fact be two distinct species in the wild).

Let’s talk Genetics, Breeder Style

I’ll state up front that I’m no geneticist, and that I’ve been known to get my terms confused. So I’ve taken the opportunity to run this by Adeljean Ho (a good friend of Dr. Matthew L. Wittenrich, and the scientist who published work in CORAL that suggests a unique genetic basis for the “Red” form of the Green Mandarin).  Hopefully he caught any errors I may have made in attempting to distill and disseminate these ideas.

Remember, I really downplay the “designer” aspects breeding of marine fish with mutations, but taking on the preservation of this wild trait has forced me to learn it.  Understanding the genetics allows a breeder who is working with “designer” fish to quite literally “create” what he or she envisions; the upshot of this knowledge is that it also levels the playing field for breeders, forcing them to turn back to producing QUALITY fish in order to differentiate themselves.  For me, the emphasis on quality, as driven by “open sourcing” the genetics of a fish, is the best route we can go if we must pursue “designer” variants going forward.

In this discussion of possible Lightning Maroon genetics, here are the important terms. We will try not use the term “gene”, because it kind of gets used interchangeably and thus will probably only confuse. The important terms here are “locus” and “allele”. “Locus” being a specific point in the genetic code where a particular pairing of alleles resides; the alleles being the pieces of genetic information, one from the father, and one from the mother, that come together at the locus to form the genetic makeup of the offspring.

We also cannot neglect the terms genotype and phenotype.  Genotype refers to the genetic “code” specifically, which is important because alleles can be present yet not “expressed” in the phenotype.  Yes, the phenotype is the outward appearance as driven by the genetics.  And this is the conundrum; due to the way certain alleles interact with other alleles, there are traits that can be masked, surpressed, or unexpressed, that is to say you won’t know a fish carries a hidden albino gene in its genotype just by looking at it (and seeing it’s phenotype).

The other important terms to remember are “homozygous” and “heterozygous”; all that really means is whether the two alleles in the loci pair are the SAME genetic code (homozygous, such as A/A or B/B), or different (heterozygous, often abbreviated as “het” for short, such as A/B). Considering the entirety of our genetic makeup, it all boils down to loci (plural of locus) and what pairing of “alleles” is inherited at each locus. Obviously, the outward result of these traits is the result of all these separate loci together, and certainly some observable traits may be governed by multiple loci, which makes it difficult to ascertain the genetics and inheritance behind them. By the same token, the possible individual alleles that can be present at a loci are perhaps infantasimal in their variation (for example, ABCDEFGHIJKLMNOPQRUSTUVWXYZ), but only two individual alleles (eg. A/Z, C/C, or B/Q) will be present in any particular locus.

That said, most all of the genetic variations that we’ve come to openly understand in fish seem to be the result of the genetic makeup of an individual loci, and from there, the combination of multiple traits at different loci  is what gives us a well-understood, massive diversity of ornamental fish varieties (as some would call them, “Designer” fish).  Freshwater Angelfish (Pterophyllum scarlare) make the perfect example as they are well understood genetically (see The Angelfish Society’s Phenotype Library). Combining multiple traits from individual loci is how we get a Pearlscale Lace Clown Veil Angelfish. Those names refer to phenotypes; outwardly discernible traits, in this case those names refer to scale structure + dark gene + stripe genetics + fin length. In the Angelfish breeding community, this would get denoted roughly as (p/p) – (D/+) – (Z/S) – (V/+).  Because we know which alleles at which loci contribute to each end result, in theory any breeder can “make” a Pearlscale Lace Clown Veil Angelfish; the breeder just needs to have the proper parents with the proper genotypes.  The breeder also knows that mating two Pearlscale Lace Clown Veil Angelfish together will result in a plethora of unique genetic combinations, 27 to be exact, all of which have their own name.  One example?  Pearlscale Blushing Superveil Angelfish, (p/p) – (+/+) – (S/S) – (V/V).

I’ll borrow notation from the freshwater Angelfish world to try to lay out the options for the Lightning Maroons, and I’ll propose that “L” will stand for the Lighting allele. “+” will stand for the wild-type, default state allele (aka. a normally striped fish). Thus, a wild fish, without any “Lightning” genetics, would be represented as (+/+). Note that in this notation, capital letters are normally used for dominant or partially dominant traits, whereas recessive traits are generally denoted using lowercase letters.  I’m going to assume right up front that “Lightning” is a trait that directly controls the “striping” of the fish.  We are going to assume here that there are only two possible alleles involved in what we are seeing, and that the Lightning Phenotype is driven by one specific allele (L in our examples) and is not in fact the result of two unique alleles coming together (eg. Lightning = L/X, wild fish being +/+).  We are also going to assume that the Lightning trait is the result of genetics at one locus only. A brazen assumption, but it seems likely at the moment.

To explain the multiple locus issue another way, we are assuming it does not take the genetics of two (or more) loci to result in the Lightning phenotype.  In the Angelfish world, there are  phenotypes like Platinum that are the resultant combination of two independent loci, and the presence of specific recessive alleles in homozygous pairings, that result in the all white Platinum Angelfish – in this case the recessive gold trait on the “dark” locus, and the recessive Philippine blue trait on the “philippine blue” locus. Independently, you’d have a Gold Angelfish, or a Philippine Blue Angelfish, but “activate” both of those recessive traits through breeding choices, and you wind up with the possibility of all white platinum offspring.  Yes, you can “make” a Platinum out of parents that are not outwardly “Platinums” themselves, and that is the beauty of understanding the genetics.  What you cannot do is use only Platinums to breed back to the wild form of an angelfish – and that is the curse of “designer” breeding (which is one reason why designer-focused breeding can get in the way of conservation minded breeding – the “ornamental” genetics can function as actual genetic contaminants…but that’s for another day).

Angelfish Phenotype Examples

Genetics in action - the large fish in the foreground is a wild Angelfish, generally presumed (+/+) unless it carries hidden recessive alleles, and called a "Silver", which is the default striping pattern in Pterophyllum scarlare. The Blue one at right is a "Blue Ghost", representing 2 doses of the Philippine Blue allele, and a single dose of the partialy dominant stripeless allele, so (pb/pb) - (S/+). The white angelfish in the back is a "Platinum", the result of a fish being both homozygous for the recessive Philippine Blue allele, as well as homozygous for the recessive Gold allele, thus (pb/pb) - (g/g).

Based on the quantity of Lightning Maroons in the very first batch of offspring, there are three possibilities for how the Lightning Maroon trait genetically functions.  It could be a recessive trait, whereby there must be two alleles for Lightning present in order for the Lightning pattern to be observed.  Lightning could in fact be a dominant trait, whereby it only takes a single dose of the Lightning allele to mask the normal stripping pattern.  And the Lightning trait could be the result of a partial (incomplete) or even codominant allele, where a double dose fish will look different than a single-dose fish, which is still different from the wild type, normally-barred fish.
The Case and Implications for “Lightning” being a Recessive Trait

So let’s look at inheritance and expression of the genetics in play. We’ll start with the easiest to understand, a recessive trait like albinism (I think we all understand how albinism works on some basic level). Another good example – the recently discovered Philippine Blue gene in Angelfish is thought to be recessive.  A fish with a single dose of this allele (pb/+) shows no real difference with the wild form.  But put on a second dose, and *Bam*, you have a Philippine Blue Angelfish.

A Blue Silver Angelfish

A Blue Silver Angelfish , (pb/pb). The angelfish breeding community is thoroughly convinced that pb is a recessive trait on its own locus.

This angelfish is a "Silver", and happens to be a sibling to the Blue Silver angelfish shown above. There is a 2/3 chance that this fish has a hidden Philippine Blue allele, denoted as (pb/+), otherwise it is wild-type in every known sense, written as (+/+). If these two fish were mated, and none of the offspring developed into blues, that would prove the 1/3 chance of this fish having no hidden blue allele.

If “Lightning” is a recessive trait (one that requires two “doses” of the Lightning allele), then the Lightning parent could only be homozygous (l/l).  A fish that is heterozygous (l/+) would appear “normal”. Thus, if our (l/l) fish is mated to a wild type (normally barred) fish with no Lightning genetics (+/+), all the offspring would be (l/+). Such a pairing would result in 0% discernible Lightning Maroons, as all offspring are (l/+) (Figure 1).

Recessive Lightning to Wild homozygous Mate = all hets = no Lightnings.

Figure 1. Recessive Lightning to Wild homozygous Mate = all hets = no Lightnings.

Thus, if “Lighting” is recessive, we know that the Lightning Maroon must be (l/l). If recessive, to have found Lightning offspring in the first generation mating, that implies that the standard-barred mate must carry a “hidden” Lightning allele, and thus be (l/+) itself. Mating (l/l) to (l/+) would give you a 50% expression rate IF (and that’s a big if) the Lightning trait is recessive. Mathematically, the door is open for this trait to be recessive (Figure 2).

Recessive Lightning to Wild heterozygous Mate = 50% Lightnings.

Figure 2. Recessive Lightning to Wild heterozygous Mate = 50% Lightnings.

Now, there is an upside if this trait is recessive; it means we got lucky. Primarily, it means I got lucky on the selection of the non-Lightning mate, because there would be no way of knowing it carried a single-dose, non-expressed Lightning gene. It would mean that the game plan of using a mate from the same island paid off. If you find a wild albino fish, you are most likely to find more albinos in the same geographic region because they’d probably be siblings. Not to mention that many of the non-albino siblings in the area could potentially carry a single albino gene as well.

The other way we will be lucky is that IF Lightning is recessive, and if the initial percentage is in fact roughly 50%, it would mean that all the siblings would then have to carry a single, non-expressed Lightning allele (because their only option from the Lightning parent is to receive a Lightning allele). This would mean that every fish in the group if mated would produce 25%, 50%, or 100% Lightning Maroons. To put it in a commercial context; if we definitively knew that this was a recessive trait, then even the normally striped offspring would be tremendously valuable to breeders, because simply mating two of those together yeilds 25% Lightning.  In an interesting twist, it seems most people expected the Lighting trait to be recessive if genetic, and assumed that we would get the results shown in Figure 1, and only in the 2nd generation would we get more Lightnings, as shown below (Figure 3).

Recessive Heterozygous F1 Offspring, Mated together, produce 25% Lightnings.

Figure 3.Hypothetically recessive heterozygous F1 Offspring, mated together, produce 25% Lightnings.

Still, I’d love to hope that this trait is recessive because it means all the siblings would then carry a hidden Lightning allele. In looking at the number of wild Lightning Maroons presumably observed (and thus caught), we know of only 2. This rarity could suggest a recessive trait, as two wild fish with hidden Lighting Genes, mating together, would produce 25% Lightnings. Given that a clownfish pair’s minimal reproductive goal is to produce two replacements, you can quickly see how a single pair of clowns, constantly churning out babies that are 25% “Lightnings”, might only yield a handful at best (remember, marine fish have been shown to suffer massive mortality in the earliest hours and days of their lives – most never even make it to settlement, and most of those, not past their first year).  Lightning Maroon babies truly stand out in the rearing tank while their normally patterned siblings are difficult to see; you can’t help but assume Lightning offspring be much easier for predators to locate. So the rare Lightning making it in the wild would fit well with a recessive trait hypothesis.

But what are the odds that I got “lucky” with the mate I selected? Impossible to say, but Occam ’s Razor suggests that the following scenarios could be more likely.

The Dominant Scenarios for “Lightning”

Let’s deal with straight up dominance. If this is a dominant trait, then you only need one “dose of the gene” to express the trait. To simplify, breeders tend to view dominant traits as being pretty uniform in their expression, and there’s no difference whether you have one dose or two. In other words, a Lightning Maroon Clownfish could either be (L/L) or (L/+) and would look the same. A good example of this, to borrow from the Angelfish community, is a trait called “Zebra”, which adds extra bars and patterning in the fins. There’s no visible difference between a homozygous Zebra (Z/Z) or a heterozygous Zebra (Z/+).

A Zebra Angelfish

A young Zebra Angelfish, straight up dominance means this fish could be (Z/+) or (Z/Z) - the only way to know is through planned and controlled matings and observing the results.

Let’s again weigh the options. If Lightning is dominant, then the non-lightning mate can only be (+/+). Why? Because any fish that is (L/+) is going to be Lightning. So in this scenario, the normal mate can only be (+/+). That leaves the Lightning Maroon to be either (L/+), or (L/L). Now, here’s where it gets interesting. If the Lightning Maroon was (L/L), we would have 100% Lightning Maroons in the offspring, because every fish could only get a ( L ) allele from the Lighting Maroon, and all (L/+) offspring would then be Lightning (Figure 4). Since we don’t have 100% Lightnings in the offspring, we can rule out the Lightning Maroon being (L/L) if this is a dominant trait.

Homozygous Lightning Father X Homozygous Wild-type Mother = 100% Lightning offspring

Figure 4. Dominant Homozygous Lightning Mother X Homozygous Wild-type Father = 100% Lightning offspring

That would leave (L/+) as our only genetic option for the Lightning Maroon, which would thus result in a roughly 50% expression rate in the F1 generation. The inheritance of the ( L ) allele from the Lightning parent is a just a coin toss, 50% of the time they get a +, and 50% a L.   Once again, the rules of genetic expression and inheritance suggest that this is a possible genetic explanation given the initial results we’re seeing (Figure 5).

Dominant Heterozygous Lightning Father X Homozygous Wild-type Mother = 50% Lightning offspring

Figure 5. Dominant Heterozygous Lightning Mother X Homozygous Wild-type Father = 50% Lightning offspring

Now, my problem with this trait being dominant starts immediately from the fact that it requires at least one outwardly visible Lightning Maroon Clownfish to be breeding in the wild in the first place (unless there is a wild-type pair that is predisposed to throwing off the odd “Lightning” mutation once in a blue moon – afterall, these traits can appear spontaneously). If this trait is dominant, then it might also suggest that this mutation ought not to be as rare as we currently are led to believe it is. And to make matters worse, it does seem that we haven’t seen much straight up “dominant” variations in ANY of our designer clownfish to date; it seems all are either recessive or the result various doses of partially dominant traits. And surprise again; looking back at the Angelfish (which happen to Cichlids, which are a closely related family to the Damselfish, and thus to the Clownfish), we see this: 0nly 1 truly straight-up dominant trait. Meanwhile, there are currently 5 known recessive traits, and 7 traits that are either partially dominant or codominant. Dominant traits just don’t seem that common in clownfishes.

What if “Lightning” represents Partial Dominance?

So what if this is a partially dominant (aka. incomplete dominant) or co-dominant trait. The difference is nuanced, but in the angelfish world co-dominance can cause “blending” of traits in certain mixes, dominant expression in other mixes, whereas partially dominant traits present more of an A/B/C result. To draw a parallel, some might say that if the Lightning trait were codominant, then a fish with a single Lightning allele should still show the white stripes “underneath” the lacy pattern of the Lightning.  I’ll dispense with codominance for the time being and just refer to this option as the partial dominance possibility.

Partial (incomplete) dominance is perhaps the most plausible and most exciting of the three options.  As the scenarios are about to play out, they suggest that the Lightning, in a partial dominance scenario, would only be the “first step”.  Partial dominance is well documented in angelfish, and the stripeless allele is a great example.  A normally striped angelfish is Silver (+/+), a single dose is a Ghost (S/+), and a double dose is a Blushing (S/S).  Take a look at a Ghost and compare it to a Blushing that happens to be showing a second partially dominant trait, the “veil” fin trait (impossible to say at this young size whether our example fish is simply veil (V/+) or super veil (V/V)).

A single dose stripeless angelfish, (S/+), aka. a "Ghost".  You can see a "Silver" (wild type, standard barred) Angelfish in the backround at right for comparision.

A single dose stripeless angelfish, (S/+), aka. a "Ghost". You can see a "Silver" (wild type, standard barred, aka. (+/+)) Angelfish in the background at right for comparison.

Blushing Angelfish

A Blushing Angelfish with two doses of the "Stripeless" allele, (S/S).

If “Lightning” is a partially dominant trait, the results in the offspring push us to only one genetic possibility. Let me step back to explain why. There are currently only 2 forms of observable pattern in the offspring; “Lightnings” and “normal”. Simply put, the Lightning cannot be (L/L) in the partial dominance scenario. If a partially dominant allele is present in a homozygous state (L/L) and mated to a wild type fish (+/+), we should get all (L/+) – something intermediate between the Lightning and the Wild form, and they should all be the same (emphasis again on the fact that there would be no Lightnings, and no normally barred fish either) (Figure 6). We don’t have that result, so (L/L) is ruled out if Lightning is a partially dominant trait.  Or is it?

Partially Dominant Homozygous Lightning X Homozygous Wild Type = 100% Intermediate Offspring

Figure 6. Partially Dominant Homozygous Lightning X Homozygous Wild Type = 100% Hypothetical Intermediate Offspring

The second consideration for parental genetics would be (L/L) x (L/+), but once again here, the (L/+) cannot look like the wild form, as (L/+) represents an “intermediate form”. Someone out there is going to say “but what if (L/+) does in fact look like the wild form?  If it did, then by definition Lightning would be a recessive trait as I described earlier (Figure 2)!  So this scenario is ruled out.

The third consideration would be (L/+) x (L/+), but then again that would mean both mates should be “intermediary” forms and roughly look the same (which they obviously don’t in our pairing). This alone is enough to scrap this mating as a possibility.   But if you’re not convinced, this hypothetical mating would also mean that 25% of the offspring would be (L/L), 50% (L/+), and 25% (+/+) – if the fact that the parents would have to look the same didn’t throw this out for you, consider that there would still have to be THREE (3) phenotypes in this batch of offspring for that proposed genetic combination in the parents to make any sense (which it can’t, because the parents are not the same).

The only way that “Lightning” works as a partially dominant trait is if the Lightning Maroon is (L/+), and the mate is (+/+). This produces a nice occurrence of 50% like the Lightning, 50% like the male parent (Figure 7). This also takes a lot of the “luck” out of the equation; we didn’t have to stumble upon a mate with a hidden allele like we would have in the recessive scenario.

Partially Dominant Heterozygous Lightning Maroon X Wild Type Male = 50% Lightning Maroons

Figure 7. Partially Dominant Heterozygous Lightning Maroon X Homozygous Wild Type Male = 50% Lightning Maroons

This also seems to be how some currently known traits may work (Picasso in Percs, maybe Snowflake in Ocellaris). If you believe that “Black Ocellaris” are a melanistic variation within Ocellaris, then “black” in ocellaris could also potentially be partial dominance…with “Blacks” having “two doses”, and when you mix Black with Ocellaris, you get “Mochas” which in all photos I’ve seen, are muddy intermediaries. The real question to be asked is what happens when you breed 2 Mochas together – do you get 25% Blacks, 50% Mochas, and 25% normal Orange Ocellaris? I don’t know that anyone has done that and tested the results yet (but I also know that I don’t believe they are the same species of fish at the moment either…you have to throw the genetics out the window when you start hybridizing)

But getting back to the Lightning; if this trait is “partially dominant”, then the most exciting part is yet to come, because it would mean that all the fish we’ve seen so far only have ONE dose of the Lightning allele (L/+), and thus, the designer breeders out there will be clamoring to mate two Lightnings together so they can discover what a (L/L) fish is going to look like.

And that’s the crazy part, because there should only be one of two things that could when we mate Lightnings together – either we’ll get 25% being something new, or we’ll just get more Lightnings. If we get 100% Lightnings, we are either looking at a recessive trait or a straight up dominant trait (or, in a less likely case the difference between a partially dominant (L/+) and (L/L) is simply too minimal to discern, and you’d then just treat it as dominant anyways).

The “Lightning Precursor” Hypothesis – Dealing with Horned and Flaked Maroons

I suppose at this point we have to step back and objectively define what we *think* a Lightning Maroon “is”.  What is the phenotype?  We have to consider the two fish that have been given that label to-date (the less familiar one being the first wild-collected Lightning Maroon from 2008).  Well, the best term I’ve heard used lately was to describe the Lightning Maroons as filigreed. Other’s commonly call the patterning “lacey” or “net-like”.  Whatever it is, the most notable place for this Lightning variation is in the headstripe.  The headstripe is dramatically wider in the Lightnings, and it is “pitted” with normal coloration.  “Horned” and “Flaked” maroons fundamentally lack this very distinct patterning and the wider headstripe it takes to make it.

The other part of the Lightning phenotype is the breaking up of the mid-stripe and tail-stripe into the lacey, interconnecting patterns that split apart and at times, reconnect.  None of the “horned” maroons show this patterning that I’ve seen, while many “horned” maroons simply exhibit broken bars or “extensions” trailing off.  Admittedly, only the most recent “Lighting Precursor” was really suggestive of the body stripping seen in the two wild Lightning Maroons, but the stripes showed a more “smooth” outline and did not reconnect (I’ve been told the other side of this fish was unimpressive) – I think this fish is better considered a more extreme form of these “Horned” Maroons being found in PNG waters.

In drafting this genetics rundown, I realized I had one other genetic possibility on the table; the notion of the “Horned” Maroons being collected in PNG potentially represented the “intermediate” form in a partial dominance scenario (eg. the hypothetical heterozygous offspring shown in Figure 6).  One such Maroon recently made the rounds in the internet being called by some a “Lightning Precursor”.

After examining the data provided publicly by EcoAquarims PNG, it seems these aberrant Maroon clowns  appear to be quite common in the waters of PNG, with various atypical Maroon clownfish being caught approximately every 11 days.  We also had other fish like Mike Hoang’s Goldflake Maroons which indeed, as young fish, had me wondering if we’d see Lightning-like traits as they grew up (sadly the best marked offspring were lost, and those that remain look no different than the “Goldflake” Maroons output by Sustainable Aquatics).

Let’s deal with the “Goldflakes” of the world first.  It turns out that abnormally spotted and overbarred Maroon Clownfish (what I’m calling “Flaked” here) are indeed commonplace in captive culture.  So far, these fish have seemed to elude genetic categorization, apparently really behaving fundamentally more like “misbarring” in other species of clownfish.  Most recently German breeder Sylvio Heydenreich shared some videos depicting some highly overbarred Maroons on the MBI website; when asked about these fish, he stated quite directly that, “Die Fehlzeichnungen lassen sich ganz leicht über die Wasser Qualität steuern.”  Or as Google likes to translate it, “The failure drawings can be controlled easily through the water quality.”  Failure drawings of course, being what is probably a literal translation for “misbarring”.  And to that end, we already are aware that misbarring in clownfish has environmental causes, not genetic causes. So as much as we like these “Goldflakes”, all observations to date suggest we think of this type of patterning as a likely non-genetic occurance.

Meanwhile, those “Horned” Maroons coming out of PNG had all of us, even me, convinced that the Lightning Maroon could be a homozygous (double-dose) example for a partially dominant trait. Simply put, the breeding results don’t really suggest this possibility because we lack the intermediaries (I would’ve expected the 100% “horned” batch to show up, like Figure 6).  Still, I do have two normally barred fish that show spots.

Note the extra spot on the back of this normally barred juvenile.

Note the extra spot on the back of this normally barred juvenile.

Is this baby a “Horned” Maroon?  Well, here’s the kicker.  There are only two ways you get hypothetical intermediates (intermediates being the proposed placement of the “horned” Maroons).  You either get 100% in the F1 batch, or the male parent has to be an intermediate itself, in this case, a “Horned” Maroon. And this is where there’s still an outside chance – the male has a single broken tailbar.  But…if this was in fact an “intermediate”, what genetics must we get in the offspring?  75% Lightnings, and 25% intermediates – NO wild-types.  Again, let me be explicitly clear – for “Horned” Maroons to be “Lightning intermediates” or “Lightningprecursors”, I would have had to encounter “Horned” Maroons  in the offspring and at a rate of 25% -or- 100%.  So…if the babies all wind up showing extra horned bars and spots as they grow up over the next few months, and the ratio of Lightnings to non-lightnings is 3:1, there could still be “hope”.  Otherwise, we have probably closed the book on the “Lightning Precursor” hypothesis that tried to link the Horned Maroons to the Lightning, at least for now.

All of that said, what I really think we’re seeing here is something much more fundamental in the Horned Maroons.  We are seeing this “flake” overbarring, a commonplace occurring in captive-bred maroons, showing up on a few random offspring.  You wouldn’t notice it in the Lightning offspring because it’s just “painted over”, but you can see it in the normally barred fish.  Years ago, breeders would have destroyed these types of fish as “culls”…that’s when the 3-bar wild-type fish was considered something to aspire to as a breeder, and not “common” and “boring” as many hobbyists may consider a wild-type clownfish today.  Given that we know of a possible causal relationship between “overbarring” and “environment”, perhaps there is something environmentally going on in the waters of PNG to show us more “environmentally overbarred”, aka. “Horned” Maroons, than perhaps we might expect in other parts of the ocean.

Or, and this is still a possibility; the “Horned” Maroons of PNG could yet represent another, distinct genetic variation.  It’s certainly possible – breeding them could give us the answers, although it may be difficult in the face of commonly-occurring “flaked overbarring” potentially giving you a fish with the same basic phenotype.

The Odds on the Lightning Pair’s Genetics

Let’s get back to the Lightnings.  If we give equal weight to all three possibilities for the interaction of the “Lightning” allele, we are left with three scenarios for the genetics of the parents.  Once again, notation here…(female first) X (male second).

Recessive, where we have (l/l) X (l/+)
Dominant, where it can only be (L/+) X (+/+)
Partial Dominance, where it must be (L/+) X (+/+)

By this alone, each has a 1/3 chance of being right.  There is a 2/3 chance, or 67% roughly, that the Lightning is (L/+).

However, for the sake of doing something interesting, what if I used the genetic ‘spread’ in Angelfish to derive an alternate baseline for the odds of a trait being dominant, recessive, or partial/codominant within the clownfish family?

Recessive = 5/13, or roughly 38%
Dominant = 1/13, or roughly  8%
Partial/Codominant = 7/13, or roughly  54%

If this was at all representative of the odds for trait expression in clownfish (and it’s really probably not, it’s just a fun way to think about it), then we have a 62% chance that the Lightning Maroon is (L/+), and within that 62%,  it would then represent a 87% chance  that the trait would be partially dominant (again, roughly 54% overall).

Overall, whether we weight the system or not, the odds remain in the rough territory of 2:1 that the Lightning Maroon is (L/+), and the mate I used is (+/+), vs the only possible alternatives of (l/l) and (l/+).  The kicker for me is when you move beyond “probability” alone, and put in the observations and the way mother nature seems to work.  I’ll get to my prognostication in a minute, but first, I must point out that this puzzle can be solved.

How are breeders going to help figure it out?

In a nutshell, this project will soon turn to the massive “cloud computing”, or in this case “crowd breeding” effort of marine aquarists who get these offspring.  It has always been my intention to get the F1 fish out to other breeders to both diversify the risk, but also to leverage the collective efforts of breeders to provide for rapid, definitive answers.  In a nutshell, anyone breeding with my offspring, you have my formal request to track your project at the MBI, and to do so openly.  You also have my request that you must track your offspring numbers and take photos of each one on both sides, because it is the headcounts and photos that will help determine the genetics in the end.  Here’s how we’ll do it (again, assuming that “Lightning” is the result of a single locus and a single allele).

We can determine (or rule out) a recessive trait by mating the non-lightning siblings together; if recessive, 2/3 of the F1 babies will carry a hidden Lightning gene. This means that picking any random 2 fish, the odds are roughly 40% that both are (L/+), so four out of 10 random pairings would yield Lightning offspring to the tune of 25%, if this is a recessive trait. The only way you get Lightnings out of pairing 2 normally-barred siblings is if this trait is recessive.

We can also determine this trait to be recessive by matings of Lightning Maroons to their non-lightning siblings. In this scenario, 2/3 of the pairings would produce 50% Lightning offspring, while the remaining 1/3 would produce nothing but normally striped fish.

We don’t need to use the siblings to specifically test for a recessive trait, but non-sibling fish present a conundrum – you have less insights onto what their genetics could possibly be.  Still, you can simply mate Lightings to unrelated white stripe maroons (and breeders out there, I will work as hard as I can to produce offspring from the other PNG White Stripe pair in the house so we have a clean PNG bloodline which we can outcross to, and Dale Prichard in the UK hopefully can contribute more, or you can look to the other PNG maroons being exported from EcoAquariums PNG now).  If the trait is recessive, then you have to consider the unknown odds that any randomly-selected, unrelated fish, could be carrying a single hidden copy of the recessive allele.

However, if the trait is partially dominant, any Lightning paired with a wild-type sibling, or any outcross (mating of a Lightning to unrelated normal fish) should yield a percentage (50%) of Lightnings in the offspring. Conversely, again, if the trait is recessive, these outcrossed matings will produce nothing but normally barred fish UNLESS, once again, you get “lucky” to stumble upon a fish with a hidden allele. But that’s the rub – you’re far more likely to find that hidden gene in the normally barred siblings.

If we get something “new” out of the Lightning X Lightning mating, it should be 25% of the “new” variety, and that would convincingly clinch the genetics as partial dominance. Sounds far-fetched? Well, in Percula, Picasso X Picasso is where we get Platinums from.  If mating Lightning X Lightning simply makes 100% Lightnings, then the trait easily falls into the category of a straight up dominant trait.

I’m a betting man if the wager is bragging rights…so my guess is…

…partial dominance.  Ultimately, my gut call is for partial dominance because it seems to be the most commonplace type of genetic trait we’ve seen in our designer clownfish, and it’s the most prevalent in a widely cultivated and well-documented group of related fish (the freshwater Angelfish). The odds also do slightly favor partial dominance.  Partial dominance may also be one of the easiest to prove – just mate two Lightnings together and see what you get. Partial dominance (and in this case, straight dominance) also requires less luck to have had the outcome I seem to have had with my initial pairing. If ever there was a project that had just about everything except “luck” on its side, it is The Lightning Project.

One last wonderful caveat – every possibility laid out above could wind up being 100% wrong.  Until we get those second generation fish produced, and aquarist start gathering the data and sharing it, we simply won’t know.

A special thanks to Adeljean Ho for acting as a sounding board and editor on this piece.  I am sure Adeljean, with his strong interest in genetics, was probably as excited about this as I am!  Thanks!

8 days post hatch.  To brine, or not to brine, that is the question.

First noticed this at roughly 1:30 PM on July 7th, 2012.  Dare you to hold your breath…

Larval offspring from the Lightning Maroon Clownfish nearing settlement.

Lightning Maroon Clownfish larvae, nearing metamorphosis

Headstripes forming on larval Maroon Clownfish spawned and hatched by the Lightning Maroon Clownfish

So this amounts to a chronological retelling of the story to date, this time with photos, starting  a couple weeks back now.  Perhaps not in as much detail as my minute-by-minute updates, but a good overview of the run to date.

June 21st, 2012

The ongoing health problems with the Lightning Maroon remained, and the left eye on the Lightning Maroon was showing slight swelling.

On a day initially planned to do a skin-scrape of the fish for further examination, I had to call things off because the fish had started going through pre-spawn motions.

By the time we had finished doing a skin scrape on some Banggai Cardinalfish downstairs, Barb & Heidi from the Great Lakes Aquarium got a super special treat, seeing the actual nest having been spawned while they were here.

Lightning Maroon Clownfish Spawning & Eggs

June 22nd, 2012

I was genuinely worried whether we’d have eggs 24 hours in.  Thankfully, they proved to be good parents and good “clownfish”; the first spawn egg eating proved to be the typical first test run that so many clownfish seem to do.  This batch, while I didn’t get a good photo of the parents, was doing well.  The swelling on the Lightning Maroon’s eye had gone away.  Phew.

June 23rd, 2012

So much for resting easy about the health of the Lightning Maroon. The eggs were developing (a fair number that probably were infertile or diseased were removed by the pair), but some funky gunk (yes, that’s the scientific term) showed up on the Lightning Maroon’s right face.  I was once again on high alert; this wasn’t pop-eye; this was more reminiscent of the mouth-rot I had to battle back a little while ago.

June 24th, 2012

So much for being on alert.  By evening, things looked so bad on the Lightning Maroon’s face that I pulled the trigger and initiated the third course of treatment with Maracyn SW and Maracyn II SW in this system.  The telltale bulge around the right eye had started to show as well.  I felt I had little other option at this point; this fish is simply too valuable to take a wait and see approach when symptoms like these show up:

The eggs were looking good and developing fast, although I took little comfort in that given the current situation with the Lightning Maroon.  The roller coaster of stress over this fish during the past couple months has been excruciating.  No doubt, there were times I pondered whether it would all be easier if the fish just passed away – of course solely a passing fancy, but when things are clearly out of your real control, it is incredibly tough to sit there and do “what you can”.  Of course, it’s a whole new level now that we are well within sight of the next major milestone in this 2+ year long project.

June 27th, 2012

June 27th represented the 4th day of Maracyn + Maracyn II treatments, and once again, it appeared I had potentially averted a crisis or loss.  The condition of the Lightning Maroon was drastically improved.  The eggs…the eggs were showing eyes?  They had the classic silvery look of clownfish eggs before they’re going to hatch.

I had been worried that these eggs would be hatching out while I was on a trip to Boston to speak at the Boston Reef Society; but now, only 6 days post spawn, I was very worried that a hatch could come sooner than expected.  The signs (and the data out there) said it was possible, sure, but maybe not likely?  Still, if I waited too long and did nothing I could miss the hatch. Conversely, if I pulled the nest too early, I could miss killing the eggs before they actually had fully developed.  Honestly though, I felt far less pressure about the decisions I was about to make than any of the disease-related issues with the Lightning Maroon; this is clownfish breeding, I can handle it.

There was really only one route to go – I had to sit and watch the tank.  The lights go off at 12:15 AM, so I got things situated for a possible hatch.  I used a small LED flashlight at the far corner of the tank as a larval attractant.

While waiting for the lights to go out, I prepared the area  with buckets and siphons to take out larvae should they hatch in the tank.

Downstairs, I prepared a black round tub to receive broodstock water and possible babies.

Lights went out, and it was time to wait.  All pumps were turned off through an extended feed timer on my Apex Lite (which would ensure they’d all come back on in the event that I somehow forgot about them and went to be).  I did have to unplug the battery backup on the Vortech…can’t have babies going through that pump either.  I’d check every once in a while, and initially got excited around 12:20 AM when I saw movement in the beam of the flashlight – until I realized it was copepods swimming around.

Many more checks turned up nothing, and I was starting to wonder if I had jumped the gun.  Multiple plans of “what next” rolled around in my head, but they all disappeared at 1:23 AM on June 28th, 2012.

That is not a copepod.  If you can’t really see it, maybe this one will help:

The moment that first baby clownfish showed up, I pulled the tile under almost complete darkness, moving it downstairs in a bucket with a lid and 5 gallons of water from the broodstock tank.  I set it up for artificial hatching, and assumed that come morning, I’d see hundreds of clownfish swimming around.  That was the hope…

June 28th, 2012

So much for hatching overnight.  There was ONE baby in the tub.  Terrific (<-sarcasm).  1 is better than none, so in the interest of keeping the one alive, I was forced to tinge the water green with a very light treatment of RotiGreen Nano, and a very small addition of rotifers (lest the baby starve).

The worst fear is that I had somehow killed the eggs in the move or prevented the hatch, which would have generally killed the eggs overnight.  There was only one way to find out.  I took a quick look at the tile.

And here’s what I saw…

They look perfectly fine.  And what a great opportunity, thanks to the advent of digital photography and Photoshop, to get a headcount.

That’s roughly 310 eggs (each color group represents me counting to 50, with the scattered red dots representing the last 10 I counted).  It’s not an exact headcount, but gives a great approximate number of eggs.  Hardly the spawn of several thousand that some Maroon Clownfish are known to put down, but I’ll take it all the same.  So very carefully, this tile went back into the black round tub…

…So long as the eggs didn’t die, there was still hope.  The rest of the tension filled day was spent fighting the urge to recheck the tile for dead eggs.  Come nightfall, I stuck with the photoperiod that the eggs had been used to, and turned the lights out in the basement a little early so that things were basically pitch black by 12:15 AM on June 29th.  Just after 1:00 AM, a quick check with the flashlight caused me to announce to the world, “Ladies and Gentleman; we’re rearing Lightning Maroon Larvae.

June 29th, 2012.

With only hours before my departure to Boston, I had to get things set up right.  As the night progessed into the wee hours of morning (that we normally still call “night”), I fired up the lights, and checked the tile:

No stragglers – that means a 100% hatch.  That means 300-ish baby maroon clownfish.  300 chances to see something really fantastic down the line.  So long as we don’t botch rearing them!

Mike Doty, a fellow aquarist who happens to live 4 blocks away from me, had been over late (or early if you want to get technical) to see how things were set up and to know where everthing was…well that and to share a beer, toasting this milestone. Mike would be completely in charge of rearing the larvae in my absence.

 

While I got my share of incredulous inquiries about that, I actually had more confidence in Mike than myself; Mike had taken a pair of extra Maroons from me, spawned and reared a couple batches, so he was perfectly qualified in my book (I’ve done clowns, but never maroons before).  We got the larval tub set up with greenwater and rotifers, and in the early afternoon I embarked on my all-day trek to Boston.

 

 July 1st, 2012

I returned home from Boston in the afternoon, anxious to see how things had gone.  Mike had kept me updated via texts during my absence and things sounded good.  The main message I got from Mike was that my three rotifer cultures had failed to keep up with demand, and he had actually depleted his as well.  I wondered, would we wind up losing this batch to starvation?!

July 2nd, 2012

I’m indeed burning through rotifers, but the cultures seemed to rebound and were producing enough for the moment.  The rotifers in the BRT were also clearing out phytoplankton pretty frequently.

Mike and I had set up a drip for the tub using a spare brine shrimp hatchery and a micro ball valve from Julian Sprung’s Two Little Fishies.  Not only is the drip good for top off, but also for introducing foods (phytoplankton) and ammonia control (CloramX) slowly.

Seeing that there were still many babies (some losses, but still many viable larvae), I took a photo for you all; your first look at what *Could be* a larval Lighting Maroon Clownfish, roughly 4 days old.

 July 5th, 2012

Things have gone well, as I’ve slowly doubled the larval rearing volume to 10 gallons, keeping a watchful eye on the ammonia alert badge as I continue to feed 4-5 gallons worth of rotifers into the tub per day.  With the warm basement temperatures (normally in the upper 60′s to lower 70′s, but lately 78F), the rotifer cultures are now roaring; I’m forced to feed them twice daily at a rate of 30 drops of RotiGrow Plus (and 30 drops CloramX).

I’ve done a couple pre-feeding rotifer enrichments with Super Selcon as well, just to keep the DHA levels up. However, today, now just before 7 days old, we reach another step in the rearing process.  Today it was decided the larvae were finally ready to feed on APBreed’s TDO, size A.  And after the second feeding, it was fair to say they are indeed consuming it.

So now we sit and wait.  Any day now, we will catch the first glimpses of stripes as these larval Maroon Clownfish go through metamorphosis and settle out into juveniles.  Most likely, I suspect that even if we have fish that will one day show the “Lightning” phenotype, we won’t see it at this stage in their development.  But at this time, it is anyone’s guess.  If you’re a betting man or woman, it’s time to place your wagers.  Our first glimpse at the possibilities are just around the corner.

Seriously, one baby found hatched in the tank, and failed to collect it.  One more baby has hatched out in the BRT (Black Round Tub), and that’s it!  It’s now 4:00 AM, so I turned on the lights on the BRT, angled it off to the side so that the light intensity is greatly diminished.  The eggs appear to be fine, so my guess is that I was tricked by a couple early hatchers, and the bulk will hatch tomorrow (Thursday, 7 days post spawn).  That’s my hope anyways, but wouldn’t it be just like the rest of this project to only get ONE baby?!  Wouldn’t surprise me in the least.

In a balancing act, I introduced a very light level of Rotifers and equally light dosing of RotiGreen Nanno into the BRT so that our lone ranger has something to do with all this time on his hands.  I went very light to hopefully avoid any contamination that could foul and kill the remaining eggs who have probably another 24 hours to hatch.

I’ve been taking photos, so at some point I’ll post a pictorial recap of tonight’s (and hopefully tomorrow’s) big event!

6-28-2012 1:23 AM – HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH HATCH!!!!!!

(after discovering  a single hatched-out baby in the flashlight’s beam, the tile was pulled under cover of darkness and moved to dedicated Black Round Tub for artificial hatching)

Moment of truth.

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Pumps are off for the next 2.5 hours (used a feed timer on the Apex to ensure it’s automatically turned back on tonight – no way am I going to screw up and leave the pump off overnight!), lights out save one flashlight for attracting larvae.  I’m still leaning towards tomorrow, and my worst fear is that the hatch out at 4 AM or something long after I’ve given up.  Now we wait.

12:20 AM – got excited when I saw movement in the flashlight beam – darn swimming copepods….

12:30 AM – ran the MP10 on battery power for a minute, then back off.  Turned out my flashlight for a bit; it’s on the opposite side of the tank, completely blocked by rockwork, but going to take no changes.

12:45 AM – ran the MP10 again for a minute, flashlight back on…no hatch yet.  HMM.  Thinking tomorrow is more likely at the moment.  But…should I pull the tile tonight? Afterall, tiles like this are shipped successfully with ZERO “incubation” performed by the parents…so as long as I don’t screw up, there’s no reason I couldn’t incubate them for the last 24 hours.  My head is leaning in that direction at the moment.

1:23 AM – that aint’ a copepod….

 

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