The Lightning Project

The ongoing saga of the PNG Lightning Maroon Clownfish Breeding Project

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Thursday evening (2-19-2015) I discovered a random small spawn from the other PNG White Stripe Maroons in the house. This pair has never been on a scheduled…they spawn infrequently, when the feel like it. As near as I can tell according to my notes here, this is only their 4th spawn.  By the evening of the 21st, the eggs were already gone, so nothing to even bother to try to hatch.  The next evening (2-20-2015), the pair of Nebula Percula I have on hand also spawned (really just noting it here as I’ve had no time to deal with their spawns!)

First, a bit of mythbusting – someone, somewhere, started some rumor that I had lost one or both of my lightning maroon clownfish pairs.  I heard about this from multiple people in early February, but never could pin down the source. Simply put, nothing could be further from the truth.  That said, there hasn’t been any production as of late…here’s why:

So nothing has been going on with the original Wild Lightning X White Stripe pair…they’re just sitting in their tank, not breeding.  I’m trying to figure out why, and here’s what I can come up with. First, when I put all the fish in the fishroom on a prophylactic course of Spectrum’s Ick-Sheild pellet, a Chloroquin-laced food, it shut down ALL my breeding pairs.  That said, most of my breeder pairs have returned to breeding…except the Lightning pair.  They had a few bad clutches and then just stopped.

Next, I had run out of their normal staple diet, Spectrum Thera-A.  So the fish were switched over primarily to feeds like Ocean Nutrition’s Formula One and Formula Two pellet as the daily staple.  Could a dietary switch account for failing spawns?  Perhaps.

Additionally, I noted tank temperatures were slightly down.  Instead of running around 80-82F, they look more like the 78F range lately.  I’m not exactly sure why this is, as I haven’t changed my room temperature nor have I adjusted their heater in any way.  Unless…unless their heater has failed and the tank is now just going down closer to the fishroom ambient temp.  I’ll have to investigate this.

Finally though, I noticed that my lights are coming on very late in the day; so I think my photoperiod is messed up and might be shorter on the tank. In fact, that seems like a logical explanation; we’re in winter, so ambient light cycles are reduced, and the length of day may be reduced as well.  In short, I may have to restore a longer photoperiod, restore warmer temps, ramp up foods and feedings, and hopefully see the pair return to active spawning.  But perhaps it’s good to give the fish a “winter break”…constant spawning certainly must take a toll on our marine fishes.

On the other front, the Lightning X Lightning pair hasn’t been spawning either, but I know why.  As readers may recall, I separated them on January 5th as the pair bond had deteriorated.  I chose to separate the female, isolating her in a breeder box from Florida Aqua Farms.  She remained in isolation for a solid 6+ weeks while the male recovered from his injuries.  Yesterday, 2-18-2015, I added her back into the main tank and watched.  At first, the male attacked her…she took it for about 1 minute, then grabbed the male by the pectoral fin, flipped him upside down, and simply held on while he struggled.  I thought for sure this was the end, but a little while later the pair was found cohabitating peacefully. 24 hours later, the pair remains in good shape, not a nick or scrape or torn fin on either fish.  This is not the first time that a “female time out” has proven to be a helpful factor in curbing excessive female aggression in my maroon clownfish pairing and breeding. I’d encourage people to keep this trick in mind with their own fishes.

Spawn #47 didn’t go anywhere and was gone after a few days. Spawn #48 was laid on 1/3/2014 by the wild Lightning X White Stripe Maroon Clownfish pair. It was a weak spawn, scattered, not a quality nest.  It certainly seems that putting the fish onto Ick Shield set them off their game.

Meanwhile, the Lightning X Lightning pair is just continuing to be in a funk. They’re not getting along, constantly bickering. This from a proven pair.  Today I found the male pretty beat up, and the female’s spiney dorsal fin is swollen and appears infected.  My plan is to at minimum segregate the pair, and possibly to move them into different quarters as well.

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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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Filials, What the F?

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It seems that at least one irate aquarist thinks I’ve made up the concept of “F1″, “F0″ and so forth. Maybe as a marketing ploy even?! Nope, sorry, they’ve been around forever.

Originally, this content was intended for inclusion in the Conservation Breeding Chapter of my half of the book, Banggai Cardinalfish, which was published last year. Most of that last chapter was cut…we were 17 pages over-length and the most esoteric parts of the book were the first to go. But, I found the text in my drafts, and I’m publishing it here, very slightly edited from what I found in my drafts.

Filials Denote Generation and/or Inbreeding

You will routinely see the use of tags like F1, F2, and F3 attached to aquarium-produced specimens of wild-type forms in the freshwater trade, most often attached to fish from private versus commercial breeders. These notations are called filials, and are meant to convey the generational distance from the last “outcrossing,” that is, a mating between two completely unrelated fish.

Officially, F0, or generation zero, represents a pairing of two unrelated fish regardless of the source, and the resultant offspring are F1, the first generation. Mate those F1 fish together and you have F2, and so forth. However, in some cases, including in the general aquarium hobby, F0 more often used exclusively to mean a wild-sourced broodstock. This slight difference in use can cause a significant level of confusion.

From an inbreeding standpoint, and for tracing the extent of inbreeding, the use of F0 to denote the parents of an unrelated mating is both practical and proper. From a conservation standpoint, breeders are often concerned with “distance from the wild.” Given the possible conservation implications, and that the general accepted practice among freshwater hobbyists is to use filials to show distance from wild genetics, Banggai breeders might consider implementing the methodology outlined here.

F+ the number of generations away from wild-sourced genetics. Thus:

  • F0 denotes a wild fish.
  • F1 is generally the progeny of a wild pairing.
  • F2 is the offspring of an F1 X F1 pair, related or not.

When fish of different generations are mated, the resultant offspring are F+1 to whichever mate is already further away. For example, F3 X F5 = F6, not F4.

Problems arise when a captive line is outcrossed back to a wild fish. This fundamentally resets the inbreeding that has occurred, so F0 x F6 should be called F1. Many aquarists and breeders may disagree, considering such a “reset” as deceptive because only half of the parentage is wild, and thus insist that F0 X F6 should be denoted F7. Some breeders sidestep all this debate, using F0 for wild fish, F1 for progeny of wild fish, and “aquarium strain” to denote everything else.

The important final message is that filials are used by different people in different ways. From a genetics standpoint, the filial is hardly the final word, because it can mean two fundamentally different things: one breeder’s F6 fish may be far more genetically solid than another breeder’s highly inbred F3 specimens, and a third breeder’s F1 fish might be 10 generations removed from the wild. If there is any doubt, a conversation with the breeder should help clarify the meaning of the filial label he or she has applied.

In the very week morning hours (eg. 2 AM) on 12-13-2014, I discovered spawn #47 from the wild caught pair.  Obviously it was actually put down on Friday afternoon (12-12-14).  Now that the fish are off the Chloroquin-laced food, it’ll be interesting to see if this clutch holds out or not.

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).

Just jotting down notes…Spawn #45 of the wild Lightning Maroon pair happened on the evening of 11-19-2014.  Meanwhile, LxL (the F1 Lightning Pair) Spawn #5 was gone by the same day….they obviously didn’t like me tiling their entire tank. I need to go by more terra cotta flowerpots…maybe they’ll like that.

So much going on and I’m NOT doing a proper job of tracking things.

I should start with the unrelated F0 PNG White Stripe Maroon Clownfish pair…they spawned sometime last week, possibly Wednesday the 12th. I didn’t make much of a fuss about documentation of it because I knew I couldn’t deal with them at any rate; I can’t remember if it was very late Sunday night, or more likely Monday afternoon, when I found the last few eggs just sitting around in the tank.  This pair, overall, is spawning quite infrequently.

The Lightning Maroon Spawn, #44, never made it to hatching.  Something happened, and the pair consumed the eggs.  Could the Lightning foundation pair be winding down or did they just have a bad spawn?  Their prior spawn produced a good amount of juveniles, which were undergoing settlement and on TDO A by last Thursday, which is when I drained them down to about a gallon, filled with clean new aged saltwater, and then turned them on to the larviculture system.

LxL spawn #4 was one I was going to let go because I wasn’t in town (I was off speaking at the Milwaukee Aquarium Society’s “Fish Bowl” event), but in short, Mike Doty decided to play with harvesting the eggs.  He took approximately 25% of the next out, using my little egg siphon, on Friday night (Nov. 14th).  I got a text while in Milwaukee…there was a hatch..and a big one.  Surprisingly, he simply did it in a 1 gallon olive jar with an air feed, sitting in a water bath with probably 75% new water.  If I had done that, every last egg would have been dead.  All credit where it’s due – Mike may well in fact rear Lightning X Lightning offspring before I do (they’re at HIS fishroom ;) )  Mike further relayed that this was a split hatch, with more eggs present Saturday the 15th, but all gone by 4 AM Sunday morning.

Yesterday, I caught wind that the F1 Lightning X Lightning pair was going to spawn again…the female was roving the tank with her ovipositor down.  I did my best to FURTHER add tile to their tank, blocking their prior spawning spot off..which caused them to move to another side of the tank’s back wall, which I then blocked off with tile. They were still going through the motions of finding a spawning site when I turned the lights off after Midnight; technically they spawned in the overnight on the wee-early morning of 11-18-2014, but if I was to be thinking about these eggs properly, then normally they’d be a 11-17 spawn.  Ultimately, they selected the ONLY section of black plastic background that I cannot get tile on (near the tank’s filter return).  Either the nest was small, or they ate most of it, and I am considering foregoing working with it.  I’m thinking that what this pair may really need is a clay pot…that might be the ticket here.  So LxL Spawn #5 has been put down, but for now, probably will be left alone.

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