Incubating Centropyge eggs and prolarvae.

Of all the marine ornamental fish possible to breed in captivity (meaning that they could be done some time),arguably Centropyge pigmy angels are the most difficult and challenging.This despite tremendous efforts by seasoned experienced breeders and well staffed and funded commercial and academic facilities.And this is not just a matter of finding the right larval food,there are previous aspects that must be adressed before we start the actual larval raising game,which are:
1-obtaining large numbers of healthy developing eggs.This was treated in:http://www.mbisite.org/Forums/tm.aspx?m=51596
2-incubating them until they become feeding larvae.
3-raising the larvae.
I will now deal with the second point.One could think that there is no need for this step,and that the eggs could be merely dumped in the larval tank,as happens with some food fish rearing.But seemingly these eggs and prolarvae need to be kept floating in undisturbed conditions.
So I am incubating the early stages in 2L white trays.I first put a black plastic sheet at the bottom,since the eggs are invisible against a light background,but then found that the hatched prolarvae show early pigmentation and are easy to see.They look very much like a well fed adult Artemia (though tiny),with the head bent downwards,and the oil globule seeming like the Artemia egg sacs.
This is a prolarva caught in the moment of hatching,see the broken "shell" (chorion) and the embryo pulling out of it:
 

 
And this is how a free prolarva looks,under a scope,you don´t see the yolk sac in direct view:
 
 
 
 

This is going to be an interesting discussion. Thanks for sharing the info.

http://www.fishtalpropagations.com/#!home/mainPage
"Making captive breeding easier."

Amaizing Luis, I am looking forward to your observatios and progress over the coming months.

I find that early prolarvae of the wrasse I'm working with are really sensitive to deteriorating environmental conditions. Given that they need to be in still water, this is a large challenge indeed. The suggestion from the recent yellow tang discussion was to keep them in small downwellers (to keep the prolarvae concentrated) in large tubs of water (to keep environmental conditions stable).
 
I also find that pelagic eggs are very susceptible to bacterial issues. Dipping the eggs into a hydrogen peroxide solution before going into a hatching container seems to help.

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

Andy,yours are 6 lined,right?.I was also concerned about WQ issues in standing water...
Oops I duplicated a pic but couldn´t edit later
Some more,this shows the yolk sac larger than the body:
 

 
And a full profile,showing the black pigmentation:
 

 
Lastly a hatching egg,close to a prolarva:
 

I am also a great fan of H2O2. I think its under utilised as it can be very caustic. I also use  it to remove parasites off new fish prior to placing in my brrodstock system. I use 1ml per litre at 12% concentration and I bathe fish for 30min to an hour.

How does H2O2 compares to formalin?

I've never used formalin so can't compare.

Luis: Yes, sixlines. http://www.mbisite.org/Forums/tm.aspx?m=34739

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

Do the prolarvae make any effort to swim or orient themselves or do they just lie there?
Do you hold the prolarvae with any light and if so do you have it on a photoperiod?
 
I've hatched a few Halichoeres eggs, mine just kind of lie around unless flow moves them. Prolarvae are really hard for me to see. I got a reusable all plastic coffee filter and set that in new saltwater, with aeration under the filter not in it. The eggs are peroxide dipped and every piece of gear is sterilized beforehand or they do not last. The prolarvae are held in the filter(s) and thus kept within the 2" I can see. That's as far as I got with the experiment before I flooded my basement, need to start again soon as the H chrysus are really going for it these days. Also need to alter my overflow, a lot of eggs get damaged with my current setup. I know it's kind of pointless anyways, but I still play with the eggs.
Thanks for posting this.
Kate

Amazing,Andy.Time to progress with the work!
See that the oil globule is placed in the front end,while it is in the rear of Centropyge prolarvae.

Kate,they just float around,wiggling ocassionally.Ambient light,14hrs L.But prolarvae don´t have working eyes,so I don´t thing this could matter.
Pelagic spawners are our last frontier,if we work as a team,we can make it!
 

2nd day.They are now on the bottom,standing head down.And most are concentrated in the corner of the tray closest to a window.So Kate,now they can  see the light and orient towards it.
Pics of the 2nd day AM:
 
Prolarvae are 2mm TL and the yolk sac is reduced.This is better seen in a lateral view.Eyes are developing,and the cloaca can be seen.
 
 

 

Nice shots.
Do you think the fact that they are concentrating in one area because of the light will affect their health and development?

Not at all,they are not so many,they are loosely "concentrated".Meaning that most of them are found around that corner.Many larvae are phototactic,move towards the light.Prolarvae exhibit this behaviour on day 2,showing that their eyes can now register the light.

very nice Luis  i havent begin with pelagic ones but you always inspire me to try somethig more so maybe soon......

Gracias,Efren!.Most pelagic spawners are families of large fish.Among the ones small enough for typical hobbyist´s tanks,we have the mandarins,the small angels and many nice labrids (wrasses).
On day 3 AM,I could see only two prolarvae.Water is full of ciliates,which means lots of bacteria=organic load=polution.
 

 
The yolk sac is almost completely reabsorved,and the oil vacuole has moved forward.The gut is completely formed but the mouth is undeveloped,and eyes are not fully pigmented.No cardiac activity could be seen,and the prolarva moves only occasionally.TL is now 2.4 mm.

Sorry to hear about the ciliates. Very cool pix though! It's hard to believe that such tiny/fragile organisms survive in the wild and become adults.

http://www.fishtalpropagations.com/#!home/mainPage
"Making captive breeding easier."

Great shots Luis!  Nice to see how you are progressing!   Do you have any pictures of your spawning set up?  Thanks much.

Thanks I had wondered. I was using a little .5 watt LED light over the "incubator". After breaking it down I used the LED light to spotlight a chalice, which burned a hole in it, so apparently it is not an appropriate light. Then I read Wittenrich's book which suggested damage from bright lights. So I had wondered what the angelfish breeders were doing.
The incubator was a small sump with shelves in it intended for bioballs and/or filter pads. Also has a float valve. The shelves hold a bowl or coffee filter in a bath for the eggs, and I can put a powerhead and heater below the shelf. It has lids. So I can keep the eggs in a dish and have high enough ambient humidity to keep evaporation at a minimum, or if I know there are no pods in the sump I can put the coffee filter on the shelf minus the bowl. Works pretty well. I aerate below the eggs, and hatch rate is miserable if I don't peroxide the gear and eggs first.
I have only hatched a few and they were pretty listless so was not sure if they were viable. I don't expect to raise them, set my goals really low. IE what foods do the parents need, what triggers spawning, how to get the eggs, how to incubate. 
It seems like they take longer than 24 hours to hatch. I know all the literature states it's 24 hours. Makes me wonder about my techniques.
 
Your photos are great! 
Thanks,
Kate
 

Kate: Do you rinse the eggs/equipment in water after you peroxide? I do my peroxide dip in a small sieve and then rinse the eggs into another container. I guess my worry would be that you might have residual peroxide in there with the prolarvae.
 
Nice photos, Luis. I'm surprised there's so little yolk left and the mouth still hasn't developed. They are going to need to eat immediately, yeah?

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

On the 3rd day,they lay on the bottom dying.I will play some more variations,but I suspect that this method,stagnant water in a 2 L tray,is not working.Some kind of more elaborated technique,that affords water exchange without surface movement must be used.See that the same problem was found by the yellow tang larval raisers.
This pic shows late prolarvae at noon of day 3.There are two constant black marks,one between the oil globule and the cloaca,and the other more distally in the dorsal fin fold.Some smaller pigmented marks show also on the yolk sac and head.
 

Hmm... Witt was telling me about an interesting concept he came up with a while ago that might work for this. I'll see if I can get him to comment.

http://www.fishtalpropagations.com/#!home/mainPage
"Making captive breeding easier."

Would it be possible to use 1 micron cloth glued into the bottom of the tray with a void underneath it that could be pressurised to have a gentle upwelling across the whole bottom surface? Mabey use a small powerhead to do this? I dont think the water volume across the whole bottom surface would be too great and could possibly suspend the fry just above the bottom. Similar to those table games where the puck is held off the table with a air pressurised bed.

Come to think of it 1 mibron bit too small...Hahah. But you get my drift.

Tal,
Certainly,Witt´s contribution would help here.I still like my tray method,where eggs can be assesed for development easily,and prolarvae progress until day 2.
After that the quality of the 2L of water deteriorates and prolarvae die,only few making it up to the 3rd day.One day more and they would be feeding larvae...
Duck,
Yes,up/downweller could give both water exchange and a quiet surface.I think Andy mentioned something along this line. 

 
That is exactly my experience. The remains of the eggs and the eggs that don't hatch fuel some really ugly water quality. I think it's plausible that bacterial population growth combined with a lack of much oxygen exchange at the water's surface causes O2 levers to drop like a rock.

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

Hi,
I rinsed the eggs in the coffee filter by dipping the entire thing in a bath of peroxide which had been warmed by being in the incubator, then yes did rinse in another dish of the incubator water, when it worked best. Figured the peroxide was probably bad for them, and did also worry that my rinse would not be enough but they did hatch and live 24 hours at least afterwards.
Really need to set this whole thing up and try again. They are spawning regularly now. It's fun to watch, and frustrating to try to take pictures of. Might have to draw out what I see as the pics are all grainy at the ISO I need to get them. They are so hyper!
I have this old aquaculture book describing raising pelagic tuna and it says the eggs will only float if the specific gravity of the water is higher than that of the egg, and the eggs should not sit on the bottom... so how high a salinity do angelfish people use? My reef tank is usually 1.0265 so thought that was plenty high but they tend to sink or at most sit midway up the water column. It describes puffer culture-the larvae are kept in still water for the first week. "It is necessary to change the water daily at first, and up to eight times a day by the end of the week." Ouch.
The food was first nauplius stage barnacles, which were cultured by sinking sticks near the surface in spring and fall in the sea, these are stored in tanks later. Exposure to air (does not say how long) followed by placing the sticks in with the larvae resulted in discharge of nauplii within 10 to 20 minutes. Pretty clever. We probably have better first foods these days though.
Think I will try resting the coffee filters of eggs on an eggcrate shelf and using a powerhead to keep the water moving, should provide more stable water quality. Wonder if it is worth using a UV unit til they are feeding to keep ciliates under control?
Do you guys have any pics or diagrams of downwellers? I see one in this book, it looks like a sump, being a long tank with baffles, and baskets of larvae are placed between the baffles, a pump takes water out at one end and dumps it in the other. Should be simple to make. Probably would not even have to be very big. But I probably would not be able to see the larvae.
Kate
 

A downweller is just a tube with mesh at the bottom. They are designed so that you can return water to the top and have it exit at the bottom, but I'm hoping that just passive water exchange is enough to get the larvae through. They really want still water. If that doesn't work, I think I would try a peristaltic pump with a tube running down to just over the mesh at the bottom (i.e., just hardly any flow at all).

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

I use 1.025,(well 1.023 right now)wanting to enhance egg buoyancy.I guess they´d float allright at 1.020 too.
My next move will be to try and move late eggs/early prolarvae into a tray with clean water...

I found a prolarva at day 3,PM,still alive amidst a ciliates soup.
2.5 TL,looking about the same as day 3,AM:
 

Frustrating and at the same time intriguing The prolarva die in the 2nd day,no matter what I do.System water,new water.With or without chlorine and/or antibiotics.Went to the extremes of moving every prolarva to a clean tray,with no improvement.I t must be a curse

I was running short of ideas,with the prolarvae dying in the 2nd day,no matter what.
Then I decided to give the eggs a formalin pre-incubation treatment (see posts above by Andy and Kate,regarding H2O2).
It definitely improved things!.Eggs seem to develop faster and most important,survival was improved,both in the original tray,as in the clean tray where I had moved some prolarvae in.In this last,there were some alive by 4th day:
 

 

 
Prolarvae are now 2.6 mm TL.Yolk sac very reduced with oil globule still present.Eyes are more developed,with some incipient pigmentation.The mouth is visible though small.Heart activity started.I think I could see a bud of pectoral fin,must be confirmed.

I think it helps to get the bacterial coating off the eggs. It likely comes back during incubation, but the dip seems to give the little guys a head start.

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

Would dosing Iodine(Iodid/Lugol's) work at all?  You'd be able to put a couple drops in and use that as an anti-bacterial agent as well.   I do think however that if the eggs were this fragile, there is no way they would ever make it in the ocean.  Is it rearing container size?  Couldn't you just use a bigger container (10-20G tank) with another "trap" inside of that so they 1) get the benefit of larger water volume and 2) keep them contained in a smaller spot?  Feeding would be a snap, as the mesh would be too small for the pods to get out, but water could still move back and forth.
 
Just a suggestion.

It seems that the eggs dip is important,and widely used,Formalin,H2O2,povidone (which I think contains iodine?).Off-shore water is probably bacteriologically much safer than our best culture condition,I guess.Yes,downwellers in a larger tank look promising.With some caveats;swimming larvae could damage against the walls of small containers and any small mesh will clog soon.

Well,we have some few larvae at day 5,fully equipped for hunting!.Large mouth,well developed refringent eyes.This point marks the end of the prolarval culturing and the start of the most challenging larval raising ordeal.
Yet I have to keep working more with this stage.I need to produce more larvae.And there is something wrong with the larvae,they don´t swim much.I guess resting on a solid substrate is not the best condition for their development,and some more elaborated culturing system must be used.
 

 
 

 
 
 

Congratulations on getting them that far, Luis.

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886

Thanks
Two larvae survived to day 6.They now look like ordinary well developed larvae.I wonder why they could be that difficult to rear.Some reddish pigment can be seen on the back:
 

 

 
A view of the underside:
 

 
and a partial profile,showing the iridiscent scales on the flanks:
 

Maybe it's just getting them to this point. Are they eating anything?

--Andy, the bucket man.
"Not to know the mandolin is to argue oneself unknown...." --Clara Lanza, 1886