Yellow banded possum wrasse breeding attempts

[F i s h y]

Small update: The first bucket experiment I can call unsuccessful. They hatched out, but I've seen my highest mortality rate yet in a first day - I think a single bubble per second on the air in the center of the bucket is enough to injure them. I think I could try to get a smaller bubble, but I think the better approach will just to be to do subsequent bucket runs with no aeration for a few days whatsoever - this is more like what it's like in the baskets, and with much more surface area and natural currents from the heating on the side, there should be enough gas exchange at least until they start getting a bit stronger.

In related-to-attempts news, I finally have a confirmed rotifer. On the fifth day, I've seen a few specks moving around in the culture, and this L strain rotifer will hopefully reproduce fast enough for me to start harvesting it a little within the week:

I left the image uncropped, because the images I took of the larvae today (start of day 4, the batch being fed 45-100 micron sieved copepods) is at the same magnification, so the size comparison is one to one. The short story for rotifers - fully grown adults are probably still too large for them to eat.

Side view, I still see the bubble, but you can see some texture to the gut too - and it doesn't look empty, though I don't see big chunks in it. This guy was feeling photogenic once I turned the brightness on the light down, it would hide near the corners of the slide wells (in the shade), but with the light down it was much happier to stay in the center of the slide with time to snap pictures. From the side, you can actually see a bit of a pectoral fin - small, but already developed around the start of day three post spawn:

And those images are half dimension still captures from the camera, whereas most of my images so far are much less sharp and noiser because they are 1080p video captures of the camera application, but it wanted to be more vertically oriented then, so I'm including one too:

Honestly, it could just be the fins slightly side on, but this is the thickest around they've looked so far.

Great pictures and good work so far. I am really enjoying the approach and the updates.

 

[DaJMasta]

Not a lot to report today, the group being raised in a basket but fed 45-100 micron food is entering their 5th day post spawn, and they look pretty much on track with previous larvae. I think these ones look a little thicker because I'm getting better at lighting the clear part (the spine is the more opaque part in the center, but isn't all their flesh.

They seem to be acting fairly normal, so they're probably eating something even though there are much fewer organisms being offered, but the advantage of the 100 micron max size blocking out a lot of copepods means the prey density of this basket is much lower. If they were getting confused by too many targets or disturbed by copepods (making them move and waste energy), there is certainly less this round. I suppose the test will be if I can find any in the middle of day 7.

Haven't been able to collect larvae for a couple of days because of evening activities, I should have a chance tomorrow if they spawn again.

Today's bad pic:

 

[DaJMasta]

After the first few hours of day 6, I caught one of maybe a half dozen that remain in this ongoing batch and took some pictures.

Probably my sharpest image of the full body, the thickness behind the head/eyes is a good sign even if it's not looking thick and the pectoral fins (I don't think they're pelvic?) are noticeably more developed. It's got pretty good control now, too, I saw a few "adjustments" in position under the microscope that involved moving less than a head length forward, but changed its orientation (pitch) somewhat. There's also a little ciliate next to him - just under his head. Hard to get a sharp image, but probably a reasonable size to be eaten.

In the same size vein, this is a somewhat blurry image of the copepod nauplius that I think is an edible size (top left)

And then this is the copepod nauplius (probably a different species) that is too large for it to eat right now:

Missed my opportunity to collect eggs today (was distracted), but got a pretty decent sized mandarin spawn later in the night. My older pair of mandarins haven't had a very high hatch rate for their spawns in the last year and change, but I'll see if I can get some out of what I collected.

 

[DaJMasta]

Lost most overnight, I think I saw one in the morning, but on the bottom of the basket rather than swimming. Most of a day shorter than the previous record, so it may not have been the large prey density that was the primary issue for the last batch, it's more likely to be nutrition.

Collected some eggs tonight (a few dozen), so here's the plan for them:
Hatch and start raising in the basket (much easier to observe)
Feed morning of day 2 (probably around 35 hours in given my Saturday schedule), primarily rotifers but with some <120 micron copepods added if density isn't good, and <100 micron copepods if it's reasonable
The goal here is to confirm food in the gut by the end of day 2 with the microscope. The next goal is to surpass the beginning of the 7th day of development, which I think will be likely if they are eating well initially. I still have no idea how long their larval stage is - weeks? months? Or even how large they may be at settlement.

I'd be looking into moving them into another container (bucket) in the second week, gradually transitioning to larger copepods and then artemia nauplii. Will probably stop feeding rotifers around the time of starting on artemia.

As I've been doing with most of my recent runs, I won't be feeding phyto to the bucket directly. Yes, it's likely that it improves nutrition of the prey organisms, but it also lets them grow up along beside them, and especially if added early on, their growth may outpace the larvae.

 

[DaJMasta]

Well I wasn't expecting to update today, but I am trying to answer a question: if you feed saltwater rotifers raised in typical salinity (10-15ppt) to a full salinity larva culture, do they die? Would they exhibit any movement of their own? It's a big salinity shock, and they are small, membrane-bound organisms, and while it's well documented that they reproduce slower in higher salinities, I don't really know about salinity shock.

This is important because larva often feed in response to movement, and most of my larval cultures run extremely low flow (not enough to keep dead rotifers in suspension, probably.) I put some in a well under the microscope and added some 35ppt saltwater, some slowed or acted a little strange, but basically all survived. I removed some more water and then added some more full saltwater and the same - they have survived and are moving around more or less as normal.

Then I took a couple in just a few drops of culture water, and added 4-5x the volume of full saltwater (closer to a feeding into a culture.) Half an hour in, I see some twitching of the rotifer, but it basically hasn't moved since a minute or so after the water was added. It may yet survive, I'll watch another half an hour, but I think this means I need to harvest some of the rotifer culture, bump the salinity up in steps (maybe try one intermediate step, for simplicity's sake), and then feed it to keep them active enough to be considered prey by the larvae.

In a related side note, I had in my notes that mandarin larvae were ready to eat at about 60 hours post spawn, but under the nice microscope, I just got a look at a 48 hour old larva with a developed digestive tract and some yolk sack still remaining. Will see if they actually try to eat anything, but they can probably eat before 60 hours.

 

[F i s h y]

Well I wasn't expecting to update today, but I am trying to answer a question: if you feed saltwater rotifers raised in typical salinity (10-15ppt) to a full salinity larva culture, do they die? Would they exhibit any movement of their own? It's a big salinity shock, and they are small, membrane-bound organisms, and while it's well documented that they reproduce slower in higher salinities, I don't really know about salinity shock.

This is important because larva often feed in response to movement, and most of my larval cultures run extremely low flow (not enough to keep dead rotifers in suspension, probably.) I put some in a well under the microscope and added some 35ppt saltwater, some slowed or acted a little strange, but basically all survived. I removed some more water and then added some more full saltwater and the same - they have survived and are moving around more or less as normal.

Then I took a couple in just a few drops of culture water, and added 4-5x the volume of full saltwater (closer to a feeding into a culture.) Half an hour in, I see some twitching of the rotifer, but it basically hasn't moved since a minute or so after the water was added. It may yet survive, I'll watch another half an hour, but I think this means I need to harvest some of the rotifer culture, bump the salinity up in steps (maybe try one intermediate step, for simplicity's sake), and then feed it to keep them active enough to be considered prey by the larvae.

In a related side note, I had in my notes that mandarin larvae were ready to eat at about 60 hours post spawn, but under the nice microscope, I just got a look at a 48 hour old larva with a developed digestive tract and some yolk sack still remaining. Will see if they actually try to eat anything, but they can probably eat before 60 hours.

I have to run 2 cultures to keep my rotifers reproducing fast enough because I keep them at 34 ppt. I also grow my larvae at that.

 

[DaJMasta]

Thanks, that's good info. I've been running my plankton cultures at 30 ppt otherwise (I've read the lower salinity helps with some phyto/copepods and it means slightly less salt in the long run anyways), so I will probably start moving the rotifers towards that and see how they fare. I've split my initial culture into a second for more production and more insurance, and I'll probably keep one primarily with 30 ppt water added and one split with some fresh and see how they do.

I fed around a pint of culture water today, primarily to the new (~20 hour post hatch) larvae, and saw at least dozens of rotifers in there (probably a hundred or two adults), sieved it with a 45 micron screen. I also fed 45-120 micron sieved copepods in a smaller quantity than the first run (maybe a third?) to get some of the other options in there. I will try to feed copepods again this afternoon before checking under the microscope in the evening for food in the gut.

 

[DaJMasta]

Last night, I took a look at a larvae under the microscope and while I didn't spend hugely long looking.... I couldn't tell if it was actually eating or not.

That forward bump is the stomach area, or at least, the gut goes above it, but some of the bump is the bubble (circle in the front) and some of it is yolk, so I can't say for sure that its food. It was taken around ~50 hours post spawn, so I compared it to earlier 52 hour post spawn images from previous runs.... and the head was in focus but not the gut so I still can't really tell.

Today, they seemed to be there, not a huge number of losses (there were some last night), and some reasonable looking hunting activity - I could swear a few looked a little easier to spot. In the evening I went to clean off the surface with a piece of paper to make them easier to see and get rid of some of the gunk... and I noticed like 8-10 dead ones on the surface. Now it's possible they were under the gunk and I couldn't see them but I think that cleaning technique may be too rough for these guys. Pulling one out to look at it, I manage to get one stuck on the side of a vial when pouring into the well slide (not the first time, so I do check), and I think I injured it by dropping a single drop of water from a pipette onto it on accident. The injury - probably a fatally broken spine - meant that it basically didn't move, so while finding focus was easy, getting an angle to see the stomach and make a good verification was not. Poor little guy, I really do try to handle them as gently as possible, but when transferring tiny volumes of water, I haven't mastered the techniques to do it yet, obviously.

There is definitely something in the forward gut area, but I don't see any bulging belly or obvious thickness over earlier runs of the same fish.

I'll keep feeding and see how they go, but I can't say for sure this run is looking better at this point.

As for the rotifers, I harvested or otherwise removed more than three pints of a one gallon culture (some was starting a second) and the next morning I ended up with more than I had the previous day, so they're certainly living up to their reproductive rate. The plan is to raise up the second culture transitioning to 30 ppt fairly rapidly (over a couple of days) and then slowly raise the salinity of the first culture with normal feeding/water changes with the hope they both survive and produce enough to supply good food.

Prey density seems reasonable in the vessels - while hard to see, I generally only get 1-2mL worth of culture water when I take a look under the scope and since beginning to feed rotifers, I've always seen at least one in that volume.

Collected some more eggs today, I will try the same basic method with rotifers only this time as a feed at around 36-38 hours post spawn. Hopefully the slightly higher density of the cultures and lower salinity shock means more baby rotifers are hatched in the basket - though with a 30 ppt culture, I may be able to pour it directly into the basket without sieving to get the maximum number of young rotifers. The thought being that the fully grown adults still look too large, so getting some density of their youngest offspring may really help increase the food of edible size.

 

[DaJMasta]

A little video from a larva a few hours into day five post spawn:


I don't think it's my imagination that it looks like his little gut has something in it. A little thicker looking than the previous day 6 video, a distinct translucence (comparable to the spine) in the gut when looking behind, a relatively active, coordinated larva, and around 10 seconds I think you can see it's little heart beating (quickly!)

While promising, it's not as far as they've gotten, it's not a sure sign of being larger/stronger/more coordinated/whatever by a significant margin, and it's far from all I've done.

I'm continuing to grow out my rotifer cultures, and the faster growing, lower salinity one turned from greenish to distinctly brown in the last day - the fast growth and then even the collection of dead adults near the surface. I'm removing some and changing water with 30 ppt saltwater as well as some phyto, so I'm trying to slow growth and reduce food to make the growth more sustainable. I've also done some sorting to try to see what size range they come in, since the adults are clearly too large. I get rid of most of the easiest ones to spot in a cup of culture water with a 100 micron sieve, but still have a decent density in what gets through. If I run that through a 45 micron sieve, most gets caught, but there are definitely a few that get through. Will probably try the 58 micron mesh tomorrow to see if I can get a decent volume in the result (otherwise you're just adding a lot of low density, dirty culture water), but the plan for the next runs is to sort the rotifers to remove the largest ones and try to make as much of the provided food edible to them as possible.

To that end, the batch I collected yesterday (hatched starting around 2:40 pm today) is going to be fed tomorrow morning with those <58 micron sieve sorted rotifers if the density is reasonable, and otherwise the <100 micron sieved rotifers. I have at least a couple dozen hatchlings and they'll be raised (at least early on) in the basket like the current run.

I didn't have any mesh sizes between 58 and 100 micron, so I placed an order for some 80 micron mesh (will take weeks to come in), and I've got some smaller S strain (probably closer to SS) rotifer cysts in my cart... but I will hold off at least a few days. Another attempt to keep parvocalanus in the long term is on the table, but since my last isochrysis came in dead, I would also need to start another algae culture (though I've only kept Iso. galbana before, and T Iso. Lutea may be a better option in any case), so that would probably be most of $100 worth of pods and algae and weeks of grow-in, best case, so I'm looking some more at rotifers first - they seem easy.

Finally, the pair have been spawning frequently, I think I collected 200+ eggs this evening. These ones have been put in a bucket with 1.5" or so of water already with the air bubbles turned off, so while the plan is a similar sorted rotifer feeding regime, these will be in their own vessel with a lot more horizontal space and surface area. They will probably be harder to monitor, but if they need more space, this may be it.

Interesting days to come!

 

[DaJMasta]

Well, the next day, those ones I thought were eating were mostly at the bottom, and by the evening they hadn't survived. I can't say for sure they weren't eating, it could have been something else, but I don't know what either. I had fed them that day prior with <58 micron rotifers, by straining the larger stuff out of the culture water and adding the culture to it. I don't think this is a great way to do it - the volume and low density are tough to add to a small basket, and it could be that culture conditions/salinity could have messed with them, but it could also just be the very small density of food was too little, or that by feeding a low density volume, it pushed enough of the food out the mesh in the side to keep the overall density too low.

I also tried that technique with the spawn from a couple days later, but I made a mistake mid feeding and overflowed the basket, probably flushing out a number of larvae. Not sure how many, but the batch fared similarly for probably the same reasons - low food density and perhaps damage to the larvae from the flow of adding the volume or the pressure against the mesh.

As for the bucket culture from the day later... larvae are getting much harder to find (as of yesterday), but I definitely saw a couple tonight. While I do think this is low numbers, it could also be that they move towards the bottom or otherwise in hard to see places, can't say for sure. I'm still feeding, 3x the first day, 2x the second (of feeding) 45-100 micron sieved rotifers, which have reasonable density in the sieve. Oddly, in the first day of feeding, I caught a larva to look at under the microscope that was around 39 hours post spawn, then I added some drops of rotifers, and while I got good confirmation under the microscope that there were rotifers there that looked like an appropriate size, I didn't witness any strikes at them, which hasn't been the case with any other larvae I've fed under the microscope.

Could be the individual, could be that the bucket culture is probably slightly cooler and their development was slowed, but odd, at least.

Was out the last nights and couldn't collect and tonight they didn't spawn, so I'm a few days out from another from day zero attempt, but the bucket larva may still be going, and I think I want to try both a more full bucket attempt (in the case that the shallow depth of this run to concentrate larvae and food is causing a problem) as well as another one in a basket so I can just get eyes on them more often and have an easier time catching them to check for food in their gut.

Also a related thing I'm working on fixing in next batches - I'm pretty confident I've been overfertilizing my phytoplankton cultures for a long while. I've actually got a batch of ~3.5 week old skunk cleaner shrimp going and the day following a small (20-30mL) phytoplankton dose, I had a big dieoff (all but six). I had sort of anecdotally seen this before in earlier runs, but I hadn't generally been using phyto recently and got the dieoff the day after the dose without any issues earlier, so the suspicion was brought to the fore. I tested some of the dense culture water (opaque at around 2 inches of depth) and it was off the charts with nitrate, but given the density of cells and the chemicals they were exposed to, I wanted to try testing just the water to be sure. Luckily, I've got a small centrifuge, so I spun up a few vials to collect the vast majority of the algae at the bottom, skimmed off the clear culture water, and retested.... >100ppm nitrate.

As a result, I won't be using phyto in the next few runs (until I get to the batch with reduced fertilizer in a few weeks), but I've cut it down to 4 mL total F/2 per gallon and will be testing the nitrates on harvest to see if they're more reasonable. Could also be a big part of the reason my main reef runs pretty high phosphate

 

[b1llvance]

I believe for other small larvae like Centropyge, prey density needs to be maintained at around 10/ml. That is what I've been told by Myecoreef in France and also by the fine folks down at the UF IRREC. From post 48 if I'm reading that right you're at .5-1/ml, which may just mean they aren't getting enough opportunity to feed.

 

[DaJMasta]

It's certainly possible, but the times when I've been closer to that, it visually looks like quite a bit (just not intuitive), and much of it has probably been too large. I should be able to get higher densities of better sizes in the basket, which I'm planning on for the next attempt, so maybe that can shed some light.

I think part of my resistance to that is just that I can't imagine natural situations where that's the case and part of it is that prey bumping into larvae absolutely do get them to move, which I think unnecessarily expends energy. It would have to be a supremely dense plankton bloom to get to that level of appropriately sized prey (rather than just phytoplankton), and then it would have to be maintained for days in the area around a larva, and while it may be that it happens more easily than I can imagine, it just seems numerically wrong to me.

Maybe once you are feeding the right size, you can no longer really see them well, so the concern of 'looking too dense' sort of falls away. I certainly haven't gotten close to that in the last few attempts or either attempt in the bucket, and I would expect that if such a high prey density was required, it probably lessened within a few days to a week's time because they become more coordinated and grow to be able to eat larger prey, and thankfully volume is a cubic function of diameter.

 

[b1llvance]

It does seem like a lot and also it is a ton of work to have copepod populations high enough to support that level of nauplii for days. The best I've ever done with a pelagic spawner is 15ish days with Centropyge potteri a few years ago. But I spent a lot of time talking to the pros who have had success. The myecoreefsolution guys in France have raised a ton of pelagic spawners and I understand skepticism with their advice as they are a for profit business and may not want to give away trade secrets. However, I also spent time at the Indian River Research and Education Center with Dr Ohs team who have raised Blue Heps, Angelfish, Copperbands, and are currently working on Candy hogfish. When I mentioned I had heard a prey density of 10/ml the response was, "At least 10, and that needs to be on average so you need to stock higher than that and then as it falls below add back to it" The other thing they really talked about was getting uneaten copepods out every night through a flush of the system water because as you stated the larvae will waste energy chasing food they can't eat.

 

[DaJMasta]

Wow, well I'll certainly strive for small vessels, then, since most of my copepod/rotifer cultures are a couple gallons total per culture and I don't have that much space for more. Not sure what to do for flushing out prey, either, but at least if I'm not feeding the copepods, they shouldn't be growing fast.

I've seen continuous drip sorts of water changes to maintain water quality and while I'd love a system to do that, I am sort of equipment limited there as well. Perhaps this is an argument for S strain rotifers, since their adult size would be the closest to in reach for the larvae, so it would be the soonest that they could eat any adults living (or fed to stay) in the culture with them, though I understand even well fed rotifers pale in comparison to copepods nutritionally.

While it's not going to be immediate and it looks like it will take two separate orders (and shipping), I think T iso Lutea and another attempt at parvocalanus is in my future. Maybe the high nitrates in the feed algae was the reason for my issues in years past.

 

[b1llvance]

I get my iso and Parvo from Chad Vossen at Vossen aquatics, if you’re in the US. Great prices and shipping and a good guy who has also been very free with information.

 

[DaJMasta]

Oh wow, they hadn't been showing in searches but I started out with one of his larva collectors. Right on both accounts - they have both, the prices are reasonable, and the shipping is cheap!

 

[DaJMasta]

I started off the week trying to catch some more eggs - and couldn't for a few days. A little was my own schedule, but then I missed a couple of spawns because they juked me - they had been consistently spawning between 8:25 pm and 8:45 pm or so, and for a few days this week, it was basically 8 pm. I missed one suspecting they would spawn that day, then I put out the collector when I saw them probably actually spawning around 8 pm one day and then disappearing into the rockwork - the pumps claimed some, but I still got a few dozen eggs. I managed to catch them the next consecutive two days, and they got back towards normal timing in that spawn, so I decided to throw everything I had at it.

The first run was that smaller spawn, planning on feeding 45-100 micron sieved rotifers and maintaining high density by keeping them in the basket. The second, a big spawn I planned on mixing in copepods with the same sieving and in a basket. The third one was a decent sized spawn that I put in the bucket, so lower prey density, but I upped the sieving to 45-120 micron to get more actual population into the water.

The first spawn hatched, I fed rotifers three times the first day with a total culture volume of over 2L fed in the course of a day (to a 100mm cube basket). That evening I did not observe any rotifers had been consumed and did not see any strikes at food under the microscope. Maybe 6-8 rotifers in the ~1mL in the slide well.

The second spawn hatched and is probably the largest number of larvae I've seen in a single spawn and started hatching as early as 17 hours post spawn. I fed more than a gallon of total culture water in the first day, all sieved, to the basket, and saw more density by eye than the first. I did not observe food in the gut but think I saw some striking behavior in the microscope slide. Density was slightly lower in the slide, maybe 5-6 things, but more than half were copepod. It looked like in the basket there was more activity of the larvae, and the larvae under the microscope seemed more energetic than the one caught from the rotifer basket.

The third spawn in the bucket hatched out in decent numbers and has been very difficult to check on by eye. I will check on feeding tomorrow (it's late, and it's now the start of day 3 post spawn), but I don't see huge volume.

There aren't many in either of the baskets left, though, in fact I haven't seen one of the second run in there today. While there could be other issues befalling them, I really don't think the foods I'm offering are going to do it. There may be one or two copepod nauplii that would work, but my cultures aren't large enough to supply it in enough quantity - maybe something to try in the future. I think, frankly, that while some of the rotifers are the right size, the larvae don't have a feeding response to their type of movement. A smaller strain would probably help convince me more, but with all the extra work (keeping them separate, changing water much more because of exploive growth rates), I think I will probably try to stress one culture into making cysts and then feed the other one in the coming week or two - doesn't seem to be worth the effort.

I did place an order for T Iso and Parvocalanus, but I haven't gotten so much as a shipping notification yet, so I don't think it's imminent. For now, I think I will continue these runs if I see larvae and not collect eggs again until I have a food solution in place (though I will keep collecting other species' larvae, as usual.)

Not the results I've hoped for, but strong enough to convince me that my methods need some freshening up.

 

[strich]

Great updates as always. Not the results or progress you're looking for, but keep up the work and at least you've got some solid next steps to try. Looking forward to hearing more!

 

[Arkayology]

Thanks for the update! We are all very interested in your work here! Keep it up!

 

[Paul B]

This is great and you certainly are putting a lot of work into it.
I used to try to raise salt water fish decades ago and I know how much work it is in a home environment. It is a tough, time consuming activity but the successes, even if small are worth it.

Good luck.