Sponges: Will it blend?

[ezrec]

Some of you may recall the old college biology experiment where the experimenter:

1) Took a sea sponge, and blended it.
2) Passes the goo through a fine cheesecloth mesh, and separated the spicules (in the mesh) from the motile cells (in the liquid).
3) Places the liquid back into the tank, and the sponge 're-forms' after a while into a single colony.

Has anyone tried that with air-bubble necrotic sponges, to try to 're-start' the colony?

 

[Drock169]

I've heard about this, I'm curious if it will actually work

 

[dendro982]

Didn't blend, but cut alive tissue from necrotic sponge, like fragging, thought it should have higher chances to regrowing - it didn't work.

I also have a sponge right now, not in a good shape, tips are dead for almost an year - may use few branches for a blending. Or it should be the whole sponge?

Some questions:
- I would rather hesitate to put such amount of blended sponge tissue in the display tank - it could be (potentially) toxic, may irritate fish's gills, may settle in undesired places, like corals or fish, and the skimmer and filtration will remove these particles.
Anybody tried to put the sponge blend in the display tank? Which sponge and how it worked?

Should this be a separate experimental tank, will be unheated tank OK (~74-78F), what else should be there, what amount of LR, flow, again, what to do with filtration and water changes to avoid removing pulverized sponge? I have spare 2.5g tank, may try.

- What amount of saltwater for blending? Mode of blending, speed.
Anything else, that I should know?

- And the last: how long will it take (regrowing, I mean)?
Wouldn't like to be involved in one more tank maintenance and monitoring water quality for an year, you know...

Open to suggestions

 

[ezrec]

The guy who initially discovered this is H.V. Wilson, from UNC in "On some phenomena of coalescence and regeneration in sponges" in the 1907 issue #5 of the Journal of Experimental Zoology

http://www.bio.unc.edu/faculty/harris/Research/sponges.htm

Some documented experimental procedures are:

* Washing with a 1% Hydrogen Peroxide solution to kill bacteria and sterilize necrotic tissue:
http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1440-169X.2005.00800.x

* Squeezing the the whole sponge through a fine cloth: http://www.biolbull.org/cgi/reprint/45/3/153.pdf

Google around some more, but shooting from the hip, I would suggest the following procedure:

* Prepare a 1% hydrogen peroxide solution by mixing 1:1 ratio of 2% Hydrogen Peroxide with distilled water. Add salt to bring the S.G. up to the S.G. of the original tank.
* In the original tank, wrap the sponge in a fine cloth ("#20 Silk"?)
* Transfer the sponge to the hydrogen peroxide sea water, and squeeze out the cyctoplasm. Discard the remainder.
- NOTE: Do not leave the sponge cells in the hydrogen peroxide bath for more than 2 minutes!
* Strain the liquid through a Poly Filter pad, and place the pad in a low-flow area of your tank for 4 hours. This will allow the cells to adhere to the pad.
- NOTE: I am suggesting Poly-Filter, because I have observed that even microscopic phytoplankton cells will adhere to it, and colonize it.
* Place the pad in a higher-flow (but not the direct output of a powerhead!) area of your tank.
* Put the strained liquid into a Quarantine Tank with some live-rock substrate, and see if you get different results than from the Poly Filter pad.

The Poly-Filter pad should act as a reasonable sterile substrate for the sponge to develop on.

This is all guesswork, but I *think* it should work.

 

[dendro982]

Good, thanks!
Some questions:
1. Type of blending - or it doesn't matter, like to the puree?
2. Blender is not sterile, and couldn't be sterilized in home conditions, other than by some wash in H2O2. How to go around this?
2ver. Or just squeeze the hard enough sponge through the fine mesh? I don't think, that this is physically possible, this is tree-shape orange spiky sponge (don't have a Latin name right now, sorry. Very common and resistant kind). May use the soft hitchhiker sponge, but the sponges of interest are the big decorative ones, right?
3. Assuming, that sponge will be blended after peroxide bath, squeezing through the fine mesh: how fine should be mesh, have no silk #20, but have 250 micron Current USA mesh filter bag, 100 and 50 micron filter pads, as well as paper coffee filters, used for tissue culture at home. What is preferable for sponge's cells size? May find some silk, but it will be with dyes.
4. Don't have PolyFilter pad too, and sorry, don't plan to buy
Which replacement will be better: Big Al's filter floss roll (thicker and more open, than any bonded pad), phosphate removing bonded pad (thin and denser, green), loose filter floss from Wal-Mart, 100 micron polyester pad, new sponge from cartridge to power filter (1/8" or 3mm thick, open cell, black)?
5. Place in low flow area: do you think, that the critters acrylic vessel with holes in the lid will work? The same, that is used in LFS to place the small shrimps or lobsters (or eels) inside the big tanks.
6. How place the pad in high flow area - in something like veggie clip? If in the crevice in the LR - the hermits will tear it apart.
7. How long will all the regrowing process be - weeks, 2 or 11 months? Most rough approximation.

Sorry, I wasn't able to check pdf, you mentioned - lost old Acrobat Reader in computer crash, and the only available now version doesn't support Firefox. So, I will rely on your word, OK?

 

[ezrec]

1. Type of blending - think 'low shear' - like squeezing through a mesh.
2. Sterilize with H2O2. Then, blend at low speed with a few quick pulses to break up the spicules, then sieve through the mesh for decorative sponges.
3. I'd try a variety of filters. Paper coffee filters may be too fine, but some undyed polyester cloth might do the trick.
4. Some sort of high-surface area superfine filter material. Polyester filter pads may work.
5. Probably.
6. You'll want to have it near where you want it to grow permanently. Sponges adapt their colony structure to the flow conditions, so you can't usually move sponges successfully. I'd put a coarse mesh basket (like those green plastic strawberry baskets) over it to keep the hermits out, if they're a problem.
7. I'd say you should start to see colony formation within a day, and growth within weeks afterwards.

The general principle here is that you want to separate the cells from the skeleton (via blending/a relatively fine mesh), then 'mount' the cells on a substrate that is similar to live rock, allow them to start re-forming their colony, then place the colony in the desired location.

Remember, I've never done this before, having never kept sponges, but it sounds like a reasonable plan to me. It's what I'm planning to do when I get my first set of sponges.

 

[dendro982]

Thanks! Will try to do that this weekend.

 

[dendro982]

Done. Let's check, if it was done right:

Original sponge, half of year ago, note the dieing ends:

Before blending it was 3"x2", trunk and the base of branches.

Procedure:
1. Peroxidized blender.
2. Prepared the 1.025 SG saltwater in 1% solution of peroxide.
3. Filled to the 2/3 the blender cup by this solution.
4. Wrapped the sponge in the double big coffee filters, while in the tank. No contact with air.
5. Removed, moved into the blender cup, removed the wet paper underwater.
6. Blended at low speed several times, in short duration each. The pulse didn't chop, then used chop mode.
7. Filtered through new 250 micron filter bag (it's finer, than brine shrimp net, but has larger mesh, than polyester fabric. Wal-Mart had only dyed kinds), in another acrylic glass.
8. Filtered this liquid through the piece of the BA filter floss roll, underlined by 50 micron filter pad, just in case if the floss is too loose, more particles will be caught by the fine polyester pad.
9. Placed to the critters box, filled by tank water.
10. Remaining liquid seemed to still be cloudy, used another 50 mk pad for one more filtering. This pad was placed onto the previous pads in the critters box.

All time of a keeping sponge in the peroxide was within 2 min.

11. The critters box was placed in the 20g established skimmed tank, fishless, but with hermits, corals and macroalgae. 4 hrs in the box to guarantee the low flow.
The only problem: box tried to flow, had to weight it down by stone.
12. Skimmer started to overflow and collect the orange-red skimmate.
13. After 4 hrs, the pads were removed from the box, placed beside the 150 gph Mini-Jet 606 (high flow, but not in direct blast), weighed down by the same Fiji LR branch.



The filtered cells looks on the pad as a slight hint of yellow-orange.

As an afterthought, the big pads are quite unsightly in the tank, may be I should use the smaller pieces, like 1"x1", only have no small funnels to hold such a small pieces.

Correct, please, if anything was done improperly.
Anyway, only piece of sponge 1"x0.5" is left now, too little for a new experiment.

 

[ezrec]

You followed the experimental procedure we're talking about to the letter, so I have no qualms with your methods.

I will be very interested in pictures after 24 hours, to see the extent of new aggregation.

I have to commend you for your boldness in this experiment. Now comes the hard parts - waiting for results, and having the strength to continue on in the face of failure - uh, I mean "Learning Experiences".

I'll see if I can source some healthy sponges locally in Pittsburgh so that I can try some variations here.

 

[dendro982]

24 hrs later, nothing happened:

Top sheet, filter pad 50 micron:


Filter floss pad:

Will continue.

 

[dendro982]

Continue:


Bottom pad, filter pad 50 micron too:

The orange piece most likely just some debris.

May be the mesh should be less, than 50 micron, or greater concentration of the cells in a small volume should be achieved, who knows...

Next update will be tomorrow morning, 48 hrs photos, if it seens reasonable. If another timing is preferable - tell me, OK?

 

[ezrec]

I'm beginning to fear that your skimmer ate the motile sponge cells.

I'll try to score some sponge large enough for 3 experiments:

* Control: Leave a piece in a tank, with no blending
* Repeat your experiment, with no changes
* Repeat your experiment, but turn off the skimmer

I've got a "waiting for speciments" request with Drs. Foster & Smith for a
"Large Sponge", and I'm calling my LFS here in Pittsburgh - no joy yet.

 

[dendro982]

Yes, it seems so.
48 hrs later - all the same...

I would like to see results of your experiment too. If it works, it will be the whole new way to work with hopeless sponges.

 

[ezrec]

It may take a while for me to acquire the specimens, but keep a watch on this thread, and I'll update it when I run the experiment.

 

[ezrec]

I've acquired a small specimen of orange tree sponge in poor shape, and will be running tests on it. I'll post some pictures and procedures tonight, when I check out the 24 hour results.

 

[dendro982]

How is it going? Still curious - big fan of sponges, but can't keep these, that I like.

 

[ezrec]

So far, all tests have been illuminating basic research, but not at all promising as propagation methods.

I performed two tests - cubing and straining.

The cubes are 1x1x1 cm, of an orange tree sponge. Each cube has at least 4 side as the original sponge exterior. Each cube was put in Poly-filter filtered aquarium water (1.024 S.G.) with a H2O2 concentration of 0%, 0.01%, 0.015%, or 0.03%, for 24 hours.

The 0.015% and 0.03% cubes had distinct bubbling after a few hours, and continued till end of the 24 hour immersion. Neither the 0% nor the 0.1% had peroxide bubbling evident. After 12 hours, the 0.03% solution had a faint 'shrimp' smell to it, but that smell passed by 24 hours.

The strained cells were squeezed through paper coffee filters, resulting in individual cells, separated from their spicules. These were also placed in seawater with the varying quantities of H2O2.

The problem seems to be that while the cells can sometimes be found to aggregate into balls of 20-100 cells, they find each other by searching via a random-walk ameboid movement over a 24 hour period, and sticking to whatever they find. All of the cellular balls in my test appear to have ceased functioning after 48 hours.

It appears that the 'aggregation' reported in the previous studies is more similar to a self-repair mechanism than a regeneration method. I expect, in the wild, it that allows cells that have been isolated in the spicule matrix due to predatory damage to re-aggregate with the main mass.

As I have yet to see (over the last two weeks) any additional tissue growth in either the control (which is a 2x1x1 cm piece of the original sponge, attached to rock) or any of the surviving test cubes , I can only assume that I am not providing the correct conditions for rapid growth.

I have a high plankton load ("pea soup" green on most days) of a whole range of sizes, a moderate current flow over the test subjects, and low light levels (to discourage algal growth in the exposed spicule matrix).

The spicule matrix on all test cubes now extends past the orange cellular matrix by 0.25mm to 1mm, and I am under the assumption that the tissue is retreating.

pH is 8.4, Temp is approx 75 - 80 F, and salinity is 1.024

Ammonia, nitrite and nitrate levels are all undetectable by my test kits.

NOTE: Orange tree sponge cells are very small - about the size of a bacteria. The phytoplankton in my aquarium is colossal in comparison - image a kitten compared to a human. This may be where I'm going wrong - I have the wrong plankton ecology.

The next piece of sponge I can get for testing, I plan to drill 3mm holes in a high-density live-rock, and inject via un-needled syringe the dissociated cells into the hole. The cells should be forced into an aggregate this way, and I hope to have better results.

 

[dendro982]

You changed the described protocol a lot

If it will work not only for repairs, but for a practical purposes, can you post more details about tanks, heating, flow, placement to prevent skimming off, and step by step procedure for one concentration, that worked the best?

Don't mind my down to the earth attitude, I'm filled to the brims by the recent allergies of gorgonians

Will watch for a future developments.

Good luck!

 

[ezrec]

All the 'strained cells' tests were performed at 78F, 1.024 S.G., in 100ML plastic cups. Cell cultures were disposed of after 48 hours. (They all seemed to be quite still after 24 hours when I checked on them, and quite dead after 48.)

On reflection, I suspect some contaminant (dilute bleach?) in the coffee filters I used to strain the cells from the spicules killed the cell cultures.

The 'cubed' tests were also performed in 100ML plastic cups, then transferred to my 55g aquarium's refugium, under the shelter of a live rock, after 24 hours. Heating on the main aquarium varies between 70F and 83F on a day-night cycle.

For the cubes, I can't really say which H2O2 concentration worked the best, since the hermit crab in my refugium decided to move all the cubes of sponge around. All of them seem to be 'live', but none seem to thrive.

 

[ezrec]

As of today, I can honestly say that all of my test sponge is dead.

On a lighter note, I appear to have some sponge growth (of a different species) on a live rock in my refugium. I'll keep an eye on it, and see if I can determine if it can be propogated via division.