Culture Journal, Species: Parvocalanus crassirostris

General
Species: Parvocalanus crassirostris
Species description:  Very small copepod, pause and dart movement
Culture source (link if possible): Dr. Andrew Rhyne, who isolated them from the ocean
If algae, CCMP # (Optional): 
http://ccmp.bigelow.edu/
Culture Establishment Date:  6/28/13
Continuation Date: 9/28/13      

Culturing Vessel Details
Salinity:  30 ppt
Temperature:   72-80F, room temp
pH:  8

Vessel description:  5 gallon bucket, then, 1 gallon HPETE wide mouth jar with hole in cap.  Current culture vessels are two 1-gallon HPETE wide mouth jars, one 10 gallon tank partially filled, and one 5 gallon bucket.
Lighting description:  ambient for the tank and the bucket, but I keep the bucket covered, so not a lot of light.  The gallon jars are in front of two undercabinet fluorescent fixtures, suitable for growing phytoplankton, although that is not necessary for the copepods.
Lighting cycle:  14day, 10 night
Aeration description:  open to room air, and airline with 1 bubble per second or so

Methodologies
Split methodology: Since establishing these cultures I have not done a lot of splitting. I feed them, and if I need them for feeding fish larvae, I take some out with a volume of water, and replace with clean saltwater, but I have no schedule of splitting.   

Culture medium description:  30 ppt saltwater, 25 ml of live Isochrysis as needed, perhaps daily. The ten gallon tank gets 3-5 ml per hour of live isochrysis, depending on the concentration of the phyto at the time, as judged by eye. If the live phyto is coffee colored, I feed 3 ml per hour, if tea colored, 5 ml per hour.

Cell count: Was shipped about 40,000. Half were used in clownfish larval tank, with rotifers, half was put in 4 gallons clean water in  5 gallon bucket for one week.  Survivors are now in a 1 gallon jar. see journal for details.
 (if known) It's really hard to count these guys, so I haven't tried in a long while.  If I take 100 ml out, and put through a 27 micron filter, they form a little brown puddle on the edge of the filter, and when I backwash into a petrie dish, there are a whole lot of them in all stages of development.

Reference links:  

Additional Information
(No Pictures or Videos in the Section Please)
Notes:  Ciliate removal seems to be important.  I first filtered through a 27 micon screen, and captured everything, including ciliates.  I may need to filter them through 53 microns occasionally to get rid of ciliates, sacrificing the smallest nauplii.
As of September, I am no longer stressing over this.  I just take what I need, replacing the water I remove, and try to remember to feed them appropriately if they are not on the peristaltic pump feeder. Ciliates are allowed to continue, with no apparent repercussions.
 
I strongly suspect that one key to the remarkably easy strategy, is that the vessels themselves have the appropriate bacteria/phytoplankton/microscopic life to handle the ammonia buildup from the copepod excrement as well as molts, dead algae, etc. 



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I'm getting some parvo from a friend, since the stuff I bought did not survive.  He first told me to increase my isochrysis culture volumes. 
 
So I remade my culture shelf, to have two shelves, each with two T5 undercabinet lights across the back.

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And expanded my culture to 8+ one gallon jars:

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By my calculations, one gallon per day is enough Isochyrsis to maintain appx. 200 gallons of a dense Parvocalanus culture, assuming a density of appx. 10,000,000 cells/mL in the harvested Isochrysis.

Quite frankly, the problem I had with culturing Parvocalanus was that I was feeding them too much Isochrysis.  Ever since I kept my feed levels down below 100,000 cells/mL per day, and more like 50,000, I've had much better success.  I've had Parvocalanus in continuous culture since early January now, using very little Isochrysis fed once or twice daily.  I'm feeding roughly 120 mL of Iso with a density of 10-12 million cells/mL to a total of about 32 liters of Parvocalanus cultures once or twice a day.
 
Excess feeding has an adverse effect on the development of the nauplii.
 
Don't get me wrong -- that's a VERY NICE Iso culture station, Kathy!  It is just a whole lot more than you probably need, unless your goal is to culture a couple of hundred gallons of Parvo.
 

You know, Jim, I agree with you as far as my experience with A. panamensis goes. I've been "practicing" with panamensis in anticipation of  the parvocalanus cultures with Isochrysis.  A very light feeding of the live stuff produces enormous quantities of copepods.  I've been feeding them lightly once a week, and the population has increased dramatically.  It's so easy.
 
The guy supplying the parvo said I need 9 gallons of Iso a week, and I'm cheating by producing only 8 gallons a week, but I really think it will be enough for my needs.  I've never successfully cultured parvo, so I am just following instructions.  My plan is to keep a bucket or two of as dense a culture as I reasonably can going, and have lots of and lots of copepods for fish larvae.  So far its not a lot of work, and pretty cheap to keep these cultures going.  I'm trying it this way, and will adapt as my needs become evident.
 
I've been following your excellent posts on culturing parvocalanus, Jim, and my experience with similar light feeding techniques for panamensis lead me to think that you are on to something.  We will see what happens once I get a thriving parvo culture.

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I'm also thinking that I can share the wealth should I find that I really have too much.

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In addition, I may curtail my rotifer culture.  I recently had a fabulous culture of rotifers crop up in one of my Iso jars just as it was getting really dark.  Perhaps I can master timing the inoculation of rotifers to have a ready supply daily, without all the floss washing and daily feeding around the clock. Just grow up a jar of Iso with an innoculation of rotifers carefully timed to peak at the same time, and there ya go.  Doing it this way would keep them more contained than the open buckets by the sink. Just a thought.  I need more space for the parvocalanus.

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Today I got a shipment of P. crassirostris.  They arrived in 2 small fish bags, about a liter each of pods and water, and another liter of air or oxygen. I am not sure which was used.  They arrived in great shape, with few mortalities.  Previous orders have been a disaster.  They came in a bottle, with only a tiny air space, and shipped 2 day air.  Arrived mostly dead both times, and I was unable to get a culture started. Why does this company think that copepods don't need air?
 
Any way, I got about 40, 000 live copepods today, and split them between one 5 gallon bucket and two 3 gallon buckets, slow airline, and about 50 ml of my darkest Iso culture, a bit less in each of the 3 gallon buckets.  If I can't culture these, I will be very surprised.

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Now I'm going to re-read Jim's Parvo thread.

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Did these come from Algagen?

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

Just keep an eye on the food levels, and if the water is visibly tinted at all at the next feeding time, either skip the feeding or just feed very lightly.  I've found that the key is to keep them fed, but just barely.

Just indulge me some speculation:  If your darkest Iso culture is anything like mine, then it is probably around 15,000,000 cells / ml.  If your 5 gallon bucket is as full as I usually fill 5 gallon buckets, then it probably has about 4 gallons of water in it.  50 ml of 15,000,000 cells added to 4 gallons = Right about 50,000 cells / ml in the bucket.  In my experience, that is an excellent feed level for Parvocalanus.

That said, your Parvo culture is probably pretty thin right now.  I would expect a dense Parvo culture to consume that level of food easily in a day, and perhaps even more quickly.  But a thin, less dense Parvo culture may take more than a day to consume that much food.

I tend to keep my cultures relatively dense, and then ramp up volume as they grow.  That way, I can be reasonably assured that the food I'm giving them will be consumed quickly.

I know that I'm probably sounding like a "broken record", but based on my previous failures, and my recent success (multiple Parvo cultures maintained for just shy of 6 months now), my fear is overfeeding.  In short, I always skip feeding on any culture that is obviously still tinted.  I generally always feed lightly once or perhaps twice a day.  I almost never let a culture go more than two days without at least some feeding, unless it is still tinted, and if that happens, I consider scrapping that culture and splitting another healthy culture to replace it.
 
Good luck, Kathy!

Thanks Jim.
Yes, I set them up at about 1 pod per ml.  My Iso is a dark coffee color. 50ml in the 4 gallon culture produced a slight tint at feeding time.  I'll try to assess color again this morning.  It's not easy to see the color in the buckets.
 
Based on my experiences with A. panamensis, I think you are right on the money with the light feeding schedule.
 
Do you find that pasteruization of the water is necessary?
 

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I use pasteruization of my copepod culture water for one reason:  To avoid cross-contamination.  I've had just about every possible combination of cross-contamination you can imagine happen in my fish room:  Rotifers in my (various species of) copepods, Apocyclops and/or Tigriopus in my rotifers and/or Moina, Moina in my Tigriopus, Apocyclops in my other copepods, etc.  Since I started pasteurizing my copepod culture water, I haven't had any such cross-contamination yet.  I currently have two rotifer species, seven copepod species, and Moina all in culture.  I think I've just finally been able to isolate the Moina again after a long time of it always being in co-culture.  If I do have a pure Moina culture, then I'll be able to boast pure cultures of all 10 animal species, and the pasteurization is a big part of that, I believe.
 
My pasteurization process is basically the same as my phyto media sterilization process, except that I don't go as extreme with the heat.  I use the same 2000 mL borosilicate media bottles I use for phyto, and microwave the water until it is at least 140F (I go to 185F for my phyto media).  This not only keeps the big bugs out, but it seems to help to keep the Euplotes populations down, too.
 
I do still get volunteer Oxyrrhis marina in any cultures that have an excess of live phytoplankton for too long, although, I tend to believe that many of the omniverous/carniverous copepods benefit from the presence of the Oxyrrhis ("trophic upgrading" see:
Breteler, WCM Klein, et al. "Trophic upgrading of food quality by protozoans enhancing copepod growth: role of essential lipids." Marine Biology 135.1 (1999): 191-198. and also: Bretler, W. C. M. K. "Continuous breeding of marine pelagic copepods in the presence of heterotrophic dinoflagellates." Mar. Ecol. Progress Ser 2 (1980): 229-233.  PDF can be found here:  http://www.vims.edu/peopl...kw/pubs/MarBiol148.pdf ).

 
Another cross-contamination issue that I've learned to watch for is "splash back".  Let's say I pour some phytoplankton into a small flask for feeding my copepods.  As I pour the phyto from the small flask into the copepod cultures, there is almost always some amount of the copepod culture that spashes back upwards after the phytoplankton media strikes the surface of the copepod culture water, regardless how gently I pour.  This "spash back" can easily reach all the way back into the small flask, and so if you aren't careful, the act of feeding can become a source of cross-contamination.  I have developed a habit of switching small flasks, or else rinsing my small flask well, between different species being fed.
 

Thanks Jim,
Two days ago, I had a wonderful hatchment of clownfish. (I've been struggling with nests that hatch over two or more days, small nests, and larvae that didn't thrive. I've offered my broodstock more variety in their feed, and I'm rewarded with bigger nests, and the larvae seem more viable. ) This nest was large, hatched all at once, and had very few mortalities. It is in 10 gallons in a 15 gallon glass aquarium, prepared in my usual way.  This time, I stocked the tank with half of my parvo culture, ciliates and all, plus live Iso, enought to significantly tint the water, for the first night feeding, blue led aquarium light on all night. I awoke to a very lively tank full of larvae! Larvae are so many, I don't think the copepods are numerous enough to keep them in food, so I added rotifers and RotiGreen and live Isochrysis.
They are doing very very well. 

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Meanwhile, I noticed that the parvo culture seems a little thin.  It is hard to tell the color of the water in the bucket, so I may have not been feeding them enough. The water always looks clear or slightly greenish to me.
 
I filtered out some with a 27 micron filter, and saw lots of ciliates along with the copepods of all sizes.  So I filtered out all the remaining culture with a 53 micron screen, and kept the filtrate as a lovely ciliate culture, moving the remaining parvocalanus to a clear one gallon jar.  I can see the color better in the jar, and can perhaps feed them Iso more appropriately, and keep this culture going.  I will split it today, so I have some insurance in case one jar dies.
 
The ciliate culture will likely have some parvocalanus nauplii in it, which I should be able to filter out in a couple of days as they grow bigger than the ciliates. Meanwhile, I can feed the ciliates to clownfish larvae or any other larvae, as it might be pretty good food for them. I plan to feed Iso to the ciliate culture as though it is a parvo culture, in other words, when I feed Iso to the parvos, I'll feed Iso to the ciliates.

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As far as keeping the Iso clean, I've been following the same practice that Jim has been using.  I pour from my culture jar to a smaller container, and then to the culture I'm feeding.  I have a stack of clean "pudding cups" from Jello Snack Packs to pour into, and just change cups with each culture.
 

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When I set up the one gallon jar, I took the remaining junk in the bucket and put it into a smaller jar.  Now a couple of days later, there's a crowd of parvocalanus in an upper corner closest to the light.  I sucked them off and added them to the gallon jar, but not before taking a drop to look at under the scope: Click on the videos.



 

 

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As expected, the ciliate bucket is full of Parvocalanus now. Meanwhile the Clean jars are not doing great, although perhaps there is some improvement.

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Last week I filtered out all the culture, except the ciliate bucket and put the 120 micron fraction into a clean jar, and the 27 micron fraction into another clean jar.
 
The nauplii in the 27 micron fraction seem to congregate on the side of the jar closest to light.  It's nice because I can keep an eye on them.  The older ones seem to be in the water column.  Now having checked the 120 micron jar, there are P. crassirostris of all sizes in there.  It looks like they've bloomed.  In the jars it is easier, than in the buckets, to see what the water tint looks like, and I think I've been feeding them more appropriately.
 
I've decided that I'll still have more than enough Isochrysis if I have 4 gallons a week, instead of 1 gallon a day. So I will use the top shelf for the copepods, and the bottom shelf for the phyto.  This will work better for the winter temperatures as the top shelf is set up with an undershelf heater. I've been having some issues with Isochrysis crashes, and the weather is warmer, and we are heading into the cooler non-air-conditioned months, when temperatures fluctuate and humidity rises a lot.  So I hope I don't regret this decision.

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Meanwhile I've started an experiment. I had a small clownfish hatch that i do'nt really need, so I set it up in a small unheated bucket--temperature in the basement is 77-80F.  Didn't sterilize. In the morning, I added isochrysis and 3 gallons' worth of the ciliate/P. crassirostris/A. panamensis  culture put through the 53 micron filter. The larvae were sticking to the sides until I moved the light to the center of the bucket.  Still some cling, but some are hunting in the middle. It probably won't work because the bucket is so small, I didn't sterilize it, and the food may not be enough.  But it would be great if it did work. There are a few rotifers in the ciliate bucket, but not many  compared to the copepods, so I hope it will work.  We will see.  All live, mostly non-rotifer food and environment.

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It's a good thing I kept that hatch.  I had a couple of batches of clowns that I thought were going great, have a bad die off, I think from too many rotifers, and now I really need that experimental group.  Any sign of die off, and they are going into a regular tank with rotifers and a greater volume of water, and the experiment will have to wait for another time.  Meanwhile I've given them another big collection of parvo and panamensis and a few rotifers.  More of them are off the wall, and either eating or dead where I can't see them. The ones I can see look robust.

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The experiment ws a complete failure as was the two batches before it. Darn, one was a double hatch of a two large nests… I got cocky and added too much rotifers and green.
 
Got another batch tonight.
 
Parvo was staging a good comeback, then I noticed today, rotifers contaminating the culture...

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Culture?  Singular?  That sucks.

Welcome to the clubI had my thriving Parvo cultures contaminated  with SM rots.Started all over again only to have them contaminated by Apo!
Trying my 3rd time... 

I must have been mistaken.  I kept the parvo culture another week, and it bloomed.  I can't find any rotifers in it. There were so many copepods of all ages in there, I put 9/10 of the culture into a sterile 10 gallon tank, half filled. with a bit of live ISO.  Today they are doing well.  I have a lot of pods.
 
I refilled the gallon jar containing the remainder of my culture with clean sterile water, and some live Iso to feed it.  There are still a lot of pods in there.
 
Meanwhile, the "ciliate" bucket is thriving with parvo and panamensis, and few rotifers if any.  I took a gallon of it, sieved at 27 microns, and looked at it under the scope before feeding it to my clownfish. Actually, there are very few ciliates in that bucket. There are copepods of all sizes, panamensis with eggs, … it all looks good, and with no water changes, only occational addition of live Iso. I keep the bucket covered with a very slow air feed.
 
Meanwhile my panamensis is completely overrun with rotifers.  I sieved it at 150 microns and put the few copepods caught into a clean sterile jar with a little ISO .  We'll see.

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Good news on the Parvocalanus!  To rid the Apocyclops of rotifers, you can try taking a split of your cleanest (fewest rotifers) co-culture, and simply starving it.  Based on my experience, I would expect the Apocyclops to outlive the rotifers in a starvation situation, but beware resting rotifer cysts.

The 5 gallon culture in a 10 gallon tank is doing well.  I've put a peristaltic pump on it to give live Iso at 2 ml per hour around the clock, and they seem to have done well over the weekend while I was at MACNA.  There does seem to be lot of molts floating, so I skimmed some of them off this morning. Very robust culture.  The gallon jars are also doing well, although the Iso seems to be getting darker in there. It is growing faster than the parvo can eat it.

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And then there is the ciliate bucket.  Originally loaded with the flow thru from the parvocalanus culture, (to get rid of ciliates in the main culture), it has since been contaminated whith Apocyclops panamensis and rotifers.  They were all coexisting in a 5 gallon bucket that got a little air and 30 ml or so of live Iso when I remembered it was there. Parvocalanus was not evident for a week or so, then began to "come back".  Now it is the dominant species, outpacing rotifers and panamensis. I would say that once "established", these copepods are hardy and prolific.
 
I put an Ammonia Alert badge in the 5 gallon culture( in the 10 gallon) tank before MACNA, and it registered a little ammonia. I squeezed out a sponge filter from another tank into a bucket, let the gunk settle and poured some of the water into the parvocalanus tank.  A few days later, the alert showed no ammonia, so perhaps it helped, or maybe the tank was just getting the right bacteria from the air and it matured at that time.  But the tank has now "cycled", so I'll be keeping the culture in there indefinitely.
 
I would guess that the ciliate culture bucket has cycled as well. I've done nothing to clean it except to remove a gallon or so of critters, and replace the water, I'm guessing, every couple of weeks.  All in all, I'm very pleased with this culture. Parvocalanus is not hard to culture at all, so far.
 

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All my parvocalanus cultures, from the gallon jars that I've ignored except to feed them, and 10 gallon tank that gets 5 ml live iso per hour and no other attention, and the "ciliate" bucket that started with every kind of critter in there, seem to keep on keeping on with populations of Parvocalanus crassirostris that are doing very well.  I should take pictures...
 

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Yeah, once I figured out how to keep from killing them (don't overfeed), they turn out to be very low maintenance, and will easily thrive.

Funny thing, I decided to take my own suggestion, and get some pictures. The Parvo in the jars got TAKEN OVER by Apocyclops, massively.  The "ciliate" culture which used to have parvo, Apo, rotifers and ciliates, is now Parvo and ciliates, and the 10 gallon culture is doing fine as a stand alone Parvo culture. This is so much fun! I am continuously surprised by what happens. 
I am having issues with Photobucket, in that videos seem to take forever to upload, and videos are the best way to see the difference between Parvo and Apo.  When I figure it out, i'll post up pix.

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finally, I was able to upload the video that proves I still have the culture going:

 

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so if ciliates creep up in your parvo, are you sieving them out or just letting them be now?

I haven't been doing anything to rid the culture of ciliates.  The parvo doesn't seem to mind. I suppose you could sieve them out, repeatedly over many weeks.  Doesn't seem worth the effort to me.

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hmmm. ok, thanks. 

Kathy, you could consider a continuous ciliate remover using an airlift tube with a small sized screen on the inlet, and an even smaller sized screen or bag at the outlet.  In other words, a 40 um screen on the inlet would allow only ciliates to pass into the airlift, and then a 10 um bag at the outflow would capture ciliates.  Every day or two you could replace the capture bag.
 
This may be too much to set up.  When my tanks are ready I will have large parvo cultures and will need to knock back ciliate populations on a continuous basis.  This may not be worth setting up on a smaller system.  The inlet screen will have a tendency to clog easily because it is so fine.
 
Just a suggestion.

I actually like having ciliates in the ciliate bucket.  I don't know if it was the ciliates or the parvocalanus that allowed me to raise Corythoichthys intestinalis to 6.5 weeks…perhaps both.

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You certainly could be right.  Parvo naups are only twice the length of many ciliates, so they are probably only 4 times the food volume.  A larvae that hunts well could be growing on ciliates just as easily as on parvo (or in combination).  
 
It just surprised me that your parvo cultures weren't taken over by ciliates given the much faster reproduction rates of the ciliates.  You are certainly doing something right.

Perhaps the spare feeding they get keeps the ciliates from taking over in that bucket.  I've done very little to keep it going, since I have better cultures of parvo elsewhere.

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It always amazes me how much I can learn about a thing when I'm trying to learn about something else entirely.  
The kreisel I set up to make a stab at raising damsels turns out to be an ideal P. crassirostris culture vessel.  Too bad I didn't take better notes.  Over a weeks' time I set it up over a mild heat source, added P. c. and live Iso (just a tint) and a bazillion damselfish eggs.  The P.c. did very well, the Iso got really almost tea colored, the damsel eggs hatched and lasted until starvation day3, whereupon they all died.  I removed some of the dead bodies but mostly left them in there.  P.c. continued to bloom and I needed no additions of Iso.  At the end of 6 days or so, I added just a few damsel eggs and watched as the Iso got thinner and the water more clear.  I added more Iso as needed.  The pods are so numerous that I sucked out all the ones I could in 500ml of water, that were crowding the side closest to the light. The concentration in 500 ml was 10 pods per ml.  All long antennaes, so they must be P.c.  I did not see any Apocyclops at all.  But they are zooming around the kreisel like I've never seen Parvo do before. I was sure I had an Apo contamination. Nope.
 
Even after harvesting these, the concentration looks unchanged. I have to make the water light tea colored EVERY DAY, as it clears that fast.

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