Culture Journal, Species: Parvocalanus crassirostris

Jim, again I see that you're doing some great work.  Your pictures are excellent quality and your noble attempt to measure algal density through a color chart may yet work out.  As someone above pointed out, deeper vessels will help give you deeper color and perhaps some kind of lighting standard would be necessary (i.e. test vessels observed under two new 40 watt 4' cool white fluorescent tubes at a height of 20" and a color rendering of ___).
 
Keep up the good work, we are learning a lot from your studies.

Time for an update.
 
My various cultures continue to do well.  Some, better than others.

In general, I've found that less is certainly better than more when it comes to feeding these copepods.  What I strive for is a level of food that allows the water to go clear within 12-24 hours.  I examine the cultures twice daily, and assess whether I consider the culture water to be "clear" or "tinted" ("tinted" means even a very slight tint -- if it is perceptible, then it is considered "tinted").  If the water is "clear", then I feed.  If it is still "tinted", then I do not feed.

It is actually quite counterintuitive:  The cultures that are fed the least (within reason) are the ones that do the best!  If a culture that is still "tinted" gets fed still more, even though it is still "tinted", then this culture will tend to go downhill, and suffer as a result. 
 
Curiously, one of the most dense cultures of Parvocalanus I have is left over in one of my 2 gallon kreisels from a  batch of Bluestripe pipefish.  The copepods continued to outpace the rate at which the pipefish fry were eating them, and after the pipefish fry settled and were removed to a growout tank, I continued to feed the copepods in the kreisel, where basically the only change was the removal of the pipefish.  That was probably two weeks ago, and the copepods remain quite dense!  I am feeding probably about 20-30 ml of a dense Isochrysis or Isochrysis + Pavlova culture twice a day to the 2 gallon fishbowl.  This keeps the water just very barely tinted with brown.

Another apparently important factor is to keep the culture from becoming too dense with copepods.  I find that once a culture gets relatively dense with copepods, it is important to split it, and to replace the harvested water with new pasteurized salt water.  There does seem to be an inhibitory effect to overpopulation, and cultures that are quite dense, if left alone without splitting, will tend to crash.  For whatever reason that I have not yet determined, the 2 gallon kreisel culture is the exception to this rule -- I have only done one 25% water change on it, about a week ago, but it seems to be able to maintain a high density level without crashing.

I have not yet taken any careful measurements of the copepod density.  For one thing, a count of "individuals" without reference to the various counts of the various life stages would be relatively meaningless.  Another issue is the relative difficulty of counting nauplii compared to counting adults, given the large difference in their relative sizes.  Still another challenge is the tendency of the copepods to congregate along the side of the container nearest the light source, which causes a discontinuity in the population density.  If the container is sufficiently stirred to equalize the density, then there is usually enough detritus in the container that also gets distributed by the stirring to make it difficult to tell the live organisms from the dead ones, the molts, and the other bits of feces, algae clumps, etc. distributed in the water column.

All that said, my best estimate of the density of my more dense cultures is that there is probably somewhere between 10 and 30 individuals per ml, roughly evenly distributed between the various life stages.  I am basing this on my observations using a test tube with two pieces of tape wrapped around it, with a 1 ml "gap" between the two pieces of tape, giving me a 1 ml window through which to observe and estimate the count I can see within that window after placing a sample in the test tube.
 

I have now been continuously culturing Parocalanus crassirostris for 90 days!  For the continuation report, I grabbed some quick-and-dirty images with the dissecting microscope at work today -- I didn't put enough work into them for them to look good, but they are good enough to verify that they are, in fact, Parvocalanus.  Here you go:
 



 
I currently have the following cultures of P. crassirostris going (listed in no particular order):
 
1)  A 1 liter Erlenmeyer flask
2)  A 2 liter media bottle
3)  A 2 liter soda bottle
4)  A 2 gallon glass drum-shaped fishbowl
5)  2 x 3-gallon plastic water carboys
 
All of them get some air from a rigid airline.  The amount of air ranges from about 1 bubble per second to perhaps 3-4 bubbles per second.  They all get a small amount of live phytoplankton (usually just Isochrysis, but also Iso + Pavlova or Iso + Pavlova + Tetraselmis when I have the others available for harvest) twice daily, unless the water is still visibly tinted from the earlier feeding.  I strive to keep the water just barely tinted enough to see that it is not clear, but no more.  If I can tell without a doubt that it is most certainly tinted with phyto, then I consider that culture to be overfed. 
 
If the cultures start looking too dense (sorry -- I know this is hard to quantify without a specific count of some sort of adults and/or nauplii and/or copepodites per ml), then it is time to split the culture by harvesting 25-50% of the culture (together with the culture water) and replacing the water removed with new, pasteurized water of the same salinity and temperature. 
 
Determination of whether or not to feed should be made after the splitting the culture, although almost always any culture healthy enough to need splitting will certainly need feeding after the split. 
 
Cultures that are both very thin with copepods and also overfed tend to have a poor recovery rate.  If you have a culture in this condition, it is probably better to split it and thereby dilute the excess food.  IMHO, it is probably better to have a culture with an overall lower number of animals but a more appropriate food concentration than a culture with a larger number of animals, but way too much food, because there seems to be something about the excess food that tends to kill off the nauplii.
 
My philosophy is to make sure that I have multiple cultures running.  If one or more of my cultures starts to develop a problem, I will generally have one or more other cultures that are doing well, and I can just toss the sickly culture(s), and replace them with splits from the healthy culture(s).

Attached Image(s)

I have a question for you, Jim. I know a while back you mentioned that algae pastes are entirely unsuitable for culturing this species of copepod, but what are your thoughts on using the reef nutrition product Phyto-feast live? It's live cells rather than the dead ones in pastes and contains Pavlova, Isochrysis, Thalassiosira, Tetraselmis, and Nannochloropsis. Are some of those too big and would just go to waste? Or do you think it could work?
 
Thanks, Heather

That is a very good question, Heather.  I simply don't know the answer.  Maybe Team RMI knows.

I do know that live Isochrysis, Pavlova, and Tetraselmis are all readily eaten by Parvocalanus.  I'm not sure about Thalassiosira.  Regarding Nannochloropsis, there is this whole issue about its tough cell wall being indigestible by many organisms.  I don't know for sure, but I suspect that Nannochloropsis may be indigestible by Parvocalanus. 
 
Whether the condition of the "live" cells in the product is sufficient to be an effective feed for Parvocalanus (e.g., the motile cells are no longer motile, having lost their flagella, as stated here:  http://reefnutrition.com/phyto_feast_live.php ) I really don't know. 
 
The Phyto Feast Live page on the reefnutrition.com website (go to http://reefnutrition.com/phyto_feast_live.php and click on the "Phytofeast FAQ" tab) links to the "viability report from the CCMP" document ( http://reefnutrition.com/pdf/CCMP.pdf ) which states that, "...Phyto-Feast Live contains live algal cells that grow and reproduce when placed in an algal culture medium."  However, the document doesn't state anywhere in it which specific species of algae grew from the sample provided, so we don't know from this document whether it was, for example, only Nannochloropsis, or if it was multiple species of algae, or all of them, that grew.  If, for example, only Nannochloropsis grew, then that is not evidence that all of the various species of algae of interest for culturing Parvocalanus are still "alive" and viable.  Also, I'm not sure what the significance is, if any, of the comment in the lower-left corner of each page that says, "Confidential Pursuant to Protective Order - Attorneys Eyes Only - Settlement" (and quite frankly, I feel my eyes have been somehow soiled by seeing this, considering how I feel about attorneys).  There are other issues with the CCMP document, such as a typographical error in the expression of the light intensity ("µmol M₂^-1 sec^-2" instead of "µmol M^-2 sec^-1").  As stated, the document indicates that the amount of light provided was increasing exponentially by the second, which is unlikely (to say the least).  The reefnutrition.com page also cites this CCMP paper as "more details" to support the statement that Phyto Feast will "...stay alive for several months", but the CCMP paper only documents that the cells were still viable "after 12 days in a refrigerator."
 
Again, perhaps Team RMI can help clarify some of these questions.  I'll see if I can get their attention to this thread.  It would be a very pleasant surprise indeed if an off-the shelf product that can be stored in the refrigerator turned out to be a feed that can be used to successfully culture Parvocalanus.  I really do hope that Phyto Feast Live is able to accomplish this.  That would be GREAT NEWS!
 

Hey Jim, great work so far and your attention to detail is staggering.  Where do you find the time???    BTW, I am gearing up to test the efficacy of Phyto-Feast Live and RGcomplete on the PC pods.  I will be running these two groups along side my control group that is being fed live Isochrysis and Tetraselmis.  The trial will be run for a week, starting today, and I will let you all know how it goes.  As you probably agree, we need for people to be able to culture this animal without live algae so that breeding of difficult species can be more easily achieved!  On a side note, I am also working on a simple methodology for culturing Tigriopus californicus and I will be presenting my results at MBI.     

That's great news, Chad, that you're doing this trial.  I certainly agree that a reproducible protocol for culturing Parvocalanus without using having to culture live phytoplankton would be a huge leap forward for the breeding community!  I really look forward to the results of your trial.
 
Anecdotally, another MBI user has commented on Facebook that his Parvocalanus sourced from AlgaGen were "doing fine" after 19 days in a 10 gallon tank with vigorous aeration ("like a boil") from a rigid airline, being fed RotiGrow Plus at a density that permitted him to see through the tank the narrow way, but did not permit him to see through the tank the long way.  He said that he doubled the water in the first week, did two or three 25% harvests during the second week, and split the culture in half the following week, and after all that had a much higher density than he started out with.  He said he is basically treating them like he would rotifers.  I have not yet tried reproducing his success, but just thought I would pass this anecdote along, since you are planning this trial. 
 
Naturally, my first thought is whether or not the organism he is culturing really is Parvocalanus, since I've made the mistake in the past of thinking a rotifer contamination was a healthy Parvo culture (until I looked more closely at them)!  I have also seen other Food Culture Journals for paracalanids here on MBI where the images of the cultured animals looked more like Tigriopus in one case, and Apocyclops in another (visible egg sacs in both those cases), and Acartia in yet a third instance.  Nonetheless, I thought I'd pass this casual report of success with Parvocalanus on preserved phyto along to you.
 
I wish you the best of success in your trials!

Well, you can guarantee that my report will not be anecdotal. 
 
Also, all species of algae in Phyto-Feast Live are viable.  Not just Nanno.  As you may assume, viability tapers off after a few weeks and Nanno is the hardiest of the bunch. 
 
Regarding feeding Nanno to Parvos.  I have run a trial with live Nanno.  They eat it and can survive with it, but reproduction shuts down.  All my adults eventually died of old age and I never saw an emergence of nauplii.  I suspect they need algae that have high levels of  DHA such as Isochrysis and Pavlova.  I also see a large increase in egg production when I add Tetraselmis to their diet.  Tet has high levels of lipids and also contains cholesterol and various amino acids.  Variety seems to be the best route.     

Thanks for all the info guys! Clayton, I look forward to seeing your report and I may attempt to copy your results if all goes well.

Assuming the supposed viability of the algal cells for at least 2 weeks, perhaps the best course of action would be to purchase fresh phyto feast live on regular intervals so ensure we don't begin feeding nonviable cells to the copepods. Any unfinished phyto could then be fed to something less picky, like tigger pods, which I have been culturing on phyto feast live for almost 2 years and don't seem to be picky at all.

 
I saw similar when trying to feed NRichPL to parvocalanus. The cultures would last up until the adults died of old age but the number of new naups steadily declined and then stopped. I assumed that the naups were starving and that the adults were eating fine but were past the point they could reproduce. But it could certainly be as you described: they just weren't getting enough nutrition to keep reproducing.

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

 
Your asking more details then the court case needed answered, thus you will not find the answers you seek, sorry.  It was not specific to which algae, just the LIVE claim over all.
 
I really don't feel like rehashing that deal again, its well documented on the internet if you are so inclined to read up on it.
 
 
PFL is made fresh every week so its rather easy to get fresh if you buy direct from us.   I can try to alert you when I know it will be shipped to a LFS near you... Fremont would be either Baja Reef (Hayward) or Neptunes (Miltpitas) .
 

 
I'm glad someone is going to try Phyto Feast as a food source but my concern, going along with what you said Jim and aside from the fact that the species viable for Parvo would need to be those growing, is that if one isn't growing, won't it will cause problems with the Parvo culture like excess build up of nutrients since you are just adding more biological matter that isn't being used up?  That and I also wonder how alive the algae in the Phyto Feast actually is by the time it gets to the end consumer.  I mean, "alive" can be used in a very relative way.  So how much of the algae is actually alive?  And I mean percent of the total biomass alive in the bottle, not which species.  If too much is dead, won't you just be adding waste to the culture?  And yes of course there will always be some waste but I'm wondering how efficient it will be and if this will cause problems over time.  I only point this out because there's no way you are adding the same amount of waste when feeding from the bottle as compared to the fresh culture (correct me if I'm wrong, that's just my logic).  I hope I don't sound nit picky but I feel like these could be important factors in the potential success of Phyto Feast as a viable food source for Parvo.

 
Any updates, Chad?

Well, I have some results.  Instead of Phyto-Feast Live, I went ahead and fed the Parvos RGcomplete.  I chose RGcomplete because of the ammonia removal component and buffering capability. 
 
So, after 7 days on RGcomplete, they are still alive and I found eggs and nauplii, which means that they are definitely eating the algae and getting enough nutrition to support reproduction.  I am also adding heterotrophic bacteria to the system.  I will be more specific about the bacteria after I try a few different brands. 
 
As expected, the control tanks outperformed the RGcomplete fed tanks.  I started all tanks with 2,000 adults. 
 
The 2 control tanks had approximately 50,000 individuals each after 7 days and were fed live Isochrysis and live Tetraselmis. 
 
The 2 RGcomplete tanks had only 5,000 individuals each after 7 days.  These tanks were fed 10 drops of RGcomplete per day.  On day 7, I did a 100% water change and will see if the next generation will perform better (creating a strain?).  I am going to continue the trials with RGcomplete and try to tweak things as the cultures progress. 
 
I will try to supply updates weekly.   

Interesting.  What is the volume of water in the tanks?

13 liters in each tank.  26C for temperature.  I used Instant Ocean mixed in deionized water and ran a salinity of 32ppt.

Excellent read and some fantastic pics Jim Welsh,
 
I was interested in your attempts to measure colour against a chart for algae density, Have you considered the Hanna pocket colour of water checker?  I haven't cracked mine open yet but would be very interested to see if this would do the job??

No, actually, I've given up on quantifying my algal densities, after a couple of weeks of watching them with a hemocytometer.  I have now learned how to "eyeball" it, and my various cultures are doing quite nicely.  Again, as I wrote above, it almost seems as though the less I feed them (within reason), the better they do.  I feed very lightly twice a day.  Sometimes I forget a feeding, and am always worried that I might be starving them, but then, I seem to get a bloom immediately afterwards!
 
I've now had Parvocalanus in culture for over 4 months, and have 7 different containers, each with a healthy, dense culture of various life stages.  I was unable (or unwilling) to sustain the Pavlova (Monochrysis) and Tetraselmis cultures, and so, I've been feeding a monodiet of Isochrysis for at least the last month.  My A. tonsa are also doing similarly well, being treated the exact same way.

It has been exactly a month since my last update of this thread.
 
My various Parvocalanus cultures continue to do well.  I did have a crash of the one 2-gallon fishbowl culture, and I also have had a gross, white bacterial bloom happen in the one 2 liter soda bottle culture, although the copepods don't seem to mind very much.  I continue to feed them very lightly, at this point only live Isochrysis, since I've lost both my Pavlova (Monochrysis) and also my Tetraselmis cultures.
 
The two, three-gallon carboy cultures have both been going for a very long time with very few changes -- not even water changes.  I did harvest about 1/2 of the copepods from one of those carboys a few days ago to feed a batch of pipefish fry, but I also just returned the culture water sieved through a 27 micron sieve back into to the carboy.
 
I've now had these copepods in culture for 5 months.  The key seems to be feeding a very, very light level of live Isochrysis, plus perhaps Pavlova and/or Tetraselmis if they are available, once or twice daily.  Overfeeding is to be strongly discouraged.  If you must err, err on the side of underfeeding.  Cultures will do very well if split when population density gets too high, but they will also do well enough if just left alone, and if you do not split.
 

Have you incorporated Andy's standpipe design?

check out Kathy's Clowns, llc website:
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Please refresh my memory about what you are referring to.

That's a good question, because I haven't seen a picture of it yet, but its a standpipe with many holes throughout its length, covered with 30 micron mesh.  It is attached to a bulkhead that enters through the bottom of the container and angles out with a valve .  Obviously this is supported on a stand of some type.  The idea is that one can open the valve to drain the container taking old water and ciliates out, leaving copepods in.  Then close the valve and  refill with pasteurized saltwater.
 
Problem I have with the design is that I catch ciliates in 30 micron mesh all the time.
 

check out Kathy's Clowns, llc website:
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the copepedites and ciliates are the same size, hows a mesh standpipe going top help?

And i either have a lot of dead ones, or lots of molts….Perhaps I am not feeding enough….
 

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Jim, do you find that you need to feed the Parvocalanus more and more frequently than the Apocyclops? My Apo cultures thrive on very light, once a week feeding, and my parvo cultures seem to need more...

check out Kathy's Clowns, llc website:
http://kathysclowns.com
Captive bred clownfish and more
(Wholesale to the trade.)

I most certainly do feed my Parvocalanus (and, FWIW, my Acartia) more frequently, but also more lightly, than my Apocyclops cultures.
 
Quite frankly, I find Apocyclops almost impossible to kill, short of abject warfare.  Parvocalanus, on the other hand, are much more temperamental.  On the whole, I have found that Parvocalanus need (1) Very light feeding, (2) Very frequently.  I feed my various Parvocalanus cultures, in general, at least once daily, and often twice daily.  As I have repeatedly said, I tend to err, if at all, on the side of underfeeding.  I have found that very light feedings, once or perhaps twice daily, is what does best.  Assuming that there is a decent population of copepods in your culture, a feed rate that would amount to about 75,000 cells of  Isochrysis per mL per day is about right.  Less food, down to even as low a rate as perhaps 10,000 to 20,000 cells of Iso per mL per day, may suffice.  Greater feed rates exceeding perhaps 200,000 or 300,000 cells of Iso per mL per day will cause problems.
 
I have found it important to learn to visually guage the amount of tinting in my cultures, and if I can detect any discernable tinting remaining in my Parvocalanus cultures when it is feeding time, then I skip this feeding, and wait.  I have also found that the cultures where I observe the excess tinting have some other issue going on, and they rarely recover by themselves.  Often, they need a large (read: 100%) water change (sieve through a 27 micron sieve, and backwash into freshly made, unused, aged saltwater, all the while avoiding any unnecessary exposure to air), and then very light feeding afterwards.
 
I hope this helps.
 

Of course, it does.

check out Kathy's Clowns, llc website:
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(Wholesale to the trade.)

updates?  have you (or anyone one for that matter) co cultured this with anything? (intentional or not?)... 

There are some in my ciliate bucket.  I'm trying to remove the rotifers from my primary culture.  Haven't checked this week.
 

check out Kathy's Clowns, llc website:
http://kathysclowns.com
Captive bred clownfish and more
(Wholesale to the trade.)

No co-cultures of anything with my Parvocalanus yet.

mmm. ok! well, I'm going to start up my Iso soon (maybe this week, if I can get an order to arrive!) and see how well I can keep that going. Then get an order of the parvo in and try my hand at it! ... It's threads like this that are well descriptive that encourage me to try my hand at it! lol... Thanks Jim!