Apocyclops panamensis Q & A

I'm sorry I missed this year's MBI workshop.  I wanted so desperately to discuss copepods with everyone.  I will not miss another, I can assure you!   
 
Now, on to my discussion/questions regarding Apocyclops panamensis.  I have been working with this pod now for 3 months, and I must say that it is incredibly durable.  I am culturing it on nothing but Instant Algae (Tet 3600 and Iso 1800), and, since the algae is non-viable, I am keen to keep pursuing this species.  I have found that this pod is euryhaline, so I have it in 15 ppt water for now, and they seem to be doing just fine.  I am working on improving feed dosing, water quality stabilization, optimal adult stocking densities and harvesting methods.  I have read everyone's threads on this animal and the information has been quite useful.  There are a number of breeders all over the world looking for copepods that will eat non-viable algae, or perhaps bacteria, so my focus has been to find the right pod and optimize mass-production protocols.  It is my understanding that people have fed these guys to larval and juvenile fish.  What about invertebrates?  Also, does anyone have any pictures/information/protocols/systems for harvesting copepod nauplii passively.  I welcome all callers! 
 
Chad

Following this. It's a pod I'm very interested in myself for a few projects I have in the works.

I'm glad you learned your lesson.
 
Seriously though, can you tell us more about how you're culturing them? Things like culture container type, how often and how much are you feeding, how difficult is it to keep the cultures from being contaminated by other things...

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

I will get you all that and more, today! 

tagging along.  I presume if you only used Tet, or only Iso, you're not getting the results you want (and if only Tet, would they come out lacking DHA)?

 
Hey Matt.  Hope you are well.  You would be correct about the lack of DHA in Tet.  So Iso is necessary, and, as usual, a feed that contains more than one species of algae is optimal.  Eventually, I would like to try our Shellfish Diet to really bolster the diet with other species of algae, including diatoms.  Also, I would like to see if these pods will consume Haematococcus, which will be very interesting.  My goal with the Haematococcus is to make them red and improve the carotenoid content with astaxanthin.  I suspect this will only work with the adults, but that won't be so bad, especially if they pass the carotenoids to their offspring.  
 
Well, I'm off to take pictures and look at the system.  I will get back to everyone with the details of my ever-evolving copepod recirculating system.
 
Chad

Apo Culture SystApo Culture TaApo Culture Tank.jpgnk 1.jpgem.jpg
 
So, as you can see, I am using a 70 qt HDPE BRT.  I have an inner standpipe surrounded by a perforated standpipe with 40 micron mesh screen covering it.  The water level is at 50L.  I have a flexible diffuser wrapped around the base.  pH probe in the tank.  300W Cobalt heater keeping the temperature at 27C. I am dose feeding with the older version of what is now the IceCap dosing pump.  So far, I have been dosing 10mls per hour from a solution that contains 2 mls of Tet3600, 2mls of Iso1800 mixed in 236mls of saltwater.  I am going to use a small scale magnetic stirrer to keep the food suspended, but I haven't found one yet. 
 
I was able to eliminate all large ciliates by performing a series of rinses through a 190 micron mesh screen.  I have not seen any contaminants in the system for 3 months now.  To keep contaminants out, I have the culture in it's own room.  I use gloves when handling and all equipment is dedicated to the culture and not shared with any other area. 
 
As you can see in the aerial image, I have made a filter floss media holder to catch unwanted organic waste.  I am wiping down the sides of the container with filter floss every evening before I go home, by the next morning, the tank is clean. 
 
I am using Dr. Tim's Eco Balance and Waste Away for obvious reasons. 
 
Once I have a protein skimmer and bio media, I am going to hook up a recirculating system. 
 
So far, I have 8K adults and 180K nauplii in the system.  The females are holding up to 10 eggs each. 
 
That's all for now!

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Regarding the floss, I checked multiple times to see if it's trapping/removing the pods.  Well, I mostly found copepodites and adults in it, but a low enough count that it's insignificant (1% of the population.  I did not find nauplii in the floss.  There is very small portion of the floss submerged and it seems to be working very well at removing all unwanted organic waste.  

You and I have the same equipment right down to the infrared thermometer!(exception is the fridge).

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

Chad have you been able to get accurate measurements on the smallest naups in the culture? I've read many times the smallest are about 70-120um but never seen it cited to anything scientific.

 
Hey Joe.  I now have a microscope in the culture room.  I have a stage micrometer and will be measuring all nauplii to see what the low end is.  I will get you that information as soon as I can.  I will also get pictures of these guys and add them to the discussion.

 
I am glad to hear that we think alike! 

Chad, you strained the culture through a 190 micron mesh to remove large ciliates and have a contaminant free culture.  Do you have small ciliates? Or do they pass through your 40 micron screen into something that eliminates them?  I didn't see a U/V or other ciliate killling mechanism attached.  
 
Am I missing something?  How do you keep down the population of small (less than 40 micron) ciliates?  Do the adult A. panamensis feed on the small ciliates?

 
Dave, before I started the culture, I poured all of my animals through the 190 micron mesh and rinsed them repeatedly.  My goal was to eliminate Euplotes, but I have yet to eliminate Vorticella (they are approximately 40 to 50 microns).  I do see protozoans in the 10 to 15 micron range, but I am not worried about them right now because they are only interested in milling around in the organic waste and will not out compete the pods for algae.  To keep the Vorticella at bay, I am simply keeping the organic waste at a minimum with the use of the floss and scrubbing the walls and heater daily.  Once I figure out the minimum size of the nauplii, I will change out my standpipe screen so that, when the recirc is set up, they will pass through it and get zapped by the UV, while retaining the pod nauplii.  I also hope to rid the culture of the 10 - 15 micron protozoans.   If this system proves to produce mass quantities of nauplii, we will dedicated a "clean room" to this animal.  I will treat the copepod eggs, while still attached to the females, with glutaraldehyde and pray that some embryos hatch so that I have a truly clean culture.  Glutaraldehyde has proven to be very useful for decontaminating rotifer eggs, so I hope it works for copepod eggs.
 
I am not sure if the pods consume small ciliates. 
 
Chad

On Friday, I had 180,000 nauplii (3.6/ml) and 8,000 adults/copepodites (0.16/ml).  Today, I took a 54ml sample of the culture to quantify the nauplii and copepodite/adults.  I now have 1.9 nauplii per ml and 1.7 copepodites/adults per ml.  A total of 98,000 nauplii and 86,000 copepodites/adults.  There are very few egg bearing females and mature males, but that number will go up as the 86K copepodites mature.  I anticipate that my nauplii numbers will jump significantly as the copepodites become sexually mature, then I can start harvesting nauplii.  I added two caps of Dr. Tim's Waste-Away today.  Once the recirculating system is set up, I plan on adding his Eco-Balance to the system for its probiotic qualities. 
 
Chad

So, with the help of Dr. Eric Henry, we were able to find what we think is a Stage 1 nauplius.  We measured its length to be 90 microns and width is approximately 65 microns.  The width measurement is just the body and does not include the appendages.   I included the photo with scale, so feel free to check my measurements.  If anyone knows what a Stage 1 nauplius looks like, please send me pictures or confirm the image is indeed Stage 1.   I believe Jim Welsh measured them at 70 microns wide, so I'm close to his measurements.  Here is the thread where Jim confirms his measurement:  http://www.mbisite.org/Fo...1&print=true 
 
Enjoy,
Chad

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Great work!

Do you use the same system for parvocalanus?

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

So right now it seems like the real question is who has used these, for what species, and what did you think?

I'm aware this species is popular with the sygnathid breeders but I haven't seen much info regarding how effective its been in larviculture. I personally plan to use it on bluestripe pipefish, PJ cardinals and a few Synchiropus species once I get it in culture.

So Chad, now that you've measured their smallest dimension at 70-90 microns not including appendages, what would that make your new standpipe screen, 80-100 microns?  I think it will be good to run through a UV for ciliate control.  That should also work for Euplotes, as I recall they are football shaped and about 40 microns on the smallest dimension.  
 
Your population seems to be fluctuating wildly.  Do apocyclops seem to produce chemical cues and restrict reproduction like some copepods?  When adults sense a lot of naups in the water the adults stop reproducing.  
 
Thanks, Dave

 
Kathy, the system is different. 
Parvos are batch cultured in inverted carboys, get 100% water changes weekly and only eat live Isochrysis.  With the Apos, I am looking to keep a continuous feed dosing system with nothing but concentrates and run a continuous culture with daily harvest of nauplii, but not all the nauplii, so that some of them mature and become the next generation of reproductively active adults.  It will take a little tweaking, but that's the general idea.  The Apo culture will also be a recirculating system.   

I'll keep everyone posted. 

Dave,  I am still going to keep the screen at 40 microns.  I have rid the culture of any ciliates over 40 microns, so keeping this screen size should help to remove the sub-40micron protozoans, while also allowing the feces and organic waste to exit the system. 
 
Regarding the population fluctuation, it's still a young culture, at least in this tank.  The nauplii are all becoming copepodites, like they should, while the sexually mature individuals are continuing to put out nauplii.  The gravid female population is very sparse, but I suspect in the next few days that the late stage copepodites will become sexually mature and begin to produce an abundance of nauplii.  I am very curious to see if reproduction and/or fecundity begins to drop as the culture reaches a certain density, like with Parvocalanus.  My current goal is to push the density just to see what happens; then start harvesting nauplii daily. 
 
Here is a population density and total population recap from the last 7 days (the culture in this tank configuration was started on 7/21)

• 7/28 - 60,000 nauplii (1.2/ml) & 27,000 adults/copepodites (0.5/ml).  There were mostly mature adults in this culture.
• 7/30 - 122,000 nauplii (2.4/ml) & 8,000 adults (0.1/ml).  Mostly mature adults with a few mid stage copepodites.
• 7/31 - 180,000 nauplii (3.6/ml) & 8,000 adults (0.1/ml).  Again, mostly mature adults with a few mid stage copepodites.
• 8/3 - 98,000 nauplii (1.9/ml) & 86,000 copepodites/adults.  Over the course of 3 days, the nauplii are now maturing into the copepodite phase.  Mostly copepodites with a dwindling population of egg bearing females and sexually mature adults.  Seeing very few newly hatched nauplii, but the majority of nauplii were in the later stages.
• 8/4 - 105,000 nauplii (2.1/ml) & 114,000 copepodites/adults.  Mostly copepodites with a sparse population of egg bearing females and sexually mature males.  Some mating observed.  Seeing newly hatched nauplii, but the majority of nauplii were in the later stages.  More nauplii going into the copepodite phase.

I predict in the next few days to see a huge increase in egg bearing females.  I'll keep everyone posted. 

 
Here is a video.  You can see that it's mostly copepodites and mature adults. 

Well, the population in the tub got hit with Euplotes contamination.  Also, I noticed that the adult females never really started bearing eggs like I expected.  I am going to tweak a few things after I decontaminate the culture; luckily, I have a backup running.  I am going to ramp up the population and then inoculate the tank and get this going again.  I have a few theories as to why the culture crashed.  One theory is that the bacteria product, Dr. Tim's Waste-Away, was digesting not only the waste in the culture, but also the non-viable algae cells before they could get consumed by the pods.  I am going to give him a call today and see what he thinks.  I think that Dr. Tim's products are great, so I am by no means pinning my failure on his product.  Just saying   It's also possible that feeding the culture once every hour is not ideal.  Maybe it needs to be fed every 30 minutes.  I may also need to increase the feed concentrations and tweak the binary feed approach.  Maybe Tetraselmis isn't ideal; I could always add a diatom.  If anyone has any input, I am open to discussion, as usual.  
 
Chad
 

I just spoke with our phycologist, Dr. Eric Henry, and he said that it is unlikely that Dr. Tim's Waste-Away bacteria invaded the algal cells.  On to the next theory. 

Chad,it looks indeed like a 1st nauplius (N1).You must play with the focus of the scope to confirm it.
N1 of all calanoid copepods is defined by three setae at the tip of A1 (antennula) and two spines at the rear end.I imagine N1 of Apo will be the same.

 
Thanks Luis!  Great information.  Not sure why I never knew that?  LOL  I guess I need to pay closer attention.  Do you have a culture going with this species?  Good to hear from you BTW.
 
Regards,
Chad

 
Chad, maybe the adult female population is too high and puts out a chemical cue to stop reproduction.  This would seem to be logical in natural conditions to prevent an outstripping of food supply.  Another theory: the naup density in your population may be the cause of such a chemical cue just as easily as your adults.  If either of these are the case you would see a quick increase in fecundity by decreasing density, so it could be easy to prove or disprove whether chemical cues are the culprit instead of the ciliates or Dr. Tim's product.

 
Hey Dave, 
 
Your theories are much more favorable than mine; I like what your saying and it's giving me some ideas to work around the chemical issue.  If I hit the maximum density with adults and/or nauplii, and want to push it higher, then maybe carbon filtration is the answer.  Carbon has been known to take up secondary metabolites of marine macro algae, and those are on a molecular level.  Maybe it could work in this case.  I have discarded my Dr. Tim's theory, and will continue to look at how the Euplotes got into the culture.
 
Chad   

Chad, my theories are just guesses, I like your idea of using carbon.  If you went to a larger container you might not even need carbon, you may reach your daily production at a lower density.  But I've always been a fan of treating copepod tanks like fish tanks with their own filtration (and very slow water turnover).
 
As to Euplotes prevention, it seems to me that ciliates will always be present in an open culture, the best we can do is try to manage their numbers.  That's why I like your UV approach.  Now how do you keep 40 micron screens from clogging every few hours?

Any updates using other pastes besides the tet and Iso yet?

 
Dave, I plan on having a bubble curtain running up the center stand pipe to keep the mesh from getting clogged.  Not sure if it will work.  I may be forced to spray the screen off every day with Isopropyl alcohol to break up the heavy organics trapped in the mesh. 
 
Chad

 
Joe,
 
I am currently ramping up my population.  I plan on trying out our SDaquarist on the pods this next go around.  It's one of our newest products.  It's essentially Shellfish Diet diluted to make room for ClorAm-X and a pH buffer, similar to how we do RGcomplete.  It contains a variety of algal species including: 2 diatoms, 2 browns, and 1 green.  I am actually really excited to use it because I had success with it on Tigriopus californicus. 
 
Chad

Again,this holds true for calanoid naups.There is at least one ref stating that N1 cyclopoids have two setae.
http://www.marinebreeder.org/forums/viewtopic.php?f=144&t=7410&hilit=apocyclops
this is an old thread showing how Apo were introduced and IDd in the hobby.Unfortunately this fundational thread ended firing a nasty scandal because of professional and commercial interests and it had to be much edited.But still it remains interesting.
And yes,since that time I keep them.Apo are "super pods"

 
Great information, Luis.  The thread was helpful. 
 
Chad

So, I set up 2 carboys to test Iso 1800 (Instant Algae) and SDaquarist (Shellfish Diet diluted containing ClorAm-X and a pH buffering component); both are doing very well.  I say very well because I see numerous females bearing the tell-tale egg sacs and an abundance of nauplii and copepodites.   I am going to write up the metrics on these cultures and get back to everyone with the details. The next step will be to scale this up and starting working on harvesting protocols.  Hope everyone at MACNA is having fun!

 

Is SDAquarist available for purchase yet? I can't find it on the website and I'm eager to try it out!

SDaquarist will be available as soon as the labels are printed and we slap a price on it.   Go to the Reef Nutrition section of the RMI website and click on the SDaquarist bottle.