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Memo for Ciona inland culture (ver. 2003/Nov.)
This is a prelimanaly version, and should be improved.
Culture itself is not final aim for the most researchers althought
it is hard task. Therefore, even preliminary experiences should
be distributed. A lot of people have been concerned with this
issue although most efforts are still unpublished, which were
informed to me by some of them. In the sense, copyrignt of this
page is owned not only by myself (=Shungo KANO) but also by the
others (=the Ciona community).
Basic matters are described in an article by Sasakura's et
al.
(PNAS | June 24, 2003 | vol. 100 | no. 13 | 7726-7730)
http://www.pnas.org/cgi/content/full/100/13/7726
(If you have a right to access, you can download a pdf file)
Some modifications are required for Ciona savignyi
because the nature is different from Ciona intestinalis.
For an example, suitable food condtion of C. savignyi
is different from that of C. intestinalis.
Culturing Condition of C. intestinalis
(this section is copy&paste of the article by Sasakura et
al.)
Metamorphosed C. intestinalis individuals were cultured
in plastic Petri dishes, 9cm in diameter & 1.5cm in height
(three to five individuals per dish) (*1)
for 10 days with 3% (vol./vol.) full-grown diatom Chaetoceros
gracilis in seawater (called as 3% GSW) (*2).
Seawater was changed every day. (*3)
This procedure is done until the atrial siphons are merged into
one.
After 10 days, the Ciona were cultured with 0.2% Microfeast
and Spirulina (MS) solution in 3% GSW(*4).
The mixture of 400 mg of Microfeast PZ-20 (Saltcreek, Salt Lake
City (*5))
and 200 mg of Jade Spirulina (Saltcreek) in 50 ml of seawater
was left for 5^10 min.(*6)
The supernatant was used as MS.
This procedure is done toward animals at the 1st ascidian stage.
After juveniles had grown to 5 mm in length,
the number was reduced (*7) (one to three individuals per dish)
and the juveniles were cultured in 500 ml of 0.2% MS in 3% GSW.
Seawater was changed every day.
The concentration of MS and the amount of seawater were increased
up to 0.6% and 1 liter, respectively,
according to the size of individuals. Culturing was done at 18C
(*8).
(Notes for *marks)
(0)
In any conditions, check if Ciona animals uptake until their
guts are full up with contents (=excrements). It is one of good
indicators for suitable feeding. Animals which lack nutrient
show lengthen siphons.
(*1)
15cm in diameter is possible to use, if you have larger aquarium.
You may think that 1.5cm in height is higher than normal size
petri dishes,
however, higher height makes floating time of the food longer.
It means that juveniles can uptake foods continuously.
(It is hard for Ciona juveniles to uptake food at the bottom
of dishes.
Floating of food is good for them as long as possible)
For the same reason, dishes with juveniles can be kept in
aquarium containing GSW with water circulation (this is an idea),
though it requires more amount of seawater and circulation could
cause detachedness of young juveniles.
(*2)
A diatom, Chaetoceros gracilis, is originally known as a food
for pluteus of Sea Urchin.
It has two advantages compared with artificial foods.
1) The size of particles is smaller than the artificial foods,
which is good for juveniles to uptake.
This species does not make long chain (cf. diatom normally make
long chains)
2) Alive plankton can maintain the quality of seawater.
The artificial foods make water pollution.
Culture of the diatom is a little bit bother, this is a disadvantage.
Now, it is possible to purchase a commercial product in Japan
(TASAKI SINJU co., LTD)
The product contains 4-5 x 10(8) cells/ ml in 1L, which costs
\20,000 (around 150euro)
it is convenient according to Dr. Sasakura.
About living planktons,
another diatom species Chaetoceros socialis or a plankton Isocrysis
can be used, too.
In my impression, Ch. gracilis is better than the others
although I have not compared the effects of these on growing
of juveniles.
(*3)
Frequent exchanges of seawater, of course, maintain seawater quality.
Further, juveniles and adults could take nourishment from
seawater as higher frequent of it apparently improves growing of the animals
according to Dr. S. Chiba and the others, and less (or no) exchange often result in bad growth and immature
although they are indeed alive. This is a reason why we prefer avoiding artificial seawater (some artificial seawater may be suitable and others are not). If supplement of nutrients could improve the situation,
we can save seawater a little bit.
(*4)
Now Dr. Sasakura and the others give additional two kinds of
powder food for fish.
This information will update later.
Anyway, multi foods should be given to animals because we do
not know which are the critical ones.
(*5)
The two products, Microfeast and Spirulina, can be imported.
At least, SZN (Naples, Italy) and Nori's lab. (Kyoto, Japan)
import the products from the USA company.
SALT CREEK, INC.
http://www.a-saltcrk.com/
It is impossilbe to visit at the site indicated, however, you
can purchase prodcuts from distributors.
About usage, see also the following site.
http://www.mbl.edu/BiologicalBulletin/MMER/cirino/CirTit.html
This was written by the members of the SZN as an electrical article.
It is useful to understand Ciona culture although the system
is opened circular system.
(*6)
These products tend to make a block.
So, vortex is required after dissolving into seawater, and then
use only supernatant after leaving for a while.
This means that supernatant contains only small particles, which
flow longer in seawater.
Big blocks of food at the bottom of aquarium often make pollution
of seawater.
(*7)
Density of animals is critical issue; high density causes less
growth of animals (even in opened circular system).
This is a limitation of inland culture, however, it is enough
to work well in small-scale culture (e.g. for insertional mutagenesis,
for mapping, and for maintanance of a mutant).
(*8)
Around 23C might be limited because the foods rot soon at higher
temperature in closed system.
Higher temperature promotes speed of the growth.
Growth is slow at less than 16C.
For some wild populations, the condition could be different from
above.
(**others)
We often observe that maturation and quality of eggs are not
good although sperms can be observed.
Some nutrients can improve it.
Dr. Sasakura uses a yeast boosted several vitamins and organic
acids,
which is commercial available (it is called as YUSI-KOUBO in
Japanese).
I myself use a fish food "Tetramin" after breaking
into powders.
Recent interesting sites
http://www.stanford.edu/group/Urchin/p2m.htm
http://www.brineshrimpdirect.com/brine-shrimp-direct-algae.html
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