Natural sea waterNatural sea water taken from Villefranche bay (Villefranche-sur-mer, France) works fine with Clytia, except for winter time when the water quality may be low.
Artificial sea waterDissolve commartially available sea salts in to RO(reverse osmosis)-filtered water and adjust the concentration at 37‰ (Measured by a salinity refractometer PR-100SA, ATAGO, Japan)
Lower salinity may cause some problems on spawning and fertilization.
Final salinity depends on brands (or even batches) of sea salts, which can be most easily explained by differences on hydration of the salts. Often the salt will be hydrated quickly. The actual salinity therefore must be measured and adjusted each time you dissolve the salt. The quality of RO-water may also affect to the embryonic development. After dissolving the salt, it is recommended to wait for a night to equilibrate temperature leaving reservoir tank in the aquarium room and to mix again before use.
- 18~20°C is the good to keep all stages considering balance of colony growth and algae growth.
- Clytia colony is perfectly healthy at 24°C and grow faster than at 20°C and metamphosis efficiency may also be better at 24°C
- 16°C is used to slow down embryonic development, particularly useful to get early gastrula stage. If eggs are fertilized at 17:00 and shift to 16°C at 2- to 8-cell stage (18:00~19:00), the embryos will be at very early gastrula stage at 9:30.
- Sex determination of colony is affected by temperature
- Female is favored if colony/jellyfish are cultured at 24°C
- Male is favored if cultured at 18°C either in colony stage or in jellyfish stage.
Range of salinity for Clytia aquarium
|Salinity||Density||Good/Bad for Clytia|
List of artificial sea salt tested and relation between weight concentration and salinity.
|Type of composition||Dissolved at||Salinity by Refractometer (‰)||Remarks|
|Sea water from Villefranche bay (01.2010)||Average 38||Usually 38.5 in Villfranche bay in winter.|
|Red Sea Salt||40 g/l (w/v)||37||Highly recommended.|
|Tropic Marine sea salt (synthetic salt mixture)||38.5 g/l (w/v)||not checked||Limited availability in France. Not tested enough.|
|Reef Crystals (synthetic salt mixture from Instant Ocean)||42 g/l (w/v)||37||Works parfectly for polyps and medusae.Gives poor results for spawning (it delays) and fertilization (low efficiency).|
Ammonia, NO2 and NO3To be tested.
Villefranche team's experiences suggest that half sea water change par 2 weeks is recommended for new Villefranche culture system or complete change in every 2 to 4 weeks for Traditional beaker culture.
Artificial sea water or natural?Artifical sea water is the first choice in most cases to avoid potential problems caused by natural sea water such as seasonal quality changes including salinity, contamination of algae or protozoans, pollution and etc.
FilteringDead artemia boosts bacteria and algae growth in the tank. Colonies also release a number of baby medusae into sea water, which will be killed and make sea water dirty unless properly cultured. It is therefore very important to get rid of them from sea water. If the culture is closed system (beaker system or nursery tank) I recommend to change sea water once a week. For water circulation system, we use 80 µm nylon mesh filter and a protein skimmer.
Artemia cultureWe use Artemia salina larvae as the food for Clytia (both polyp and medusa). Olnly 1.5~4 dph (days post-hatching) larvae, instar II to instar IV stages, are appropriate as the food. Younger larvae, especially newly hatched instar I nauplii are not digestible by polyps and kill them, when used several days. (Occasional use of instar I is OK.) To have appropriate quality of artemia every day. I recommend to start artemia culture three times a week.
MaterialsArtemia dried eggs (available from aquarium shop) A tall plastic bottle with pointed bottom and valve. (A separatory funnel is good) Air pump and tubing with sold plastic pipe at the end.
ProcedureThe timing of schedule is indicated to make T=0 for the day of hatching (= dph days post-hatching).
Setting up culture (T = -1 day)Put 1~2 ml (depends on requirement) of artemia eggs in the and add SW (~1 liter). Put the aeration tube in the tank. Make sure that the end of the aeration tube comes to the bottom otherwise eggs stay at the bottom of the funnel. keep the funnel in 30°C otherwise warm the culture with a lamp etc.
Collecting newly hatched instar I larvae (T=0)Separate hatched artemia from cysts and unhatched eggs. This process must be done within the day 0 ( when the it is in instar I stage) because instar I larvae go to the bottom of the funnel while most of cysts come to surface or are attached to the side of the funnel. Separation will be harder in later stages because both cyst and artemia lavae come to surface.Take the aeration tube out of the funnel slowly not to distrub the cysts attached to the side of the funnel. Wait for 5~10 minutes until artemia larvae comes to the bottom. Open the valve of the funnel to take settled artemia larvae to a small beaker (normally 20~50 ml). Wait another 10 minutes to collect artemia as much as possible (repeat a few to several times). Artemia suspension still have a bit of cysts. In order to separate perfectly, put them in a flat dish and light it from the side. Artemia basically moves to the side of the dish. You can easily separate artemia by a pipette from cysts. Separated artemia will be transferred to a large beaker with clean sea water and keep at 18°C with good aeration.
Using larvae (T= 1.5 days)It is highly recommended to use older Artemia larvae (T=1.5~4 days). Temporarily stop aeration of the beaker for Artemia stock (see above) and wait for a few minutes. Artemia will be concentrated by phototaxis. Then collect a part of them to a smaller beaker as an immediate stock and wait their phototaxis. Now the artemia is ready to be used as food. No areation is necessaly when they are stored at low density.
Organizing scheduleHere is an example of Artemia culture schedule. 1/2/3 indicate the lot of Artemia.
|Culture Start||Culture 1 start||Culture 2 start||Culture 3 start|
|Instar I selection||1||2||3|
Traditional culture method
- 5 liter glass beaker
- Artificial or natural sea water (see more detail in Sea Water section)
- Motors (5~6 rpm, 12 or 24 volt)
- Motor mount: to fix the motor on the beaker.
- Power supply (AC adaptor)
- Pedal (about 5 cm x 10 cm plastic plate)
- It is recommeded to use disposable materials for making pedals so that you can change to new pedals if it is highly contaminated with algae. We make them with transparent plastic casing of "Grainer Bio-One EasyLaod micropipette tips" and disposable "pastette".
- Soft silicone tubes to connect the pedal to motor
- Dish (φ10 cm x 5 cm)
- Glass pipte (φ 8 mm) to transfer medusae
Common tank set upA tank consist of a 5 liter beaker, slow speed motor, a plastic plate (pedal) connected to the motor with a silicone tube. Put hot water in the beaker and leave it one to two overnights to get rid of remaining chemicals for manufacturing when you use it for the first time. The motor and the pedal are to make constant water current, which is necessary to keep medusae swimming all the time and critical for them to alive. It is recommended to use low-voltage motor (12 or 24 volts) with gears to reduce the rotation speed to 5~6 rpm. Motor should have power cable and appropriate (hopefully water-resistant) connector to power supply. Each tank should have fixed volume of sea water to check evaporation easily. For example, 3.5 liter of sea water is put into 5 liter tank in Villefranche laboratory. Put motors on top of the beaker and connect pedals to motor so that pedals mix sea water slowly and continuously. Adjust the hight of the pedal by changing the length of silicone tube. We normally adjust it to come to near the surface of sea water.
Configuration for Jellyfish (Medusae)Only and most importent point for jellyfish is the density. You can grow a few to several hundreds of baby medusae (up to a few mm in diameter) in a tank. For bigger baby jellyfish (4~5 mm) the number drops to 100~200. 80/tank is maximum for young (but immature) jellyfishes with diameter up to 10 mm. For adult jellyfish, it is highly recommended not to exceed 40 in a tank and 30/tank is ideal. This number is determined so that all jellyfish can catch food without making the sea water dirty at least 3~4 days. Jellyfish growth seems to be affected directly by the density. This is because jellyfishes don't grow even we feed enough and change sea water often, if there are too many in a tank.To transfere meduse to the tank, use a glass pipette for large medusae or plastic pipette (similar to pastour pipette) for smaller medusae. Medusae - especially adult - are fragile and it is not recommeded to add water to a beaker which contains medusae already, despite they are strong enough to resist the pipetting.
Configuration for Colony
Transplanting a colony to a new beakerColony is sessile form of Clytia and normally stay on solid materials such as inner wall of the glass beakers. You can easily split colonies as a cut of polyp, which then attach to its new habitat. Cut existing polyps at the bottom of the vertical stolon using scissors and wash in a glass dish by pipetting to get rid of algae and protozoans as much as possible. Add 500~1000 ml of sea water in a new beaker and put 5~10 polyps (numbers depend on strain) in it. Try to disperse polyps in the tank. Then leave the tank at least 1 nights (for healty strain) or up to 3 days (weak strain like Z4C2). Don't touch the beaker while you wait them to attach to the glass.
Once They attached...Once they attach to the beaker, set up temporary culture system. The polyp cuts attach very weakly to the glass therefore there is no need to add sea water. Set up the motor and pedal so that only several millimeter of the pedal is immersed to sea water. Feed on them artemia. Try to reduce artemia as little as possible because the sea water can't be changed when the polyp is newly attached. It is also recommended to flash artemia directly nearby the polyp using a Pasteur pipette. Polyp will be stably adhered to the glass within one week. Then you can wash or change sea water like as the existing polyp colonies.
We collect baby medusae by leaving colonies in this tank up to 2 nights. This is because...
- Polyps eat quicker than baby medusae. If there are babies and polyps in the same small tanks, babies can never eat their first food.
- Leftover of the food (dead artemia) will stay inside and attach to the polyps, which accelerate algae growth.
- Simply set up another nursery tank and transfer polyps to it.
- Take out all babies using a pasteur pipette and grow them on a dish at least for a few days. Clean the nursery tank and re-install the sea water.
- Connect the aeration tube.
- Fill the tank with sea water to 90~95%.
- Install colony plates
- Fill the tank to 100% (to maximize the water circulation).
- Adjust the aeration speed.
- 2 Days later, take out the polyp plates and fill up with sea water
- (first 3~4 days or for baby medusae up to 2~3 mm): Relatively strong. Some bubbles will travel 10~15 cm on the surface before they disappear. See right tank of the photo above.
- (Until 7~9 days or for baby medusae up to 3~ 4 mm): Relatively weak. Bubbles do not travel and disappear at the edge of the tank. See left tank of the photo above.
The speed has to be strong enough not to let the baby medusae staying on the bottom but not too strong to avoid the damage. Usually choose minimum speed enough to rotate baby medusae. We normally keep baby medusae up to 3~4 mm in diameter in this tank. Once babies are grown to this size, any speed of bubbles will be harmful. It is then necessary to transfer them to Kreisel tank.
Nursery tank (dram) model 2010
|Dimenstion||φ250 (240) x 80|
|Target of culture||Collecting baby medusae and grow-out (up to 3~4 mm)Nursely for very young medusae|
Villefranche culture system
Kreisel tank for polyp
- Eliminate chemicals attached to the tank by soaking it in sea water for a few days before the first use.
- Place the tank in the position. Water outlet pipe is to be inserteda hole on exhaust water collection pipe.
- Place T-shape nozzle near the cannel between main- and sub-chamber.
- Connect the nozzle to water-supply and open the valve.
- Adjust the water speed to 5~10 ml/sec.
- Leave water running for a while until dusts etc are all gone.
- Place glass plates in the tank using plate holders.
Kreisel tank for jellyfish
- Equilibrate the inner surface of the tank for at least 3 weeks leaving it in the running sea water or by using it for polyp colony culture before using it for medusa culture. This is particularly important to use any plexiglass tank for Clytia medusa culture. Jellyfish is quickly damaged (within several hours) in a new tank. It is most likely because of physical damages by the solid surface of the tank such as hydrophobicity of the material or residual chemicals attached. The toxic surface is neutralized by the 3 weeks of wash.
- Attach filter holder with 1 mm nylon mesh and hold with clips. Confirm the mesh is not wrinkled when it is wet.
- Place T-shape nozzle near the cannel between main- and sub-chamber.
- Adjust the position of the T-nozzle position. Nozzle opening should be about 3~6 mm below the level. Nozzle should be as close as the filter holder.
- Connect the nozzle to water-supply and open the valve.
- Adjust the water speed to 2~3 ml/sec. Speed can be checked either by seeing the water speed coming out from the nozzle or by confirming that jellyfish passing by the nozzle is not contracting.
- Add jellyfish (up to 35 medusae/tank)
Kreisel-tank model 09
|Dimenstion||100 mm width. 200 mm x 200 mm for the tank "diameter"|
|Filter||1 mm nylon mesh|
|Target of culture||Medusae (3~4 mm or larger) Polyps (several or more gastrozoids)|
|Weight||Over 2 kg (7 kg with water)|
|Capacity||3.7 liter (main chamber) + 1 liter (sub chamber) = 4.7 liter (overall)|
|Note||2 to 3 weeks of water circulation is required before use for medusae culture. It is most likely because of the surface of the material is hydrophobic and medusae will be damaged. 2 to 3 days will be enough for polyps.|
|Future modification||8 mm thick plexiglass is unnecessarily thick and heavy. Change to 5 mm thick plexiglass.Sub-chamber can be smaller. The bottom of the sub-chamber will be just below the window between main and sub camber.|
Colony plate suspender
Large plateGlass plate: 140 mm x 160 mm x 3 mm
Small plateDouble size glass slide: 50 mm x 76 mm glass slide holderG
Making transplants of Clytia colonies
When you make transplants?
- Colonies are large enough and can afford to take several polyps.
- To change substrate for the colonies, especially to switch to a larger plates etc.
- There is no backup colonies for the strain.
- A few weeks before sending off the strain.
1. Confirm the polyps are well enoughEfficiency will be lower if the polyps are starved. Feed enough to make them good in shape. Polyps should be big enough and catch artemia without helps.
2. Cut out polyps with a fine scissorIsolate individual polyps by cutting stem of polyps at proximity of stolon (bottom of the stem). Some part of stolons in a colonies may not be attached to the substrate and growing vertically. In this case you can easily cut the stolons by scissors without damaging. Isolate a short piece of stolons with 2~3 well formed polyps.
3. WashFlush polyps with sea water together using a pipette (opening 3~4 mm) to get rid of algae as much as possible.
4. TransplantPlace isolated polyps on the target substrate in sea water and leave them quiet for 1~3 nights. Several pieces will be necessary for a large substrate (100 square-centimeter or more) or a few of them for a glass slide. Time required to attaching polyps depends on the strain. Well-growing Z4B polyps will attach in a night while Z4C2 polyps usually takes a few days. Confirm attachment by gently blowing sea water over the transplanted polyps.
5. Start normal cultureSlowly add sea water to the beaker (in case of beaker culture) or transfer the substrate to a culture tank. The attached polyps will extend stolons in good conditions of nutrition. Feeding therefore is very important for new transplants to let them attach stably. Feed artemia as soon as possible if the polyps are still capable to eat. Often the they degenerate polyps to give priority in extending stolons then reform a new polyps later on. Reformed polyps are usually smaller and the timing is unpredictable. Check such new polyp formation every day and feed very carefully. Bisected artemia (tail half) is good for small polyps. The new transplant can be treated as normal colony once they make 2nd or 3rd polyps in it.
Collecting eggs/sperms and fertilization
Transferring jellyfish into dish
Ovulation of eggs occurs 2 hours (110~135 minutes) after the light is turned on, after minimum 3 hours of dark. Sperm release may occur a bit earlier and mechanical shock may induce sperm release earlier. Transfer jellyfish to 10 cm dish to collect eggs and sperms.
Sperm: transfer male jellyfish (5~10) into a deep 10 cm dish with 200 ~ 300 ml sea water within 1 hour after the light is on.
Eggs: transfer female jellyfish (max 30/dish) just before the ovulation. (90~100 min after the light is on.) This is not to leave jellyfishes for long time in a dish at high density.
- Max 30/dish for 1 hour
- Max 12/dish up to several hours
- 5~8/dish for overnight incubation in a dish
Collecting eggs (for microinjection purpose)
For microinjection, you may transfer eggs to smaller dish (2 cm ~ 3 cm diameter) using a mouse pipette or a pasteur pipette. This step can be done before or after transferring jellyfish back to culture tank. Please remember that rate of irregular development increases if eggs are fertilized > 60~70 minutes after spawning. It is therefore often necessary to collect eggs as quick as possible if microinjection (or other manipulation before the fertilization) is planned. Procedure (a) is recommended for this purpose. It is also used to collect as many eggs in the dish as possible. Procedure (b) is easier if you don't do microinjection and if you don't need to collect all eggs in the dish. Procedure (c) is mixture of (a) and (b); begin with procedure (a) first. Then once you get enough eggs to inject in 15~20 minutes, switch to strategy (b) and go for injection while you are waiting to concentrate eggs on the shaker.
Collecting eggs during ovulation (jellyfishes are still in the dish).
This can be done to collect as many eggs as possible or as quicker as possible in order to microinject into a lot of eggs. Jellyfish swimming keep eggs suspended in sea water, making egg collection a bit painful. Start egg collection under binocular as soon as you see the first several eggs released into the dish. Normally we use mouth pipette to collect if allowed. Try not to pick jellyfish with the pipette.
Collecting eggs after taking out jellyfish from the dish back to the culture tank.
Once ovulation is over (or there is enough eggs released into sea water), you can take out jellyfish from the dish to culture tank. Then collect eggs into other dishes quietly. If there is no manipulation/microinjection is necessary, you can fertilize eggs in this dish (without collection eggs) by adding male sea water (see step (3).) In this case, you can collect fertilized eggs/embryos after confirming the first cleavage.
Mixture of a) and b): for a lot of injection (600 or higher)
Use (a) approach to collect eggs just enough for microinjection in the next 15~20 minutes (3~400 if you are experienced). Then switch to (b) approach, meanwhile you perform the first series of injections . When you finish the injection to these eggs (15~20 minutes later), there are concentrated eggs in the center of the dish ready to transfer to the injection chamber, to continue microinjection. The point of this mixed approach is to save time to collect eggs to spend more time to do microinjection.
You can concentrate eggs at the center of the dish by leave the dish on a rotary shaker for 10~15 minutes. The speed of rotation to efficiency concentrate eggs depends on dishes and volume of sea water. The concentrated eggs are ready to transfer into an injection chamber.
Fertilization has to be done with in 60~70 minutes after spawning (3 hours ~ 3 hours 10 minutes after the light ON.) Rate of cleavage error goes higher if you wait longer.)
Take out male jellyfish from dish and quantify approximately the amount of sperm with dark-field illumination with the dissecting microscope. Take sea water (male seawater) from the bottom of the dish and put it into egg dishes. Use plastic transfer pipette for cell culture.
Amount of sperm is OK when you estimate 20~40 spermatozoids are swimming around an egg. Amount of sperm will be dependent on the number of eggs, size of the dish etc. Usually 3~5 ml of sea water taken from the bottom of 10 cm dish with 10~12 male jellyfish is optimal for 300~400 eggs in 3 cm dish. Too much spermatozoid results in polyspermea and cleavage error. (Eggs from old jellyfish (= 3 weeks after maturation or older) tend to have polyspermea.)
If you simply need to get fertilized eggsPut 10 female + 1~2 male in a 10 cm dish and leave it on the shaker. This approach is also useful if you need to fertilize midnight or early in the morning using timer-controlled lighting.
- Sort out healthily dividing embryos at 4- or 8-cell stage. Cleavage error occurs to some embryos, especially eggs from older embryos.
- Transfer embryos to plastic dishes coated with 2% agarose/MFSW (see materials). 300~400 embryos/3.5 cm dish will will be maximum.
Add 1/2000~1/4000 volume of PS (penicillin/streptomycin SIGMA P0781, 10,000 unit/ml for penicillin and 10 mg/ml streptomycin) to sea water if your culture more than 24 hours. It is to avoid bacterial growth, which induces metamorphosis. Changing sea water once in the second day may help to prevent bacterial contamination.
- Incubate at 18~20°C. Agitation is not important. 16~20°C is useful to slow down development (blastula~early gastrula).
- 3~4 days old planula larvae
- GLWamideII (GNPPGLW-NH2) 1 mg/ml
- Normal (1 x 3 inch) or wide (2 x 3 inch) glass slides, properly marked with diamond pen (not a marker pen).
- 10 cm petri dish
- 1 ml syringe with 25G needle (to smash artemia)
- Add x1/1000 GLWamide II (final 1 ng/µl) in MFSW (prepare freshly).
- Prepare stable and non-disturbing bench/desk. Use a black plastic bag if the surface is not a dark color. It facilitates to see planulae.
- Put a glass slide into a plastic dish and place on the stable place. (Keep separate the slide from the wall of the dish.)
- Quietly pour 2~3 ml of GLWamideII-MFSW for a normal slide (4~5 ml for double-width slide) using a transfer pipette.
- Collect planulae with mouth pipette and put on the glass slides. Planulae will stop swimming immediately (5~10 seconds) and will undergo metamorphosis.
- Leave over a night.
- Next morning, transfer slides to a culture tank. Primry polyp is safe out of sea water for short time (5~10 seconds) for transferring from the petri dish to the tank.
- Some primary polyps start to eat 1~2 days after metamorphosis. Feed smashed artemia, passed through the 25G needle 2~3 times, to primary polyps for several days. Normal feeding will be OK once they form bigger polyps.
Important note: Primary polyp is smaller than normal polyp. They often degrade the polyp body after eating for up to a few times. This is because the primary polyp cannot simply change its body size and a colony makes bigger polyp through degrade-reform process. Reforming new (and bigger) polyp however fails quite often. Only small population of primary polyps, for example 10%, will grow to colonies more than 2 polyps.
Trouble shooting for polyp stage
Polyp doesn't grow
- Too little food.
- Check amount of food. Confirm most of gastrozoid (feeding polyp) catched at least one Artemia
- Too many jellyfish cohabit in polyp tank.
- Check number of medusae cohabit in the same tank. Usually jellyfish catch Artamia quicker than polyp and there might be not enough Artemia left for polyp even you put enough food.
- Algae contaminated
- Clean glass surface where polyp is living.
- Sometimes Cutting stolon may help to induce its growth, especially by removing most heavily contaminated area of the polyp, making new "growth cones".
- Sea water got dirty
- Colony doesn't grow well when the sea water has not been changed for more than 4~5 weeks.
- Changing at least half sea water is required for each month.
- Amount of food was diminished after too much feeding
- Too much food often rises gonozoid/gastrozoid ratio. In normal condition it should be 1 but it goes up to 5 or 10 depending amount of food because one gastrozoid can get enough nutrition for massive medusa production. If the amount of food suddenly drops in this stage, gastrozoid wouldn't get enough food for medusae production and would sacrifice polyp growth. (Medusa bud passed "critical point" can't stop the development, while younger medusa before the point can stop and degenerate. Volker Schmid's unpublished observation in Podocoryne.)
Polyp suddenly died
- This doesn't happen so often but most likely you have been feeding too young Artemia for several days.
- Young Artemia contains high amount of lipids undigestible for Clytia.
Troubleshooting for jellyfish
Medusa (jellyfish) diesWhy my Clytia jellyfish dies even they are not so old ( up to 4 weeks)?
- Bacterial, protozoan predator is contaminated in the culture.
- Sea water is quality is low
- Salinity is not in the right range (35~36‰)
- Ammonium/NO2/NO3 increased
- Change sea water more often
- Natural sea water is polluted
- Use artificial sea water
- Too little food
- Too much food
- Sea water quality might have been bad by overfeeding
Medusa doesn't grow
- Not enough food
- Too high jellyfish density
- Reduce number of jellyfish. For appropriate range of jellyfish density, see here.
- Manubrium (oral part) is degenerated and can't eat
- This happens sometimes. There is no way to recover. Discard and start new culture.
Medusa becomes flat rather than "hemispherical"
- Check sea water quality
- Most likely salinity is too low
- You can recover them relatively easily if you react quickly.
Medusa becomes like a ball
- Check sea water quality
- Most likely salinity is too high
- You can hardly recover jellyfish when they became a ball. Discard and start new culture.
Medusa float on the surface of sea water
- Salinity suddenly went higher, most likely after sea water changes.
- There is no problem as long as the salinity is within the range.
- Jellyfish can adapt quickly (5~10 min) depending on the difference.
- To avoid surface tension kills jellyfish, it's better to mix with shakers or glass pipettes etc until they adapt to new sea water.
Trouble shooting for ovulation fertilization and development
Female medusae don't spawn eggs
- Medusae too young
- Check gonads and confirm that they have oocytes
- Light/Dark cycle is not controlled properly
- Minimum 3 hours of dark environment for 24 hours/cycle
- Short illumination (< 1 min.) doesn't induce ovulation
- Oocytes may not be mature if you change the timer setting earlier. (jet lag)
- Jellyfish will adapt to the new cycle in one day.
- Not enough food
Female medusae spawns. But the ovulation takes too long (more than 30 min)Normally ovulation takes 10~15 min.
- Salinity is low. Check it in the right range.
Egg size is small
- Not enough food. Amount of feeding may have dropped in the last 1~2 days.
- Medusae are too young.
Too few eggsNormally one medusa spawns at least several eggs (100~200 from 30 medusae). You can expect 10~20 eggs/medusa.
- Medusae are too young
- Medusae are too old. Medusae that have spawned for more than 2 month might be replaced by younger medusae.
- Not enough food. Amount of feeding may have dropped in the last several days.
Eggs don't get fertilized (no or few eggs start cleavage)In any case, check the amount of sperm under binocular with dark-field illumination. See below depending on the results.
- In the dish of male jellyfish before mixing with eggs. (BI)
- After eggs are placed in the male sea water / sea water from male jellyfish dish is poured over eggs. (AI)
There is no spermatozoid (AI)
- Male medusae are too young or too old.
- Check male gonad under microscope. They usually have two layers: inner transparent layer and outer opaque layer. The outer layer will be disappeared or diminished when they get too old. The outer layer is a sign that males are actively producing spermatozoids.
There are a bit of sparmatozoids (AI, BI) or little spermatozoid (BI)
- The cause of this problem is the same as for the previous question
- Increasing number of male medusae may simiply resolve this problems.
- If you are adding sea water from male dish to a dish with eggs. (Sperm-to-Egg method)
- Do the other way around: transfer eggs into a dish with male medusae. (Egg-to-Sperm method)
- If you don't need to control the timing of fertilization
- Put both male and female in a dish from the beginning.
- See also next question
There are plenty of spermatozoid but egg don't get fertilized
- The quality of eggs is bad
- Confirm the salinity is in the right range. (This may affect to the quality of eggs.)
- Change sea water.
- If the treatment above don't help
- Change to new female if they are old.
- Male jellyfish are old (The quality of the spermatozoid may not be good.)
- Change to new males