Assorted tank sizes in the X. tropicalis room.


Aquanode, UV and drum filter controllers. The self-cleaning drum filter is housed inside large white fiberglass container on the left. Particulate and carbon filter units are shown in the foreground.

X. tropicalis room reservoir. The large heating element is seen on the bottom of the reservoir in the foreground. The biological filter is inside the center section. Pumps keep the biological filter material in constant circulation.


Reverse Osmosis (RO) system in the X. tropicalis room. The plastic RO water holding tank is on the right.


More racks of tanks in the X. tropicalis room.

HUSBANDRY

Fertilization
We, and others, have noticed a marked difference in the viability of X. tropicalis sperm compared to X. laevis sperm. This can result in very poor fertilization rates in vitro. This is helped by using bovine serum albumin (BSA; 0.1% w/v) in all solutions and rinse glassware and plasticware with BSA containing solutions immediately prior to use. Not all batches of BSA are suitable for embryo handling - a fact we discovered in our early attempts at nuclear transfer. We recommend testing new lot numbers for toxicity prior to routine use in the lab. We now use L15 media plus 10% calf serum (instead of MMR or MBS) for homogenization of testes. Fertilization is performed in 0.1x MMR and the eggs are dejellied with 2% cysteine in 0.1x MMR pH 8.0. Use X. tropicalis testes as soon as possible after harvesting. The viability of sperm drops at a remarkable rate. If you keep these things in mind, fertilization rates are as good as in X. laevis.

Note: Thanks to the Amaya, Grainger and Harland labs for tips on in vitro fertilizations of X. tropicalis.

Tadpole Husbandry:
Our current method for raising tadpoles, both X. laevis and X. tropicalis, is to allow embryos to grow in static tanks until metamorphosis. As soon as the embryos reach froglet stage they are transferred our flow-through re-circulating system. Tadpoles are raised in plastic tanks in X. tropicalis system water (~500 uS marine salts, neutral pH). The tadpoles are raised in a separate nursery room kept at 24oC and on a 12/12 light/dark cycle. Each tank is aerated with a small airstone at a slow rate; enough to break the surface of the water but not high enough to generate a significant water current in the tank. We feed tadpoles at least three times a day, seven days/week, with a suspension of Sera Micron. As we have a large number of tanks, we make up a fresh suspension in a lab squeeze bottle to facilitate the speed of feeding. We add enough food to slightly cloud the water - the amount varies with the density and age of tadpoles. A good rule of thumb is that the tadpoles should clear the water in less than 20-30 minutes. We prepare the food fresh prior to each feeding using sterile X. tropicalis system water. Any left over food is disposed of and is not saved for the next feeding. Once a week we add a few drops of Fishtamins (vitamins for fish - available at pet stores) to one regular feeding. We perform a 50% water change once a week with fresh sterile system water. The tanks will grow some algae. We do not clean the tanks as the algae keeps the ammonia levels in check, and the larger tadpoles seem to eat it.

Froglet Husbandry:
Juvenile frogs are transferred to the regular system as soon as possible after, or during, metamorphosis. We feed baby frogs NASCO frog brittle in the smallest pellet form. For very young frogs we break up the small pellets using a short burst in a coffee grinder; just enough to break each small pellet into three or four pieces. Juvenile frogs are fed three or more times a day for maximum growth rate. Enough food is added so that the frogs will clear the tank in 5-10 minutes. The young froglets grow at a remarkable rate and one must be careful to sort frog according to size as the larger froglets will attempt to eat the smaller ones.

Adult Frogs:
Adult frogs are feed twice a week (Tuesdays and Fridays) with NASCO frog brittle. For Nasco Frog Brittle ingredients see http://www.nascofa.com/prod/Static?page=xen_brittle&seqid=1.
Females are primed in the evening prior to egg harvest and placed in a large static container filled with fresh system water. After egg harvest, the females are placed in a static tank again overnight before being reintroduced to the system. This allows the frogs to shed all their eggs before reentering the system. This practice helps to decrease the load on the mechanical and biofiltration systems.

Frog Identification:
We currently identify founder frogs by housing in separate 2-5 gallon tanks.

MeadLab Aquarium
The frog systems were designed, built and maintained by Marine Biotech, MA (see links). The frogs are housed in two 400 sq. ft. rooms in separate aquaria, one for X. laevis and one for X. tropicalis. A 200 sq. ft. preparation/microscope room also serves as the tadpole nursery. All system parameters are constantly measured and adjusted by a central computer (Aquanode) in each room. The automatic controller performs all system functions including maintenance of system parameters and daily water changes. Each system room has it own reverse osmosis (RO) water purification system that feeds a 220 gal holding tank. Each day we perform two 5% water changes (10% water change per day). Each system has an in-line, self-cleaning, 20 um drum filter, a 1,000 gal reservoir that houses the bio-filtration media, a 20 um particulate standing filter, an activated carbon filter and two serial 100 watt UV sterilizers. The Aquanodes constantly monitor the following water conditions; temperature, pH, salinity, dissolved oxygen (DO), oxidative reduction potential (ORP) and system water pressure. The Aquanodes adjust the temperature, pH and salinity by dosing from reservoirs and by turning off or on in-line heaters and chillers.