TITLE: Housing and Care for Hymenochirus boettgeri
AUTHORS: Tamilie Carvalho, Catherine Si, Rebecca A. Clemons, Evelyn Faust, Timothy Y. James

[DESCRIPTION]
This protocol provides an overview of housing and care procedures for H. boettgeri in a controlled laboratory environment, encompassing feeding at all life stages and the maintenance of water quality in both a closed recirculating system (standard aquarium) and a static closed system (used for individual housing). Housing can also be done in a semi-open recirculation system (ZebTec, Tecniplast) that is compatible with zebrafish.

[GUIDELINES]
Conclusion

This protocol offers comprehensive guidance on housing and caring for H. boettgeri in a laboratory environment, including instructions on aquatic systems, environmental conditions, feeding, and water quality maintenance. The equipment used in this protocol can be updated as needed, as long as the basic requirements for animal safety are maintained. Adhering to these procedures ensures the well-being and safety of the frogs throughout various experiments and research studies.

[STEPS]
SECTION: Housing
1. Juvenile or adult frogs
SECTION: Housing
1.1. Animals can be kept in groups or individually as needed. House animal groups in 38 L glass tanks (standard system; Figure 1A) with a density of at least 1.5 L of water per individual. House animals individually in 1 L plastic tanks (static system; Figure 1B).  
SECTION: Housing
1.2. Keep the tanks covered to prevent animal escapes.
SECTION: Housing
1.3. Maintain a minimum 1 cm space between the lid and the water surface for frogs to access the surface for breathing.
SECTION: Housing
1.4. Enrich standard tanks with 1 cm layer of gravel substrate, artificial plants, and PVC pipes. Add at least gravel substrate to static tanks.
SECTION: Housing
1.5. Sterilize all enrichment items with 1% Virkon for 20 min and rinse thoroughly with sterile water.

SECTION: Housing
1.6. Ensure all enrichments are free from rough surfaces or sharp edges.
SECTION: Housing
1.7. Maintain a 13:11 light-dark cycle.
SECTION: Housing
2. Eggs and tadpoles
SECTION: Housing
2.1. Keep eggs and tadpoles inside net breeder tanks to prevent them from being preyed by larger animals or getting sucked into the filter.
SECTION: Housing
2.2. Place a Net Breeder tank inside the standard tank. Position the Net Breeder near the surface so that eggs and tadpoles can access the water's surface.
SECTION: Housing
2.3. Maintain up to 80 eggs or 30 tadpoles of similar age in each Net Breeder tank. This number can be updated if larger breeding tanks are used.
SECTION: Housing
2.4. Larger tadpoles may prey on smaller tadpoles or eggs, so relocate tadpoles by size to another tank if necessary.
SECTION: Water Conditions
3. Fill the tanks with RO water.
SECTION: Water Conditions
4. Adjust water conductivity to 900 ± 100 µS/cm by adding 0.5 1655 of formulated synthetic sea salt mixture.

SECTION: Water Conditions
5. Adjust pH levels to 7 ± 0.4 by adding sodium bicarbonate when necessary.
SECTION: Water Conditions
6. Maintain a constant water temperature, ranging from 20 °C to 25 °C.

SECTION: Water Conditions
7. Position the aquarium filter in the middle of the tank for effective water circulation.
SECTION: Water Conditions
8. Initiate the biological cycle by adding 1 ml/L of nitrifying bacterial suspension (ProLine® Aquaculture Bacteria Concentrate).

SECTION: Monitoring
9. Monitor pH and conductivity three times per week.
SECTION: Monitoring
10. Check nitrogenous compounds once a week in cycled tanks (i.e., established nitrifying bacteria in sufficient abundance to maintain ammonia and nitrite values near 0 and increase nitrate values).
SECTION: Monitoring
11. Check nitrogenous compounds at least three times a week in tanks with cycling in progress.
SECTION: Monitoring
12. Always check parameters before water changes.
SECTION: Water Changes
13. In cycled tanks, ammonia should be ≤ 0.25, nitrite should be 0, and nitrates will steadily increase, necessitating water changes.
SECTION: Water Changes
14. Perform a 50% water change in cycled tanks when nitrates exceed 40 ppm.
SECTION: Water Changes
15. In non-cycled tanks, perform a 50% to 70% water change when necessary (i.e., ammonia  1 ppm, nitrite  1 ppm, or nitrate  40 ppm).
SECTION: Water Changes
16. Condition water temperature, pH, and conductivity before water changes. If water changes coincide with feeding days, perform them at least 4 hours after feeding.
SECTION: Feeding
17. Juvenile and adult frogs
SECTION: Feeding
17.1. Feed the frogs ad libitum twice daily three times per week.
SECTION: Feeding
17.2. In the morning, feed with newly hatched brine shrimp.
SECTION: Feeding
17.3. At least 5 hours later, feed with frozen gamma-irradiated bloodworms, or small-granule fish food.
SECTION: Feeding
17.4. Remove uneaten food from the tank at the end of the day.
SECTION: Feeding
18. Tadpoles and newly metamorphosed frogs
SECTION: Feeding
18.1. Begin feeding tadpoles when they start free swimming (typically 1-2 days after hatching).
SECTION: Feeding
18.2. Provide ad libitum daily feedings of powdered fry food (Sera® Micron Nature) for one week.
SECTION: Feeding
18.3. Transition to a diet of newly hatched brine shrimp ad libitum twice daily, which should continue for at least 4 weeks post-metamorphosis. Afterward, switch to the same diet as juvenile and adult frogs.
SECTION: Feeding
18.4. Remove uneaten food from the tank at the end of the day.
SECTION: Health Monitoring
19. Continuously observe and monitor the health and behavior of H. boettgeri.
SECTION: Health Monitoring
20. Inspect all animals daily for signs of disease (e.g., lethargy, red spots, bloating, abnormal floating or swimming patterns, and lack of righting response).
SECTION: Health Monitoring
21. Isolate any sick animals from healthy ones.
SECTION: Health Monitoring
22. If euthanasia is necessary, immerse the animal in a solution of tricaine methanesulfonate (MS-222; 0.5 g/L; Syndel, Ferndale, WA) for at least 15 minutes. The time required in MS-222 solution may vary per animal.