Animal Maintenance Systems: Xenopus tropicalis
- ↵2Correspondence: mhorb{at}mbl.edu
Abstract
Modular recirculating animal aquaculture systems incorporate UV sterilization and biological, mechanical, and activated carbon filtration, creating a nearly self-contained stable housing environment for Xenopus tropicalis. Nonetheless, minimal water exchange is necessary to mitigate accumulation of metabolic waste, and regular weekly, monthly, and yearly maintenance is needed to ensure accurate and efficient operation. This protocol describes the methods for establishing a new recirculating system and the necessary maintenance, as well as water quality parameters, required for keeping Xenopus tropicalis.
MATERIALS
Reagents
Ammonia Freshwater and Saltwater Test Kit (API, Chalfont, PA)
Bleach (6.0% NaClO)
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Dilute with Type II water to make a 10% bleach solution (final concentration of NaClO is 0.6%).
Dechlorinator (Na2S2O3; ProLine brand Pentair AES, Apopka, FL)
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This contains both the anhydrous (CAS# 7772-98-7) and pentahydrate salt (CAS# 10102-17-7) molecules. 1.6–2.6 ppm of Na2S2O3 per 1 ppm of chlorine is typically sufficient to dechlorinate water.
Ethanol (190 proof)
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Dilute to 70% with Type II water.
Nitrate NO3 Freshwater and Saltwater Test Kit (API, Chalfont, PA)
Nitrifying bacteria (ProLine brand, Pentair AES, Apopka, FL)
Nitrite NO2 Freshwater and Saltwater Test Kit (API, Chalfont, PA)
Reef salt (Seachem Laboratories; Madison, GA)
Sodium bicarbonate (NaHCO3; ProLine brand, Pentair AES, Apopka, FL)
Type II water (ASTM International 2018)
Virkon Aquatic (DuPont, Wilmington, DE)
Xenopus tropicalis frogs
Equipment
Air Pump, 40 LPM (Alita Industries, Inc., Baldwin Park, CA)
Orion Star A211 pH Benchtop Meter (Thermo Scientific, Beverly, MA)
PINPOINT Water Hardness Meter (American Marine Inc., Ridgefield, CT)
Recirculating Aquatic System (Iwaki Aquatic Systems and Services, Holliston, MA)
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There are several companies that sell recirculating aquatic systems, including Aquaneering, Tecniplast, and Aquatic Enterprises.
Scrub pads
METHOD
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Each section below should be treated independently; these should not be viewed as consecutive procedures as the numbering may imply.
Establishing a New Recirculating System
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1. Starting with a new or sterilized system, rinse the biomedia (provided by the system manufacturer) with Type II water and place in the biofilter.
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2. Disable the water effluent exchange and UV sterilization, and keep the carbon filter empty.
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It is necessary to initially limit filtration and sterilization functions and allow nitrogenous waste products to build up sufficiently to support healthy growth of the biomedia.
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3. Allow flow from a single tank on the end of each rack and add 5–10 frogs to the system.
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Larger systems require more frogs. This number of frogs is suggested for a system with ∼500-L capacity.
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4. After 24 h, disable the water flow but keep the biomedia aerated using an external air pump.
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5. Add nitrifying bacteria to the biofilter.
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Larger systems require more nitrifying bacteria. Follow the instructions outlined by ProLine.
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6. Wait 1–2 h before restarting the water flow.
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During this brief period, the frogs present in the system should be kept in their tanks. The absence of water flow should not be detrimental to their health.
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7. Perform daily NH3/NH4++, NO2−−, and NO3−− level measurements using the relevant test kits.
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Wait for NH3/NH4++ levels to measure at least 1–2 ppm.
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8. Initiate UV sterilization and water exchange, and add carbon to the appropriate filter housing. The levels of NO2−− and NO3−− should begin to increase.
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Flow to 1–2 additional tanks can be started. Additional tanks can be filled as needed.
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9. Allow for NH3/NH4++ and NO2−− levels to reduce to 0.5 ppm before adding more frogs to the system.
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The entire process can take 2 wk to 2 mo. Nitrifying bacteria can thrive when water parameters are consistent. If dosing reservoirs are not present, sodium bicarbonate can be used to stabilize pH. Once NH3/NH4++ and NO2−− levels reach the desired level, gradually add frogs to the system while monitoring water quality parameters. Care should be taken to not open flow to too many tanks at once as this can drain the sump below the set safe level and result in an automatic system shutdown.
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Daily Water Assessment and Maintenance Required for Proper Operation
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10. Record system readings. See Table 1 for age-appropriate guidelines for water temperature, pH, conductivity, and frog density.
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This allows the user to deduce trends and identify potential issues that may be occurring.
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11. Inspect the UV bulb.
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Check system display to confirm the UV bulb is on and functioning correctly. Replace the bulb if necessary.
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12. Regularly scrape internal sides of the tanks with scrub pads to remove any algae buildup.
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Algal buildup will vary depending on several factors including lighting and dissolved nutrients. Excessive buildup should be removed as needed.
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13. For systems that use stand pipes with overflows, shake the stand pipes to clear them. Tanks with only an overflow bulkhead will require daily active removal of detritus to keep them clean.
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14. Exchange 10% of the system water.
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Modern recirculating aquatic systems can be programmed to perform water exchange automatically. Make sure that the automatic setting is kept at 10% daily exchange.
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15. Measure pH and conductivity dosing reservoirs daily.
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Use sodium bicarbonate to buffer and regulate pH and sea salt to control conductivity.
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16. Observe carbon and mechanical filters for buildup of waste and replace as needed once passage of water through them is significantly impeded.
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17. Observe individual tanks for accumulation of waste and film on the bottom and sides, and sanitize tanks that are particularly dirty.
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To clean tanks, scrub and rinse with Type II water, spray with 10% bleach and let sit for 1 h, rinse again with Type II water, spray with 70% ethanol and let sit for 1 h, rinse once more with Type II water, and allow to air dry.
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Water-quality parameters for Xenopus tropicalis
Weekly Water Assessment and Maintenance Required for Proper Operation
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18. Assess NH3/NH4++, NO2−−, and NO3−− levels with the relevant API test.
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Acceptable measurements are as follows: NH3/NH4++ = 0 ppm (0–0.5 ppm), NO2−− <1 ppm, NO3−− <40 ppm, alkalinity >40 ppm. If levels are out of range, perform a 25% water exchange. Repeat water change each day until values reach acceptable levels.
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19. Test pH and conductivity with a reliable external probe as a reference for internal system probes.
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If the external and internal probes are not in agreement, clean the system probe and check that it is correctly reading calibration solutions. Recalibrate the probe if necessary.
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20. Measure temperature with an external thermometer.
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Heaters and chillers are used to keep the temperature stable. Measuring with an external thermometer confirms that the system is calibrated to correctly engage heaters and chillers as necessary to keep the water temperature stable. Recalibrate or replace the system thermometer if it is not in agreement with the external thermometer.
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Annual Water Assessment and Maintenance Required for Proper Operation
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21. Replace UV bulbs and check to ensure that the quartz sleeve is intact.
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22. Replace pH electrodes.
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23. Remove and clean return pipes.
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24. Replace all rubber tubing.
System Sterilization
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System sterilization is necessary to restore full operational capabilities following an outbreak of disease.
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25. Disable all system components including the water pump, biomedia agitators, and probes.
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26. Discard biomedia or sterilize in a 10% bleach solution.
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27. Scrub the inside of the sump and associated parts and remove all detritus.
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28. Remove and dispose of all filter pads, cartridges, and carbon.
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29. Disassemble and scrub all the tanks and accessories.
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30. Rinse the tanks with water, coat with 10% bleach, and let stand for 1 h.
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31. Rinse the tanks with water, coat with 70% ethanol, and let stand for 1 h.
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32. Rinse the tanks with Type II water and let air dry.
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33. Soak accessories in 10% bleach for 1 h.
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34. Soak accessories in 10 g/L solution of Na2S2O3 in Type II water for 1 h.
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35. Soak accessories in 70% ethanol for 10 min, rinse, and air-dry.
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36. Drain 75% of the sump volume according to the manufacturer's instructions.
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37. Reassemble the system, putting back all the tanks, filter housings, and new rubber tubing. Leave the filter housing empty, and do not add any carbon to the system.
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38. Start the system.
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39. Add bleach to the system.
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Ensure the final concentration of NaClO in the system is 0.06%.
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40. Reduce the effluent rate to its lowest setting.
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Do not reduce the daily water exchange rate to <1%.
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41. Wait 2–3 d, then increase the daily water exchange rate to ∼50%.
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This will begin removing the bleach from the system.
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42. Begin testing the pH of the system after a week.
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43. Add Virkon to the system once the system water pH matches the influent water pH.
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The concentration of Virkon should be 3.2 g per 1 m2 of estimated internal surface area of the entire system.
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44. Reduce the effluent rate back to its lowest setting.
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Virkon can cause foam accumulation and in some cases, may cause the system to overflow. To help mitigate this, limit agitation of the water and spray 70% ethanol directly on the foam to help disperse bubbles.
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45. Wait 7 d then increase the daily water exchange rate to ∼50%.
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This will wash the Virkon out of the system.
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46. Allow the system to run for 2–3 d, replace all rubber tubing, and follow the protocol for establishing a new recirculating system described above to stabilize the system and allow for housing animals.
Discussion
Optimal eggs and embryos are produced by healthy laboratory population of Xenopus tropicalis. Regular monitoring of water-quality parameters, as well as daily monitoring and visual inspection of the animals, is crucial for prevention and early identification of developing conditions that may be detrimental to overall colony health. The use of tap water is discouraged, and starting with a good water source providing Type II water will aid in maintenance of optimal water parameters as well as limit the chances of introducing pathogens and toxic contaminants. Municipal tap water may also be basic and thus may add the additional complication of needing to use acid buffers to aid in stabilizing the pH. The stability of the parameters will also be affected by the animal maintenance system configuration, with recirculating systems being more cost effective and efficient than flowthrough systems and also requiring much less maintenance than static systems. Furthermore, animal health is best when the accumulation of nitrogenous waste is low, thus the initial effort made to establish a stable biofilter in a recirculating system is absolutely necessary. The Nitrosomonas and Nitrobacter bacteria in the biofilter respectively oxidize the toxic ammonia into toxic nitrite and then the nitrite into nontoxic nitrate (Hem et al. 1994). Following establishment of a stable ammonia/nitrogen cycle, regular maintenance and tests of water parameter levels will aid in maintaining the animals in good health (McNamara et al. 2018). Nonetheless, sterilization of the entire system may be necessary if addition of new frogs to the system also introduces pathogens that negatively affect their quality of life. Once sterilized, the system can be put back into operation by again following the steps for establishing a functional and stable biofilter.
