axolotl husbandry - university of kentucky · axolotl newsletter number 25 6 axolotl larvae housing...

Fall 1996

1

Contents

Amphibian Maintenance Facilitiesat Sogang University, Korea..................... 3

Wonsun Kim

Axolotl Larvae Housing Methods ............. 6Cheryl A. Nugas

Corwin Lab Axolotl Protocols ................. 10Anne Hennig

Axolotl Housing and Care atSt. Mary’s College of Marylandand What is an Axo-condo?. .................. 13

Karen Crawford

Raising Axolotl Larvae ........................... 14Peggy Egar

Raising Axolotl Larvae ........................... 15Linda Muske

Northcutt Lab Larval RearingMethods: 1996 ................................ ...........15

R. Glenn Northcutt and Linda Barlow

Inexpensive and Low MaintenanceFacility for Housing Axolotls .................. 16

M. Moody and G. Thibaudeau

Housing and Feeding Larval Axolotls ..... 18Carl Schneider

Axolotl Care at IndianaUniversity Northwest ............................. 19

Timothy Stabler

Maintaining Larvae ................................ 19Katia Del Rio Tsonis

Simple Brine Shrimp Hatchery .............. 20Brent Mundy

Terrestrial Axolotl Care SheetAmbystoma mexicanum). .........................22

Brent Mundy

Compendium of

Axolotl

Husbandry

Methods

1997

Reprinted from the AxolotlNewsletter, Issue 25

Axolotl Newsletter Number 25

2

Fall 1996

3

Amphibian MaintenanceFacilities at SogangUniversity, Korea

Wonsun KimDepartment of Life Science

Sogang UniversityCPO box 1142

Seoul 100-611, Korea

*Department of AnatomyUniversity of Wisconsin

351 Bardeen, 1300 University AveMadison, WI 53706-1532, U. S. A.

*address effective until January, 1997

Maintaining a large number of axolotls canbe problematic due to the time needed formaintenance and space needed for containers.Here, I would like to briefly describe how wehave overcome some of these problems in ouraxolotl colony facility.

1. Water supply and treatment proceduresWe use charcoal-filtered tap water that has

been aerated overnight after filtration.

2. Container types and sizesA. For larvae up to 4 cm in size

We use custom-made compartmentalizedcages to prevent cannibalism amongsmall larvae.

B. For larvae larger than 4 cm in sizeAnimals are housed individually in roundplastic food storage containers. The sizeof the container is dependent on the sizeof the animal.

C. For adult animalsFor large stock animals, we use largeaquaria connected to a continuously cir-culating filtering system (Figure 2). Onefiltering unit is composed of eight aquaria(in two rows of four each on steel shelves)and one filter box. The aquaria are con-nected by pipes that lead to a commonfilter box, located beneath the shelf. Eachtank has a separate inlet valve whichcontrols inflow, and each row of tanks

has a master flow valve. The filtering sys-tem is designed to circulate waterthrough the filter box beneath theaquaria. Within the filter box is a microhabitat which consumes organic materi-als from food debris or excretion. Exceptfor while feeding and retrieving uneatenfood (about 4 - 5 hours a day), water iscontinuously circulated. The whole sys-tem requires complete cleaning only oncea year.

3. Food supplyYoung larvae (up to 1 month from hatching)

are fed freshly hatched brineshrimp.Larvae and adults are routinely fed chopped

beef heart, occasionally supplemented withbeef liver.

4. Daily routineTypically, animals are fed in the morning,

and cleaning is done in the afternoon.

A. Small larvae:The compartmentalized inner cage isslowly raised and put into a clean outercage with fresh water in it. Any trappedfood or droppings are siphoned out.

B. Medium sized larvae:Water in the plastic container is ex-changed for clean aerated filtered water.

C. Adult axolotls:For the animals housed in the aquaria,uneaten food is retrieved and water cir-culation is resumed.

5. Lab space usedAll animals are in a single room measuring

8 m (L) X 5 m (W) X 2.5 m (H). Room tempera-ture is kept at 20 o C, with12 hours light and12 hours dark cycle.

6. Time required per day/ weekFeeding and cleaning 20 cages, 100 plastic

containers, and the aquaria takes about 1 to2 hours for two people per day. However, thisvaries with the number of animals. Keepingstock animals requires no more than 30 min-utes, since it is semi-automated.


4

<1> inner cage

Material: acrylic plate, 3 mm thickDimensions: 40 cm (L) X 23 cm (W) X 5 cm (H) with 50 cellsCell size: 4.5 cm (L) X 3.7 cm (W) X 5 cm (H)The bottom plate of the each cell has 9 holes (1mm in diameter)

<2> outer cage

A plastic storage box (44 cm (L) X 27 cm (W) X 11 cm (H)) from a local store is used to holdwater.

<3> setting

The outer cage is partially filled with filtered water, and the inner cage is slowly lowered into it.The water level is adjusted to a depth of 3.5 cm. Larvae are placed into individual cells. Once set,this unit can be stacked or put onto shelves for better ventilation and lighting.

A

B

C

Figure 1. Compartmentalized Cage

A: top view, B: side view, C: bottom view

(inner cage)

40 cm

5 cm

1 mm hole9 holes/cell

23 cm

Fall 1996

5

<1> Aquarium

Material: glass plate 5 mm thick (oven-tempered glass plate with a hole of 3 cm in diameter in thebottom for overflow pipe) Dimensions: 40 cm (L) X 40 cm (W) X 30 cm (H) for 6 - 8 animals

<2> Filtering system

Components: PVC pipe, PVC valve, PVC filter box, electric water pumpFilter box dimensions: 180 cm (L) X 30 cm (W) X 30 cm (H) for 8 aquaria

Circulation Motor with capacity of 35 L/min

Acrylic cotton layer

Drain

FilteredWater

Water level

Valve

Figure 2. Filtering System for Adult Axolotl

Pipe size: inlet water pipe (3 cm OD), drain water pipe (7 cm OD)

inside pipe in aquarium (3 cm OD), outside pipe in aquarium (7 cm OD): direction of water flow

wateroutlet

Coarse sand & gravel


6

Axolotl Larvae HousingMethods

Cheryl A. NugasSusan V. Bryant Laboratory

Developmental Biology CenterUniversity of CaliforniaIrvine, CA 92637-2275

Water supply and treatment

Axolotls are housed in two locations: thebreeding rooms (located away from the generallab) and a more general access room locatedin the laboratory. In the breeding rooms, thewater is purified through a carbon filter sys-tem and stored in a large tank. The water ispumped through lines to each animal con-tainer. There is no routine treatment of thewater other than filtering with carbon filters.

The general animal room houses variousstages of axolotls, embryonic through adult.All axolotls in this room live in Holtfreter’ssolution, a buffered salt solution adapted fromthat used by the Indiana University AxolotlColony. The embryos live in 20% Holtfreter’ssolution, and both the larvae and adults arehoused in 40% Holtfreters solution. The waterfor the embryos and larvae is purified throughthe Millipore filter system. The adult’s watersupply comes from distilled water suppliedfrom the building’s filtering system.

Bowl types and sizes

General animal room in laboratory:

Prehatchlings (embryos) are kept in clearplastic containers obtained from the TriStatePlastics company.

Size: 5" x 7" x 3 1/2" ( W x L x H)

How many animals per container: Thesmaller the number of eggs per container thebetter for development and hatching. We putapproximately 20-30 per container.

How much 20 % Holtfreter's solution: Themaximum amount of liquid this containerholds is 1L. We keep approximately 400-500mls of 20% Holtfreter's in each container ofthis size.

Larvae are kept in clear plastic cups pur-chased at the local grocery store, Albertsons.

Brand Name: Solo Cups.Size: 3 1/2" x 2 3/4" (D x H).Cost : $1.00 per package of 10 cups.

Size and number of larvae housed: One ani-mal per cup, approximately 1.5 cm to 6 cmlong.

How much 40% Holtfreter's solution: Wekeep approximately 150 mls of 40%Holtfreter's in each cup.

Secondary container: According to UCI ani-mal protocols, experimental animals are re-quired to be housed in a secondary containerto prevent any loose animals in the laboratory.

We use two different types of secondary

Modified Holtfreter’s Recipefrom the IU Axolotl Colony as communicated to the SVB Lab by S. Duhon 10/4/94

Recipe is for modified 40% Holtfreter's. Dilute 50/50 with filtered water for 20% concentration.Use 20% for embryonic (prehatchling) stagesUse 40% for everything else

For 45 gallons: 2 tablespoon MgSO4

.7H20

2 1/2 Teaspoon (or 1 scant tablespoon) CaCl2

1 teaspoon KCl240 cc NaCl27 grams Trizma (7.4) fish grade

Dissolve the above recipe in milliQ H20 to volume of 3000 mls. This stock makes enough solution

to fill 9 Nalgene carboys (5 gallons each x 9 = 45 gallons total).

To make 40% Holtfreter’s: Add 300 mls of Holtfreter’s stock to a 5 gallons of milliQ H20.

To make 20% Holtfreter’s: Add 150 mls of Holtfreter’s stock to 5 gallons of milliQ H20.

Fall 1996

7

containers to hold our clear plastic cups: clearplastic sweater-type boxes and longer, whiteplastic mouse cages.

Clear Sweater boxes:Size: 9 1/2" x 12" x 3 1/2" (W x L xH) Each holds 9 cups.

White boxes:Size: 10" x 16" x 5" (W x L x H)Each holds 12 cups.

Larger larvae–adults: Larvae bigger than 6cm and large adult axolotls that are not usedin the breeding colony are kept in the animalroom in the laboratory.

Company name: TriStar PlasticsCatalog number: (Part Number)395CSize: 3 1/2" x 7" x 3" (W x L x H)

How many animals per container: 1How much 40% Holtfreter’s: The maximum

amount of liquid the container holds is 600mls.

Basement Breeding Rooms:Container size: 24" x 24"Number of animals per container: 1(when not breeding)Company container purchased from:Home Depot

Cost per container: $25.00How many per room: 9 containersper room.

Temperature

The breeding room is kept at 16-18°C. Thesecond room is kept between 18–20°C

Food supply

Young axolotl larvae (from just after hatch-ing to 4 cm) have a diet consisting exclusivelyof baby brine shrimp. Axolotls love brineshrimp and readily eat them. As the larvaegrow, we mix frozen ADULT brine shrimp intotheir diet.

Older axolotl larvae (4 cm and up) are fed amixture of fresh baby brine shrimp and frozenadult brine shrimp. However, some peoplebegin to feed their animals fish pellets at thisstage. We have a supply of fish pellets that aresoft and moist, easy to crush and easily bro-ken down in the water. We try to get them toeat fish pellets as soon as possible in order forthem to grow up quickly.

Adult axolotls are fed fish pellets exclu-sively. We use various sizes and forms of pel-lets. The pellets range in size from 2.4 mm to4.0 mm and come in a variety of texturesvarying from soft-moist pellets to a dryer and

Sources of food

Fish pellets: Rangen Inc.115- 13th Ave. SPO Box 706Buhl ID 83316-0706800-657-6446

Soft Moist pellets: 1.6 mm $34.00 for 40 lbs; 2.4 mm $31.50 for 40 lbsTrout pellets (harder): 4.0 mm $13.50 for 50 lbs; 4.8 mm $27.50 for 100 lbsFreight cost: $13.49 per box; Handling fee: $5.00

Brine shrimp: Bayou Aquatics.1908 S Lake PlaceOntario, CA 91761909-947-4575

We use brine shrimp eggs and adult frozen shrimp grown in Utah.

SO PRO80 Frozen adult brine shrimp: $9.70 per 21 lbsOcean Star International Brine shrimp eggs: $32.00 per 32 oz containerPO Box 643Snowville, UT 84336 USA


8

harder pellet for the adults.Daily routine

Researchers are responsible for the mainte-nance of their own research animals. How-ever, in general all embryos and larvae arecleaned daily. The larvae are fed at a mini-mum of 1 hour and maximum of 2-3 hoursprior to cleaning. Adult animals are fed andcleaned three times a week in both the generalanimal room and basement breeding rooms.

Lab Space Used

Basement rooms: The university has desig-nated a floor to house all research animals inthe building. The SVB lab has two rooms onthis floor.

The SVB lab has obtained special permis-sion to house some research animals inside asmall room within the laboratory. The tem-perature of this room can be adjusted as de-sired.

Time required per day/week

The total time required to clean eachresearcher’s batch of pre-hatchlings and lar-vae is approximately 30 minutes. It takes afew minutes to feed each batch of animals,and this is done at least 1 hour before clean-ing, but no more than 2-3 hours, in order forthem to get a chance to eat. We do not leavefood in the animals’ containers after cleaning.This helps to cut down on the number of ani-mal deaths from bacterial or fungal infectionscaused by dirty containers.

Adult animals both in the general animalroom in the laboratory and in the basementbreeding rooms are fed and cleaned threetimes per week. It takes approximately 1 1/2hours to feed and clean both basement rooms.It also takes 1 1/2 hours to feed and clean theadult animals in the laboratory. This work ismainly done by undergraduate researchersand by workstudy students. We believe thatthis is the optimal amount of cleaning that theadults need to remain healthy. We have veryfew bacterial and fungal problems with ouradults.

Cleaning Techniques

General access room in laboratory:The animal is placed in a fishnet set in a

secondary container filled with the proper so-lution. Each cup, plastic box, and lid isscrubbed with a dishcloth in hot water andrinsed briefly with distilled water. The con-

tainer is refilled with the proper solution, andthe axolotl is placed back in its container.

All utensils that come into contact with theanimal,the dishcloth, net, and secondary con-tainer, are briefly dipped in bleach and rinsedoff thoroughly. This bleach solution ischanged daily.

Bleach Solution: 1 cap full of bleach in 3 L ofdistilled water.

Holtfreter’s solutions are kept in 5-gallonNalgene carboy containers. We have separatecontainers for the prehatchling (20%Holtfreter’s), larvae (40% Holtfreter’s solutionwith milliQ H20), and adult (40% Holtfreterswith DI H20) solutions. Each week everyNalgene carboy is rotated, and the used con-tainer is cleaned.

Cleaning protocol

• Rinse each piece with hot waterand scrub with sponge.

• Soak for 5 minutes in dilutedbleach solution.

• Soak for 5 minutes in diluted So-dium Thiosulfate solution. (5 Tbs.per full sink)

• Rinse thoroughly with tap water 5times

• Rinse thoroughly with milliQ water• Let air dry completely.

The animal room floor is swept and moppeddaily with a diluted bleach solution and airdried. Weekly, the sinks are cleaned withscouring powder (Comet) and rinsed thor-oughly. Table counters are rinsed with 70%EtOH after each use.

Basement cleaning protocol

The basement animal room is cleaned threetimes per week. Each container is drained andcleaned with a sponge. Then the tanks arefilled with fresh carbon-filtered water. Thereare no special solutions added to the water.There are no chemicals used to clean the ani-mal containers. Sponges are cleaned by dip-ping them in a diluted bleach solution, rinsedthoroughly, then air dried.

Brine shrimp collecting protocol

Brine shrimp eggs are collected four timesper week: M, W, F and Sat. In general, theprotocol is as follows:

• 2.5 mls of Utah brand brine shrimp eggs

Fall 1996

9

• 30 mls of rock salt• 1 ml of Pen/Strep• 1 L of MilliQ H20.• 1 L bottle• 1 glass rod with a rubber stopper

This solution is air-bubbled in a 23°C waterbath for 48 hours.

The next step is to separate the brineshrimps from the egg shells. It is important toinsure that all egg shells are separated fromthe brine shrimp, because the axolotls cannotdigest the egg shells and will die if allowed toingest them.

Step 1:Pour contents from the bottle into a gravy

strainer. Keep one end of the gravy strainertilted by propping it up. Allow contents tosettle and separate, usually 5 minutes. Whathappens is that the egg shells will float to thetop while the live brine shrimp will sink to thebottom corner of the gravy strainer. Using abrine shrimp collecting net, gently pour ourthe live brine shrimp from the bottom of thestrainer into the brine shrimp net. Do not col-lect the top part of the solution, this containsthe brine shrimp egg shells. This step allowsmost of the egg shells to be discarded. Pourout the rest of the solution that contains theshells into the sink and rinse out the gravystrainer thoroughly. Invert the net into thegravy strainer and rinse out net with 40%Holtfreters, collecting the shrimp and solutioninto a clean gravy strainer.

Step 2:Utensils:

• Large Plastic petri dishes with90% of the side covered withblack electrical tape. (This createsa window.)

• Transfer pipettes• Light source

Brine shrimp are attracted to light. We usethis phenomenon to further separate the brineshrimp from the egg shells by creating an en-vironment with only one light source, the win-dow. The shrimp will swim toward the light,and the egg shells will sink to the bottom of

the petri dish. Place the light source approxi-mately 12 inches away from the petri dish. Fillthe petri dish with some 40% Holtfreter’s so-lution. Using a transfer pipette, suck up somebrine shrimp from the gravy strainer. Gentlyand without creating waves, pipette out thebrine shrimp into the middle of the petri dish.The shrimp will swim to the side of the light.After some minutes, a clump of orange brineshrimp will collect at the bright end of thepetri dish. Using a separate transfer pipette,suck up this orange clump of brine shrimp.Using the light or a white paper towel as thebackground, inspect the transfer pipette tosee of there are any egg shells in the pipette.You should just see orange brine shrimpswimming in the pipette. An egg shell will bebrown. Continue this process until all theshrimp is collected. You can create as manypetri dishes as needed. We use two or threepetri dishes. Dispense the brine shrimp in acup until all the shrimps are collected. Whenfinished the clean brine shrimp solution ispoured into a clean brine shrimp net. The netis inverted into a 1L beaker and rinsed outwith diluted rock salt solution. The brineshrimp is covered and an airline is placed in-side the beaker. The shrimp is aerated untilneeded.

Diluted Rock Salt Solution:50 mls of Saturated Rock Salt solution450 mls of milliQ H20.

Saturated Rock Salt Solution:500 mls of milliQ H20Rock Salt, enough to supersaturate the

water

When brine shrimp are needed, pour theproper amount into a cup. Using a brineshrimp net, pour the contents from the cupinto the net. Invert the net and rinse out con-tents from the net into the cup withHoltfreter’s solution (the proper concentrationdepends on the type of animal being fed). Thebrine shrimp live longer when swimming in arock salt solution; however, when feeding theanimals we remove the rock salt solution andplace the shrimp in Holtfreter’s solution. Thisway, we do not change the content and saltconcentration in which the axolotls live.


10

Corwin Lab Axolotl Protocols

Anne HennigAnatomy and Neurobiology

Boston University School of MedicineBoston, MA 02118

Reagents

Axo Water

Distilled water, dechlorinated tap water, orcommercially available spring water can beused. Deionized distilled water is not recom-mended due to expense and lack of trace nu-trients.

400% Holtfreter’s Stock (HFR)

KCl 2.30 gCaCl2.2H2O 4.29 gMgSO4.7H2O 8.90 gNaCl 126.72 g

Packets can be prepared ahead of time andstored in Ziploc bags at room temperature in adry place. Dissolve salts in axo water and ad-just volume to 8L. For 100% HFR, dilute 1:4in axo water; for 50% HFR, dilute 1:8; for 20%HFR, dilute1:20.

0.07% Benzocaine Stock (10X)

Ethyl p-aminobenzoate 0.7 g(Sigma # E-1501)

Dissolve in 1L distilled water (takes severaldays at room temperature with stirring). Storeat room temperature.

Anesthetic HFR Solution (0.007% Benzocaine)

400% HFR 500 mlbenzocaine stock (10X) 200 mlHEPES (free acid) 2.384 gddH20 to 2L final volume

Check pH and adjust to 6.8 - 7.0 with 1NNaOH. Autoclave if desired (not necessary foranesthesia of less than 18 hours or for ampu-tations) and store at room temperature.

Brine Shrimp

NaCl 22.5 gbrine shrimp eggs 1.5 g

(Bonneville Artemia International

Inc., Salt Lake City, UT has givenbest results. Various brands frompet shops have been less satisfac-tory.)

Packets can be prepared ahead and storedin small Ziploc bags at room temperature. 18-24 hours before feeding, mix with 1L axo wa-ter in glass or clear plastic container until salthas dissolved. Aerate and incubate 6-12inches from incandescent light source.

Hatching Axo Larvae

Keep eggs in the solution in which theywere shipped, at room temperature and out ofdirect sunlight, until hatching begins.

As axos hatch, transfer them to 20% HFRusing a wide-bore pipet. (We cut the tips offplastic transfer pipets using a razor blade orscalpel.) For eggs containing healthy axoswhich have not hatched within 24 hours ofthe rest of the batch, gently tear open the eggmembrane using two No. 5 forceps to releasethe axo. (Otherwise axo will be permanentlybent.) Transfer to 20% HFR.

The next day, transfer the larvae to 50%HFR. Axos need to eat daily at this stage, butif there are too many brine shrimp they willkill the axo larvae. (There will be a lot of brineshrimp in the gills of the dead larvae.) There-fore, feed a few brine shrimp at a time, andclean out the dish when the axos’ bellies arefull. Be sure egg shells have been removedfrom the brine shrimp.

After 7 - 10 days, increase the amount ofbrine shrimp given, and decrease feeding fre-quency to every other day.

Older Larvae

Place larvae into containers of fresh 50%HFR when they are received. (We use shallow4 - 10 cup Rubbermaid refrigerator disheswithout the tops.) Larvae less than 1.5 cmlong can all be placed in the same container.If brine shrimp are available, feed new larvaeas soon as they have recovered from shipping(when movements become rapid). Otherwise,start a fresh batch of brine shrimp immedi-ately and feed the next day.

Feed every 2 - 3 days, being careful to re-move brine shrimp egg casings before placingin axo dishes. Clean a few hours after feeding,when bellies are full. Remove any dead (unre-sponsive and no heartbeat) larvae as soon aspossible. “Sick” larvae can sometimes be

Fall 1996

11

saved by transferring to small containers of100% HFR. They can be returned to 50% HFRafter recovery (usually takes several days).

When axos get to be 1.5 - 2 cm long, sepa-rate them to avoid nipping and cannibalism.They can be housed in Rubbermaid 12-oz.shallow containers without lids, plastic delidishes, or other similar containers which havenot been used for toxic chemicals. These canbe kept in stackable plastic or wire closet or-ganizing bins which provide airflow space be-tween levels, to save bench space. As the axosgrow, transfer to larger containers. Theyshould have sufficient space to swim around,and the level of 50% HFR should be sufficientthat they can stay completely submergedwithout having to duck their heads.

“Stressed” axos (recently operated or ampu-tated, sick, or after long periods of anesthesia)should be kept in 100% HFR during recovery.

Feeding

The day before feeding, prepare brineshrimp as specified in Reagents.

Remove airstone from 18 to 24-hour-oldbrine shrimp. Allow 10-15 minutes for shellsand unhatched eggs to settle. Brown casingswill be seen floating on brine surface and atbottom of container. Pink brine shrimp movein jerks and congregate close to the lightsource.

Wet a fine fishnet and place in an emptycontainer below the level of the brine shrimpcontainer. Position rigid end of a siphon tubein the shrimp container near the swimmingshrimp congregation and away from egg cas-ings. Start siphon by applying suction to theother end of the tube until fluid level extendsbelow the bottom of the shrimp container.Allow fluid to flow through the net into theempty container so the brine shrimp collect inthe net. Avoid transfering egg casing material.When sufficient shrimp have been collected,stop the siphon.

Avoid feeding egg casings to newly hatchedlarvae (less than 3 months old) because theycan not process the casing material. For smalllarvae, we transfer shrimp to a second con-tainer of salt solution (22.5g/L axo water) andallow any egg casings to settle a second timein front of the lamp before collecting theshrimp for feeding.

Rinse the shrimp collected in the fishnetwith axo water to remove the salt, then trans-fer a small amount to each axo dish using aplastic transfer pipet or spatula. When all

axos have been fed, rinse inverted net to re-move any remaining shrimp. Clean siphon ofshrimp and salt deposits. Clean brine shrimpcontainer by rinsing with tap water and wip-ing any residue with a paper towel. Invert todrain, and turn off incandescent light.

Allow several hours for axos to feed (belliesshould be swollen with pink mass in gut),then discard any uneaten brine shrimp andclean the dishes.

Larger axos (older than 4 months) can befed small pieces of liver. Clean dishes 30 min-utes to 2 hours after the axo finishes feeding.(If disturbed too soon, axos will regurgitate,but if left too long the uneaten liver will reallystink.)

Cleaning

Transfer axos to holding dish containing50% HFR, avoiding debris as much as pos-sible. This can be done using a wide-boretransfer pipet to suck up the axo head-first (fitshould not be tight, but also not loose enoughto allow the axo to turn around). Alternately,pour off as much dirty HFR and debris as pos-sible and then gently pour the axo into theholding dish. (Axos larger than 3 cm or somay try to jump out; be prepared for this. Ifthe axo escapes, keep it wet with 50% HFRuntil it can be scooped up and returned to adish.) For very small axos, pour dirty axo wa-ter through a clean brine shrimp net to con-tain any escapees.

Discard any dirty HFR remaining in thedish, and scrub out residue with a clean pa-per towel. Use a separate paper towel for eachaxo dish, and clean the newest axos last toavoid transferring any diseases. Refill the axodish with fresh 50% HFR and return the axos.Wipe out the holding dish after each use.

Between cleanings, check for evaporation (asalt ring around the dish above the surface ofthe HFR). If this occurs, clean as above butrinse dish with axo water (not HFR) to dissolvethe salt before adding fresh HFR.

Anesthesia

Prepare anesthetic HFR as specified in RE-AGENTS section. Larvae are sensitive to boththe pH of the solution and the concentrationof benzocaine. For small larvae (less than 6months after hatching), further dilute anes-thetic HFR solution with 100% HFR.

The axo should be completely immobilewithin 10-15 minutes of immersion (it can be


12

turned onto its back without twitching). Itshould recover completely within 20 minutesof removal from the anesthetic solution, andits heart rate should remain steady at ap-proximately 1 beat/sec throughout the periodof anesthesia. If the heart rate drops, discon-tinue anesthesia, remove axolotl to 100% HFRand allow it to recover.

Keep the axo in 100% HFR for a period oftime (several hours for short periods of anes-thesia, or during the period of regeneration orhealing if axo has been operated on.) Feed andclean according to usual schedule. Axos canbe fed as soon as they have recovered fromanesthesia.

If euthanasia is necessary, add 0.4 g NaClto 100 ml stock Benzocaine (0.07%). Whensalt has dissolved, place axo in solution untilheart stops beating completely.

Acknowledgments

These procedures are based on work by theauthor as well as on past work by PaulaBorden, Jay Jones, and Kenneth Balak in theJeffrey Corwin Laboratory at the University ofVirginia, and on important advice receivedfrom Karen Crawford and from the staff of theIU Axolotl Colony over the past twelve years.

Fall 1996

13

Axolotl Housing and Care atSt. Mary’s College of Maryland

andWhat is an Axo-condo?

Karen Crawford, Ph.D.Department of Biology

St. Mary’s College of MarylandSt. Mary’s City, Maryland 20686

Our Colony

At St. Mary’s College of Maryland we rou-tinely maintain a breeding group of about 10adults around 2 years old, 12-15 animalsaround a year old and anywhere from 100–300 larvae. Animal care is performed by re-search students working in my lab or, whenfunding is available, an animal caretaker.Holiday times, exam times, odd times, we all(family members included) pitch in to feed andclean.

Food and Water

All animals are fed three times a week, andtheir Holtfreter’s solution is changed within 3but not more than 6 hours after feeding. Toprevent cannibalization, young larvae arereared individually, in 8 oz. SweetheartÆ icecream and food cups, S308, (approximately$50.00/1000) in 25% Holtfreter’s solution.Larvae are fed fresh-hatched brine shrimp andthen switched to frozen brine shrimp as soonas they will self feed. Larger larvae are main-tained on frozen brine shrimp and adults arefed small cubes of fresh thawed beef liver.Larger animals are housed in 64 oz. polyethyl-ene disposable FisherbrandÆ multi-purposecontainers (11-840E) or larger polystyrenemammal animal boxes. Matings are set upand newly fertilized embryos are maintainedin 10% Holtfreter’s solution to minimize exo-gastrulae in the developing embryos. We makeup 20 liters of 100% Holtfreter’s solution at atime and then dilute it as needed with housedistilled water. Because of temperature fluc-

tuations in our science building, a cold roomset at 18°C is used to house the animals.

Anesthetic

To anesthetize animals for amputation ortreatment we immerse the animals in a0.007% solution of Benzocaine, Ethyl p-amino-benzoate (SigmaÆ), dissolved in 25%Holtfreter’s solution. For convenience, a0.07% stock solution can be made and storedon the shelf at room temperature. To makethis stock solution, Benzocaine must first bedissolved in a small amount of 100% Ethanolbefore being added to the Holtfreter’s solu-tion. Benzocaine also works well for fish andworms, however the concentrations may needto be modified.

The Axo-condo

To minimize the amount of space requiredfor my colony in the lab and cold room, I havedesigned a special rack to house trays of lar-vae. The standard lunch trays found in mostcafeterias accommodate 12 of the waxed pa-per cups we use to hold larvae. These traysare handy to use when organizing experimen-tal groups or transporting animals and areeasy to clean. To acquire a rack designed tohold lunch trays, I went through the companywhich provides our college with racks. Work-ing over the phone, I was able to design arack specific to my needs. The “Axo-condo”rack stands 65 inches tall and is 22.5 incheswide and 29 inches deep. It moves easily onwheels and has 11 shelves spaced 5 inchesapart. Each shelf holds two trays of 12 ani-mals. In this way we are able to move or store264 larvae in the floor space of little morethan two lunch trays. To discourage animalsfrom hopping out of their cups, plastic ceilinglight “egg crate” panels are cut to size withheavy shears and placed over the top of each12 cups/tray. These covers minimize animalloss and are easily cleaned. The initial cost ofthe rack in 1991 was about $150 with ship-ping a surprisingly high additional $150. Re-gardless and despite the cost, I cannot imag-ine functioning without this useful cart andcould easily use another.


14

Raising Axolotl Larvae

Peggy Egar2969 Somerton Avenue

Cleveland Heights, OH 44118

I had some 1000+/- hatchlings left overfrom the 14 January spawnings that I set upfor the Cleveland State University BiologyDepartment’s Development course. I kept thelarvae in groups of 25–30 in cut-off, plasticgallon water/milk jugs. These were fed dailyon newly hatched brine shrimp until mid-May. Setting up the brine shrimp (in largefinger bowls of saltwater under goose-neckeddesk lamps) and feeding the larvae only tookabout 30 minutes/day and that includedcleaning a few bowls each time. The difficultpart comes in trying to switch over to frag-mented salmon chow.

The Biology Department allows me to useone of their cold rooms (15 degrees), and I havethat lined with shelves. Some of the shelvesare on wheels, and, in addition, I have a handcart for moving dirty animals out to the sinkin the lab for cleaning. Near the sink, I keep a30-gallon plastic garbage can filled with tapwater to allow the chlorine to evaporate over-night. I used to treat the water but no longerbother and have not noticed a difference.

Once the larvae are about an inch long, Iisolate them into small and medium-sizedplastic butter tubs which are labeled with bitsof tape. I have an inexhaustable supply ofplastic dishes. My Physical Therapy and Occu-pational Therapy students are more than hap-py to bring them into class, and many of thestudents have axolotl pets for their children.

Once isolated in the small dishes, the lar-vae either start eating the broken up bits ofsalmon chow or start shrinking in size. At thattime they become lollypops for the adults orbegin growing by leaps and bounds. The iso-lated larvae take lots longer to clean and feed,but are all stored, stacked up on the shelvesin the coldroom. I usually spend 4–5 hoursevery other day cleaning, feeding, or doingexperiments.

The animal-care fellow for the Biology De-partment is now ‘into’ axeys too. I just gavehim 50 adults today to sell to a wholesale guy.The money he gets he uses to buy shrimpeggs or frozen brine shrimp to feed the larvalaxeys that he raises in large groups up in the

animal facility. He then supplies me with asmany 3" larvae as I need to run my experi-ments. This is just a hobby with him, but it isa neat way for me to get the older axolotls offmy hands when they are no longer useful formy experiments. My stock of adults is down toabout 150 now, but I have plus or minus 500larvae from 1 to 3 inches long, plus an almostinexhaustable supply from my friendly ani-mal-care fellow.

I keep the salmon chow in another garbagecan in the cold room, so there is no problemwith roaches. If I’m in a hurry to grow-upsome larvae for use, I’ll hand feed them stripsof beef liver cut from slightly thawed liverstored in freezer on picnic plates in baggies.Many of the staff and students around CSUnow have axey pets and feed them this way.When they accidentally get spawns, they usu-ally bring them into me to raise. There willsoon be more axolotls in Cleveland than thereare researchers!! I contribute them to theclassrooms of as many area science teachersas I can reach with my tales about the mar-vels of axolotls.

I often have 4–6 year adults in my colonyand have not noticed a damaging effect of theplastic dishes. The square cut-off plastic gal-lon milk cartons fit nicely on shelves and arejust high enough to prevent isolated animalsfrom jumping out. They hold slightly less than1/2 gallon water. I can usually clean about 70adult dishes with my 30 gallons ofdeclorinated water. So any individual adultaxey usually gets cleaned at least once everytwo weeks and fed 2-4 pellets every other dayor so.*

Acknowledgments

I learned about all I know about axolotlsfrom spending 6 months in Hugh Wallace’slab in Birmingham, England (Jan –June,’80).He’s really great and certainly the mostknowledgable person imaginable when itcomes to any kind of amphibian. He dug apond in his back yard and now ‘grows hisown’ newts and frogs.

* These very infrequent changes are probablypossible because the animals are kept at 15°Cand in the dark. If in doubt, monitor ammonialevels. The editor.

Fall 1996

15

Raising Axolotl Larvae

Linda MuskeDepartment of BiologyUniversity of Virginia

Charlottesville, VA 22903

In our experience, the biggest hassle withsmall larvae has to do with food; Raising livebrine shrimp (or any live food animal) is a nui-sance. Getting babies to switch food items asthey grow is another hassle.

We have experimented with a variety of fro-zen food — brine shrimp of various sizes,worms of various types. Axolotls love wormsand will readily gobble up living ones. Likeother amphibians, their visual system is high-ly sensitive to moving, but not necessarily todead, immobile prey items. But keeping liveworms around is a pain, and they are not al-ways available. We have found that frozenworms are readily accepted by axolotls so longas they are first trained to identify the frozenworms (black or red —available at pet stores)as food. It seems pretty clear that axolotls use

odor, as well as visual cues to localize andidentify food, and they very quickly learn toassociate a particular odor with food. We soakthe frozen worms in fresh water to defrostthem, then offer them to the axolotl on theend of a forcep. It helps to move the wormsaround and bring them in contact with theaxolotls nose. Within a few trials the axolotlsidentify dead, limp worms as food, and you nolonger have to wave them across their face.

We have applied the same training tech-nique to inducing young larvae, previouslyraised on live brine shrimp, to accept deadfood. The worms need to be cut up a lotsmaller (obviously), and the training takessomewhat longer, but it is worth the invest-ment: More larvae survive, grow and thrive ifthey are encouraged to eat in this way, theydevelop faster if they are eating on a regularbasis, and you don’t need to mess with livingfood. We repeat the training process when itcomes time to wean the larvae onto salmonpellets, and in very short order they learn tofeed on their own.

Northcutt Lab Larval RearingMethods: 1996

Linda BarlowNeurosciences

University of California at San DiegoLa Jolla, CA 92037

Embryos are raised in egg capsules tohatching in large finger bowls of artificialfreshwater (AFW) at room temperature (20oC -28oC over the course of the year).

AFW Recipe: CaSO4 50 mg/LMgSO4 5 mg/LKCl 4 mg/LNaH2PO4 1 mg/Lin dH2O

Feeding of larvae

At stage 43, we begin to feed the axolotllarvae brine shrimp nauplii that we hatchfrom commercially available encysted eggs(found at any pet store). We raise the brineshrimp according to the instructions on the

packet. Before we give the shrimp to the lar-vae, the shrimp are quickly rinsed in AFWusing a fine-meshed net. Larvae are fed daily,and their water is changed every day.

Once the larvae are 10 days old (x = 1.7cm), we begin to feed them finely chopped tu-bifex worms on a daily basis, and their wateris changed every day. We get tubifex weeklyfrom the pet store, and keep them in tap wa-ter in a bowl in the refrigerator.

By the time the larvae reach 4 - 5 weeks (~4cm), they are fed intact tubifex worms. Waterchanges continue every day.

Larval / juvenile density

For the first 3 weeks, approximately 50 lar-vae are kept in each large finger bowl. After 4weeks (x = 3.6 cm), the density of juveniles isreduced to 5 - 6 per bowl. By ~7 cm, only 2 -3animals are in each bowl, and that number isreduced to 1 animal per bowl once the juve-niles reach approximately 9 cm (9 weeks). Wetypically raise 12 -15 animals to adulthood. Ittakes about an hour or so a day to handle 20animals including getting their food from thepet store.


16

Inexpensive and LowMaintenance Facility for

Housing Axolotls

M. Moody and G. ThibaudeauBiological Sciences

Mississippi State UniversityMsState, MS 39762

In order to maintain axolotls in the labora-tory we have constructed an inexpensive eas-ily maintained system from plastic containers,PVC pipe, rubber tubing, and picnic coolers.Stagnant water and all waste materials areremoved by siphoning, and then tanks areflushed with aged tap water. This procedure isperformed following feedings every 2–3 daysand provides for an efficient low-maintenancemethod of caring for axolotls. Labeled photo-graphs of the system are included, and a morecomplete description follows:

Large axolotl tanks: The larger axolotls arekept in clear plastic rectangular containers(22 x 36 x 14 cm) partitioned in half by 8-mm-diameter plastic mesh glued in place withclear silicon sealant. Two adult axolotls arehoused in each container. A drain hole wascut and a protective grating was inserted inthe front of each container. This drain main-tains a constant water depth of about 5.5 cmand the grating prevents axolotls from escap-ing into the drainage pipe.

Small axolotl tanks: The smaller axolotlsare kept in clear plastic rectangular contain-ers (16x30x8 cm) partitioned in half or thirdsby 1 mm diameter plastic mesh glued in placewith clear silicon sealant. Two to six axolotls(depending on their size ) are housed in eachcontainer. A drain hole was cut and a protec-tive grating covered by a 2 mm diameter meshwas inserted in the front of each container.This drain maintains a constant water depthof about 4 cm and the mesh size of the gratingprevents axolotls from escaping into thedrainage pipe.

Water replacement system: Two large Ig-loo coolers which hold 40 liters of water eachare mounted on a sturdy shelf approximatelytwo feet above the axolotl tank water level.Half-inch PVC pipe connects each of the cool-ers’ drain holes to a valve before converginginto a single 0.5" PVC pipe with a series of 3/32" ID adjustable aquarium valves. Clearaquarium tubing (two per tank) supply eachaxolotl tank with fresh water at a rate of about4 ml/s when one cooler valve is fully open.The tanks rest on a Plexiglass-covered ply-wood bench tilted slightly forward. Excessrunoff water is collected in a trough cut fromlarge PVC pipe at the front of the bench and iscarried to a drain which eventually emptiesinto a sink.

Additional information: We currentlymaintain 6 adult axolotls (15-19 cm long) in 3large axolotl tanks and 6 juvenile axolotls (4-10 cm long) in 2 small axolotl tanks; however,

the system is easily expanded to ac-commodate additional axolotl tanks.The Plexiglas covered bench onwhich the axolotl tanks rest mea-sures 42" wide x 38" deep and re-sides on top of a lab bench 46.5"above the floor. The shelf on whichthe water storage tanks rest mea-sures 38" wide x 26" deep and is lo-cated 24" from the ceiling. The entiresystem occupies a space 66" wide x38" deep x 52" high, not includingthe sink located conveniently adja-cent to the system. The sink servesas both a source of water for refillingthe water storage tanks and a drainfor flushed or excess runoff water.

Care of the axolotls: The axolotlsare maintained at room temperaturein ambient room lighting conditions.They are fed 4-5 pieces of frozen beefor pork liver that is slightly thawed(as it is easier to cut when semi-fro-

Fall 1996

17

zen) and cut with a razor blade. Theadult axolotls are fed pieces approxi-mately 10 x 10 x 5 mm in size. Afterfeeding the axolotls, their tanks aresiphoned to clean out waste materialand then flushed with fresh waterfrom the water storage tanks. Thewater used to refill and flush theaxolotl tanks is untreated tap waterwhich has been allowed to age forthree days in the water storage tanks(uncovered) in order to remove thechlorine. After cleaning the axolotltanks, the water storage tanks arerefilled using a rubber garden hoseattached to the spigot of the nearbysink. The entire feeding/cleaning/refilling procedure takes approxi-mately 45 minutes and is repeatedevery three days.


18

Housing and Feeding LarvalAxolotls

Carl SchneiderPsychology Department

Indiana University of PennsylvaniaIndiana, PA 15705

1. We house axolotl larvae individually inbowls that have the water changed every otherday. We use glass bowls that are 9 cm wideand 3.5 cm deep. They are filled 2/3 full withwater (solution: see 5 below).

2. The animals that we house usually rangein size from 2 to 4 cm.

3. We feed the animals brine shrimp twice aday at 08:00 and 17:00. Once a week we feedthem white worms from a colony that we keep.

I think the simplest way to go is feeding thelarvae brine shrimp. They are easy to brew ina separatory funnel that is aerated from thebottom and you get a good hatch in 24 hours.

4. I usually house 24 to 30 animals at atime, but I have housed as many as 80 duringa big experiment. I always house the animalsindividually.

5. Holtfreter’s solution. The recipe I useconsists of the following:

To 6 liters of water add:

300 ml Sodium Chloride6 g Potassium Chloride

12 g Calcium Chloride32 g Magnesium Sulfate

To another 6 liters of water add 24 gramsSodium Bicarbonate. The 12 liters are mixedtogether in a carboy. The chemicals dissolvebest if the water is hot.

White worm colony

I have a 3-pound coffee can thatcontains potting soil (about 2/3 full). Ibury a slice of bread in the dirt once aweek (whole wheat) and keep the soildamp. It is important that the bread stayburied or you get mold if it’s exposed toair. I started with about 10 worms and in2 weeks I had a colony. The best way toget the worms for a feeding to theaxolotls is to make the soil very wet.They come to the surface and can bepicked off. You could feed them toaxolotls everyday, but you would need abig supply for a lot of animals. I have acolleague who used to keep a largeaquarium full of the worms to feed fish. Ithink a larva that is 2 cm long could eatsix to eight 1.5 cm worms a day. I usethe worms primarily for reinforcementduring learning experiments. They canlast in a bowl of solution for about 24hours. If they die the salamander won’teat them. It could also become verytedious picking the worms off the pottingsoil. You can’t do it with forceps withoutpicking up some dirt. I always transferthem first to a bowl of clear water usingforceps. I tease them away from the dirt(they tend to clump with each other),then I pull them into a stopper and squirtthem into their destination.

I make 24 liters at a time and store it in a30 liter carboy I keep another 30 liter Carboywhere I mix 1 liter of the above stock solutionto 12 liters of water. I used to use a 1:6 ratioof stock to water, but I have found that 1:12works just as well, and I don’t have to mix upthe stock solution as often.

Fall 1996

19

Axolotl Care at IndianaUniversity Northwest

Timothy StablerDepartment of Biology

Indiana University NorthwestGary, IN 46408

At Indiana University Northwest, about twodozen animals are in the axolotl colony atpresent. Eight of these are proven breeders,and the remaining are from 1995 spawnings.In the present building, care was taken tohave all animal care facilities in one location.

The main animal care room is in the shapeof an “L” with the long leg measuring about 8feet by 30 feet and the short leg measuringabout 9 feet by 14 feet. In the long leg are tworacks to hold cages for warm-blooded animals.These racks have been modified so that eachrack shelf is considered an animal room, andeach is connected to an air vent leading out-side the building. In fact, a different speciescan be placed on each shelf and still be inexcellent shape for anyone’s inspection. Sucha facility allows the remainder of the room tohold a rack with the axolotl containers on it.In the elbow of the “L” is a large sink andwork area, which is used for the warm-blooded animals. At the end of the “L” is astorage room measuring about 10 feet by 10feet. Tall storage racks on each side holdmouse food, cages, shavings, axolotl bowls,and other equipment.

This large room has a Tork timer (modelW202) set for 12 hours light and 12 hoursdarkness.

Off from the short leg of the “L” are twosmaller rooms, one of which measures about9 feet by 12 feet and the other about 9 feet by9 feet. Each room has a door, which may beclosed and locked if necessary. The smallerroom has a still in it (Barnstead Fistreem II)which empties into a 30 gallon holding tank.The tank is equipped with a switch that shutsthe still off when full.

The larger room has a sink and work areaalong one long wall with a table against theopposite long wall. On the table is a 30 gallonplastic tank with spigot (see Cole PalmerHPDE H-06317-71). Distilled water and saltsto make a 50% Holtfreter’s solution are keptin this tank and used for axolotls, regardlessof age. The rack with the axolotls is movedfrom the larger room into this room for chang-

ing and feeding. The bowls are changed in thesink and placed on the table. After the bowlshave been filled with solution, the animals arefed pellet food, as used by the I.U. AxolotlColony. The bowls are then returned to therack and the rack returned to the larger room.The total time needed for the colony forchanging and feeding is about a half-hour.

Larvae are also kept on the rack althoughtanks for brine shrimp are set up in the stillroom, where light for the brine shrimp will notbother any other animals.

The smaller rooms have their own lights notassociated with the timer in the large room. Itwas discovered that if the solution tank werekept in the larger room, an algae problem de-veloped with the extra light. In the smallerroom with no lights on except for feeding andchanging, there is no such problem.

When Biology first moved into this buildingabout 6 years ago, the colony was about 100animals—all adult and non-breeding. Chang-ing and feeding took a little longer but stillwent smoothly with this set-up.

Maintaining Larvae

Katia Del Rio TsonisDepartment of BiologyUniversioty of Dayton

Dayton, OH 45469-2320

We use 1x Holfreter’s solutionmade in deionized water. We changethe axolotls every other day, butsometimes they go from Friday toMonday without a change. We keepthem in Tupperware containers usu-ally at least 5 times the size of theanimal. We feed them the salmonpellets from Rangen, but they likeliver more. They are fed 3 times aweek. The containers are cleanedwhen the water gets changed with ableached and well-rinsed brush.

Our animals are housed onshelves in a small room with a sink.We spend about 2- 3 hours a weekon maintenance depending on thenumber of animals and whetherHoltfreter’s solution needs to bemade.


20

with a permanent marker. Empty the waterback out.

Position the air pump so that the air hosereaches the bottom of the inverted bottle. Thediagram shows how the setup should look.

Operating the Hatchery

Rinse out the bottle, clean with a brush ifnecessary, make sure the drain spout is se-curely closed, and place it in the stand in in-verted position. Add about a cup (about 350ml) of hot tap water through the hole at thetop. Use a funnel to add three tablespoons(45 cc) plain (uniodized) table salt —NaCl— tothe hot water. Swirl to dissolve the salt. Fillthe bottle the rest of the way to the half-fullline with cold tap water. Next, add 1/4 - 1teaspoon (2-5 cc) brine-shrimp eggs (cysts) tothe bottle, depending on how many larvae youneed to feed. For fewer than 20 larvae, 1/4teaspoon (2 cc) should be plenty. Swirl thebottle gently to mix the eggs and brine. Placethe air hose through the hole so that itreaches the bottom of the inverted bottle.Make sure that the air hose is positionedproperly and bubbling vigorously because theshrimp will not hatch well unless the water isagitated continuously.

Most of the shrimp should hatch within 24to 48 hours. The length of the cycle dependsupon the temperature. The hatched shrimpare orange and can be easily seen through theplastic bottle. When most of the shrimp ap-pear to have hatched, remove the air hose andhold the bottle over a clean shallow pan. Aftermost of the gray shells have floated to the sur-face, open the drain to empty the newlyhatched shrimp and brine into the pan. Closethe drain before the last of the brine and theshells enter the pan.

Manipulating the Cycle

To shorten the cycle raise the temperatureof the system by putting the apparatus in awarmer location or directing a lamp with alow-wattage bulb at the hatchery. Alterna-tively add more hot water initially to start witha warmer mixture.

To lengthen the cycle place the hatchery ina cooler location or use less (or no) hot waterinitially.

Troubleshooting

The percentage of shrimp that hatch de-pends on the temperature and length of the

Simple Brine Shrimp Hatchery

Brent MundyIU Axolotl Colony

Bloominton, IN 47405

Constructing the Hatchery

Construct a simple shrimp hatchery out ofa two-liter clear plastic cola bottle, the pull-upcap from a 32 oz (1 liter) dishwashing deter-gent bottle (or sports drink bottle), an aquar-ium air pump, a 3-4 ft (about 1 meter) longpiece of tubing that fits onto the air pumpoutlet to use as an air hose, and a stand ofsome kind to support the bottle in an invertedposition. A heat lamp with a 40-watt bulb isoptional.

First, empty and clean the two-liter colabottle with hot water. Use a sharp knife to cuta hole 1 to 1 1/2 inches (about 3 cm) in di-ameter in the bottom of the cleaned bottle.Next, rinse the pull-up cap from the detergentbottle very well with warm water. The threads

on the cap and the two-liter bottle should becompatible. Screw the cap onto the two-literbottle.

This apparatus will be used inverted. Thepull-up cap serves as a reclosable drain at thebottom. Fill the bottle half-full of water anddraw a line across the bottle at the water line

Fall 1996

21

cycle, on whether the brine has been continu-ously and vigorously agitated, and on the qual-ity of the shrimp eggs used. If, after checkingthat temperature and agitation are appropri-ate, only poor hatches are obtained, considerchanging to a different brand or supplier.

Collecting Shrimp to Feed

Place a large coffee filter across the top of awide-mouthed jar or similar container. Collectlive, swimming brine shrimp from the pans bysucking them up in a large pipette (turkeybaster). Do not suck up dead brine shrimpfrom the bottom of the pan. Avoid getting anyfloating shells by placing the tip of the pipettejust below the surface of the water. For easiercollecting, place a light at one end of the pan.The shrimp are phototropic and will swim to-ward the light, conveniently gathering them-selves together.

Squirt the shrimp and brine into the filter

to strain the shrimp out of the brine. Discardthe brine, then wash the shrimp off the filterand into the container with axolotl water. Dis-tribute the shrimp suspended in axolotl wateramong the bowls of larvae with the pipette.Feed very young larvae just enough to maketheir bellies orange. Feed larger larvae gener-ously to forestall cannibalism. It may take afew days of feeding and observing the resultsto get a sense of how much to feed.

Care for Larvae

Small larvae (< 2 inches or 5 cm long) arefed brine shrimp. Change the water and feedthem daily. Keep their bowls clean but neveruse soap. If necessary, use a little baking sodaand salt mixed together as a cleaning agent(two parts baking soda to one part salt).Scrub, then rinse thoroughly. As the larvaegrow, split them up into additional bowls.Keep similar sized larvae together.


22

Terrestrial Axolotl Care Sheet(Ambystoma mexicanum)

Brent MundyIU Axolotl Colony

Bloomington, IN 47405

Metamorphosis

Transforming from an aquatic salamanderto a terrestrial salamander is very stressful forthe axolotl. Once it is noticed that an axolotlis metamorphosing it should be removed fromits regular habitat and placed in a covered onegallon container. The container should belarge enough for the animal to fit comfortably.A few rocks should be placed in the containerfor the animal. Add just enough water to thecontainer so that the axolotl’s back is covered.

Over the course of a few days, the animalwill molt and shed quite a large amount ofskin, which will look like black sheets. Theskin should be removed on a daily basis. Dur-ing metamorphosis the axolotl will absorb itsdorsal fin, grow eye-lids, absorb its gills, loseits slime coat, and begin breathing air. As theanimal progressively becomes more terrestrial,the amount of water that is in the containercan be reduced.

After the animal switches over, a period ofadjustment will occur. Limbs that were onceused for swimming must now learn to walk onland. At first the axolotl will spend most of itstime in the water, but as it learns to use itslegs, travel on land will become more com-mon. It is not uncommon for a newly meta-morphosed axolotl to go off of food for sometime. This fast may last for up to two months.

Habitat (Vivaria)

A wide variety of habitats can be created forthe terrestrial axolotl. The only steadfast re-quirements are that the animal has access towater, so that it can totally submerge itself ifit so desires, and a dry land area. A very basicdesign could consist of a 10 gallon aquariumand a large water bowl for the axolotl. Obvi-ously more sophisticated setups can also becreated. Below is the outline of an advancedsetup:

A large sturdy aquarium will need tobe divided in a 30% water, 70% landsplit; a piece of glass or Plexiglascan be glued using aquarium sili-cone cement in the tank in order to

make a shore. Slant the partition tomake a slope that the axolotl canclimb to leave the water. Fill the wa-ter portion with an inch of aquariumgravel (Note: gravel should be largeenough that the animal cannot in-gest it. Put two inches of coarsesand or washed aquarium gravel inthe bottom of the land portion of thetank for drainage. On top put sterilepotting soil or peat moss. The soilshould be loose so that the axolotlcan burrow if it chooses. Furnishpieces of bark, rocks, or clay potshards to make hiding places for theaxolotl. You can plant small plantsin the soil if you like, but theyshould be large enough so that theaxolotl will not destroy them.

You will need a fitted lid for the tank. Thelid will keep live food inside the tank. The lidis also a useful place on which subdued light-ing can be placed.

Temperature

Salamanders need a relatively cool environ-ment. The terrestrial axolotl should be keptbetween 70-75 °F. (15-24 °C.). If possible pro-vide a temperature gradient so the axolotl canthermoregulate by moving back and forth be-tween warmer and cooler areas. This is easilyaccomplished by designating one side of thetank as the warm side, usually the dry side. Aslight gradient of only a few degrees is needed,which can be created using lights.

Lighting

Light is necessary for the regulation of theaxolotl’s seasonal clock. Do not rely on sun-light, which could raise the temperature of thehabitat too much. Use a broad-spectrum lightconnected to an appliance timer. If you planto try breeding the axolotl, you may wish toincrease and decrease the number of hours oflight based on the changing photoperiod of theaxolotl’s native environment near Mexico City.

If possible, the light should be subdued andindirect. A hiding place should be provided forthe animal so that it is able get out of directlight if it wants.

Ventilation

Ventilation is important to prevent the at-mosphere inside the tank from becoming foul

Fall 1996

23

and to reduce the growth of organisms in thewater and soil. Drafts, however, should beavoided. This can be accomplished by using ascreen cover over the tank.

Water Filtration

To keep the water clean, either filter it or doa complete water change every other day.Choose a filter appropriate to the volume ofwater in the tank.

Food

Larger prey items should be dusted with avitamin powder that is available for herptiles(found in pet stores). Worms, small spiders,pillbugs, beetles, earthworms, small milli-pedes, aphids, small moths and other night-flying insects are suitable for more terrestrialaxolotls. In winter, crickets, mealworms, whiteworms, and tubifex worms can be purchased.Mealworms should be fed only as a supple-ment as their hard outer covering is difficultto digest. Tubifex worms can be fed in a shal-low dish with a little water in it.

Feed daily only as much as the axolotls caneat. Leftovers can be left in the tank if theyare still alive, but hold off feeding more untilthey are gone. A wide variety of food will in-sure a more balanced diet. For the most part,terrestrial axolotls need live food, becausethey rely on movement to detect their prey.You may be able to train them to eat raw beefor dead prey. Crickets, earthworms, meal-worms and the like can be grown at home orpurchased, if collecting prey is a problem.

Handling

Handling should be kept at a minimum. Ifyou need to handle an axolotl, be sure yourhands are cleaned and free of soap residues.

Wet your hands first, then scoop up and cagethe axolotl in your two hands without squeez-ing it. Use gloves (wet them first) or a net ifyou prefer. Be sure to wash your hands thor-oughly when you are done handling the axo-lotl, since amphibians can carry Salmonellaand other diseases.

Mixing Species

A note on keeping different species togetherin one tank: many species cannot tolerate thetoxins produced by other species. Puttingthem together may result in the deaths of oneor more species. The Axolotl Colony has beensuccessful in housing terrestrial axolotls andtiger salamanders of similar size together.

References

Bjorn, Byron. 1988. Salamanders and Newts:A Complete Introduction. TFH Publica-tions, Neptune City, New Jersey.

Obst, Fritz Jurgen, et. al. 1988. The Com-pletely Illustrated Atlas of Reptiles andAmphibians. TFH Publications, NeptuneCity, New Jersey.

Kaplan, Melissa. Salamanders and newts.World Wide Web Site: “http://www.sonic.net/~melissk/sallies.html”.

Mattison, Chris. 1992. Keeping and BreedingAmphibians. Sterling Publishing Inc.,New York

Zimmermann, Elke. 1983. Reptiles and Am-phibians: Breeding Terrarium Animals(Care, Behavior, Reproduction). TFH Pub-lications, Neptune City, New Jersey.

Breen, John. 1974. Encyclopedia of Reptilesand Amphibians. TFH Publications, Nep-tune City, New Jersey.

Scott, Peter. 1981. Axolotls. TFH Publica-tions, Neptune City, New Jersey.


24

To 8 liters distilled, dechlorinatedtap, or spring water add:

2.3 g potassium chloride4.29 g calcium chloride8.90 g magnesium sulfate126.72 g sodium chloride

For 100% dilute 1:4, for 50% 1:8,for 20% 1:20.

Cheryl Nugas (Holtfreter’s stock)

To 3 liters milliQ water add:

2 tablespoons magnesium sulfate2.5 teaspoons or scant tablespooncalcium chloride1 teaspoon potassium chloride240 cc sodium chloride27 g Trizma (7.4) fish grade

For 40% add 300 mls stock to 5 gal-lons milliQ water.

Wonsun KimWonsun KimWonsun KimWonsun KimWonsun Kim

Use charcoal filtered tap water thathas been aerated overnight afterfiltration.

axolotl husbandry - university of kentucky · axolotl newsletter number 25 6 axolotl larvae housing...

Documents