CCaarroolliinnaa™™ RReeppttiilleess aanndd AAmmpphhiibbiiaannss:: CCaarree aanndd CCuullttuurree This booklet provides a brief description of the general characteristics and care requirements of the amphibians and reptiles supplied by Carolina Biological Supply Company. Throughout the booklet, you will find Carolina item numbers in parentheses following the mention of a product that we carry. If you have questions about any of our products, please call 800.334.5551. Note:Under no circumstances should any animal be released into an area where it is not native. If you no longer need or want a classroom pet, investigate alternatives such as sharing them with other classes, giving them to other caring individuals, or disposing of them as humanely as possible. Safe Handling of Reptiles and Amphibians Whenever handling any reptile or amphibian, or any part of their habitats, be sure to wash your hands thoroughly with soap and warm water afterwards. The feces of these animals may harbor Salmonella, the bacterium responsible for the disease salmonellosis. Immunocompromised individuals, the elderly, and children under five years of age should avoid contact with reptiles and amphibians. These animals should not come in contact with food preparation areas, such as the kitchen sink or countertops. Additionally, reptiles and amphibians should not be allowed to roam freely in your home. With minimal precautions, both reptiles and amphibians are safe for classroom use. For more information, please refer to the Center for Disease Control’s December 12, 2003, article: “Reptile-Associated Salmonellosis—Selected States 1998–2002” (http://www.cdc.gov/). CAROLINA BIOLOGICAL SUPPLY COMPANY 2700 York Road Burlington, North Carolina 27215 Original copyright 1993 Second Edition ©2007 Carolina Biological Supply Company Printed in USA CarolinaTM Reptiles and Amphibians: Care and Culture Amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Terrestrial Salamanders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Aquatic Salamanders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Frogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 Bullfrogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Xenopus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Fire-Bellied Toad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 Giant Toad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 Green TreeFrog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 Dwarf AquariumFrog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 Amphibian Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 Salamander Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Rana Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Other Frog and Toad Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Xenopus Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 Amphibian Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Acclimating the Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Metamorphosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 Reptiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 Turtles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 Snakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 Lizards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 Preparing a Vivarium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29 3 Amphibians Amphibians are a class of “cold-blooded” (poikilothermic or ectothermic) vertebrates including caecilians, frogs, toads, and salamanders. The females lay their eggs in or near water since the larval stage is usually spent in the water. The name “amphibian” derives from the Greek words amphimeaning “both” and biosmeaning “life,” or “living both on land and in water.” Amphibians have smooth or warty skin with no scales but with many mucous glands that keep the skin moist. These glands may be modified to exude poisons for self-defense. Although few amphibians are dangerous to humans, we recommend that you wash your hands thoroughly each time after handling amphibians. With only a few exceptions, amphibians lack claws on the toes. Terrestrial Salamanders Keep salamanders in a covered vivarium if you are going to keep them for more than a few days. They can be held temporarily in a slanted container with a small amount of water at one end. The temporary container should have a tight-fitting lid that allows air circulation. Salamanders require high humidity and cool temperatures. Hot, dry conditions cause salamanders to become much less active than normal. Sprinkle water on the leaves of plants and the side walls of the tank every other day, and maintain the temperature at 18–21°C (64–70°F). A small fluorescent bulb placed near the vivarium provides adequate light without creating excessive heat. Feed the salamanders mealworms, crickets, whiteworms, earthworms, or soft- bodied insects of appropriate size. Eating a variety of foods keeps the salamanders healthy. Remember that overfeeding causes stress and that larger food organisms may harm the salamanders if they are present in large enough numbers. Remove dead and uneaten food after the salamanders have finished eating. Provide fresh drinking water at all times. If you are raising terrestrial salamanders from larvae, you will need an aquatic habitat. The larvae have gills, which they will gradually lose over the course of several weeks. You must provide something for the larvae to crawl onto as their lungs develop, and you will eventually convert the aquatic habitat into a vivarium as described above for Keep salamanders (14-6070) in a vivarium with high humidity and cool temperatures. the adult salamanders. 4 Aquatic Salamanders Necturusis an aquatic salamander found in rivers, lakes, and streams throughout North America. Necturusis neotenic, retaining the larval form throughout life. Keep Necturusin a large container of water that has a tight-fitting lid to prevent escape. Provide hiding places for the salamanders by piling rocks to form crevices and caves. Spring or pond water (16-3380) is best, but tap water can be used if it is treated with a water conditioner capable of removing both chlorine and chloramines, as well as Necturus(14-6030) is a large neotenic detoxifying harmful ammonia (67-1985). salamander. Salamanders should be housed individually to prevent fighting and spread of disease. Although Necturusis neither poisonous nor aggressive, handle them carefully to avoid bites. To prevent temperature shock when transferring Necturus, place the shipping bag in or beside the holding tank until the temperature of the water in the shipping bag matches the temperature of the water in the holding tank. This may take 1 or 2 hours. The best temperature at which to hold Necturusis 4–10°C (39–50°F), but they can be held at room temperature (20–26°C [68–79°F]). Necturuswill eat earthworms, tadpoles, small grass frogs, and small crayfish. Waste materials and uneaten food must be removed from the tank daily. Newts Newts (14-6050) are aquatic salamanders that do not retain external gills as adults. Newts can live in aquaria or any other containers of water. Most often, the newts shipped from Carolina Biological will be broken-striped newts (Notophthalmus viridescens dorsalis). A floating platform must be provided so they can crawl out of the water and rest. Newts are non-territorial and non-aggressive, so several may be housed in one container. Newts eat small tadpoles, small insects, worms, and bits of raw meat, or canned, all-meat dog food. Do not foul the water with too much food, and remove all uneaten and dead food promptly. Frogs Carolina ships frog eggs, live frogs for breeding, and frogs for general use. When the frogs arrive, empty the box into 15–25 cm of cool water (10–15°C [50–59°F]) in a deep container (such as a bucket or plastic trash can) and wash the frogs free of the packing material, which kept them moist in transit. We recommend that you 5 use the frogs for experimental purposes as soon after arrival as practical; the shock of shipping is hard on frogs and reduces their defenses against endemic microorganisms. Reddened legs on a frog may be a symptom of environmental shock. Even healthy frogs may be traumatized by shipment and arrive with reddened ventral surfaces; we can professionally pack healthy frogs, but we cannot control their exposure to stressful conditions or handling while in transit. We do guarantee to ship healthy frogs, but the recipient must provide reasonable care. Be sure to tell us the expected use date of the frogs so that we can package and ship accordingly. If you need to maintain the frogs for some time, be prepared to provide very good care. If an arriving frog is to be added to a tank with other frogs, we recommend that you quarantine the new frog for several days before adding it to the established tank. Generally, illness in frogs results from unclean habitats, malnutrition, or excess stress. Lethargy, thinness, and cloudy eyes are all signs of illness. Any frog you suspect of being sick should be quarantined. Quarantined frogs can be kept temporarily in shallow water in aquaria or plastic containers. Keep the water 2–5 cm deep and provide a platform or rocks so the animals can get completely out of the water. Use chlorinated tap water to reduce bacterial growth, and change the water daily. Very sick frogs can be treated with an antibiotic such as tetracycline. Many home remedy suggestions may be found both in literature and online, but it is best to take any sick animals to a veterinarian for treatment. Carolina ships both northern and southern grass frogs (also known as leopard frogs), depending on the season. We recommend a maintenance temperature of 10–15°C (50–59°F) for northern grass frogs and 15–20°C (59–68°F) for southern grass frogs. Outside these temperature ranges, the frogs tend to have more The mature southern grass frog, Rana sphenocephala(left), is generally larger than the northern grass frog, Rana pipiens, as illustrated by these two females. 6 disease problems. Most of the grass frogs shipped from Carolina Biological are northern grass frogs. These frogs tend to be less active than their southern counterparts, making them well suited to captivity. You can differentiate between the two species by examining their spots. The northern grass frog has dorsal spots outlined with a light color, while the southern grass frog has solid dark spots. Adult frogs will eat most animals that move above the water and are not too large, such as sowbugs, flies, mealworms, crickets, caterpillars, moths, and earthworms. A variety of food items may be offered to your frogs. Frogs should be fed in a dry area to allow them to capture the food easily and to prevent food organisms from drowning and fouling the water. Offer food daily. Bullfrogs Bullfrogs are also available from Carolina. Bullfrogs should be maintained at 20–25°C (68–77°F). Most often we ship Rana catesbeiana, but this may vary with availability. Adult bullfrogs eat a variety of foods, from crickets to small turtles, snakes, and even other bullfrogs—basically any moving thing of edible size. Excess food items and waste materials should be removed often, as tank cleanliness is essential for bullfrog health. Bullfrogs are native to the eastern United States but can be found in the wild as far west as the Rockies. Bullfrogs are known for their ability to colonize new areas and endanger native species. For this reason, never release any bullfrog or their progeny into the wild. Male bullfrogs have a loud, bellowing call. If you plan to house a single bullfrog for any period of time, an aquarium size of at least 20 gallons is suggested. Five additional gallons should be allotted for each individual added. Floating plants, such as those found in Carolina’s Floating Plants Set (16-1790), are a good addition to any bullfrog tank. Bullfrog, Rana catesbeiana 7 All stages of bullfrog development, from egg to adult, are available. For tadpole care requirements, please refer to the section Amphibian Larvae, in this manual. Bullfrog Eggs (200–300) 14-6540 Pack of 6: 14-6550 Bullfrog Tadpoles (Up to 2”) Pack of 12: 14-6552 Pack of 50: 14-6554 Pack of 6: 14-6560 Bullfrog Tadpoles (2” to 4”) Pack of 12: 14-6562 Pack of 25: 14-6564 Pack of 12: 14-6470 Bullfrog Tadpoles (Hind Limb Bud Stage) Pack of 100: 14-6472 Pack of 6: 14-6570 Two-Legged Bullfrog Tadpoles Pack of 25: 14-6572 3”: 14-6530 Adult Bullfrogs 4”: 14-6532 5”: 14-6534 Breeding Frogs Either northern or southern grass frogs can be obtained year-round. A certain number of female southern grass frogs reach sexual maturity during every season, so we can ship mature southern grass frog females any time except during winter months when it is too cold to collect them. During the winter, northern grass frogs are available for breeding experiments. Northern grass frogs normally reach sexual maturity in the fall after a summer of feeding. Mature frogs are collected in late fall and held at low temperatures (8–12°C [46–54°F]) during the winter months with little harm. Southern grass frogs cannot withstand such cold temperatures. In the wild, southern grass frogs ovulate throughout the year, while northern grass frogs ovulate only in the spring. In either species, ovulation can be induced in mature females by artificially increasing the level of pituitary hormones. The eggs can then be stripped from the uterus by hand, as described later in this manual. The female’s pituitary level is increased by injecting whole frog pituitaries, frog pituitary suspension, or a pituitary progesterone mixture into the body cavity. Carolina offers preinjected frog sets, pituitary extracts, gravid female frogs, mature females with eggs (14-6350), and mature males (14-6356). The species of frog supplied depends upon the season. The Preinjected Frog Set (14-6300) includes one injected female frog and two male frogs for sperm. The Pituitary Extract (14-6330) includes six dried frog pituitary glands for inducing ovulation and yields the best results when used with the northern grass frog. Gravid female frogs (14-6340) have been injected with pituitary extract and are available individually. 8 In addition to frogs and appropriate containers, the following materials are needed to demonstrate induced ovulation: *heavy scissors (62-1775) *dissecting needles (62-7231) *fine scissors (62-1810) culture dishes (at least 12) (74-1004) *forceps (62-4504) hypodermic needle *medicine droppers (73-6903) syringe *Carolina offers a wide variety of dissecting instruments and can customize a dissecting kit for you if needed. All instruments that come in contact with the eggs must be biologically clean. Glassware should be washed in hot, soapy water and rinsed thoroughly. Avoid glassware that has been used with fixatives. Chromium-plated instruments should not come in contact with living frog eggs. Pond water or springwater known to support frog eggs is used for culturing (see Amphibian Eggs, in this manual). Tap water may be used if it is treated with a water conditioner (67-1985). Distilled water does not contain the salts necessary for normal development of the eggs. The water used should be at room temperature. Removing Pituitary Glands Carolina offers preinjected frogs from November to March, as well as prepared dried pituitary glands, but you may wish to collect pituitary glands yourself. To remove the pituitary gland from a frog, first cut off the frog’s upper jaw and head, using heavy scissors and cutting along the line indicated in the following figure; this will kill the frog. The brain and part of the medulla will be intact. Insert the point of the fine dissecting scissors into the back of the cranial cavity. Keep as far as possible to the right side to avoid damaging the pituitary gland. Cut through the bone. Withdraw the scissors and cut along the left side in the same manner. This should release the floor of the cranial cavity as a small flap. Pull the flap up with a pair of forceps to expose the pituitary, which will be either adhering to the flap or lying on the ventral surface of the brain . Pituitary Cut lines 9 Site of Injection The pituitary gland is a small, kidney-shaped organ often embedded in a mass of white lymphatic tissue. Remove the lymphatic tissue by carefully rolling the gland on a damp paper towel. Do not rupture the gland, as it will lose much of its effectiveness if broken. Place the gland in a small dish with 1 mL of aged tap water or springwater. When all pituitaries are removed, draw them into Site of injection the syringe with a small amount of water. Put a needle on the syringe and inject the pituitaries intraperitoneally. Be sure that the pituitaries do not cling to the sides of the syringe. It is best to inject through the wall of the lower abdomen, directing the needle anteriorly. Be careful not to damage the ventral abdominal and lateral veins or the internal organs. For best results, a female northern grass frog should be injected with six whole pituitary glands at once; this is best done at some point between November and April. In October, November, and December, ovulation requires 72 hours. During January and February, ovulation occurs in 48 hours, while in March and April, eggs can be obtained in 24–36 hours. Female northern frogs that have ovulated should not be held in the refrigerator more than 48 hours. The female southern frog also requires six pituitary glands for artificial induction, but the injections are split into two sessions (as described in the following section, Using the Pituitary Extract). Place the injected female in a container in a quiet, dimly lighted, undisturbed area. When the eggs are ready for release from the ovary, the lower abdomen will bulge on both sides when the upper abdomen is pressed. When the female is ready to release eggs, pith a male frog and remove the testes. The testes are fairly large, oval, yellowish bodies on the dorsal surface of the peritoneal cavity posterior to the kidneys. The testes should be thoroughly chopped or crushed in about 10 mL of pond water. Sperm activity begins approximately 5 minutes after release from the testes and can be observed with a compound microscope. It may be desirable to strip the eggs from a single female in several batches Artificial induction of eggs from a frog (e.g., for two or three different embryology laboratory sections). If so, simply remove a few eggs at each session. To strip the eggs, hold the female firmly in the palm of one hand while holding her legs in the other hand. With 10