1
Q
- Amphibian is derived from the greek word “amphibios”. What does it mean?
A
“double life” describes the aquatic larval stage and postmetamorphic terrestrial lifestyle of many amphibians
2
Q
- What 3 orders are found in the class Amphibia?
A
Gymnophioma (caecilians), Caudata (salamanders), and Anura (frogs)
3
Q
- What are the 3 groups of Caudata?
A
Sirens (eel-like amphibians), primitive salamanders (hell benders and related species), advanced salamanders (mudpuppies, amphiumas, axolotls, newts, and many terrestrial species)
4
Q
- What is neotony?
A
Condition where salamanders retain larval characteristics while becoming fully functional reproducing adults
5
Q
- Which salamander species demonstrate neotony?
A
Hellbenders (Cryptobranchus sp.), Ambystoma mexicanum (Axolotl), Ambystoma tigrinum (tiger salamander)
6
Q
- Frogs can be divided into 3 groups. What are they? Give examples.
A
Primitive frogs-family Discoglossidae (fire bellied toad-Bombina orientalis and midwife toad-Alytes obstetricans), Transitional frog-family Pelobatidae (spade foot frog-Scaphiopus), Advanced frogs-family Pipidae (Pipa and Xenopus) and 16 other families (Bufonidae, Dendrobatidae, Hylidae, Leptodactylidae, and Ranidae)
7
Q
- How can you tell males from females in Rana catesbiana (bull frog), R. grylio (pig frog), and R. clamitans (bronze frog)?
A
Male-the typanum is larger than the eye and , in females, the tympanum is the same size as the eye.
8
Q
- How has Xenopus laevis (African clawed frog) been used in research?
A
Originally used in pregnancy tests (assays). Injection of pregnant woman’s urine into the dorsal lymph sac of a female Xenopus would induce the frog to lay eggs. Used in cell and develomental biology. Can inject with commercially available hormones to ensure that the frogs reproduce year-round. Also Xenopus have been the subject of developmental toxicology investigations using FETAX assays (frog embryo teratogenesis assay: Xenopus). Xenopus has also been used in molecular biology by injecting oocysts with foreign DNA or messenger RNA to allow expression of genetic material.
9
Q
- List other amphibians used in research.
A
Mudpuppies (Necturus)-comparative anatomy, axolotls (Ambystoma mexicanum)-developmental research, axolotls and newt-limb regeneration, frogs (bull frogs-Rana catesbiana and leopard frogs-R. pipiens)-physiology and also effects of emetics because they regurgitate easily, and antinociceptive (pain relieving) effects of analgesia
10
Q
- What are the most important considerations when choosing primary enclosures for amphibians?
A
Should be constructed of impermeable, easily sanitized material; able to withstand multiple cage washings; adequate height to accommodate behavioral needs of climbing and jumping species; and fitted with nonabrasive lids
11
Q
- What environmental conditions are amphibians susceptible to?
A
Dessication if environment is too dry and acidic pH (soil or substrate)
12
Q
- Many amphibians will die from exposure to chlorine in water. How can water be dechlorinated?
A
1) Age open containers of water for 24-48 hours.
2) Add sodium thiosulfate to water.
3) Pass tap water through activated carbon filters.
4) Aerate the water.
13
Q
- Cloramines, more toxic than chlorine, are sometimes added in municipal water systems. How are these removed?
A
Use an unused activated charcoal system. Alternatively, add sodium thiosulfate which reacts with the chloramines to form ammonia. The ammonia can be removed with zeolites.
14
Q
- Most amphibians have thermal zones lower than reptiles. What are the preferred zones for tropical and temperate amphibians?
A
Tropical species-21-29o C (70-85o F), temperate species-18-22o C (65-72o F)
15
Q
- If salamanders of the genus Plethodon are maintained at temperatures over 20 C (68 F), what will happen?
A
Prevents animals from assimilating food rapidly enough to meet the need of the increased metabolic rate.
16
Q
- What type of lighting and light:dark cycles are recommended?
A
Direct exposure to bright light should be avoided. Use full-spectrum bulbs. Provide shelter to escape light. 12:12 light cycles are adequate unless breeding or trying to mimic natural habitat.
17
Q
- What relative humidity is recommended for most amphibians?
A
80%
18
Q
- Animal room airflows my dry out wet environments. How can this be alleviated?
A
Decrease room airflows and house in primary enclosures with minimal openings
19
Q
- How often should the primary enclosures of amphibians be cleaned?
A
Singly housed terrestrial animals with ample floor space can be cleaned every 2 weeks with spot cleaning as needed. Group housed should be cleaned more frequently as needed.
20
Q
- Can phenols be used to clean amphibian enclosures?
A
No, phenols are toxic and can be absorbed through the skin.
21
Q
- Normally amphibians have a protective mucous layer on the skin and have the ability to secrete toxins through the skin when threatened. What is the proper way to handle these animals?
A
Glove should be worn that are free of powder and moistened with dechlorinated water. Do not use abrasive paper towels. Nets of soft fine mesh can be used. Small terrestrial can be restrained in 1 hand. Larger salamanders should be grasped behind the head and around the pectoral girdle with 1 hand and around the pelvic girdle with the other hand. Do not pick up by the tail as it will release! (predator avoidance)
22
Q
- What are 5 methods of identifying amphibians?
A
Individual color patterns on the animals. Toe clipping-ineffective in long term studies because the toes regenerate. It also requires anesthesia, sterilized instruments, and hemostasis. Tattooing and freeze or chemical branding has been tried but it is not permanent. Glass or plastic beads have been sewn with nonabsorbable sutures to amphibians but they must be secure to a muscle mass or they will be shed. Lastly, passive integrated transponders have been implanted intracoelomically.
23
Q
- How long should amphibians be quarantined for long term studies?
A
2-4 weeks minimum and longer is wild caught
24
Q
- What are some zoonotic diseases that may be carried in amphibians?
A
Atypical mycobacterosis (Mycobacterium fortuitum, M. marinum. M. xenopi), Salmonella, and Chlamydia psittaci
25
25. What are the 2 primary types of skin glands present in amphibians? What do they secrete and why?
Mucous glands secrete a slimy protective layer which prevents mechanical damage to skin, facilitates retention of body fluids, and provides a barrier against pathogens. Granular glands synthesize and secrete a variety of compounds that protect against predators, as well as chemicals that have antibacterial and antifungal properties. Defensive compounds can have neurotoxic, cardiotoxic, myotoxic, hallucinogenic, hypotensive and vasoconstrictive activity. Toxic alkaloids are found in poison dart frogs (Dendrobatidae), Bufo marinus, and other species can cause vomiting, respiratory paralysis, and sometimes death.
26
26. What are the antimicrobial peptides called that are secreted in the skin of Xenopus laevis?
Magainins which inhibit G+ and G- bacteria, several fungi, and some protozoal species.
27
27. What other chemical compounds can be secreted by granular glands?
Pheromones -courtship and mating
Dermorphins -potent opioid and endogenous analgesic
Bioadhesives -temporary entrapment of predators or adhere to female during mating
28
28. Frogs have pronounced ribs. T/F?
False, ribs are absent or greatly reduced.
29
29. What is the primary respiration surface of most amphibians?
skin
30
30. What forces air in and out of lungs in amphibians which have lungs?
Movement of buccopharyngeal floor
31
31. How many chambers are found in larval and adult amphibians?
Larval-2 chambers, adult-3 chambers
32
32. What anatomical feature may impact the pharmacokinetics of drugs with hepatic excretion?
Hepatic portal veins drain the rear half of the amphibian’s body.
33
33. The lymphatic system of amphibians drains directly into the venous system. The lymph is first collected into lymph sinuses. Where is the most accessible place to inject a substance that would be transported to the venous circulation?
Dorsal lymph sacs which lie SC over sacral area and lateral to midline
34
34. Adult amphibians are herbivorous and larval stages are carnivorous. T/F
False, adults are carnivorous and larval may be herbivorous, omnivorous, or carnivorous.
35
35. How dos Xenopus prehend food?
They direct food in with their front legs but many other species use their well developed tongue.
36
36. Can amphibians vomit?
Yes and often use vomition as a defensive mechanism. Some frog species evert part of the stomach during regurgitation.
37
37. What type of kidney is found in salamanders and frogs?
Mesonephric-lacks the ability to concentrate urine in excess of plasma levels
38
38. Do amphibians secrete ammonia or urea?
Aquatic amphibians secrete ammonia and terrestrial amphibians secrete urea.
39
39. How many cranial nerves are present in amphibians?
10, the hypoglossal nerve (cranial nerve XII) is formed by branches of the first 2 spinal nerves.
40
40. A lateral line system (similar to fish) is well developed in larval amphibians and is retained by adults of many aquatic species. What is its function?
The linear arrangement of neuromasts on the head and along the body detect changes in water pressure and currents and function in locating prey.
41
41. Can amphibians detect color?
Yes, 2 types of rods (red, green) are responsible for color sensitivity.
42
42. How do amphibians detect odors?
Vomeronasal (Jacobson’s) organ
43
43. Do amphibians live longer in northern or southern climates?
Northern
44
44. How many years have Xenopus laevis been documented living ?
Fifteen (15) years
45
45. What 2 types of blood cells are nucleated in amphibians?
Red blood cell and thrombocytes
46
46. What is the preferred choice of food of most adult amphibians?
Carnivorous
47
47. If only raw meat and organs are fed to growing amphibians, what may be the result?
Calcium deficiency
48
48. Most whole vertebrate prey constitute a balanced diet. Why is this?
Bones-Ca, P, and magnesium, liver/kidneys-vitamins, pancreas-zinc, thyroids-iodine
49
49. Crickets (and other insects) lack a Ca-rich skeleton. How can the crickets be treated to supplement Ca?
Dust a vitamin-mineral mix on crickets before feeding to amphibians or feed insects with a diet that is vitamin-mineral rich (gut loaded) before feeding to amphibians.
50
50. How often should amphibians be fed?
Daily to twice weekly, depending on species, age of animal, and ambient temperature
51
51. Should amphibians be group housed?
No, especially terrestrial species, because they are territorial. They mark territories with fecal pellets and granular gland secretions and will fight to defend territories.
52
52. Sexual dimorphism exists in many amphibian species. What are some male characteristics?
Male bullfrogs, pig frogs, and bronze frogs have a tympanum larger than the eye. Vocal sacs of male frogs become larger and more pigmented during breeding season. Cloacal glands in salamanders become swollen which results in enlarged cloacal lips. Male plethodontid salamanders develop hedonistic glands on the chin which are rubbed on the female during courtship. Most male frogs develop keratin pads on their thumbs to better grip females during amplexus. Certain frog and salamander males develop teeth to abrade the skin of females. It allows introduction of chin gland secretions into the female’s bloodstream.
53
53. What is amplexus?
Prolonged breeding embrace
54
54. Do frogs and salamanders have internal or external egg fertilization?
Frogs have external fertilization and salamanders have internal.
55
55. What is unusual about care of the young in the Surinam toad (Pipa pipa)?
After a circuitous amplexus, the fertilized eggs land on the back of the female where the skin covers them until metamorphosis when froglets emerge.
56
56. What is unusual about care of the young in the midwife toad (Alytes abstricans)?
The male wraps egg strands around his legs and keeps them moist until hatching.
57
57. What unusual reproductive behaviors are seen in the Darwin’s frog and Rheobatrachus?
The male Darwin’s frog takes the newly hatched tadpoles in his mouth and holds them in vocal sacs until metamorphosis. The Rheotrachus female swallows fertilized eggs. Eggs and tadpoles live in her stomach until metamorphosis. During this time all gastric activity ceases due to release of prostaglandin.
58
58. What regulates reproductive behavior?
Arginine vasotocin
59
59. How should axolotls be bred?
Hold males and females separately for 1 week at 72o F then place them together in a shaded tank at 54o F. Breeding and egg deposition will occur within 2 days. After oviposition, remove eggs and incubate in separate containers.
60
60. How does Xenopus tropicalis differ from X. laevis?
X. tropicalis is smaller, has half the haploid genome size per nucleus and has 20 chromosone pairs (X. laevis has 36), generation time is 4-6 months, females lay 3000 oocytes/ovulation, and the embryos are less variable in development
61
61. What is the natural habitat of Xenopus sp.?
Central to South Africa. Found in rivers, lakes swamps, ditches, and wells. It appears to prefer still, cloudy water.
62
62. Distinguish between male and female Xenopus.
Adult males have dark inner surfaces on front limbs (nuptial pads). Females have large cloacal papillae and are larger than males.
63
63. What are some components of Xenopus skin secretions?
Thyrotropin releasing hormone, caerulein, xenopsin, and magainins (antimicrobial compounds)
64
64. How does Xenopus eat?
They shred prey with rear claws and use their front feet to shove food into their mouth (no tongue).
65
65. How does Xenopus detect smell?
Two distinct olfactory cavities. One detects scent in water and the other detects airborne odors.
66
66. How does Xenopus respire?
They must come to the surface to gulp air because cutaneous respiration is not as well developed as in other species. Even tadpoles develop and use lungs as well as gills for breathing.
67
67. How does Xenopus handle waste products during periods of drought?
Xenopus produces urea instead of ammonia. They revert back to ammonia as soon as ample water is available to dissipate it.
68
68. What is the life span of Xenopus?
Ten years in the wild and 15 years in the lab
69
69. Xenopus tanks can be made of what types of materials?
Fiberglass, glass, plastic, or stainless steel
70
70. Do Xenopus tanks need covers?
Yes, Xenopus will jump out if the water level is low or if startled.
71
71. Clawed frogs have been shown to prefer opaque containers to clear containers because they do not feel threatened. What are other things that can be added to the frog’s environment that will allow the frog to hide?
Partially cover tanks or add retreats such as PVC pipe, stainless steel rabbit feeders, styrofoam, sponge, round ceramic tiles, terra cotta pots, and sandstone.
72
72. If static water systems are used, how often should the water be changed?
After the animal has been fed to remove excess food and defecated material.
73
73. What are 2 other types of water systems in addition to static?
Flow through-nonrecirculating and flow through-recirculating
74
74. What process should water go through before adding the frog tank?
Dechlorination
75
75. Population density within the Xenopus tank is a critical factor in growth and productivity. What are suggested stocking densities?
One frog/3 liters water to 4 frogs/5-10 liters
76
76. What is the optimum temperature range for water in the Xenopus tank?
20-22 C (68-72 F)
77
77. What is the recommended light:dark cycle of breeding year-round?
12/12
78
78. How should Xenopus be handled?
They should be handled with a soft net or gloves to prevent skin abrasion and disrupting the protective mucous membrane.
79
79. What are 3 methods that have been used to identify individual animals?
The first is sewing a bead on a thread to the skin of the frog. The thread or suture must be anchored in a muscle mass to prevent sloughing. The second method is to graft pale skin from the ventral surface of the frog to the back of the frog in the desired pattern. Thirdly, microchips can be injected into the dorsal lymph sac.
80
80. Historically, Xenopus were fed beef liver, beef heart, or ground meat. These were not nutritionally complete. What is used now?
Commercially prepared foods-salmon chow, trout chow, and sinking frog food
81
81. How often should clawed frogs be fed?
2-5 times/week
82
82. Additional treats include earthworms, mealworms, chick embryos, tubifex worms, gold fish, and crickets. What is a possible complication of feeding too many crickets or mealworms?
Intestinal obstruction
83
83. At what age do Xenopus reach optimum egg production?
2-3 years and continue for several years
84
84. How often are Xenopus females bred in the laboratory?
Every 1-4 months (ideal induced breeding interval)
85
85. How are Xenopus induced to ovulate and breed?
HCG (human chorionic gonadotropin) is injected into the dorsal lymph sacs of both the male and female in 2 separate injection 1-5 hours apart. Males receive 400 units/injection and females get 150-250 on the first injection and 650-850 units in the second injection. Both frogs are placed into FETAX solution and allowed to breed. If frogs are bred in containers with false bottoms, the fertilized eggs can be removed safely.
86
86. How can the jelly coat be removed from the frog embryo within the eggs?
Swirl in 2% cysteine solution at pH 8.1 for 1-3 minutes, decant, rinse embryos with FETAX solution 5-8 times
87
87. What are the components of FETAX solution and what does FETAX stand for?
NaCl-625 mg/liter, NaHCO3-96, KCl-30, CaCl2-15, CaSO4.H2O-60, MgSO4-75, mixed with deionized, distilled water. FETAX stands for “frog embryo teratogenesis assay: Xenopus”.
88
88. What do Xenopus tadpoles eat for the first 4 days after hatching?
They do not feed while they are absorbing the yolk. They start eating by Day 5.
89
89. What do Xenopus tadpoles eat in the wild?
The bulk of the diet is plankton suspended in water.
90
90. What are Xenopus tadpoles fed in captivity?
They can be fed strained baby food green vegetables or algae powder; nettle powder, baker’s yeast, and bonemeal in a 7:2:1 ratio; or pelleted food to larger tadpoles.
91
91. What is the recommended stocking density for Xenopus tadpoles?
1-10 tadpoles/liter of water
92
92. What type of environment should be given to tadpoles undergoing metamorphosis?
Shallow water to prevent drowning
93
93. What anesthetic agent is usually used on female frogs to surgically harvest oocytes and in what concentration?
Tricaine methane sulfonate (MS 222) 0.5-2.0 g/liter, buffered with sodium bicarbonate 0.42-1.05 g/liter
94
94. How is frog skin prepped for surgery?
Apply dilute povidone iodine or chlorhexidine solution. Do not use scrub that will disrupt the mucous layer.
95
95. What analgesics have been used in leopard frogs and will probably work in Xenopus post op?
Morphine (10 mg/kg intracoelomic q 12 hrs), flunixin (25 mg/kg intracoelomic q 4 hrs), butorphanol (25 mg/kg intracoelomic q 12 hours), or xylazine (10 mg/kg intracoelomic q 12-24 hrs).
96
96. How often can oocytes be harvested surgically?
Rest frogs at least 1 month between surgeries. The IACUC at UT allows a maximum of 6 survival surgeries/frog.
97
97. Blood samples from amphibians should collected in lithium heparinized tubes. Identify blood collection sites in frogs and salamanders. (Use 26-27g needles.)
Midventral abdomenal vein in frogs and salamanders, ventral caudal vein in salamanders, lingual venous plexus beneath tongue in frogs (use heparinized capillary tubes), and cardiocentesis in frogs and salamanders (should be anesthetized)
98
98. What are other diagnostic techniques used in amphibians?
Fecal exam, bacterial swabs, impression smears, skin scrapings, abdomenocentesis, biopsy, radiographs, fluoroscopy, endoscopy, transillumination
99
99. Name common routes of injection.
Subcutaneous, intramuscular, intracoelomic, and dorsal lymph sac
100
100. How are amphibians most commonly euthanatized?
Overdose of MS 222 or other immersion anesthetic at concentration exceeding 3 g/liter of buffered water
101
What is the primary etiologic agent of atypical mycobacteriosis
Mycobacterium marinum
102
What was the first organism isolated from amphibians with red leg?
Aeromonas hydrophilia
103
Bacterial causes of skin sloughing, discoloration, ulceration in Necturus
Pseudomonas, Aeromonas, Proteus spp.
104
Clinical signs of mycobacteriosis
wasting, pneumonia, dermal nodules and ulcers, loss of diving reflex, distended abdomen
105
Clinical signs of Cryseobacterium infection
weight loss, edema, septicemia, ascites petechia, dyspnea, uveitis, corneal edema, incoordination
106
Zoonotic agents that infect amphibians
Mycobacterium, Cryseobacterium, Salmonella
| Chlamydophila
107
Clinical signs of salmonellosis
anemia, lethargy, anorexia, diarrhea (infects intestinal tract)
108
Clinical signs of chlamydiosis
lethargy, bloating, dysequilibrium, subcutaneous edema, hydrocoelom, bloat, and erythema and patchy depigmentation of skin
109
Lucke herpesvirus
affected species
pathogenesis
clinical signs
Ranid herpesvirus 1 (RaHV-1)
Lithobates pipiens
renal adenocarcinoma, exp. in warm months
nonclinical until advanced stages, then emaciation, lethargy, ascites, death
110
Ranavirus
pathogenesis
clinical signs
Iridoviridae, DS DNA
eosinophilic INIB in RBCs or basophilic ICIB in stomach glands
infects skin then viscera
emaciation, erythema, ulcerations
111
Major nematode parasites of frogs
```
Pseudocapillaroides xenopi (xenopus epidermis)
Rhabdias (frog lung worm)
```
112
Target organs of Polystoma, Gyrodactylus, Nematotaenia
Polystoma (trematode) - bladder
Gyrodactylus (trematode) - skin/gills
Nematotaenia (cestode) - intestinal tract
113
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Bd
causative agent
target organ
mortality rate
necropsy findings
```
Batrachochytrium dendrobates
skin (keratinophyte)
high mortality
hyperkeratosis, dysecdysis, secondary infections
Amphibians
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