18. Amphibians biology, diseases and models

Ambystoma mexicanum (axolotyl)

Ambystoma tigrinum (tiger salamander)

Anaxyrus (Bufo) americanus (american toad)

Rhinella marina (cane toad)

Dendrobatid spp. (poison dart frogs)

Lithobates pipiens (leopard frogs)

Lithobates catebeianus (bull frog)

Xenopus laevis (African clawed frog)

What is the research use of axolotls and newts?

Regenerate limbs, tail, jaws, skin, spinal cord, brain, heart apex

Joint regeneration, blastema function, and scar-free healing

Oocytes used in Breast cancer research

What is the research use of Tiger salamander (Ambystoma tigrinum)?

Vision and retinal research

Agrochemical and pesticide toxicity

Animal model for acetic acid wiping response - antinociceptive effects of analgesics

L. pipiens (leopard frog)

Animal model for teaching physiology?

Lithobates catebeianus (bull frog) and Lithobates pipiens (leopard frogs)

Amphibian that has had genome sequenced and has 10 chromosomes

Xenopus tropicalis

What characteristics do family Pipidae (african clawed frogs, Xenopus laevis) have?

(5th toe much longer than tibia)

No tongue or reduced tongue

No eyelids

Convex cornea

What is FETAX=Frog embryo teratogenesis assay?

Developmental toxicology research

96-hour test

Count developmental malformations

early stage (mid-blastula) whole embryos to measure the potential of chemicals to cause mortality, malformation and growth inhibition

What animal’s eggs are the animal model used in FETAX?

Xenopus laevis

What animal model has been used for pregnancy testing in humans? How does this work?

Xenopus laevis

inject suspect pregnant person’s urine into dorsal lymph sac and the frog will lay eggs if the human is pregnant

Why should you be cautious with natural substrates?

Can harbor chytrid fungus and viruses

What type of amphibians are the most territorial?

terrestrial

provide barriers and retreats

What size should gravel be so that axolotls do not ingest it? Can cause GI blockage

>3cm in diameter

What kind of water can be provided to an amphibian?

-dechlorinated / dechloraminated

-reconstituted reverse osmosis or deionized

What are the 4 ways water can be dechlorinated?

-open container of water and age for 24-48hr

-aerate water

-add sodium thiosulfate

-pass water through activated carbon filter

How do you remove chloramines from water?

-toxic and do not evaporate

-sodium thiosulfate or activated charcoal filter

Caution should be taken when using sodium tiosulfate to dechlorinate water. Why?

-releases ammonia

-zeolites can remove the ammonia

Hard water is higher in ___ and ___. Species used to hard water that are then housed in soft water may develop ______.

-calcium and magnesium

-edema and renal problems

What solution is an alternative to hard water?

Holfreter’s solution

Axolotls prefer hard or soft water?

hard

What pH should the water be at for amphibians?

6.8-7.1

What is the most common gas associated with gas bubble disease in amphibians?

nitrogren

When using a flow through water system with axolotls the flow thorugh rate matters. If it’s too ___ it can lead to ____.

high

skin lesion

Amphibians are ____ and rely on behavioral thermoregulation to maintain core body temp.

ectothermic

Electrical outlets should be ____ to prevent electrical shock

ground fault interrupted

What is effective against chytrid fungus?

-Heat (140F), dessication

-virkon, benzalkonium chloride, 2% bleach, 70% ethanol

What is effective against ranavirus?

-chlorohexidine

-3% bleach

-1% virkon

-all need 1 min contact time

What disinfectant should you never use with amphibians? Which ones should you not use with dendrobates sp. (poison dart frogs) specifically?

-Phenolics

-iodine based disinfectants

What should be worn when handling amphibians?

-powder free gloves moistened with dechlorinated water

What species eject a toxin when parotoid gland is pressed

Rhinella marina (cane toad)

How to ID amphibians?

-color pattern, photos

-tattoo, freeze brand, chemical brand - not permanent since they shed skin

-glass or plastic beads sutured to muscle layers

-passive integrated transponder (PIT) tags

-visible injected or implanted elastomers

-toe clipping - debatable, doesn’t work if regenerates (only 1 foot, avoid specific toes critical for locamotion, IACUC approval, anesthetics/analgesics)

Quarantine of amphibians?

-house separately, handle last, treat for parasites, lasts 4-6 weeks

-2-3mo for Xenopus if wild caught

Zoonotic amphibian dz - causes self limiting cutaneous lesions on fingers and hands

Mycobacterium marinum (fortuitum, xenopi, chelonae, gordonae, lifandi, avium, ulcerans, szulgai)

Potentially zoonotic amphibian dz but no documented amphibian to human transfer

Chalmydiophila psittaci

fragile lungs of amphibian lacking alveoli

2 types of glands in amphibians. What are their purposes?

• Mucous glands: slimy protective layer secreted to prevent mechanical damage, facilitate retention of body fluids, barrier against pathogens

• Granular: synthesize and secrete variety of compounds that protect against predators (pheromones) and have anti bacterial/ fungal properties (peptides- dermorphin (opiod analgesic))

  • Parotoid gland: toads (Bufonidae), as well as fire salamanders, defensive secretions

  • Toxic alkaloids, poison dart frogs (Dendrobatidae, Rhinella marina)

What is the function of Nuptial pads?

Males of several species develop keratinized epidermal thumb pads

Regrown each mating season

What is the function of "Seat patches" (Drink patches) in amphibians? What amphibians develop them?

-These are a specialized area of permeable skin in the ventral abdomen and hindlimbs, that is pressed against wet substrate and through the action of arginine vasotocin and aquaporins, increase permeability and facilitate absorption of water

-hylid, bufonid, ranid frogs

amphibian with cartilagenous skeleton

salamanders

Frogs lack ribs and instead have a ___ which is fusion of post sacral vertebrate and ___ which is a fusion of the tibia and fibula

urostyle

tibiofibula

What is the predator avoidance mechanism called tail autotomy?

allows the tail to break off through a fracture plane if grasped. Some newts, axolotls and other species can also regenerate limbs, jaws and ocular tissues.

What adult amphibians can retain their gills?

Axolotls

Amphibian lungs lack

alveoli

If intubating a frog know that:

Trachea is short

Bifurcates close to glottis- intubation*

Amphibians have a ____ chambered hear that becomes ____ in adults

-2

-3 (paired atria and single ventricle

What is the importance of the first pass mechanism in amphibians?

Hepatic portal vein drains blood from rear of amphibian’s body and can affect drug metabolism (quicker, first pass effect greater)

What is the importance of the dorsal lymph sacs in frogs?

-paired large sinuses that are SC over sacral area lateral to midline

-injections in there go straight to venous circulation

dorsal surface of x. laevis with skin removed showing lymph hearts that drain dorsal lymph sacs into venous system

male bullfrog tympanum is larger than females and larger than the eye

Amphibians are ___ with short GI tracts. They are opportunistic feeders and will cannibalize larvae.

carnivores

Why should you not feed raw meat or organs to young amphibians?

calcium deficit

How often should you feed amhibians?

X laevis 2-3 x a wk

Axolotls daily

Larvae daily, or multiple times daily – more than adults

What is a common defensive mechanism of amphibians?

vomiting

frogs can evert stomach

amphibians have ___ kidneys meaning that they lack the ability to concentrate urine in excess of plasma levels. What kind of “urine” do amphibians excrete (depends on where they live).

-opishtonephric

-aquatic - ammonia

-terrestrial - urea

-arboreal - uric acid

What is the plasma osmolarity of amphibians?

Plasma osmolarity is 200 mOsm/kg (mammals 290)

Amphibian Ringer's solution

Xenopus excrete ammonia in aquatic environment and can excrete _____ during drought

urea rather than ammonia

What is the function of Lateral line system?

Linear arrangements of neuromasts on head and along body

Detect changes in water pressure and currents

Function in locating prey

Lateral line system on x. laevis and typical skin shedding on right hind limb

What do frogs use to hear high frequency sound versus low frequency sound?

High frequency sound through tympanum

Low frequency sound through forelimbs and cranium

What amphibian has terrestrial and aquatic olfactory receptors?

X. laevis

What kind of RBCs and thrombocytes do amphibians have?

nucleated

What kind of fertilization do salamander hav compared to frogs?

-salamanders are internal

-frogs are external

What is more important to reproduction photo period or temp?

temp

Larval amphibians all lack what?

eyelids

What hormone and nutrient is needed for Metamorphosis?

Controlled by hypothalamic-pituitary-thyroid axis

Requires thyroid hormone and iodine

What is the developmental stages of X. laevis? hatching, metamorphosis, sexually mature and optimum egg production?

3 day after spawning: hatch

2 months: metamorphosis

8 months: sexually mature

2-3 years: optimum egg production

X. laevis tadpole hovering in characteristic head down fashion

Strange physiological differences of X. laevis

-lack tongue and eyelids

-convex cornea (adapted for air vision)

-two seperate olfactory cavities (one water and one airborne)

-lateral line system dorsally and ventrally retained in adult animals

-gulp air bc cutaneous respiration not well developed

-during drought produce urea rather than ammonia

How deep should water be for X. laevis?

5-20cm water depth

What is the tank density for X. laevis tadpole and adult frogs?

-50 tadpole / L

-guide says 1 frog/2L (BB says 1 frog/3L)

What is the housing water temperature for X. laevis?

21-22°C

Light dark cycle for X. laevis?

12/12

X. laevis are ___ during feeding. Do not clean tanks until ___ post feeding or they may regurgitate food.

-aggressive

-1hr

What is the blood collection method in X. laevis?

Blood collection by cardiocentesis

Lack other good vessel options

What is the recommended water temperature for Xenopus laevis, Dendrobates auratus, Ambystoma mexicanum, Xenopus tropicalis?

Xenopus laevis: 21-22ºC (68-72ºF)

Dendrobates auratus (poison frogs): 22-26ºC

Ambystoma mexicanum: 15-18ºC

Xenopus tropicalis: 24-25ºC

What sanitation method can be used to prevent chytrid fungus?

hot water > 60 C

1% Virkon, 1% benzalkonium chloride, 2% bleach, and 70% ethanol

No phenolics in amphibians

When handling amphibians, what precaution should be taken when choosing gloves?

Gloves: powder free, moistened with dechlorinated water

Tadpole morbidity and mortality noted with latex and nitrile gloves

When do X. laevis metamorphasize and when are they mature? Prime egg production when?

-2mo

-8mo

-2-3yr

What induces egg laying in X. laevis? Rest period between collections?

-hCG

-1-3mo

When do X. laevis hatch?

3d after spawning

X. laevis tadpoles have function ___ and ___.

lung and gills

group housed x. laevis

surgical oocyte harvest in X. laevis

What should you use to prep frog for oocyte harvest? Why?

-MS222 - anesthesia

-sterile saline or chlorhex/benzylalkonium sparingly for prep - other destroy protective mucous barrier

Signs of stress/sickness in axolotl

float, rub, roll, dart - usually sedentary bottom dwellers

Signs of stress/sickness in X. laevis?

float, reluctant to dive

Where can you collect blood on an amphibian? What about xenopus?

• Midventral abdominal vein, frogs and salamanders

• Ventral caudal vein in salamanders

• Venous plexus beneath tongue in frogs

• Xenopus lack all 3, cardiocentesis utilized

Where to give injections in amphibians?

IM, IC, dorsal lymph sac (SC caudodorsal over pelvic area, IC - intraceolomic off midline in lower abdomen)

How to euthanize amphibian?

MS222, benzocaine, pentobarb

Normal ovaries in X. laevis have ____ color same as the liver

black pigment

What pathogenic mycobacterium spps are found in amphibians?

M. fortuitum

M. marinum

M. chelonae

M. xenopi

M. marium is pathogenic in zebrafish and M. chelonae is the most commonly found mycobacteria in zebrafish

How is mycobacteriosis transmitted in amphibians?

Transmission through direct contact, contaminated water, or fomites

What clinical signs are associated with mycobacteriosis? zoonotic

Wasting in spite of a good appetite

Nodules or ulcers on skin surface

Loss of diving reflex/distended abdomen