Laura Gallaugher, DVM, DACLAMClinical Laboratory Animal Veterinarian
Vertebrates ScalelessAmphibios “double life”p
Aquatic larvae & terrestrial adultsClosely associated with aquatic or very moist environments
ALAT, 30, 2009 Introduction
**Ectotherms: Cold‐bloodedBody temperature is regulated by the Body temperature is regulated by the external environment
ALAT, 30, 2009 Introduction
h l h dPhylum: ChordataClass: Amphibia (4300 species)Order(s): Gymnophiona, Caudata & Anura
Developmental research **pRegenerate body parts
Ease of breeding
**g
• Large embyrosFull development visible
Near extinction in the wild
ALAT, 2009 Introduction
Tiger Salamander g(Ambystoma tigrinum)Ophthalmology Limb regeneration
Red Spotted Newt (Notophthalmus viridescens) Leg and lens Leg and lens regenerationALAT,30, 2009 Introduction
3800 Species3 pRanidae (True Frogs)Bufonidae (True Toads)Bufonidae (True Toads)Pipidae (Clawed Frogs)
Anatomy for jumpingAnatomy for jumpingNo tail (Order Anura)
**Bullfrog (Rana f g (catesbeiana)Physiology and acousticsMotion sickness in space travel
Leopard frog (Rana pipiens)H diHormone studies
ALAT, 30, 2009 Introduction
XenopusXenopus tropicalistropicalis
XenopuspAfrican clawed frogEntirely AquaticEntirely AquaticOriginally used in pregnancy assaysp g y y
Urine → dorsal lymph sac → eggs
XenopusXenopus laevislaevispp
ALAT, 30, 2009 Introduction
**Cell and molecular biologyCell and molecular biology
Foreign DNA or mRNA is expressed in oocytes
ALAT, 30, 2009 Introduction
Metamorphosis: 2 monthspSexual maturity: 8 monthsOptimum egg production: 2‐3 yearsp gg p 3 yBreeding females: max. 1/month ideally 1/4months
•Mature oocytes are darkly pigmented in the animal hemisphere & light pigmented in vegetal hemisphere
Nonsurgical: manual expressiong pSurgical: MS‐222
Paramedian incision in lower abdominal quadrantFrogs should be rested for 1 month between surgery
Maximum of 6 surgeries
Developmental researchpDevelopmental toxicologyFETAX (Frog Embryo Teratogenesis Assay: X )Xenopus)
Test material added to a solution containing Xenopus embryosp yEmbryos checked for developmental abnormalitiesM t litMortalityMalformationGrowth inhibition
**Mucous Glands
Slimy protective layerGranular Glands
Head & shouldersCompounds & chemicals Poison dart frog
Defense compounds (neurotoxins, hallucinogenics)
( h b h f h )Magainins (inhibit growth of pathogens)Pheromones
Skin sheddinggPermeable abdominal skin for absorption of waterwater
Skin = primary respiratory surfacep y p yPlethodontids (lungless salamanders)
Larvae use gillsgLungs:
No diaphragm (single cavity)p g g y
Lymphatic Heartsy pDorsal lymph sacs in frogs
SQ over hindendQInjected substances go directly to venous circulation
L t l li tLateral line systemLarval & aquatic
High vs. low frequency detection
Mouthl k ll d l dXenopus lack a well developed tongue
Vocal sac in male frogsG GI Tract
CarnivorousSh t GI t tShort GI tract
Vomiting as a defense mechanism
****Like BirdsUrine, Feces and Reproductive substancespXenopus sexing
Female Male
Salamanders & NewtsSalamanders & Newtsmostly cartilaginousLimb regenerationg
AnuransRibs absent/greatly reducedg yUrostyleTibiofibula
F > MF > MColoration Anura:Anura:
Males: Tympanum > eyeFemales: Tympanum = eyee a es: y pa u eyeMales large colorful vocal sacsMale nuptial padsp p
Amplexus grip
****
Male Female
****
Nuptial pads, bufonid Amplexus
Simple → elaborateSimple elaborateHula dancing, phermone trails & vocalizations
Salamanders: Internal fertilizationFrogs: External fertilization
Midwife toad (Alytes obsetricans)Darwin’s frogGastric brooder (Rheobatrachus)
Heart
LiverLungs
Spleen
Small IntestineP
Fat Bodies
Pancreas
Fat Bodies
Large Intestine
http://www.biokids.umich.edu/critters/2744/Caudata/specimens/
Stomach Liver
Oviduct Small Intestine
Ovary w/ Eggs
**
ALAT, 2009 Introduction
**Neotenic: Retain Larval Neotenic: Retain Larval Characteristics throughout life
Northern climates > Southern latitudesLarger & aquatic > smaller & terrestrialXenopus laevis has been documented to live 15 yearsAge determined by counting layers in bone
Photo of Brady Barr w/ giant salamander (National Geographic Channel)
**Moist, powder‐free gloves
Skin toxinsFine‐meshed nets, jarsNo tails!Eye protectionNever restrain larval forms or caudate w/ gills around the
kneck
**Salamanders & frogs:g
Pectoral girdle & pelvic girdle/legs
Mycobacterium fortuitum, M. marinum, M. Mycobacterium fortuitum, M. marinum, M. xenopiSalmonellosisChlamydia psittaci, Chlamydia pneumoniaeRare allergies to skin secretionsRa e a e g es to s sec et o s
**Unique markings (Photographs)
l d dToe‐clipping discouragedFreezingSki l iSkin transplantationBeadsP i i t t d t d (PIT)Passive integrated transponders (PIT)
Usually from the ventral tail vein (salamanders) y ( )or abdominal vein (frogs)
26‐27ga needle, midway between pelvis & sternum
FecalImpression smears, skin scrapingAbdominocentesisRadiology
No restraint neededExtremity settingBarium sulfate
R bb hRubber catheter10‐15ml/kg PO
SubcutaneousSkin overlying shoulder/pelvis
IntramuscularF li b l i fForelimb muscles in frogsBack muscles in salamanders
IntracoelomicIntracoelomicOff midline, lower abdomen
Oral GavageOral GavageBall‐tipped needles or soft catheters
**2007 AVMA Guidelines on EuthanasiaAcceptable:
Overdose of inhalant anestheticCO COCO2, CO
Barbiturate overdose (IC or DLS)MS‐222 overdose (3g/L)MS‐222 overdose (3g/L)Double Pithing
Pithing does not work well for Xenopus d/t g pthe difficulty in bending the head forward to expose the atlanto‐occipital space
http://www.treknature.com/gallery/photo128926.htm
**The health of the captive amphibian is p pdirectly proportional to the “health” of their environment
TerrestrialTerrestrialSemiaquaticAquaticAquatic
ALAT, 2009 Anatomy & Physiology
Impermeable, easily sanitizedp , yHeight to allow for climbing & jumpingFitted nonabrasive lidsFitted, nonabrasive lids
Pharmacal Research
Terrestrial Species:pMoist substrate
moistened sphagnum
Eastern mud salamander
mossheat‐treated soil w/ leaves ( H) (pH) moistened filter paper/paper towelspaper/paper towels
MistingShallow dishesShallow dishes
**Semiaquatic species:Semiaquatic species:
Sloping floor
Hiding placesBark
http://www.amphibiancare.com
BarkPVC pipesDarkly colored cagingDarkly colored caging
http://www.xenopus.com/husbandry.htm
**Chlorine
Age 24‐48hrsAerationSodium thiosulfateActivated carbon filters
ChloraminesActivated charcoal filter (unused)( )Sodium thiosulfate
CopperCopperOxygenAmmoniaAmmonia
Sodium thiosulfate + choramines = ammoniaZeoliteseo tes
Test kitspHp
6.8‐7.0
**Static
Frequent cleaningFrequent cleaningLower humidity
RecirulatingRecirulatingFilters remove debris & nitrogenous waste
Flow‐throughFlow throughDeclorination built‐in
**Pheromones!Terrestrial: q2wksAquatic: prevent water foulingq p g
i.e. Xenopus, cleaned after feeding (3x week)Avoid cleaning solutionsg
Dilute bleach (1:32 ratio w/ water)Rinse thoroughly
**lTNZ tropical vs. temperate
Tropical species – 21‐29 ° C (70‐85°F)T t 8 °C (6 °F)Temperate – 18‐22°C (65‐72°F)Not too hot!
Hi h h idit 8 %High humidity: 80%Lighting
f ll l th tfull wavelength spectrumShelterBreeders & species differencesBreeders & species differences
Generalists!Wide species varietyVariety depending of life stage
Sight feedersMoving food is preferred over stationary
TimingNocturnal
Ca/Phosphorus balance1:1 or 2:1
**Adults: Carnivores
Vertebrate & invertebratesCrickets & mealworms
Vitamin/mineral mix dusting or “gut load” insectsVitamin/mineral mix dusting or gut load insectsDaily‐twice weekly
Larval salamanders: CarnivoresLarval salamanders: CarnivoresTadpoles: HerbivoresLarval amphibians are feed more frequently than p q yadults
Amphibian DiseasesAmphibian Diseases
Postural changesgDecreased avoidance responsesDecreased righting reflexg gChanges in skin colorAbdominal Distentionbdo a ste t o
Physical Exam:Physical Exam:Observe animal & primary enclosureExamine skin, eyes, nares & oral cavityAbdominal palpation is difficultWeigh and assess BCS
Aeromonas hydrophiliay pTransmission: water & can be normal GI floraStress & immunosuppression predispose to colonizationpredispose to colonization
Clinical Signs:Ulceration of skin (legs & abdomen) Ulceration of skin (legs & abdomen), lethargy
LATG 15, 2007, Frogs
Treatment: Culture & SensitivityyTetracycline (50mg/kg PO BID) Aminoglycosides (5mg/kg IM q48hr) Aminoglycosides (5mg/kg IM q48hr) Chloramphenicol (50mg/kg IM, IC q 24 hr)hr)Gentamicin (2.5‐3mg/kg IM q72hr)
Pseudocapillaroides xenopip p
Transmission: eggs are ingested w/ l h d ki sloughed skin Clinical Signs: rough, thickened skin w/ pittingpittingopportunistic infections
LATG 15, 2007, Frogs
Colonizes pre‐existing skin lesionsCottony Cottony
http://clawedfrogs.tripod.com/id31.html
Intestinal impactionpIngestion of gravel/sandClinical Signs:Clinical Signs:
Lack of fecal outputBloatinggLethargy +/‐ writhingg
May require surgical intervention
http://clawedfrogs.tripod.com/id31.html
DehydrationDehydrationClinical Signs: dull, dark skin w/ dry sticky mucusTreatment:eat e t:
immersion if mild dexamethasone & fluids if in shock
Batrachochytrium dendrobatidisP i f id d l b l Primary cause of widespread, global amphibian decline
http://clawedfrogs.tripod.com/id31.html
Environmental SensitivityyDouble jeopardy
Research TractabilityResearch TractabilityLab, outdoors and mesocosms
I di E ti tiImpending ExtinctionOver the past 30 years, Amphibian deaths
“ h i i f represent “the greatest mass extinction of land vertebrates since the dinosaurs”
Small wetland vs. larger wetlandsgSmall are critical breeding habitatsLarge are valued by the general public
E i l iEnvironmental contaminantsFreshwater is a scarce resource
Disease and Global ChangeDisease and Global ChangeDisease outbreaks at high‐altitudesDue to atmospheric pollutants?p p
ILAR 48(3), pp. 270‐277.
Upon receipt of amphibians transfer to clean p p pcage with shipping H20House separately do lastHouse separately, do last2‐4 week minimumT t f it f l & b t i l Test for: parasites, fungal & bacterial infections
Which of the following statements is gFALSE?
A All amphibians have a cloacaA. All amphibians have a cloacaB. All adult amphibians are carnivorous.C All hibi h li l C. All amphibians have a slime layer. D. All amphibians require some moisture in
htheir environment.
Which drug is appropriate for euthanasia of g pp pamphibians?
A. LidocaineB. Tricaine methanesulfonate (MS‐222)C. GentimicinD. Isoflurane
What is the scientific name of the African clawed frog?
A Xenopus laevisA. Xenopus laevisB. Rana catesbeianaC X t i liC. Xenopus tropicalisD. Ambystoma tigrinum
What are two noninvasive methods for identification of amphibians?
A Photographs toe clipping A. Photographs, toe clipping B. Toe clipping, cage cardsC T tt i d C. Tattooing, cage cards D. Cage cards, photographs
Regarding their diet, larval amphibians g g , pare:
A HerbivorousA. HerbivorousB. FrugivorousC O iC. OmnivorousD. Carnivorous
With a flow‐through water caging g g gsystem, what must be removed from the incoming water?g
A. Nitrogen B Chlorine B. Chlorine C. Sodium D. Oxygen
In general, what is thought to be good g , g genvironmental enrichment for amphibians?p
A. Hiding places B Resting boards B. Resting boards C. Balls
hD. Chew Toys
What is the process of transforming p gfrom an aquatic animal with gills into a terrestrial animal with lungs called?g
A. TerreformingB Gill depletionB. Gill depletionC. Pneumotransition
hD. Metamorphosis
What is neoteny?yA. The process of a tadpole losing its gills
developing lungs and moving to the land developing lungs, and moving to the land B. A deformed (mutant) salamander C Wh l d h t it C. When a salamander reaches maturity
without undergoing metamorphosis b l dD. A newborn salamander