Mycological Society of America

Batrachochytrium Dendrobatidis gen. et sp. nov., a Chytrid Pathogenic to Amphibians

Author(s): Joyce E. Longcore, Allan P. Pessier and Donald K. Nichols
Source: Mycologia, Vol. 91, No. 2 (Mar. - Apr., 1999), pp. 219-227
Published by: Mycological Society of America
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Mycologia,91(2), 1999, pp. 219-227.
© 1999 by The MycologicalSocietyof America,Lawrence,KS 66044-8897
Issued 15 March 1999
Batrachochytriumdendrobatidisgen. et sp. nov.,
a chytridpathogenicto amphibians
JoyceE. Longcorel
DepartmentofBiologicalSciences,University
ofMaine,
Orono,Maine 04469-5722
Allan P. Pessier2
Donald K. Nichols
DepartmentofPathology, National ZoologicalPark,
SmithsonianInstitution,Washington,DC 20008
Abstract: Captive and wild frogs from North and
Central America and Australia recentlyhave died
with epidermal infectionsby chytridiomycete
fungi.
We isolated a chytridiomyceteinto pure culturefrom
a captive,blue poison dart frogthat died at the Na-
tional Zoological Parkin Washington,D.C. Using this
isolate, we photographed developmental stages on
nutrientagar,examined zoospores withtransmission
electron microscopy,and inoculated testfrogs.This
inoperculatechytriddevelops eithermonocentrically
or coloniallyand has thread-likerhizoids that arise
fromsingle or multipleareas on the developingzoo-
sporangium. The taxonomicallyimportantfeatures
of the kinetosomalregion of the zoospore indicate
that thischytridis a member of the Chytridialesbut
differsfromother chytridsstudiedwithtransmission
electronmicroscopy.Its microtubuleroot,whichbe-
gins at kinetosome triplets9-1 and extends parallel
to the kinetosomeinto the aggregationof ribosomes,
is distinctive.Histologicexaminationof testfrogsre-
vealed thatthe pure cultureinfectedthe skinof test
frogs,whereasthe skinof controlfrogsremainedfree
of infection.The fungusis described as Batrachochy-
trium dendrobatidisgen. et sp. nov.
Key Words: Chytridiales,
chytridiomycosis,
Chytri-
diomycota,frogs,fungus,ultrastructure,
zoospore
INTRODUCTION
During 1996-1998, juvenile blue poison dart frogs
(Dendrobates azureus), green-and-black poison dart
frogs(D. auratus), and White'streefrogs(Litoria ca-
erulea) at the National Zoological Park (NZP) died
Accepted forpublicationNovember9, 1998.
1 E-mail:longcore@maine.maine.edu
2 Currentaddress:
Departmentof Pathology,Zoological Societyof
San Diego, San Diego, CA 92112-0551
219
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with a unique skin disease associated with the pres-
ence of sphericaleukaryoticorganismslocated in the
epidermis. Electron microscopyof the affectedepi-
dermis revealed that the intracellularorganismsin
the skin produced zoospores characteristicof mem-
bers of the Chytridiomycota (Pessier et al 1999). His-
tologicallysimilarchytridshad been observed in the
skin of frogsfromNZP as earlyas 1988 in a single
White'streefrog.Initiallyand in a subsequentreport
of an infectionin arroyotoads (Nichols et al 1996)
the infectiveorganismwas not recognized as a chy-
trid.Chytridshave also been observed histologically
in the skinof frogsthatdied at severalotherUS zoos
and research institutions(Nichols et al 1998) and
have been hypothesizedto be the proximatecause of
declines ofwildpopulationsof amphibiansin Central
America and Australia(Berger et al 1998). Although
zoosporic members of the Kingdom Fungi are well
known as parasitesof protozoans and invertebrates,
none previouslyhad been reportedfromtissueof liv-
ing vertebrates(Sparrow 1960, Powell 1993).
We have isolated the infectiveorganismfromthe
epidermisof a blue poison dart frogand herein de-
scribeitsmorphologyand the ultrastructural features
of its zoospores. Because thalliof manychytridiomy-
cetes are simple and provide few characters,ultra-
structuralfeatures of the asexually produced zoo-
spores are evaluated to make taxonomic decisions
(Barr 1990). The developmentof the thallusand the
ultrastructure of the zoospores of the frogpathogen
differdistinctlyfrom those of other chytridialean
genera and species; therefore,we describe this or-
ganism as a new genus and species.
MATERIALS AND METHODS
in thehost.-Complete
Histology necropsieswereperformed
on dead frogs;tissueswereprocessedforhistologic
exami-
nationas previouslydescribed(Pessieret al 1999). After
paraffin
embedding, 5-6 Lmsectionswerestainedwithhe-
matoxylinand eosin.
Isolation
and culture.-Initial
attemptstobaitforthefungus
byplacingsnakeskin withwaterand debrisfromthecon-
tainersin whichinfected frogshad livedwereunsuccessful
and the snakeskin rapidlybecomeovergrown withoomy-
cetes.One hindlegfroman infected bluepoisondartfrog
thathad recently died was double baggedin plasticand
shippedovernight on ice packstoJ.Longcore'slaboratory.
220
Pieces of loose epidermiswere removedfrombetweenthe
toes of the frog and placed on culture dishes containing
PmTG nutrientagar (1 g peptonized milk,1 g tryptone,5
g glucose, 10 g agar, 1 L distilledwater,with400 mg strep-
tomycinsulfateand 200 mg penicillin-Gadded afterauto-
claving (Barr 1986, 1987). While viewingwith X30 stereo
magnificationand substage lighting,small pieces (0.5-1
mm2) of infectedskin were dragged throughagar witha
sterileneedle to remove bacteria and yeastcells. Cleaned
pieces of skinwere transferred to a plate of PmTG nutrient
agar and incubatedat room conditions(20-23 C). Sporan-
gia developed on the frog skin but did not release zoo-
spores or continue developmentof a colony.A group of
these sporangiawas added to PmTG broth.After16 d the
brothwas opalescent fromthe growthof the fungus.Since
the isolationof the cultureused in thisstudyothercultures
have been isolatedbytransferring groupsof wk-oldsporan-
gia that formed on the original isolation plate onto TGhL
agar (16 g tryptone,4 g gelatinhydrolysate, 2 g lactose, 12
g agar,1 L distilledwater).To produce zoospores forTEM
and to growzoosporangia for lightmicroscopy,we spread
approximately1 mL of broth containingthe fungusonto
cultureplates containing2% tryptoneagar or TGhL agar.
Maximum temperaturefor growthwas determinedby in-
oculatingflaskscontaining50 mL of TG broth (1% tryp-
tone and 0.3% glucose; Gauriloffet al 1980) with0.5-1 mL
of broth containingisolate L-197 grownat room tempera-
ture.Controlsweremaintainedat room temperature.Thalli
at differentdevelopmentalstageswere photographedwith
a Nikon Labophot-2microscopeusingphase lenses and Ko-
dak Tri-XPan 400 film.
Sterile,distilledwaterwas added to culturesof isolate L-
197 growingon 20 agar plates to induce dischargeof zoo-
spores.The liquid containingthe zoosporeswas pooled and
zoospores were prepared for transmissionelectronmicros-
copy (TEM) with a sequential glutaraldehyde-osmium
troxidemethod (Barr 1981,Longcore 1992). Serial sections
were cut witha diamond knifeand placed on carbon-sta-
bilized, pioloform-coatedslots. Sections were viewed on a
PhilipsCM-10 microscopeat 80 kV.
Infectionoffrogs.-To attemptto fulfillKoch's postulates,
five2-3 mo-old blue-and-yellow poison dart frogs(Dendro-
batestinctorius)were obtained froma dealer who did not
have a historyof chytridiomycosis or skin disease in his
frogs.Frogs were separated into 2 groups; 2 frogswere in-
oculated dailyforup to 30 d with0.1 mL of swirled,pure
broth culture of isolate L-197, applied to the hind legs.
Three frogswere used as controlsand were inoculatedsim-
ilarlywith0.1 mL of sterilebroth.Frogs thatdied or were
euthanizedwere processed forhistologyas above. The fun-
gus was isolatedfroma testfrogusingthe isolationprotocol
above.
TAXONOMY
Batrachochytrium Longcore, Pessier et Nichols gen.
nov.
Thalli monocentriciaut colonici. Ex una ad complures
rhizoidesstirpes;rhizoidessimilesfilae. Quodque segmen-
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MYCOLOGIA
tum colonici thalli efficienszoosporangium.Zoosporangia
cum una aut compluribusinoperculatisfluxispapillis; na-
tantes zoospores sphaericas aut paene ovatas. Ribosomata
aggregata.Numerus guttularumolei; assuescata ad mem-
branam microcorporisatque nexa ad peripheramriboso-
mati aggregati.Kinetosomataradicula facta de aggregatis
microtubulis;assuescataad (microtubula)triplica9-1 atque
extentiaparallela ad kinetosomain ribosomatumaggrega-
tionem.Kinetosomaannexa ad centriolamnon-flagellatam
cum innexisfibris.Egens rumposomatoet occluso regionis
Habitatum:Efficiensmonocentricaet colonica
transitantis.
sporgangiain stratocorneo amphibianum.
TYPE. Batrachochytrium dendrobatidis Longcore,
Pessier et Nichols.
Thalli monocentric or colonial. One to several rhi-
zoidal axes; rhizoids thread-like. Each segment of co-
lonial thallus forms a zoosporangium. Zoosporangia
with one or more inoperculate discharge papillae;
swimming zoospores spherical or slightly ovate. Ri-
bosomes aggregated. Numerous lipid globules; asso-
ciated with sheet of microbody and nested in periph-
ery of ribosomal mass. Kinetosomal root consisting
of a group of microtubules; arising near triplets 9-1
and extending parallel to kinetosome into ribosomal
core. Kinetosome attached to nonflagellated centri-
ole with overlapping fibers. Lacking rumposome and
transition zone plug.
Habit. Forming monocentric and colonial sporan-
gia in epidermis of amphibians.
Batrachochytrium dendrobatidis Longcore, Pessier et
Nichols sp. nov. FIGS. 2-30.
Descriptioad genus. Sporangia in purum cultumangu-
te- lata ad sphaerica;ad 40 pLmdiametraex una ad complures
papillas. Longitudo flagelli19-20 tpm.
TYPE. FIGS. 2-30.
Description as for genus. Sporangia on nutrient
agar up to 40 pLmdiam with 1-several discharge pa-
pillae. Flagellum 19-20 .tmlong.
Diagnosis based on isolate L-197 from a blue poi-
son dart frog, NZP #97432, 14 Sep. 1997. Also ob-
served: isolate L-198 from a black-and-green poison
dart frog, NZP #97471, 3 Oct. 1997; isolate L-206
from a White's tree frog, NZP #98149, 11 May 1998
and isolate L-203 from a false tomato frog (Dyscophus
guineti), Bronx Zoo, New York, #C6-98, 20 Jan. 1998.
Etymology. Greek; batracho = frog and chytr=
earthen pot. The species name is from "Dendrobates,"
the genus of frogs in which the organism was respon-
sible for high mortality in a captive population and
from which the culture was isolated to make the type
photographs.
RESULTS
Observations in the host.-The stratum corneum (su-
perficial epidermal layer) was thickened, up to 2-5
 LONGCORE ET AL: B.
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f.
FIG. 1. Haemotoxylinand eosin stained section of Ba-
dendrobatidis
trachochytrium in epidermisof naturally
in-
fectedblue poison dartfrog.Arrowindicatesopen sporan-
gium,arrowheadsindicatecolonial sporangia;top of photo
is surfaceof skin;youngerstages of the fungusare in the
deeper layers.
times normal. Within the thickenedstratumcorne-
um were large numbers of spherical (7.0-15.0 ,um
diam) chytridthalli(FIG. 1) (Pessieret al 1999). Thal-
li were most numerous in the skin of the ventralab-
domen, hind limbsand feet.Severalprofilesof thalli
were observedwithinthe skinincludingmonocentric
thalli,colonial thalliwiththininteriorwallsand zoo-
sporangiawithprominentdischargepapillae (FIG.1).
One of the 2 frogsthatwere inoculatedwithisolate
L-197 died on the 23rd d of inoculationand the sec-
ond experimentalfrogwas found dead on the 31std
of the experiment.Two controlfrogs(the thirdcon-
trolfrogdied of metabolicproblemsunrelatedto the
experimenton the 10th d afterinoculationsbegan)
were euthanized 5 d afterthe last experimentalfrog
died. Complete necropsiesperformedon all frogsre-
vealed thatonlythe 2 frogsthatwere inoculatedwith
B. dendrobatidis had histologicevidence of skin dis-
ease and chytridthalliwithinthe epidermis.We iso-
lated B. dendrobatidis fromone of the artificially in-
fectedfrogs.
Morphologyin pure culture.-Motile zoospores are
nearlysphericalor somewhatovate,varyin size (com-
monly3-5 pumdiam) and lack prominentlipid glob-
ules (FIG. 2). Zoospores may become elongate and
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DENDROBATIDIS GEN. ET SP. NOV. 221
are frequentlyelongate (FIG. 3) when firstreleased
fromthe zoosporangium.Frequentlyone to manycy-
toplasmicextensionsprojectand retractfrommotile
zoospores (FIG. 4). A posteriorflagellum(19-20 Ixm
long), dark bodies, which are the lipid globules and
microbody,and a shadowyarea, which is the aggre-
gation of ribosomes,are visible with phase micros-
copy,particularly when zoospores are flattenedon a
of
layer agar under a coverslip(FIGS.4, 5).
On PmTG or 2% tryptonenutrientagars mosten-
cystedzoospores do not formcell walls;theylyserath-
er than developing into sporangia. On TGhL agar,
however,some zoospores formgermlingswithfine,
thread-likerhizoids.Rhizoids mayextend froma sin-
gle area (FIG. 6) of a developing zoosporangium,or
fromseveralareas (FIG. 7). On nutrientagar,zoospo-
rangia increase in size over the next 4-5 d (FIGS.8-
10) and when maturehave one to severalprominent
dischargepapillae, depending on the size of the spo-
rangium(FIGS.10, 11). Rhizoidsare thread-like(FIG.
8) and maybe shortand bushyor sparselybranched
and extend long distancesfromthe sporangium.Dis-
charge papillae formduring the growthof the spo-
rangium,and wallsat the tipsof papillae break down
and formplugs thatdeliquesce and release zoospores
(FIGS. 10-12). The wall at the edge of the papillar
opening often appears slightlythickened (FIG. 13).
Most thalli develop in the described manner,how-
ever,during earlydevelopmentsome thalliformin-
ternalsepta (FIGS.14, 15). Each cell formedbythese
septa develops as do the zoosporangia described
above and formsits own discharge papilla. Thus, a
group of sporangiacan growfroma singlezoospore.
This typeof developmentis most similarto the cat-
egory of developmentreferredto as "colonial" by
Barr (1990), and we referto it by that name. The
colonial thallithatdevelop on nutrientagar are anal-
ogous to the thalliwithinternalwallsthatformwhen
the fungusdevelops in epidermal cells of the host
(FIG. 16). Restingspores were neitherproduced on
nutrientagar nor seen in host tissue.
Isolationand culture.-Singlezoospores usuallyfailed
to develop on the nutrientagar thatwe used, how-
ever,groups of sporangia deposited onto agar from
broth culture readilyformed colonies, even on nu-
trientagar thatdid not supportthe growthof single
zoospores. This "group effect"also occurred during
isolation attempts.Single or small groups of sporan-
gia that developed on cleaned pieces of skin failed
to continue development,whereas larger groups of
sporangiacontinuedgrowthand formedcolonies. In
broth culture isolate L-197 develops fastestat 23 C.
Growthoccurs at 28 C, althoughat a slowerratethan
at lower temperatures.Broth culturesthat failed to
222 MYCOLOGIA

FIGS. 2-16. Developmentof isolate L-197 of Batrachochytrium in pure culture.2. Shape of motilezoospore.
dendrobatidis
3. Elongate amoeboid zoospore. 4. Fine cytoplasmicextension (arrow) fromsurfaceof zoospore. 5. Zoospore flattenedon
agar surfaceunder coverslip;black dots are probablylipids and microbodyand grayshadowyarea is the aggregationof
ribosomes.6. Germlingwithrhizoidsfromsingle area of thallus.7. Germlingwith3 rhizoidal axes. 8 and 9. Thread-like
rhizoidson developingsporangia. 10. Large zoosporangiumwith2 dischargepapillae (arrows). 11. Wall of tip of papilla has
begun to deliquesce. 12. Zoospores dischargingthrough2 papillae (arrows).13. Nearlyemptyzoosporangiumshowingslight
thickeningof wall at tip of papilla. 14. Group of thalli;one thalluswithinternalseptation(arrow). 15. Colonial thalluswith
severalwalls (arrows). 16. Appearance of isolate L-197 in skin of blue-and-yellow dart frog.Colonial thallus (arrow) and
monocentriczoosporangium (arrowhead) in epidermisof host thatwas experimentally infected.Scale bar = 10 ,Lmforall.

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 LONGCORE ET AL: B. DENDROBATIDIS GEN. ET SP. NOV.
make substantialgrowthafter2 wk at 29 C grewwell
afterflaskswere placed at 23 C. PmTG and reduced-
strengthTGhL have been used successfullyas isola-
tion media, and TGhL has been the best growthme-
dium. During attemptsto isolate Batrachochytrium
from5 frogs(4 fromNZP and one fromthe Bronx
Zoo), otherfungialso grewon cleaned pieces of skin.
These included hyphomycetes,a spherical, multi-
pored species of Rhizophydium; and the oomycete
Aphanomyces.
Zoosporeultrastructure.-Theultrastructural view of
the zoospore is dominated by a core of aggregated
ribosomesand a single posteriorflagellum(FIG. 17).
Endoplasmic reticulummore or less surroundsthe
ribosomalmass and also is found withinit (FIGS. 18,
19); a cone of ribosomessurroundedby ER extends
to the kinetosome (FIGS. 17-19). Lipid globules and
microbodies lie just outside the ribosomal core and
often are nested partlywithin it (FIGS. 17, 18). A
sheet of microbody,whichis usuallylinear in outline
(FIGS. 17, 18), abuts or partlywraps lipid globules,
but seldom entirelyencloses them. One or more
groups of lipid globules and microbodymaylie any-
where around the peripheryof the ribosomal core
(FIGS.17, 18). Cisternaeof endoplasmicreticulumdo
not consistentlysurround or abut lipid globules.
Nine lipid globules,organized into twoclumps,were
in one zoospore that was analyzed from serial sec-
tions.Mitochondriaare scatteredaround the edge of
the ribosomalaggregation(FIGS.17-20), and maybe
nested partlyor completelywithin it. Some mito-
chondria abut lipid globules and microbody (FIG.
17). The nucleus is cupped towards,and nested in,
the mass of ribosomes (FIGS. 17-19). Often, small
clumps of ribosomesenclosed byER lie on the outer
side of the nucleus (FIG. 17).
The cytoplasmthatis exteriorto the core of ribo-
somes and major organelles contains clear and
dense-coredvesicles (FIGS.17, 19) and a single Golgi
apparatus (FIGS. 18, 19), which is not in any fixed
area of the spore. The cytoplasmicextensions that
can be seen withlightmicroscopyalso are seen with
TEM (FIGS. 18-20). These extensionscontain dense
cytoplasmwith few vesicles or other substructure
identifiablewithour fixation(FIG. 20).
Flagellar apparatus.-The kinetosome from which
the flagellaraxoneme extendslies in the posteriorof
the zoospore and connects to the plasma membrane
with9 interconnectedprops (FIGS.17, 19, 21, 24, 25)
similar to those in other members of the Chytridi-
omycota (Barr and Hadland-Hartmann 1978, Barr
1992). A nonflagellatedcentriole (nfc) lies parallel
to the kinetosome (FIGS. 21, 27, 28), and two of its
tripletsare connected to triplets6 and 7 of the ki-
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223
netosome with overlappingconnectingfibers(FIGS.
27, 28) for most of the length of the nfc. Fibrillar
materialalso extendsfromtriplet5 but does not con-
nect directlyto a tripletof the nfc. The transition
zone of the kinetosome contains concentricfibers
(FIGS.23, 24), transitionalfibers(FIG. 23) and a ter-
minal plate (FIG. 19) but no transitionzone plug. A
root consistingof a group of microtubulesarises at
the side of the kinetosomenear kinetosometriplets
9 and 1 (FIGS. 27, 28) and extends towardthe inte-
rior of the cell, parallel with the kinetosome (FIGS.
21, 27-30). Two-8microtubuleshave been identified
in differentseries of sections.The microtubuleroot
is embedded in a cone of ribosomessurroundedby
ER and extends into the mass of denselypacked ri-
bosomes (FIGS. 18, 21, 29, 30). Because of the diffi-
culty in distinguishingmicrotubules among ribo-
somes,we could not determinethe exact numberof
microtubulesor the ending point of the root.FIGURE
31 presentsa schematicviewthe ultrastructure of the
zoospore.
DISCUSSION
We firstidentifiedBatrachochytrium as a chytridiomy-
cete fromelectron photomicrographsof zoosporan-
gia containingzoospores withinthe skinof a White's
tree frog (Pessier et al 1999). Our identification
was
based on the presence of flagellarprops,discoid cris-
tae in the mitochondriaand aggregatedratherthan
dispersed ribosomes. This conclusion has been
strengthenedby the more thoroughexaminationof
the zoospores frompure culture. Charactersassoci-
ated withthe flagellarapparatusare the mostreliable
of the ultrastructural charactersforgroupingchytrid
taxa at the ordinaland genus level (Barr 1990, 1992).
The flagellar apparatus of Batrachochytrium has a
nonflagellatedcentriolethatis parallel and attached
to the kinetosome,and a microtubuleroot thatorig-
inates fromthe side of the kinetosome.These char-
actersare diagnosticof the order Chytridiales.Barr's
emendation of the Chytridiales(1980: 2388) states
"... zoospore microtubules extend from one side of
the kinetosome in a parallel array,theyusually ex-
tend to the side of a rumposomewhichis on the lipid
globule surface." The microtubuleroot in Batrach-
ochytrium is similarto microtubulerootsin otherchy-
tridialeanzoospores in thatit arisesnear kinetosome
triplets9-1 (Barr and D6saulniers 1988). It differs,
however,in that other chytridialeanroots that arise
from this position leave the kinetosome at nearly
rightangles and extend to a part of the microbody-
lipid globule complex (MLC) (Powell 1978, Powell
and Roychoudhury1992). In Batrachochytrium, the
microtubuleroot leaves the kinetosomein a parallel
224 MYCOLOGIA

(18)
®

4'.
=>Si _

'kI 6Us : .

FIGS. 17-20. TEM of zoospores of Batrachochytrium dendrobatidis.Abbreviationsused: ce, cytoplasmicextension; ER,

endoplasmic reticulum;G, Golgi apparatus; K, kinetosome;L, lipid globule; M, mitochondrion;mb, microbody;mt,mi-
crotubules;nfc,nonflagellatedcentriole;N, nucleus; P, prop; tp, terminalplate; Va, vacuole; Ve, vesicle. 17. Longitudinal
section of zoospore withposteriorflagellumattached to kinetosome;showingarrangementof lipid globules and associated
microbody,mitochondriaand nucleus around mass of ribosomes. 18. Lipids and microbodynear kinetosome; cone of
ribosomes surrounded by ER around microtubuleroot. 19. Longitudinal section of zoospore showingGolgi apparatus in
cytoplasmoutside of mass of ribosomes and cytoplasmicextension.20. Cytoplasmicextension.Scale bars = 1 uim.Bar for
17 also for 19.

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 LONGCORE ET AL: B. DENDROBATIDIS GEN. ET SP. NOV. 225

t .,- t .X)

!'r
9
aw.-
>i
-,.· g.

FIGS.21-30. Kinetosomalarea of B. dendrobatidis. 21. Longitudinalsectionof parallel kinetosomeand nfcshowingover-

lapping connecting fibers (arrow) and microtubuleroot (arrowhead) extendinginto ribosomes.22-30. Serial sections1-8
and 12. 22. Axoneme where 2 centralmicrotubulesbegin. Doublets are numbered followingthe conventionof Barr and
Desaulniers (1988). 23. Concentricfiber(arrow) and transitionalfibers(arrowhead); asteriskindicatesmicrotubuledoublet
or triplet#1.24. Connection of props (arrow) to plasmalemma.25. Props (arrow). 26. Beginningof tripletsof kinetosome.
27. Fibrillarconnectionfromkinetosometriplets6 and 7 to nonflagellatedcentriole;beginningofrootmicrotubules(arrows)
near triplets9, and 1. 28. Ribosomes surroundingroot microtubulesnear triplets8-2. 29. microtubules(arrows)in cone of
ribosomes.30. Microtubuleswithincone of ribosomessurroundedby ER. Scale bar = 0.5 ,umforall.

directionand extends into the aggregationof ribo-
somes. Althoughthe zoospores of Lacustromyces hie-
malisLongcore (1993) and Rhizophlyctis hardenUeb-
elmesser (Roychoudhuryand Powell 1992) contain
microtubuleroots thatextend into the core of ribo-
somes, neitherof these roots arises fromthe triplet
9-1 position.Rather,in both of these species, the mi-
crotubule root that arises fromthe 9-1 position ex-
tends towardthe MLC.
An MLC containinga group of lipid globules and
no rumposome ratherthan a single large lipid glob-
ule witha rumposome,althoughunusual forthe Chy-
tridiales,is also characteristic which
of Lacustromyces,
also was placed in the Chytridiales(Longcore 1993).

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226 MYCOLOGIA

phlyctis. Rhizophlyctis spp., however, do not have

thread-likerhizoids.We have not yet traced the in-
fection process in the skin cells of frogs,but if the
zoospore encystson the outside of the cell and the
nucleus and contentsenter the cell via a germ tube,
as it does in other endobiotic species (endogenous
development),then the classical genus would be En-
Although the thallus of B. dendrobatidishas
tophlyctis.
fewinvariablemorphologicalfeatures,this species is
not likelyto be confused with any species of Ento-
phlyctis, Rhizophydium, or Rhizophlyctisbecause its
zoospore is distinctiveeven at the lightmicroscopic
level. In addition, some Batrachochytriumthalli are
colonial, both in the host and in pure culture.Care-
fulobservationis necessary,however,so thatcolonial
development is not confused with development of
thallifromzoospores thatdo not exit the parentzoo-
sporangium. Classical chytridialeangenera, which
relyheavilyon typeof development,have littlepre-
dictive value for phylogeny (Barr 1990, Longcore
1995). Consequently,ultrastructuralcharacters are
needed to describe new genera in the Chytridiales.
FIG. 31. Summary diagram of Batrachochytrium zoo- Based on its capabilityto formcolonial thalliand its
spore with flagellumand kinetosomalarea enlarged. Ab- distinctivezoosporic ultrastructure,a new genus is
breviationsused: ce, cytoplasmicextension;ER, endoplas-
mic reticulum;G, Golgi apparatus; K, kinetosome;L, lipid appropriatefor thisfungus.
globule; M, mitochondrion;mb, microbody;nfc,nonflag- Chytridsthat are histologicallyindistinguishable
ellated centriole;N, nucleus; P, prop; Va, vacuole. Organ- fromB. dendrobatidis occur on a range of amphibian
elles surroundingthe ribosomal mass and cytoplasmicex- hosts in North American zoos (Pessier et al 1999,
tensionsare not consistently
found in anyparticularpartof Nichols et al 1998), in wild populations of amphibi-
the cell. ans in Central America and in captivityand in the
wild in Australia (Berger et al 1998). Whether the
populations of Batrachochytriumthat affectamphibi-
We do not infer,however, that possession of this fea- ans fromthese widespread areas will be attributable
ture indicates that the two genera are closely related. to the same species is yet to be determined.Berger
Their kinetosomal characters suggest that they are et al (1998) studied the zoospore ultrastructureof
not. The distinctive overlapping of the kinetosome the chytridfrom frog tissue from Central America
and nfc connective fibers, and the origin of the con- and Australia.They found no microtubuleroot as-
nective fibers from triplets 6 and 7 of the kinetosome sociated with the kinetosome and presented a zoo-
are features that occur in species with a Rhizophydium spore diagram that portraysthe nonflagellatedcen-
sub-type of zoospore (Barr 1980, Barr and D6saul- triole at an angle and unconnected to the kineto-
niers 1988). These features and the lack of a transi- some. Althoughthese are importanttaxonomicchar-
tion zone plug, which is also absent in the Rhizophy- acters,we do not interpretthe differencesbetween
dium zoospore, suggest that Batrachochytriummay be theirfindingand ours as a differencein actual ultra-
related to species with a Rhizophydiumsubtype of zoo- structure.The zoospores studied by Berger et al
spore. Batrachochytriumand Rhizophydium,however, (1998) werewithinzoosporangia and were fixedwith-
have dissimilar MLCs, and no species of Rhizophy- in the frogtissue.Thus the zoospores mayhave been
dium is known to have colonial thalli. immature and not optimally fixed. In addition, serial
When grown on agar, Batrachochytriumthalli de- sections were not examined. These differences in
velop endogenously. The nucleus remains in the en- methods most likely account for Berger et al not ob-
cysted zoospore and the encysted zoospore enlarges serving a microtubule root or the connection and
to form the zoosporangium. When rhizoids extend parallel orientation of the kinetosome and the non-
from only one area of the sporangium, the organism flagellated centriole. The zoospores studied by Ber-
looks like a species of Rhizophydium (Sparrow 1960, ger et al were otherwise similar to those of B. den-
1973); however, when sporangia develop with several drobatidis. Because zoospore ultrastructure is not
rhizoidal axes, the classical genus would be Rhizo- consistently useful at the species level and scant mor-

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All use subject to JSTOR Terms and Conditions
 LONGCORE ET AL: B. DENDROBATIDIS
phology exists on which to base species differences
in this organism, differences among species may
need to be detected using molecular methods.
Batrachochytriumseems to be a widespread and
ecologically significant genus of chytrids. Important
questions remain about how the amphibian patho-
gen persists in nature, and whether it lives outside of
a host. We have not seen resting spores in the labo-
ratory,although they may occur in nature. Our ability
to culture Batrachochytriumsuggests that it may be
capable of growing in nature as a saprobe. Also, de-
velopment of the fungus does not cease when the
host dies. In the laboratory at least one generation
of sporangia has developed on skin removed from
dead frogs, before bacteria and oomycetes overran
the skin. When in pure culture, Batrachochytrium
grows on boiled snakeskin (keratin), and the fungus
may be able to live saprobically on keratin in nature
if other components of the ecosystem limit the
growth of bacteria and oomycetes.
ACKNOWLEDGMENTS
We thankDrs. TraceyMcNamara and Michael Linn of the
Wildlife Conservation Society for providingthe infected
false tomatofrog.Mr. Paul Miles, owner of The Boa Barn,
graciouslydonated the poison dart frogsused in the rein-
fectionstudy.We gratefullyacknowledgeDr. Tina Passman,
chairof the DepartmentofModern Languages and Classics,
Universityof Maine for translatingthe Latin diagnosis.A
Senior Post-doctoralFellowship(97-3545A)fromFriendsof
the National Zoo (FONZ) supportedDr. Pessier.
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