diversity Review Effects of Emerging Infectious Diseases on Amphibians: A Review of Experimental Studies AndrewR.Blaustein1,*,JennyUrbina2 ID,PaulW.Snyder1,EmilyReynolds2 ID, TrangDang1 ID,JasonT.Hoverman3 ID,BarbaraHan4 ID,DeannaH.Olson5 ID, CatherineSearle6 ID andNatalieM.Hambalek1 1 DepartmentofIntegrativeBiology,OregonStateUniversity,Corvallis,OR97331,USA; [email protected](P.W.S.);[email protected](T.D.);[email protected](N.M.H.) 2 EnvironmentalSciencesGraduateProgram,OregonStateUniversity,Corvallis,OR97331,USA; [email protected](J.U.);[email protected](E.R.) 3 DepartmentofForestryandNaturalResources,PurdueUniversity,WestLafayette,IN47907,USA; [email protected] 4 CaryInstituteofEcosystemStudies,Millbrook,NewYork,NY12545,USA;[email protected] 5 USForestService,PacificNorthwestResearchStation,Corvallis,OR97331,USA;[email protected] 6 DepartmentofBiologicalSciences,PurdueUniversity,WestLafayette,IN47907,USA;[email protected] * [email protected];Tel.:+1-541-737-5356 (cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1) (cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7) Received:25May2018;Accepted:27July2018;Published:4August2018 Abstract: Numerous factors are contributing to the loss of biodiversity. These include complex effects of multiple abiotic and biotic stressors that may drive population losses. These losses are especiallyillustratedbyamphibians,whosepopulationsaredecliningworldwide. Thecausesof amphibianpopulationdeclinesaremultifacetedandcontext-dependent. Onemajorfactoraffecting amphibian populations is emerging infectious disease. Several pathogens and their associated diseases are especially significant contributors to amphibian population declines. These include the fungi Batrachochytrium dendrobatidis and B. salamandrivorans, and ranaviruses. In this review, weassesstheeffectsofthesethreepathogensonamphibianhostsasfoundthroughexperimental studies. Such studies offer valuable insights to the causal factors underpinning broad patterns reported through observational studies. We summarize key findings from experimental studies inthelaboratory,inmesocosms,andfromthefield. Wealsosummarizeexperimentsthatexplore theinteractiveeffectsofthesepathogenswithothercontributorsofamphibianpopulationdeclines. Thoughwell-designedexperimentalstudiesarecriticalforunderstandingtheimpactsofdisease, inconsistenciesinexperimentalmethodologieslimitourabilitytoformcomparisonsandconclusions. Studiesofthethreepathogenswefocusonshowthathostsusceptibilityvarieswithsuchfactorsas species,hostage,lifehistorystage,populationandbiotic(e.g.,presenceofcompetitors,predators) andabioticconditions(e.g.,temperature,presenceofcontaminants),aswellasthestrainanddoseof thepathogen,towhichhostsareexposed. Ourfindingssuggesttheimportanceofimplementing standardprotocolsandreportingforexperimentalstudiesofamphibiandisease. Keywords: amphibianpopulationdeclines;experiments;pathogens;Batrachochytrium;ranavirus 1. Introduction RapidratesofbiodiversitylosshavesupportedthenotionthattheEarthisheadingtowardasixth majorextinctionevent[1–3]. Currentspeciesextinctionratesarehigherthanpre-humanbackground rates,suggestingthisbiodiversitycrisisislargelyattributedtoanthropogenicchanges[1–6]. Although numerous species from all taxonomic groups are affected, amphibians are at the forefront of this Diversity2018,10,81;doi:10.3390/d10030081 www.mdpi.com/journal/diversity Diversity 2018, 10, x FOR PEER REVIEW 18 of 33 1. Introduction Diversity2018,10,81 2of49 Rapid rates of biodiversity loss have supported the notion that the Earth is heading toward a sixth major extinction event [1–3]. Current species extinction rates are higher than pre-hcurmisiasn[ 3b,7a,c8k].gTrohuenirdp oraptuesla, tsioungsgeasrteindge ctlhinisin bgiomdoivreerrsaiptyid clyristhisa nis thlaorsgeeolyf baitrtdrisbourtemd atmo manaltshr[8o]p.oLgiekneic changes [1–6]. Although numerous species from all taoxthoenromgriocu gprso,uapms parhei baifafnecsteadre, aamffepchtiebdiabnys amreu latitp tlhee ffaocrteofrrsoncot notfr tihbiust icnrigsitso [3p,o7,p8u].l aTthioenir dpeocpluinlaestio[9n]s. are declining more rapidly than those of birds or mammThaelss e[8in].c Lluikdee ohtahbeirt agtrdoeusptsru, actmiopnh,icboianntas marine aanfftesc,tceldim bayt emchualtnipglee, foavcetor-rhs acrovnetsrtiibnugt,iningv taos ipvoepsupleactiieosn, declines [9]. These include habitat destruction, contamipnarendtsa,t ciolinm,aatned chinafnegceti,o ouvsedr-ihsaearvseess,tianlgl,o ifnwvahsiicvhe mspaeyciweso, rpkreinddateipoenn, adnednt ilnyfeocrtsioyunse rdgisisetaicseasll,y altlo oaf fwfehcitch may work independently or synergistically to affect amamphpihbiibaina npoppouplualtaitoinosn s[9[–91–21]2 ](F(Figiguurer e1)1.) .SoSomme eoof fththe erereseseaarcrchh wwee ssuummmmaarrizizee bbeeloloww ffooccuusseedd oonn hhooww a particular pathogen alone affects a host, whereas somea sptuadrtiiecsu aladrdpreasthseodg ehnowal oan peaathffoegctesna mhaoys tb,ew ahfeferecatesds obmy oetshteurd vieasriaadbdlerse sthseadt mhoawy iantpearathcot gweinthm paaythboegens. affectedbyothervariablesthatmayinteractwithpathogens. Figure 1. Potential abiotic and bFiiogtuicr efa1c.toProst ethnatita mlaabyi ointifcluanendcbei ohtoicstf–apcatothrsogtheant dmyanyaimnflicuse innc aemhopshti–bpiaanth dogiseenasdey snyasmteimcssi.n amphibian diseasesystems. Among the major threats to amphibians are emerging infectious diseases (EIDs). Several prominent pathogens and associated EIDs affect amphibian populations worldwide. Batrachochytrium dendrobatidis (hereafter referred to as Bd) is a pathogenic fungus that causes amphibian chytridiomycosis [13–15]. Among the major threats to amphibians are emerging infectious diseases (EIDs). Several prominentpathogensandassociatedEIDsaffectamphibianpopulationsworldwide. Batrachochytrium dendrobatidis (hereafter referred to as Bd) is a pathogenic fungus that causes amphibian chytridiomycosis[13–15]. Thisdiseasecancausepopulationdeclines,localextinctionsandcontribute to species extinctions [8,16,17]. A related yet highly divergent fungal pathogen that also causes amphibian chytridiomycosis, Batrachochytrium salamandrivorans (hereafter referred to as Bsal), is a newly discovered pathogen primarily infecting salamanders [18]. Iridoviruses of the genus Ranavirus (hereafter referred to as Rv) have been implicated in declines and mass mortalities of amphibians [19–23]. Teacher et al. [22] stated that populations can respond differently to the virus and emergence can be transient, catastrophic, or persistent with recurrent mortality events. Althoughamphibiansarehoststoanassortmentofpathogens/parasites,includingbacteria,viruses, fungi, water molds and helminths [13,24–27], we focus on Bd, Bsal and Rv, given accumulating evidenceoftheirpotentiallydevastatingeffectsonamphibianpopulationsworldwide. Inparticular, we focus on reviewing the literature that report the results of experiments (manipulation of key variables[28])conductedwithBd,Bsal,andRvconcentratingonpapersthatusedliveamphibian hosts. Giventhecomplexityofthesehost–pathogensystems,experimentalapproachesarecrucialfor disentanglingpotentialmechanismsdrivingpatternsoftransmissionandexaminingvariationinlethal andsublethaleffectsduetohostspecies,hostlife-historytraits,pathogenstrain,hostpopulations,and environmentalconditions. Prior to 2009, relatively few studies of amphibian diseases employed standard experimental designs[28](Figure2). Since2009,therehasbeenasurgeintheuseofexperimentstodeterminehow diseasesaffectamphibians. Experimentaldesign,methods,andinterpretationvary;thus,itisusefulto summarizetheseaspectstoassessgenerality. Oneproblemwithexperimentalworkonamphibian Diversity 2018, 10, x FOR PEER REVIEW 3 of 48 Diversity2018,10,81 3of49 diseases has been the lack of standardization in experimental methods. Here, we present a synthesis of experimental studies and attempt to address some of the issues regarding the lack of diseaseshasbeenthelackofstandardizationinexperimentalmethods. Here,wepresentasynthesisof standardization and difficulties in generalizing about the dynamics of the host–pathogen systems we experimentalstudiesandattempttoaddresssomeoftheissuesregardingthelackofstandardization focus on. anddifficultiesingeneralizingaboutthedynamicsofthehost–pathogensystemswefocuson. Figure2.ThenumberofexperimentalstudiesofBatrachochytriumdendrobatidis(Bd),B.salamandrivorans Figure 2. The number of experimental studies of Batrachochytrium dendrobatidis (Bd), B. (Bsal)andRanavirus(Rv)byyear. salamandrivorans (Bsal) and Ranavirus (Rv) by year.  SSuummmmarayryo fofP PatahtohgoegnenL iLfiefeH Hisitsotroireises  BBaattrraacchhoocchhyyttrriiuumm ddeennddrroobbaattiiddiiss FFiirrsstt ddeessccrriibbeedd bbyy LLoonnggccoorree eett aall.. [[2299]],, BBdd iiss aa ffuunnggaall ssppeecciieess iinn tthhee pphhyylluumm CChhyyttrriiddiioommyyccoottaa tthhaatt hhaass mmuullttiippllee hhoossttss oonn eevveerryy ccoonnttiinneenntt wwhheerree aammpphhiibbiiaannss eexxiisstt [[1155,,1166]] aanndd hhaass bbeeeenn aassssoocciiaatteedd wwiitthh nnuummeerroouuss ppooppuullaattiioonn ddeecclliinneess aanndd ssoommee eexxttiinnccttiioonnss [[3300––3322]].. RReecceenntt eevviiddeennccee ssuuggggeessttss tthhaatt tthhaatt tthhee ssoouurrccee ooff BBdd wwaass ttrraacceedd ttoo tthhee KKoorreeaann ppeenniinnssuullaa,, wwhheerree oonnee lliinneeaaggee,, BBddAASSIIAA--11,, eexxhhiibbiittss tthhee ggeenneettiicc hhaallllmmaarrkkss ooff aann aanncceessttrraall ppooppuullaattiioonn tthhaatt sseeeeddeedd tthhee ppaannzzoooottiicc eemmeerrggeennccee [[3333]].. OO’’HHaannlloonn eett aall.. [[3333]] ddaattee tthhee eemmeerrggeennccee ooff BBdd ttoo tthhee eeaarrllyy 2200tthh cceennttuurryy,, ccooiinncciiddiinngg wwiitthh tthhee gglloobbaall eexxppaannssiioonn ooff ccoommmmeerrcciiaall ttrraaddee iinn aammpphhiibbiiaannss.. BBdd hhaass aa ccoommpplleexx lliiffee ccyyccllee tthhaatt ccoonnssiissttss ooff aa ffrreeee--lliivviinngg iinnffeeccttiioouuss aaqquuaattiicc zzoooossppoorree ssttaaggee aanndd aa nnoonn--mmoottiillee zzoooossppoorraannggiiuumm ssttaaggee.. MMoottiillee zzoooossppoorreess aarree cchheemmiiccaallllyy aattttrraacctteedd ttoo kkeerraattiinn iinn aammpphhiibbiiaann hhoosstt,, ssuucchh aass kkeeraratitninizizeedd lalravravla jlawjaw shsehaethasth osr okrerkaetrinaitzineidz eedpiedpeirdmearlm laaylelrasy oerf sadofulatd aumltpahmibpiahni bsikainn s[3k4in,3[53]4. ,I3n5f]e.cItniofenc tcioann claeandl etaod htoypheyrpkeerrkaetorasitso saisndan hdyhpyeprpelrapsliaas ioafo tfhteh edderemrmala llalayyeerr, ,eerroossiioonnss aanndd uullcceerraattiioonnss ooff tthhee sskkiinn,, aanndd ddiissrruuppttiioonn ooff tthhee eeppiiddeerrmmaall cceellll ccyyccllee [[3300,,3344––3377]].. TThhee iinnaabbiilliittyy ttoo rreegguullaattee iioonnss tthhrroouugghh tthhee sskkiinn mmaayy lleeaadd ttoo ccaarrddiiaacc aarrrreesstt [3[388].]. CClliinniiccaall ssiiggnnss ooff cchhyyttrriiddiioommyyccoossiiss iinncclluuddee lleetthhaarrggyy,,l alcakcko foafp papetpiteet,itaeb, naobrmnoarlmpaols tuproes,tluorses, olforsisg hotfi nrgigrehfltienxg, cruetfalnexe,o ucsuetarnytehoeums ae,rayntdheinmcare, aasnedd sinkcinresalsoeudg hskining [s3lo7u].gHhionwge [v3e7r],. nHotoawllevinefre, cnteodt aanlli minafelsctaerde saynmimpatolsm aartei cswymhepntoimnfaetcitce dw.hOenn cienwfeicttheidn. tOhnecheo wsti,ththine zthoeo shpoosrt,a tnhgei azomoastpuorreanangida dmeavteulorep apnadt hdoegveenloicpz poaotshpoogreensicth zaotoasrpeorreelse athseadt aorue trseidleeasthede hoouststiidneto ththe ehaoqstu iantitco etnhve iaroqnumatiecn et.nvironment.  BBaattrraacchhoocchhyyttrriiuumm ssaallaammaannddrriivvoorraannss TThhee rreecceenntt iissoollaattiioonn aanndd cchhaarraacctteerriizzaattiioonn ooff tthhee ffuunnggaall ppaatthhooggeenn,, BBssaall mmaayy eexxppllaaiinn ssoommee aammpphhiibbiiaann ppooppuullaattiioonn ddeecclliinneess.. FFoorr iinnssttaannccee, ,tthhee ddrraassttiicc ddeecclliinnee ooff fifirree ssaallaammaannddeerrss,, SSaallaammaannddrraa ssaallaammaannddrraa,, iinn tthhee NNeetthheerrllaannddss,, GGeerrmmaannyy,, aanndd BBeellggiiuumm,, hhaass bbeeeenn lliinnkkeedd ttoo BBssaall [[3399––4411]].. AA ssttuuddyy ccoonndduucctteedd bbyy MMaarrtteell eett aall.. [[4422]] pprrooppoosseedd BBssaall oorriiggiinnaatteedd iinn EEaasstt AAssiiaa aanndd ccooeexxiisstteedd wwiitthh ssaallaammaannddeerrss tthheerree ffoorr mmiilllliioonnss ooff yyeeaarrss. .TThhee inintrtroodduucctitoionn oof fBBsasal ltoto EEuuroroppe eisi shhypypotohtehseisziezde dtot ohahvaev eococcucrurerrde ddudeu teo Diversity2018,10,81 4of49 toalackofbiosecurityintheinternationalpettrade[42]. AlthoughBdandBsalinfectionsresultin lethalskinerosion,thepathogenicmechanismofBsalisnotwellunderstood. Bsalproducesmotile zoospores,containcolonialthalli,andproducegerminationtubesinvitro[18]. Studieshaveassessed thepresenceofBsalinvariousamphibianpopulationsinNorthAmerica(e.g.,[43–45])andChina[46] utilizingseveralmethods(phalangeshistology,nestedPCR,qPCRandduplexqPCR),butitspresence hasyettobeconfirmedinthosepopulations. Givenitshighlethality,increasedfieldsurveillanceof thesenaïvepopulationswillbecriticaltocontainthepotentialspreadofthisnewlyisolatedpathogen, particularlyinNorthAmerica,aglobalbiodiversityhotspotforsalamanders[47–50]. Ranavirus Rvsareagroupoflargedouble-strandedDNAvirusesinthefamilyIridoviridaewithfish,reptile, andamphibianhosts[51]. ThefirstRvwereisolatedfromLithobatespipiensin1965[52]. TheGlobal RanavirusReportingSystem(https://mantle.io/grrs/map),createdtoaidintrackingRvoccurrences andstudies,showsRvtobefairlywidespreadinCanadaandtheUSwestoftheRockyMountains. ThistoolisintendedtofacilitatecommunicationamongresearchersconcerningRvdetectionandto accelerateresearchandmanagementofthediseasethreat. The genus Rv is composed of 6 identified viral species, three of which infect amphibians (Ambystoma tigrinum virus (ATV), Bohle iridovirus (BIV), and Frog Virus 3 (FV3)) [51]. Although the effects of Rv are well documented, little is known about the genetic basis for virulence across isolates[53]. FV3andATVinfectmanyamphibianspecies,buttheseisolatesaremostvirulentwithin theanuransandurodelans,respectively,fromwhichtheywereisolated[54]. Laboratoryexperiments haveshownthatintroducedRvisolatesmaybesignificantlymorevirulentthanendemicstrains[55]. Amphibians become infected with Rv by physical contact, dermal exposure to contaminated water,ordirectingestionofvirions[56,57]. Infectioncanoccurinasshortasaonesecondofdirect contact with an infected individual of the same species [56] or 3 h of contact with contaminated water [58]. Empirical studies confirming its potential effects in amphibians are limited [56,59–61]. FishcanalsobeinfectedwithRv,butsusceptibilitytoRvinfishesappearstobelow,thoughthereis potentialforfishtotransferRvtoamphibiansinhabitatswheretheyoverlap[62,63]. Rvsinfectionscancausecellapoptosisandtissuenecrosiswithinafewhours[51,64]. Common indicators of Rv infection include erratic swimming, lethargy, erythema, skin sloughing, loss of pigmentation, lordosis (excessive inward curvature of the spine), and ulcerations [65,66]. Lesions and hemorrhages associated with fatal cases of Rv occur in internal organs, particularly the liver, kidney,intestine,spleen,andreproductiveorgans[25,67,68]. However,theprecisemechanismsofRv disseminationwithinthehostarerelativelyunclear,especiallyattheearlieststagesofinfection. A recentstudydemonstratedthatFV3infectioniscapableofalteringthebloodbrainbarrierinXenopus laevistadpoleseventually,leadingtoRvdisseminationintothecentralnervoussystem[69]. Deathcan occurwithoutexternalsignsofinfection[70]. 2. Methods TheeffectsofBd,Bsal,andRvfoundinexperimentalstudiesaresummarizedinTable1. Our searchwasconductedviatheWebofScienceandsupplementedwithaGoogleScholarsearchusing the keywords “Batrachochytrium dendrobatidis + amphibians”, “Batrachochytrium salamandrivorans + amphibians”,and“Ranavirus+amphibians”,respectively. Duplicatesandnon-experimentalstudies wereremovedandtheremainingstudiesweredocumented. Studiesthatexaminedinteractiveeffects (i.e., pesticide + pathogen) were included, but only the effect of the pathogen independently was reported. TheBdsearch(1999–2017)resultedin1207hits,ofwhich110wereexperimentalstudies. TheBsalsearchresultedin41hits,ofwhich5wereexperimentalstudies. TheRvsearch(1992–2017) yielded269hits,ofwhich33wereexperimentalstudies. Ifonepublicationexaminedmultiplespecies orhostlifestages,eachspeciesandlifestagewasreportedseparately(Figure3). Diversity2018,10,81 5of49 Diversity 2018, 10, x FOR PEER REVIEW 5 of 48 Figure 3. Trends in all articles published on Bd (top) and Rv (bottom) in the literature over time. Figure 3. Trends in all articles published on Bd (top) and Rv (bottom) in the literature over time. Publicationswerecompiledusingthesearchstrings“Batrachochytriumdendrobatidis+amphibians”and Publications were compiled using the search strings “Batrachochytrium dendrobatidis + amphibians” “ranavirusandamphibians”intheWebofSciencedatabase,fromwhichduplicatesandarticlesthat and “ranavirus and amphibians” in the Web of Science database, from which duplicates and articles wereunrelatedwereremoved. TheBdsearchyieldedatotalof1207hitsandtheRvsearchyielded that were unrelated were removed. The Bd search yielded a total of 1207 hits and the Rv search 269hyiitesl.ded 269 hits. 3. Re3s.u Rletssults Results from experimental studies are summarized below. We presented general trends across Resultsfromexperimentalstudiesaresummarizedbelow. Wepresentedgeneraltrendsacross studies according to the response variable (e.g., physiology, behavior) and/or source of response studies according to the response variable (e.g., physiology, behavior) and/or source of response variation (e.g., life stage, virus strain). We then focused on interactive effects and summarize the variation (e.g., life stage, virus strain). We then focused on interactive effects and summarize the experimental work with each pathogen in combination with natural or anthropogenic environmental experimentalworkwitheachpathogenincombinationwithnaturaloranthropogenicenvironmental stressors. Below, we provide a summary of patterns and gaps in the accumulated experimental work stresosonr sh.osBt–eplaotwho,gwene dpyronavmidices aofs uBmd,m Basrayl, oanfdp aRtvte rannds athnedir gaamppshiinbiathne haocsctsu. mSpuelcaitfeicd reesxupletsr iomf ental workexopnerhimosetn–tpaal tshtuodgieesn adrey ndeatmaiilecds oinf TBadb,leB 1s aaln,da nddataR svuamnmdatrhizeiinrga tmhep nhuibmibaenr hoof sptasp.eSrps epcuibfilcisrheesdu, ltsof expesruimrveivnotarslhsitpu adnides liafer estdageetas ialered siunmTmabarleiz1eda innd Fdigautraess u4–m6.m arizingthenumberofpaperspublished, survivorshipandlifestagesaresummarizedinFigures4–6. Thenumberofexperimentalstudiesconductedonhostsatdifferentlifestagesvaried,withmost studiesofBdconductedinhostsaftermetamorphosisandmoststudiesofRvconductedwithlarvae (Figure4). TheonlyexperimentalstudieswefoundonBsalwereconductedwithpost-metamorphic Diversity 2018, 10, x FOR PEER REVIEW 6 of 48 The number of experimental studies conducted on hosts at different life stages varied, with most Diversity2018,10,81 6of49 studies of Bd conducted in hosts after metamorphosis and most studies of Rv conducted with larvae (Figure 4). The only experimental studies we found on Bsal were conducted with post-metamorphic hohsotsst(sF (igFuigruer4e) .4)E. xEpxepreirmimenetnatlasl tsutduideisesa nadnds usruvrivviavlasl hsohwowededc leclaerard idffieffreernecnecsews withithh ohsotslti fleifset asgtaege (Fi(gFuigruesre5s a5n adn6d) .6M). Moroeroevoevr,etrh, ethdeo dsoesoef opfa pthaothgoegneand amdimniisntiesrteedreddu druinrginsgu ssucescpetpibtiilbitilyiteyx epxepriemrimenetnstiss is alsaolsiom ipmoprotarntatnint iinn tienrtperrpetrientginrge sruesltuslt(sF i(gFuigreur7e) .7). 3.13..1B. aBtraatcrahcohcohcyhtryiturmiumde dnednrdobroabtiadtiisdis HoHsot–spt–apthaothgoegnedny ndaymniacmsaicrse ianrfleu einnfcleudenbcyemd abnyy fmacatonrys (fFaicgtuorres 1()F.iFgourree xa1m). pFloe,r beioxtaicmvpalrei,a bblieost,ic suvcahriaasbtlhese, spurechse ansc tehoef ppreresednactoe rosf, pdreendsaittyoros,f dheonsstistya nodf hcoosmtsp aentdit icoonmapmetointigonp aamthoongge npsa,tmhoagyenasff, emctay hoasftfescuts hceopstt ibsuilsictye,pmtiboirltiatyli,t ymaonrtdalpitayt haongde npaltohaodgsen[7 1lo–a7d4]s. [L71a–b7o4r]a. tLorayboarnadtofirye ladnedx pfieerldim eexnptesrhimaveents shhoawvne tshhaotwabni othtiactf aacbtioortsici nfflacuteonrcs iningflBude–nhcoinsgt dBydn–ahmosicts dinycnlaumdeiccsl iimncaltued,ese calsiomna,tael,t isteuadseo,nr,e saoltuitrucede, avraeisloabuirlcitey ,aavnadilatebmiliptye,r aatunrde [t7e5m–7p7e]r.atEuxrpe er[i7m5–e7n7t]a.l sEtxupdeierismfoenutnadl dstousde-idese pfeonudnedn tddoifsfee-rdeenpceensdinent dedvieffloerpemnceenst ,inin fdeecvtieolnoplomaedn,ta, nindfmecotirotna liltoya,din, daincda timngoritnaclirteya,s ienddiincafeticntigo ninvcrireualseendc einafsescoticoiant evdirwulietnhce inoascsuolucimateddo swei[t7h4 i,7n8o–c8u0l]u(mFi gduorsee 7[7).4E,7x8p–e8r0i]m (eFnigtsuhrea v7e). cEoxnpfierrmimedenttesm hpaevrea tcuornefairsmaecdr itteicmalpmereadtuiartei nags a facctroitricianl mBdeddiaytninagm fiaccst.orF ionr Bedx admynpalem,iAcsn. dForer eextaaml.p[l7e5, ]Afnodurned ett haal.t [7h5o]s ftofurongds thhaotu hsoesdt firnogwsa hromuesred teminp weraartmureers t(e2m2p◦Cer)aetxuhreibsi t(e2d2 s°Cig)n eifixchaibnittleydl oswigenrifmicoarnttallyit ylotwhaenr mthoorsteahliotyu sthedanin thcoosoele hroteumsepde rinat ucoreosler (17te◦mCp).eIrnatfuecrteiso n(17in °pCo).s It-nmfeecttaiomno irnp phoicsta-mmpethaimbioarnpshciacn ambepchliebairaends wcahne bnet ecmleapreerda twurheesna treemepleevraatteudres abaorvee etlheveantoedte dabBodveth tehrem naolteodp tBimd uthmerrmanagl eop[7t7im,8u1–m8 4r]a.nge [77,81–84]. SoSmoemeex peexrpimereimnteanltsatul dsiteusdiilelus stirllautestsrtartaei ns-tdreapine-nddeepnetnidnefenctt ioinnfeocutticoonm oeust[c1o5m,3e4s,8 0[,1855,–3848,8]0,,w85h–il8e8], otwhehrilset uodthieesr shtuavdeierse hvaevaele rdevneoaleedff encot eafsfseocct iaastseodciwatietdh wstirtahi nstrdaiifnfe dreifnfceeresn[c8e9s, 9[08]9.,90W].h Wethheetrheorr onro ntot strsatrinaind idffiefrfeernecnecseasr aerde edteectetecdtedca cnand edpeepnedndo nonth tehaem amphpihbiibainanh ohsotsstp sepceieciseus suesdedin ine xepxepreimrimenetnsts[9 [19]1.]. CoCmompapraartaivtieves trsatrinaine xepxepreimrimenetnstsa loalnogngw withitho bosbesrevravtaiotinoanlala mamphpihbiibainans usruvrevyesysa raereu suesfuefluli nin inivnevsetisgtiagtaintigngth tehere lraetliaotniosnhsihpispbs ebtweteweenenh ohsotspt oppouplautliaotniontr etnrednsdasn adnBdd Bvdi rvuilreunlceencvea rviaartiioatni.onF. oFror exeaxmamplpe,leP, iPoivoivai-aS-cSoctottet teat l.al[.9 [29]2l]i nliknekdeda nano bosbesrevrevdedR aRnaancaa csacascdaadeape oppouplualtaiotinond edcelicnlieneto toa ak nkonwown,n, hihghiglhyliyn finecfeticotiuosu,sa,n adndle tlhetahlaBl dBsdt rsatirnaitnh rthoruoguhgmh umltuilptliepllein leinseosf oafn aanlyaslyess.esI.n Ionn oeneex epxepriemriemnetn,at,d audlutlt RaRnaancaa sccaasdcaaed,aeex, peoxspeodsetod tthoe tBhde sBtdra sintrcauinlt ucureltdufrreodm fraosmit eau snitdee urgnodinerggaoihnogs tap hoopsut lpatoiopnuldaeticolinn ed,ehcalidne, sighnaidfi csaignntliyficloawntelyr slouwrveirv asulrravtievsa,lc roamteps,a creodmtpoatrheods teoe txhpoossee edxtpooasesdtr taoin a fsrtormaina fsriotemw ai tshitea wstaitbhl ea hsotasbtle pohpouslta ptioopnu[l9a2ti]o.nT h[9is2]B. dThsitsr aBind asltsroaidni saplsloa ydeidspglraeyaetde rgirmeamteurn iomtomxuicniotytoixniceixtyp eirni mexepnetrailmaessnatayls a[s9s2a]y.s Ex[p92o]s.u Erxeptoosuenred etom eincdvesm. inco vvse. lnsotvraeiln sstrcaainnsa clasno aalfsfoec atffheocst thsousrtv siuvravli.vDalo. dDdoidndgitnogntoent aelt. a[l9. 3[]93fo] ufonudnd susruvrivvaivladli fdfiefrfeenrecnesceins icna cpatipvteiv-ber-ebdreAdl Aytleystems umleutleentseinsseisx epxepriemriemnetanltlaylleyx pexopseodsetdo ttow towBod Bsdt rsatirnasin,as, lao cloaclal MMallaolrlocarcnanst rsatrinain(T (FT5Fa51a)1o) roar hay hpyeprevrivruirluenletnBt dB-dG-PGLPLst rsatrinain(U (KUTKvTBv)B.)T. oTaodasdesx epxopsoesdedto ttoh tehBe dB-dG-PGLPL strsatrinaihna hdahdi ghhigehremr omrotarltiatylittyh athnainn dinivdiidvuidaulsalesx epxopsoedsetdo ttoh ethMe aMllaolrlcoarncasntr satirnaionr ocro ncotrnotlroglr oguropu[p93 []9.3]. FigFuigruer4e. 4T.h Tehneu nmubmebreorf oefx pexepriemriemnetanltaslt usdtuiedsiecso ncodnudcutecdteadt aat sain sginleglleif elifseta sgtaeg.eO. bOtabitnaeindefdr ofmromdi rdeicrtect cocuonutsntfsro fmromTa Tblaeb1le. 1. DiDveirvseirtysit2y0 1208,1180, ,1801, x FOR PEER REVIEW 77o fo4f 948 Diversity 2018, 10, x FOR PEER REVIEW 7 of 48 Figure 5. Effects on survival in experimental studies. These data are direct counts from Table 1. Figure5.Effectsonsurvivalinexperimentalstudies.ThesedataaredirectcountsfromTable1. Figure 5. Effects on survival in experimental studies. These data are direct counts from Table 1. l a val viv rvi u r su d s ed ce uc du ed re hr th i wt i w . p xp. ex f e o f % o % FiFgiugruer6e. 6P. ePrecercnetnagtaegseosf oefx epxepreimrimenetnstssh sohwowinigngre rdeudcuecdedsu sruvrivviavlaalt aat sai nsignlgellei fleifset asgtaeg.eT. hTehseesde adtaataar aere Figure 6. Percentages of experiments showing reduced survival at a single life stage. These data are peprceercnetangtaegsefsro fmromTa Tbaleb1le( 1E x(Epxepriemriemntesnstsh oshwoiwnginrged reudceudcesdu rsvuirvvailv/atlo/ttaolta#l o#f oefx epxepreimrimenetnstws withithsu sruvrivviavlal asapasen racenen nedntpadgopeinosit fn)r.to)m. Table 1 (Experiments showing reduced survival/total # of experiments with survival as an endpoint). DDiffieffreernecnecseisn inm metehtohdoodloolgoygyc acnanc ocmomplpicliactaeteo uoruirn itnetreprrperteattaiotinono fotfh tehree rseuslutsltfsr ofrmomco cmompapraartaivtieve Differences in methodology can complicate our interpretation of the results from comparative stsratrianinex epxepreimrimenetnst.sF. oFroer xeaxmamplpel,eB, dBdd odsoasgaeg,es,i steiteo fosft sratrianinis oisloaltaiotino,na, nadndst sratrianinp apsassasgaignigngh ihstisotroyryca cnan strain experiments. For example, Bd dosage, site of strain isolation, and strain passaging history can iniflnufleunecnecoe uotucotcmomeseosf osft rsatrinainex epxepreimrimenetnst[s1 [51,856,8–68–88,984,9–49–69].6]. influence outcomes of strain experiments [15,86–88,94–96]. AAcccucmumulualtaintignge veivdiednecnecseu sguggegsetssttsh tahtasto smomehe ohsotsstp sepceiecisevs avrayryin inth tehireisru ssucsecpetpibtiibliitlyityto toB dB.dS. oSmomee Accumulating evidence suggests that some host species vary in their susceptibility to Bd. Some spsepceiecsiecsa ncapne rpseisrtsiwsti thwiinthfe cintifoenct[i9o7n] a[9n7d] oatnhder sotehxepresr ieexnpceermieonrctea lmityorratapliidtyly raafpteidrlByd aefxtepro sBudr ee[x8p6o,9s7u–re species can persist with infection [97] and others experience mortality rapidly after Bd exposure 10[08]6.,9V7a–r1ia0t0i]o. nVianrisaktiionnc ionm spkions ictoiomnp,ionscitliuodni,n ignckluerdaitning kaberuantidna anbcue,nddiasntrcieb,u dtiisotnri,bauntdiotnh, iacnkdn etshsi,ckmnaeyss, [86,97–100]. Variation in skin composition, including keratin abundance, distribution, and thickness, afmfecatyt haeffedcetp tthhe, odfetphtehz, ooof stphoer ez-oporsopdourcee-dprgoedrumcienda tgioenrmtuinbaetwiohni cthubcea nwahffiechct cthane saefvfeecrti tythoef sienvfeecrtiitoyn of may affect the depth, of the zoospore-produced germination tube which can affect the severity of aminofnecgtiaomn pahmiboinagn ahmospthsi[b3i5a,n1 0h1o].stDs i[f3fe5r,1e0n1c]e.s Dinifftehreenabceilsi tiyn otfhaem abpihliitbyi aonf sapmepciheisbitaonm sopuecnitessu tfofi mcieonutnt infection among amphibian hosts [35,101]. Differences in the ability of amphibian species to mount ensduoffcirciineonlto egnicdaolcrreisnpoolnosgeisc,apl arertsipcuolnasrelys, sptraerstsicruelsaprolyn ssetsr,esms aryesaplsoonpselasy, maaroyl eal[s1o0 2p–l1a0y5 a]. rFoulret h[1e0rm2–o1r0e5,]. sufficient endocrinological responses, particularly stress responses, may also play a role [102–105]. Furthermore, habitat preference may influence host susceptibility to infection [106,107]. Future Furthermore, habitat preference may influence host susceptibility to infection [106,107]. Future Diversity2018,10,81 8of49 Diversity 2018, 10, x FOR PEER REVIEW 8 of 48 habitat preference may influence host susceptibility to infection [106,107]. Future research should research should consider amphibian life-history traits, particularly of species that do not seem to be consideramphibianlife-historytraits,particularlyofspeciesthatdonotseemtobesusceptibletoBd susceptible to Bd infection, to better understand differences in host susceptibility and will be useful infection,tobetterunderstanddifferencesinhostsusceptibilityandwillbeusefultotargetspecies, to target species, which may act as reservoirs for the pathogen. whichmayactasreservoirsforthepathogen. FigFuirgeu7r.eT 7h. eTehfef eecftfeocftB odf Bddos deo(sine l(oing lzoogo zspooosrepso)roens)s ounrv siuvravl.ivTahl.e sTehdesaeta daartea dairree cdtirceocutn ctosufrnotms frToambl eT1a.ble Exp1e. rEimxpeenrtismtheanttsu stehamt uulstiep lmeudlotispelele vdeolsseo lremveulslt ioprl emsturlatiinpslew setrreaienxsc lwudereed .eRxcelduudceedd. sRuerdvuivcaeldm seuarnvsival momrtaelaintys omfohrotasltistye xopf ohsoesdtst oexBpaotsreacdh tooc hByattrriaucmhowchaystrsiiugmn iwficaasn stilgynhifiigchanertltyh hanigchoenr ttrhoalnm coornttarloitly m.Horetrael,ity. weHdiesrpel,a wyeth deimspilnaiym tuhme ,mfiirnstimquuamrt,i lfeir,smt eqduiaarnti,lteh, imrdeqduiaanrt,i lteh,iardn dqumaarxtiilme,u amndz omosapxoimreudmo szeoroesgpaordrein dgose hosrtesguarrvdiivnagl .host survival. AnAinm pimorptoarnttandtr idvreirveorf hoof shto–psta–tphaotgheongeinn teinratecrtaiocntisoniss hiso shtobset hbaevhiaovri[o7r2 [,17028,1,10089,1]0.9B].a sBkaisnkgi,nfgo, rfor exaemxapmlep,mle,a ymbeaya nibned icaanti oninodfidcaisteioanse ionff ecdtiiosneainsea minpfheicbtiiaonns [1in10 –a1m12p].hAiblitaenresd t[h1e1r0m–1o1r2eg].u laAtoltreyred behthaevrimoro(rie.eg.u,blaethoarvy iobreahlafveivoerr ()i.me.a, ybeahidaviinocralel afreivnegr)B mdainyf eacidti oinn .cHleoawrinegv eBr,df einvfeercbtieohna. vHioorwdeevpeern, fdesver onbsepheacvieiosra nddepliefnedsst aogne [s1p0e8c,1ie1s3 ]a.nAdd dlifitei osntaaglley ,[i1t0h8a,1s1b3e].e nAsdudgigtieosntaeldlyt,h aitt hagasg rbegeeanti osnugbgeehsatveido rtshat canagingcreregaasteioBnd bephraevviaolersn ccaen. Tinhcures,assec hBodo plirnegvaslpeencciee.s Tmhauys, bsechmooolrienga tspriesckietsh manaya mbep hmiboriaen ats priescki ethsan witahmspohliitbairaynl isfpeesctiyelse sw[i1t0h9 s]o.lTithairsyp lrifeed sictytiloens [d1e0p9e].n Tdhsisst prornedgilcytioonn tdheepaesnsdums sptrtoionngtlyh aotni nthfeec atesdsuhmospttsion shetdhaitn ifnefceticoteuds zhooostssp sohreesd. iRnefeccetniotuws ozrokosshpoowress.t hRaetcesnpti lwloovrekr sinhfoewctsio tnhadto sepsilnloovteorc icnufrecintioanll dhooesst sn,ot sugogcecustri ning athlla htoasstpse, cstusgogfelsitfienhgi sthtoarty a(sep.gec.,tsb oodf ylifsei zhei)staonrdy b(ee.hga.,v bioordayl isnizteer) aacntido nbseh(ea.vgi.o,rinalt eirnstpereaccifiticons com(ep.ge.t,i itniotner)sbpeetcwifeice ncohmosptestmitiaoyn)d brievtewienefne chtioosntss mevaeyr idtyriivne hinofsetcctoiomnm seuvneirtiiteys i[n1 1h4o]s.tI ncofemctmeduntaitdiepso [le1s14]. havInefedcetmedo tnasdtrpaotleeds halatveere ddemacotinvsittryatleedv eallst,erwehdi achctimviatyy lbeevealns, iwmhpicohr tmanatyi nbed iacna tiomrpoofratanntit- ipnrdeidcaattoorr of behaanvtii-oprre[7d2a,t1o1r5 b].ehWavhiioler [r7e2d,u11ce5d]. Wachtiivleit yrecdauncemda akcetivtaitdyp coalens mleasksev tiasdibploeleasn dlestsh uvissilbelses aantdr itshkufso lress preadt artiisokn f,osrl upgregdisahtiobne,h salvuigogrischan behhinadvieorra cnanin hdiinvdideru aanl’ sinadbiivliitdyutaol’ess acbaiplietya tpor eesdcaatpioe na epvreednat.tiHona neveetnt. al.H[1a1n5] eotb asel.r v[1e1d5B] do-binsefervcteedd Btoda-dintfaedcpteodle stosaede ktiandgproelfeusg esemekoirnego frteefnugthea mnootrhee orfstpeenc itehsatne sotethde.rP asprreicsies etatel.s[t7e2d]. dPeamrriosn estt aral.t [e7d2]t hdaetmwohnesntratateddp othleast wwhereen etxapdopsoeldest woeornel yexvpiosuseadl ptore odnaltyio vniscuuael sp,ruendiantfieocnt ecdues, induinviidnfueacltsedp oinsidtiiovnideudatlhs epmossietlivoensedfa trhthemersferlovmes tfhaertphreerd farotomr tthhae nprinedfeacttoerd thananim inafles.ctCedar aenyiemtaalls..[ C99a]rey obseet ravl.e [d99th] oatbspeorsvte-md tehtaamt poorspth-micettoaamdosrepxhpico tsoeaddtso eBxpdowseedre toh Boldd winegreth heoilrdbinodg itehseoiru btoodfiwesa otuert omf owreater thamnourne etxhpaons uedneixnpdoisveiddu inaldsi.vIidnuoanlse. sItnu odnye, fsrtougdsyt,h farotghsa dthante vheard bneeevnere xbpeeonse edxptoosBedd dtois Bpdla dyeisdplnaoyed signnoifi sciagnntifaivcaonidt aanvcoeidoarnactter aocrt iaotntratocttihoen ptoa tthhoeg penat,hwohgeerne,a wsphererveaiosu psrlyeviinofuecstleyd infrfoecgtsedas fsroocgiast aedsswociitahted patwhoitghe pna-fthreoegefrno-gfrseea fmroagjos rai tmyaojofrtihtye otifm thee[ t8i3m].e [T8h3i]s. Tinhdisi cinatdioicnatoiofnp ooft epnottieanlltyiallelya rlneaerdnebde hbaevhiaovriaolral avoaivdoaindcaentcoe Btod Badn danpde rphearphsapotsh oetrhpear tphaotgheongsewnsa wrraanrrtasnftusr ftuhertrheexrp elxopralotiroanti.on. Differences in Bd susceptibility are dependent on amphibian life stage, with juveniles and adults usually being more susceptible than embryos and larvae, most likely due to increased keratin distribution and abundance after the larval stage [80,116]. Bd infection in tadpoles rarely results in mortality (see [15,86,98], but has generally been related to reduced foraging efficiency and food intake Diversity2018,10,81 9of49 Differences in Bd susceptibility are dependent on amphibian life stage, with juveniles and adultsusuallybeingmoresusceptiblethanembryosandlarvae,mostlikelyduetoincreasedkeratin distributionandabundanceafterthelarvalstage[80,116]. Bdinfectionintadpolesrarelyresultsin mortality(see[15,86,98],buthasgenerallybeenrelatedtoreducedforagingefficiencyandfoodintake inlarvae[117–120]. Inpost-metamorphicamphibians,Bdinfectionismanifestedinthekeratinized epidermis; thus, the effects of foraging efficiency are dependent on the locality of infection. For example, in adult salamanders (Plethodon cinereus), Bd-infected individuals displayed increased feeding behaviors in comparison with uninfected individuals, a behavioral modification that has beensuggestedasastrategytooffsetthecostsassociatedwithimmuneactivation[121]. Body size may also be a factor in host susceptibility to pathogens [122]. Experiments have shownthatindividualsizemaybeaninfluentialfactorinBdsusceptibility[116]. Garneretal.[79] showedthatsmallertoads(Anaxyrusboreas)weremorepronetoBd-inducedmortalitycomparedwith largerindividuals. Experimentsonhost–Bdinteractionshaveaddressedphysiologicalstressresponses. Inbothfield andlaboratoryinvestigations,Bdsignificantlyelevatedphysiologicalstresshormone(corticosterone) levelsinamphibianhostsofmultiplespecies[102–104,123],thoughthereisnoevidencethatexposure toendogenouscorticosteronealtersamphibiansusceptibilitytoBd[104]. DifferentstrainsofBdelicit significantlydistinctivehormonalstressresponsesfromtheirhosts,withmorevirulentstrainsresulting in higher corticosterone levels [123]. New methodologies, such as a non-invasive stress hormone assay[102],enhancethevalueoffieldstudiescoupledwithexperimentallaboratoryinvestigationson physiologicalstressresponse.Thedynamicsbetweenstressresponseandchronicdiseasemanifestation warrantfurtherexploration. 3.2. Batrachochytriumsalamandrivorans Duetoitsrecentdiscovery,therearefewexperimentalstudiesdocumentingtheeffectsofBsalon amphibianhosts(Table1b). Bsalprimarilyaffectsnewtsandsalamandersratherthananurans. The commonmidwifetoad(Alytesobstetricans),aspeciessusceptibletoBd,didnotexperienceanyclinical signsofBsalinfection[18]. Further,Marteletal.[42]showedthattenanuranstestedwereresistantto skininvasion,infection,anddiseasesignswhenexposedtoadoseof5000zoosporesofBsal. Studies conductedwithBsalonpotentialurodelanhostsdemonstratedthatresponsesvariedacrossspecies andwithinthesamegenus. BsalinducedlethaleffectsonLissotritonitalicus,theItaliannewt,whereas noinfectionordiseasesignsweredocumentedinL.helveticus[42].TheresultsofBsal–hostexperiments show that Bd and Bsal differ in how they show the effects of exposure to these pathogens [18,42]. Experimentallyinfectedfiresalamanders,Salamandrasalamandra,experiencedataxia,ararelyreported signinexperimentalstudieswithBd. Thestudyalsoidentifiedthreepotentialreservoirspecies,the Japanesefirebellynewt(Cynopspyrrhogaster),theChuxiongfire-belliednewt(Hypselotritoncyanurus), andtheTamDaosalamander(Paramesotritondeloustali),asindividualsofthesespecieswereableto persistwithorclearinfectioninsomecapacity[42]. Bsaltransmissiondynamicsarenotyetwelldocumented. Inastudyexaminingtransmission between infected and naïve hosts, Martel et al. [18] found that two days of shared housing in salamanders resulted in infection and mortality of formerly naïve hosts within one month. All experimentalworkdoneregardingBsalhasusedonlyonepathogenisolate,asmallrangeofdoses, andfewsourcepopulationsforeachspeciestested(Table1b). Becauseexperimentsconductedon Bd–hostdynamicsshowthatresponsesareheavilydependentonspecies,population,pathogenisolate, temperature,andexposuredose,futureresearchshouldconsiderhowthesefactorsinfluenceinfection dynamicsintheBsalsystem. 3.3. Ranavirus Experimental studies have shed light onto the comprehensive effects of Rv on amphibians worldwide(Figure3;Table1c). ExperimentalRvmortalityisinfluencedbyavarietyoffactorsmost Diversity2018,10,81 10of49 notably, exposure method. Ingestion of Rv infected carcasses result in infection transmission and reducedsurvival[57,124]. ExposuretoRvviawaterinducedvariableratesofmortality,withmost studiesshowingslowerratesofmortalitywhentransmissionoccurredviawater,comparedtowhenit occurredviaingestion[70,125]. Hovermanetal.[126]foundthatinfectionandmortalityrateswere greaterfortadpolesthatwereorallyinoculatedwithRvcomparedtothoseexposedviawaterbath. AggressiveinteractionsmayserveasanefficienttransmissionrouteofRv[56]. Cannibalisticbehavior may be harmful to the individual exemplifying the behavior because of disease transmission, but anexperimentalstudyshowedcannibalismcanresultindecreasedcontactratesbetweennaiveand infectedindividualsinthepopulation[56]. Additionally,experimentshavesuggestedthatnecrophagy mayserveasacommonrouteofRvtransmission,shiftingtransmissionfromdensity-dependentto frequency-dependent[56,57,124,127,128]. TemperatureinfluencesRvinfectivityandsurvivalratesinhosts[129,130]. Whenexposedtothe Rv,ATV,larvalAmbystomatigrinumsalamandersexperiencedhighersurvivalrateswhenexposedat 26◦Cthanthoseexposedat18◦Cand10◦Cwithvirustiterbeinghigherincoolertemperatures,and viralreplicationrateswerehigherathighertemperatures[130]. Similarly,Echaubardetal.[129]found thattheprobabilityofRvinfectionincreasedatlowertemperatures(14◦C),butthattheeffectswere isolateandspecies-dependent. Itiscriticaltotakeacomparativeapproachtoexperimentallyinvestigatespeciesvariationin susceptibilitywithregardstoRv. Understandingtherelativesusceptibilityofhoststoapathogen isimportantforpredictinghost–pathogendynamics. CoevolutionbetweenRvsandtheirhostshas beenhypothesizedtobeadrivingforcebehindhostvariationofsusceptibility[131]. Hovermanet al.[132]discoveredawiderangeoflethaleffectsamong19larvalamphibianspecies,whichresulted inmortalityratesspanningfrom0to100%. TheirstudyshowedthatanuransinthefamilyRanidae weretypicallymoresusceptibletoRvthantheotherfivefamiliestested. Previousexperimentalworkhasdemonstratedinfectionandvirulencevariationamongisolates andRvspecies[54,125,132,133]thoughphenotypicvariationamongRvisolatesisnotwellunderstood. Schock et al. [54] determined that FV3 and ATV Rv species vary in their ecology and restriction endonucleaseprofiles,eventhoughtheyhaveidenticalmajorcapsidprotein(MCP)genesequences. Their results further emphasize the importance of characterizing isolates beyond MCP sequence analysis. Cunningham et al. [125] detected differences in tissue trophism and pathology between twostrainsofFV3-likeRvsincommonfrogs(Ranatemporaria). Schocketal.[133]revealedthatATV strainsdifferedinvirulence,butthiswasdependentupontheoriginofthesalamanderhost. Similarly, Hoverman et al. [132] showed that mortality rates were ~50% greater with a Rv isolate obtained fromanAmericanbullfrog(Lithobatescatesbeianus)culturefacilitycomparedtoFV3. Theseresults highlighttheimportanceofcontrolledexperimentalstudiestoelucidatepatternsofdifferentialhost susceptibilitywithregardstoRvisolatesandspecies. Experimentalandobservationalfieldstudieshaveshownthatlate-stagelarvaethatarenearing metamorphosis are the most susceptible to lethal effects of Rv infection [60,61,105,134,135]. When exposedtoATV,metamorphosedAmbystomatigrinumlarvaewerefivetimeslesslikelytobeinfected thanthosethatremainedatthelarvalstage[70]. ExperimentalstudiessuggestthattheeffectsofRv aremorelethaltolarvaethananyotherhostlifestage. Inanexperimentalstudyexaminingseven amphibianspeciesatvariousdevelopmentalstages,Haislipetal.[136]observedthatmortalityand infection prevalence were greatest at the hatchling and larval stages in four of the species tested comparedwithfrogsundergoingmetamorphosis,andthattheembryowastheleastsusceptiblestage, possibly due to the eggs protective membranous properties. Similarly to what has been observed withBdinfections,life-stagevariationinsusceptibilityhasbeenattributedtochangesthatoccurin thehypothalamic–pituitary–interrenalaxis(thecentralstressresponsesystem)aroundthetimeof metamorphosis,whichhelpstomediatetheimmunesystem[137]. Hostgeneexpressionvariationmay contributetolife-stagedifferencesinsusceptibility. Andinoetal.[134]foundthatlarvaeexperienced greaterinfectionratesandpossessedloweranddelayedexpressionofinflammationassociatedantiviral