Is Radon Emission in Caves Causing Deletions in Satellite DNA Sequences of Cave-Dwelling Crickets?


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Is Radon Emission in Caves Causing Deletions in Satellite DNA Sequences of Cave-Dwelling Crickets?

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Is Radon Emission in Caves Causing Deletions in Satellite DNA Sequences of Cave-Dwelling Crickets?
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PLOS One
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Allegrucci, Giuliana
Sbordoni, Valerio
Cesaroni, Donatella
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DNA, Dolichopoda Cave, Crickets ( local )
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The most stable isotope of radon, 222Rn, represents the major source of natural radioactivity in confined environments such as mines, caves and houses. In this study, we explored the possible radon-related effects on the genome of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae) sampled in caves with different concentrations of radon. We analyzed specimens from ten populations belonging to two genetically closely related species, D. geniculata and D. laetitiae, and explored the possible association between the radioactivity dose and the level of genetic polymorphism in a specific family of satellite DNA (pDo500 satDNA). Radon concentration in the analyzed caves ranged from 221 to 26000 Bq/m3. Specimens coming from caves with the highest radon concentration showed also the highest variability estimates in both species, and the increased sequence heterogeneity at pDo500 satDNA level can be explained as an effect of the mutation pressure induced by radon in cave. We discovered a specific category of nuclear DNA, the highly repetitive satellite DNA, where the effects of the exposure at high levels of radon-related ionizing radiation are detectable, suggesting that the satDNA sequences might be a valuable tool to disclose harmful effects also in other organisms exposed to high levels of radon concentration.

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RESEARCHARTICLEIsRadonEmissioninCavesCausingDeletions inSatelliteDNASequencesofCave-Dwelling Crickets?GiulianaAllegrucci * ,ValerioSbordoni,DonatellaCesaroniDepartmentofBiology,UniversityofRomeTorVergata,Rome,Italy * allegrucci@uniroma2.itAbstractThemoststableisotopeofradon,222Rn,representsthemajorsourceofnaturalradioactivity inconfinedenvironmentssuchasmines,cavesandhouses.Inthisstudy,weexploredthe possibleradon-relatedeffectsonthegenomeof Dolichopoda cavecrickets(Orthoptera, Rhaphidophoridae)sampledincaveswithdifferentconcentrationsofradon.Weanalyzed specimensfromtenpopulationsbelongingtotwogeneticallycloselyrelatedspecies, D . geniculata and D . laetitiae ,andexploredthepossibleassociationbetweentheradioactivity doseandthelevelofgeneticpolymorphisminaspecificfamilyofsatelliteDNA( pDo500 satDNA).Radonconcentrationintheanalyzedcavesrangedfrom221to26000Bq/m3. Specimenscomingfromcaveswiththehighestradonconcentrationshowedalsothehighestvariabilityestimatesinbothspecies,andtheincreasedsequenceheterogeneityat pDo500 satDNAlevelcanbeexplainedasaneffectofthemutationpressureinducedby radonincave.WediscoveredaspecificcategoryofnuclearDNA,thehighlyrepetitivesatelliteDNA,wheretheeffectsoftheexposureathighlevelsofradon-relatedionizingradiation aredetectable,suggestingthatthesatDNAsequencesmightbeavaluabletooltodisclose harmfuleffectsalsoinotherorganismsexposedtohighlevelsofradonconcentration.IntroductionRadonisaradioactivegasoccurringnaturally.Itispartofthenormalradioactivechainofuraniumandrepresentsthedecayproductofradium.Itisararegasandusuallymigratesfreely throughfaultsandfragmentedsoilsandmayaccumulateincavesand/orwater.Themoststableisotopeofradon,222Rn,hasahalf-lifeofabout4daysandduetothischaracteristic,itsconcentrationdecreaseswithincreasingdistancefromtheproductionarea.Groundwaterhas generallyhigherconcentrationsof222Rnthansurfacewaterbecausetheradoniscontinuously producedbytheradiumpresentintherocks.222Rncanbesignificantlyhighinhotsulfur springwaters[ 1 ].Duetothesecharacteristics,222Rnrepresentsthemajorsourceofnaturalradioactivityinconfinedenvironmentssuchasmines,cavesandhouses.Typicaldomesticexposuresareabout100Becquerelpercubicmeter(Bq/m3)indoorsand10 – 20Bq/m3outdoors[ 2 ]. PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 1/11 OPENACCESS Citation: AllegrucciG,SbordoniV,CesaroniD (2015)IsRadonEmissioninCavesCausing DeletionsinSatelliteDNASequencesofCaveDwellingCrickets?.PLoSONE10(3):e0122456. doi:10.1371/journal.pone.0122456 AcademicEditor: JoshuaB.Benoit,Universityof Cincinnati,UNITEDSTATES Received: September26,2014 Accepted: February13,2015 Published: March30,2015 Copyright: ©2015Allegruccietal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: Allrelevantdataand sequencenumbersarereportedinthepaper. Funding: ThisworkwassupportedbytheRegione Lazio(DipartimentodelTerritorio),Italy,grant number:B4886/05.Thefunderhadnoroleinstudy design,datacollectionandanalysis,decisionto publish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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Concentrationlimitsofradonfordomesticareasarevariableanddependontheorganization; theEuropeanUnionestablishedtwothresholdvalues,onefortheoldhouses(400Bq/m3)and oneforthenewones(200Bq/m3),whiletheUS-EPA(2007)putthelimitatconcentrationof 74Bq/m3.Studieshavedemonstratedasignificantanddose-relatedexcessoflungcancerin radon-exposedminers(NationalResearchCouncil1988)andseveralecologicstudieshave foundincreasedratesofleukaemiainregionswithelevatedlevelsofradoninhomes[ 3 , 4 , 5 , 6 , 7 ]. Incaves,radonconcentrationisknowntovarywithinanextremelywiderange[ 8 , 9 ].Naturalcavesofvolcanicorigincanbecharacterizedbyexceedinglyhighlevelsofradonbecauseof thepresenceofuraniumandthereforeofthedecaychainproductsofuraniumseries[ 10 , 11 ]. Artificialcavesascellars,Etruscangraves,andRomancisternsareoftenbuiltwithtuff,atype ofrockconsistingofconsolidatedvolcanicashejectedduringavolcaniceruption.Insuchenvironments,radonconcentrationmaybeveryhigh. TheoccurrenceofawidespectrumofradonconcentrationinItaliancaves,andthepossibilitytofindsomeofthesecavesconstantlyinhabitedby Dolichopoda cavecrickets(Orthoptera, Rhaphidophoridae),ledustoevaluatetheseinsectsasasuitablemodeltostudytheeffectsof radononcavelife. Dolichopoda cavecricketsarestrictlydependentuponcavesandseveralpopulationsinhabit cave-likehabitats,suchasrockcrevicesandravines,cellars,catacombs,aqueducts,Etruscan tombsandothersimilarman-madehypogeanenvironments.Theyhavelongbeenstudiedin ourlaboratoryfromawidearrayofgeneticandecologicalaspectsaddressedtounderstand theirevolutionandphylogeny[ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ].Apreliminarystudy,carriedoutthroughtheCometassay,suggestedastatisticallysignificantdose-effectincreaseof DNAdamageinspecimensof Dolichopoda fromradon-pollutedcaves,especiallyforthebrain cells[ 22 ]. Dolichopoda populationsandspecieshavealsobeeninvestigatedforprocessesofmolecular evolutionofsatelliteDNA(satDNA),[ 23 , 24 , 25 ].SatDNAisaclassofnon-codingDNAtypicallyorganizedinlargehomogeneousarraysoftandemlyarrangedrepetitionunits.These unitsareusuallylocatedintheheterochromaticpartsofthechromosomesintheregionsclose tothecentromeresandtelomeres.Repeatsizecanvarylargelywithinandbetweenspecies fromonlyafewbasepairsuptoseveralthousandbasepairs[ 25 andreferencestherein].Three specificsatDNAfamilieshavebeencharacterizedfor Dolichopoda species,twoofthembeing species-specific( pDo102 and pDsPv400 )andone( pDo500 )occurringinall Dolichopoda species [ 23 , 24 ].Apotentialhammerhead(HH)ribozymeisembeddedwithinthe pDo500 tandemly repeatsatDNA[ 26 , 27 ].Inthepresentstudy,weexploredwhetherincreasinglevelofradon-relatedionizingradiationcouldinduceanincreasedmutationpressureatthelevelofthe pDo500 tandemlyrepeat satDNA.Weconsideredtwospecies, D . laetitiae (Menozzi1920)and D . geniculata (Costa, 1860)that,asdemonstratedinpreviousstudies[ 13 , 14 , 15 , 18 , 19 ],aregeneticallycloselyrelated.Populationsampleswerecollectedincavesshowingvariousamountofradonconcentration,inordertoinvestigatethepossibleassociationbetweenradon-relatedionizingradiation andthelevelofpolymorphisminthe pDo500 tandemlyrepeatsatDNAMaterialsandMethodsThisstudywasformerlyapprovedbyRegioneLazioinItaly(DipartimentodelTerritorio),asa contributiontotheknowledgeofradoneffectsoninsectsconstantlysubjectedtoradioactivity. Noneofthefieldsurveysinthepresentstudyinvolvedendangeredorprotectedspeciesandno permissionwasnecessaryforthestudiedareas.SpecimensfortheDNAanalysiswerecollected Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 2/11

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insevendifferentcavesshowingdifferentradonconcentration( Table1 , Fig1 ).Sequencesof pDo500 tandemlyrepeatssatDNAwerederivedfromsamplesbelongingtotwopopulationsof D . laetitiae andtofivepopulationsof D . geniculata .Wealsoretrieved pDo500 satDNAsequencesfromGenBankforpopulationsamplescomingfromotherthreecaveswhoseradon ’ s concentrationvalueswereknown:twopopulationsamplesof D . geniculata ,[ 25 , 28 ]andoneof D . laetitiae [ 25 , 29 ].See Table1 fordetails.RadonmeasurementsThepresenceofradonincaveswasdetectedbytheAlfatrackdetectorLR115.Thisdetector hasaparticularfilmcapableofmeasuring222Rnconcentration.Itsworkingisbasedonthe principlethattheradon'salphaparticlesleavetracesonafilmcoatedwithathinlayerofgelatin.Ithastobeplacedinastableanddrylocationforanadequatetime.Inthisstudy,theAlfa trackdetectorLR115waslocatedatthecenterofthecaveinallconsideredsitesexceptforthe Pastenacave(PAS).Thelatterisalargecavesubdividedintwodistinctroomsandthespecializedequipmentwaslocatedatthecenterofeachroom.Followingthemanufacturerinstructions,theAlfatrackdetectorwasleftinthecavesforamonth,toobtainthemeasureofthe Table1. Dolichopoda populationsamplesincludedinthisstudy. SpeciesCodeLocalityAveragehourly concentrationBq/ m3± SE Samplesize/ Reference GenBankAccessionnumbers D . laetitiae MTR1Sulfurmine,CanaleMonterano,Roma,Latium. Latitude:42°6'47";Longitude:12°27'47" 25997 ± 5207individuals,17 clones/Present paper KM598457-KM598475 MTR2Cellar,CanaleMonterano,Roma,Latium. Latitude:42°6'47";Longitude:12°27'4" 2677 ± 13412individuals,95 clones/Present paper KM598476-KM598570 PSCPoscolacave,Priabona,Vicenza,Veneto. Latitude:45°39'02";Longitude:11°21'42" 2004individuals,4 clones/(23,29) GU322289-GU322292 D . geniculata PRAPraieCave,Lettomanoppello,Pescara,Abruzzo. Latitude:42°14'22";Longitude:14°3'18" 9825individuals,5 clones/(23,28) GU322284-GU322286 PTVPertusodiTreviCave,Filettino,Frosinone, Latium.Latitude:41°87.11'81.03";Longitude:13° 28.22'84.43" 1320010individuals,32 clones/(28) KP399737-KP399768 TUSTuscoloCave,RomanAqueduct,Frascati, Latium.Latitude:41°47'57.37";Longitude:12° 41'47.25" 1906 ± 767individuals,11 clones/(23and Presentpaper) GU322316-GU322319KM598585KM598591 ACPFiumeCopertoCave,Sermoneta,Latina,Latium. Latitude:41°51.74'49.56";Longitude:12° 99.38'41.14" 1305 ± 913individuals,7 clones/Present paper KM598447-KM598453 AUSAusiCave,Prossedi,Latina,Latium.Latitude:41° 50.99'20.72";Longitude:13°27.41'24.99" 1047 ± 3795individuals,5 clones/(23,28) GU322149-GU322153 CLPReginaMargheritaCave,Collepardo,Frosinone, Latium.Latitude:41°75.91'53.45",Longitude:13° 36.71'19.97" 221 ± 356individuals,8 clones/(23and Presentpaper) GU322194-GU322199KM598454KM598456 PASPastenaCave,Pastena,Frosinone,Latium. Latitude:41°49.76'23.9";Longitude:13° 49.10'18.89"13,4910188941,4976239 2385 ± 11910individuals,19 clones/(23and Presentpaper) GU322261-GU322265KM598571KM598584 Radonradioactivitymeasurements(Bq/m3)andGenBankAccessionnumbersarealsoreported. doi:10.1371/journal.pone.0122456.t001 Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 3/11

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averageconcentrationofradonbasedontracesleftbyalphaparticles(certificatenumbers from13843to13849,inaccordancewithU.SEPANationalRadonProficiencyProgram EPA — CFARecommendedTestReportFormat).LaboratoryproceduresGenomicDNAwasextractedfromlegmusclesusingtheSigma-AldrichGenEluteMammalian genomicDNAMiniprepKit,followingtheinstructions. PDo500 satDNAsequenceswereamplifiedwiththefollowingprimers,5'-GTTTTACACGTTCACTGCAG-3'and5'GACACATTGATGAGACTGCAG-3'[ 24 ].ThePCRconditionswereasfollow:95°Cfor3minutes,followedby30cyclesofdenaturationat95°Cfor 30seconds,annealingat50°Cfor30seconds,elongationat72°Cfor30secondsandonefinal elongationstepat72°Cfor2minutes.TheobtainedPCRproductswereclonedusingthe pGEM-TEasyVectorkit(Promega).PositivecloneswereselectedthroughPCRamplification usingthereverseandforwardM13primers.TheobtainedPCRproductswerepurifiedusing theenzymaticdigestion(ExoSAP-IT,Affymetrix,U.K.)andsequencedusingtheABI-3730GeneticAnalyzer.AlignmentwascarriedoutusingClustalX1.81[ 30 ].DataanalysesSatDNArepeatpolymorphism,consideringtheestimatesofnucleotidediversity( )andthe averagenumberofnucleotidedifferences(K),wasinvestigatedusingDNAspsoftware[ 31 ]for eachsampledpopulation.DNAspwasalsousedtoperformslidingwindowanalysisinorderto detectregionsofhighsequenceconservation.Wecarriedoutthisanalysisbyconsideringsites withalignmentgapinthewindowlength.Thewindowsizewassetto30withstepsizeof5. Theanalysiswasperformedonboththecompletealignmentforeachpopulationandonconsensussequencesforeachspecies. Insertion-deletionpolymorphismwasalsoanalyzed,usingthemultiallelicoptionin DNAsp.Thetotalnumberofindelevents,theaverageindellengthperevent,thenumberof indelhaplotypesandtheindelhaplotypediversitywerecalculatedforeachpopulation. Toinvestigatepossiblerelationsamongtheradonconcentrationincaves,theamountof satDNApolymorphism,andpossiblythetaxonomicstatusofeachpopulation,twotypeof Fig1.Samplingsitesof Dolichopoda populationsconsideredinthisstudy(seealso Table1 ). Different colorsrefertodifferentspecies:pinkto D . laetitiae andblueto D . geniculata . doi:10.1371/journal.pone.0122456.g001 Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 4/11

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Fig2.Slidingwindowanalysesofthe pDo500 satelliteDNAinpopulationsof D . laetitiae and D . geniculata includedinthisstudy. Theanalysiswas performedforeachpopulationof D . laetitiae (A), D . geniculata (B)andonconsensussequencesforeachspecies(C).Thevalueofnucleotidediversity( ) wasobtainedbyaslidingwindowsizeof30withstepsize5. doi:10.1371/journal.pone.0122456.g002 Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 5/11

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multivariateanalyseswerecarriedout.Inparticular,multivariateordinationof Dolichopoda populationsamplesbasedonpolymorphism ’ smeasureswasstudiedbyFactorialCorrespondenceAnalysis(FCA),[ 32 ],usingXLSTAT2014.Theradonconcentrationincavesandthetaxonomicstatusofeachpopulationwereconsideredassupplementaryvariables.Inthisway,these twovariableswerenottakenintoaccountforthecomputationoftherepresentationspaceand theircoordinateswerecomputedaposteriori. Amultivariatemultipleregressionanalysis(MANOVA)wascarriedoutusingPASTsoftware [ 33 ]tocompareradonconcentrationincavesandthetaxonomicstatusofeachpopulation withthemeasuresofsatDNApolymorphism.Inparticular,thefrequencyofpolymorphicsites (PSF)andindelsites,theaveragelengthofindelandthehaplotypediversityperpopulation werelogtransformedandconsideredinthisanalysis.Inordertoexcludethepossibilitythat ourresultswereinfluencedbytheunbalancedsamplesize,wecarriedoutaLinearMixed Model,usingXLSTAT2014,consideringtheenvironmentalradioactivityasexplanatoryvariable andthelengthofindel,calculatedforeachindividual,astheresponsevariable.Radioactivity measures(Bq/m3)werelogtransformedandconsideredasfixedeffect.Totestthenon-independencyofdatafromthesamecave,eachcavepopulationwasconsideredasarandomeffect. Likelihoodratiotests(LRT)werecomputedusingtherestrictedmaximumlikelihood(REML) method,asimplementedinXLSTAT2014.Thesignificanceofthefixedeffectwastestedbya LRTbetweenthefullmodelandanullmodelcomprisingonlytheinterceptandthe randomeffects. Table2.Polymorphismestimatesinsampledpopulationsof Dolichopoda cavecrickets. PopulationPSFSIFALIHIFHTFHIDHTD ± Stand.Dev.K ± Stand.Dev. ± Stand.Dev. MTR10.1250.63544.0000.2630.9440.5260.993 ± 0.0218.719 ± 0.5850.020 ± 0.003 MTR20.2830.76057.6250.1790.6480.3440.962 ± 0.0134.838 ± 0.1900.012 ± 0.001 PSC0.0330.0092.0000.7501.0000.8331.000 ± 0.1778.000 ± 1.3330.018 ± 0.003 PRA0.0730.0452.3330.8001.0000.9001.000 ± 0.12618.200 ± 1.7420.041 ± 0.009 PTV0.1220.64518.7650.6560.8150.9720.988 ± 0.01613.160 ± 0.5920.030 ± 0.003 TUS0.1340.0281.3000.7271.0000.8911.000 ± 0.03914.782 ± 0.9920.033 ± 0.005 ACP0.0550.0021.0000.2860.8570.2860.952 ± 0.0968.000 ± 0.9430.017 ± 0.004 AUS0.0680.0131.2000.8000.8000.9000.900 ± 0.16114.100 ± 1.5330.031 ± 0.006 CLP0.0680.0071.0000.3750.8750.6070.964 ± 0.0779.321 ± 0.9420.020 ± 0.003 PAS0.1390.0653.3330.4740.8950.7780.988 ± 0.0219.462 ± 0.5920.022 ± 0.003 PSF:Frequencyofpolymorphicsites;SIF:Frequencyofindelsites;ALI:Averagelengthofindel;HIF:Frequencyofindelhaplotypes;HTF:Haplotype frequency;HID:Indelhaplotypediversity;HTD:Totalhaplotypediversity;K:Averagenumberofnucleotidedifferences; :Nucleotidediversity.Standard DeviationsarealsoreportedforHTD,Kand . doi:10.1371/journal.pone.0122456.t002 Table3.Estimatedvalues,standarderrors( SE ), t -valuesandsignificance( P -values)obtainedbyLikelihoodRatioTest(LRT)offullvs.reduced models,forthecoefficientsoffixedeffectsinLinearMixedModels(LMMs)fortheaveragelengthofindel(ALI,seetextfordetails). FixedeffectEstimate SEt -valueLRT df LRT 2P -value Intercept-50.57136.769-1.375 log radioactivity20.16810.3771.944113.4530.053 doi:10.1371/journal.pone.0122456.t003 Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 6/11

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Results RadonmeasurementsTable1 showsthemeanvaluesofnaturalradioactivityforeachoftenmeasuredcaves.The highestlevelsofradioactivitywerereportedinMTR1andPTVcaves,showingvaluesof25,997 and13,200Bq/m3,respectively.Allothercaves,exceptforCLPandPSC,showed222Rnconcentrationmeasuresrangingbetween982and2,700Bq/m3,whicharemuchhigherthanthe lowestthresholdvalueestablishedbyEuropeanUnionfortheradonconcentrationindwellings (400Bq/m3).OnlyCLPandPSCcavesexhibitedradonconcentrationlevelsclosetothethresholdestablishedforhumanhousing.SatelliteDNAWeanalyzed163satDNArepeatsofthe pDo500 familyfromsevensampledpopulations,each representedwith4 – 95sequences.Thelengthofthe pDo500 sequencesrangedbetween458and 481bp.Thetotalalignmentconsistedof500positions.Theaveragenucleotidecomposition Fig3.FactorialCorrespondenceAnalysisonpolymorphismmeasuresof Dolichopoda populationsanalyzedinthisstudy. Ordinationofpopulations ontheplaneisdescribedbythefirsttwoaxes.Populationsamplescomingfromcaveswithhighradonconcentrationaredisplacedintodifferentporti onsof theordinationplane. doi:10.1371/journal.pone.0122456.g003 Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 7/11

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wasT=35.2%,C=23.9%,A=21.6%andG=19.3%.Theestimatedtransition/transversion bias(R)was0.79. Resultsfromslidingwindowanalyses,carriedoutseparatelyforeachpopulationandeach species,indicatedthatpopulationsamplescomingfromcaveswiththehighestradioactivity showedalsothehighestnucleotidediversityinthepeaksoflocalmaxima,regardlessofthespecies(MTR1in D . laetitiae andPTVin D . geniculata , Fig2Aand2B ).Resultsfromslidingwindowanalysescarriedoutonconsensussequencesforeachspecies( Fig2C )indicatedhigher nucleotidediversityin D . geniculata ( rangedfrom0to0.1)thanin D . laetitiae ( ranged from0to0.07). Table2 showstheestimatesof pDo500 satDNApolymorphismwithinpopulationsamples. Frequencyofpolymorphicsites(PSF),frequencyofindelsites(SIF),andaveragelengthof indel(ALI),areremarkablyhigherinpopulationsamplescomingfromthethreecaves,with thehighestradioactivitymeasures:MTR1andMTR2housing D . laetitiae andPTVhousing D . geniculata .OnespecimenfromMTR1,fivefromMTR2andfivefromPTVshowedalsoclones withveryshortsequencesrangingfrom102to378bp.Theseshortsequenceswerecharacterizedbyalargegapinthemiddleofthe pDo500 satDNAsequence.Inparticular,deletioncoveredtheregionbetweenthefirst10basesof pDo500 andtheposition296inthealignmentand didnotoverlaptheregionofthepotentialhammerhead(HH)ribozymeembeddedwithinthe pDo500 satDNA[ 25 ; 26 ].Theseshortsequencesweretakenintoaccountonlywhenindelpolymorphismanalysiswascarriedout. Multipleregressionanalysis(MANOVA)revealedasignificantcorrelationbetweenthepolymorphismestimatesandtheradonconcentrationincave(F=4.02,Wilk Â’ slambda=0.040; P=0.033).Inparticular,thefrequencyofindelsites(SIF,p=0.008)andtheaveragelengthof indel(ALI,p=0.004)werestatisticallysignificantcorrelatedwiththelevelsofradioactivityin cave.Ontheotherhand,thenucleotidediversity(K,p=0.057)andtheaveragenumberofnucleotidedifferences( ,p=0.078)showedahightendencytobedependentfromthetaxonomic statusofeachpopulation. ResultsobtainedfromLMManalysis,carriedoutforeachindividual,showedasignificant regressionline(P=0.05; Table3 ),indicatinganincreaseofthelengthofindelrelatedtotheradioactivitylevelsmeasuredinthecaves. Fig3 reportsresultsfromFCAwiththefirsttwoaxesexplainingtogether95.12%ofthe totalvariance.Thefirstaxisclearlyseparatespopulationsamplessubjectedtohighradioactivity fromalltheothers.DiscussionInrecentyears,theimpactofchemicalsandphysicalpollutantsonthefunctionalityofDNA hasbeeninvestigatedinmanyanimalspecies[ 34 ].Mostofthestudiesevaluatedthebiological responsetotheagentsconsideringgenemutation,chromosomeaberration,sisterchromatid exchanges,DNAdamagebyCometassay,micronuclei[ 22 , 35 , 36 , 37 ].Inthisstudy,thepossiblebiologicalresponsetotheenvironmentalradioactivitywasinvestigatedbyconsideringa specificcategoryofnuclearDNA,thesatDNA,andtwogeneticallycloselyrelatedspecies.We usedtwospecies,inordertoverifyif,regardlessofthespecificgeneticvariability,theycould havethesamebiologicalresponsetotheenvironmentalcontaminant. Theanalysisofvariabilityshowedthat D . geniculata ismorepolymorphicthan D . laetitiae , asexpected[ 25 ];( Table2 , Fig2 ).However,wefoundsignificantcorrelationsbetweensome polymorphismestimatesandradonconcentrationincavesinbothspecies,regardlessofthedegreeofvariabilityexpressedbyeachone.Inparticular,bothMANOVAandFCAanalyses( Fig3 ) revealedthattheindelpolymorphism(SIF,ALI; Table2 )issignificantlycorrelatedwithradon Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 8/11

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concentration(Bq/m3; Table1 );whilethenucleotidediversityandtheaveragenumberofnucleotidedifferencesappeartobespeciesdependent.Inparticular,thetwolocalitiesMTR1, hosting D . laetitiae ,andPTV,hosting D . geniculata ,showed222Rnlevelshigherofonetotwo ordersofmagnitudethantheothercaves( Table1 ).ThisveryhighradioactivitycanbeexplainedbythepresenceofsulfurspringsinMTR1andbyboththeverylowcirculationofair andthepresenceofnumerousfaultsandfracturesinPTV.Samplescomingfromthesetwo sitesshowedanaveragelengthofindels(ALI; Table2 )greaterthanthatobservedinother caves.Highlevelsof pDo500 polymorphism(SIF,ALI,PSF; Table2 )weredetectedalsoin Dolichopoda samplesfromMTR2site,althoughthelattershowedaradioactivitylevelofoneorder ofmagnitudelowerthaninMTR1andPTVcaves( Table1 ).Thisresultmightbeexplainedby assumingthatsamplesfromMTR1andMTR2belongtothesamepopulation.Indeed,theselocalitiesareverycloseandarelocatedintheNaturalReserveofMonterano,MTR1isanoldsulfurmine,MTR2isacellarinanoldruinedhousebuiltwithtuffandislocatedat100meters awayinfrontofMTR1,beingseparatedbytheMignoneriver.Thetwocavesaresurrounded bywoods,anoptimalenvironmentfor Dolichopoda that,atnight,exitthecavetoforageand movearound.ResultsfrommtDNACytochromeOxidaseIsequencesshowedthatindividuals comingfromtheoneortheothersiteareverysimilar,showingatmostasingletondifference (notpublisheddata)and Dolichopoda cavecricketsshowgenerallyacertaindegreeofmtDNA variabilitybothbetweenandwithinpopulations[ 19 , 20 , 21 ].Therefore,itisreasonabletoconsiderthesetwositesashostingasinglepopulationandtoexpectthatindividualsof Dolichopoda transferfromonecavetoanother,beingdefinitelysubjectedtothesameradondose. TheLMManalysis,carriedoutforeachindividualtoattempttocorrectforourunequal samplesize,confirmedtheseresults( Table3 ),suggesting,again,thattheradioactivitylevels measuredinthecavesappeartoberesponsibleforthegaps Â’ lengthobservedinoursamples. ThebiologicalsignificanceofsatDNAhasbeentheobjectofseveraldiscussionandgenerally,basedonthediversityofsatDNAinnucleotidesequences,lengthofrepeats,genomicabundance,aspecificfunctionhasnotbeenyetassignedtothisgenomicregion.However,anumber ofpossiblefunctionshavebeenhypothesized[ 38 ]andmostofthemarerelatedtoheterochromatinand/orcentromereformationandfunction. Samplesfrompopulationsinhypogeanenvironmentswiththehighestradioactivityshowed alsothehighestfrequencyofindelsitesandcloneswith pDo500 repeatsequencesshorterthan thestandard,butthatareintegeratlevelofHHribozymeregion.Previousstudiessuggested thattheHHregionofthe pDo500 sequencefamilyhasafunctionalrolein Dolichopoda cave cricketsalthoughitsfunctionisunclearandremainstobeinvestigated[ 27 ].Ourdataseemto supportthehypothesisthattheHHribozymeregioncouldhaveanimportantrole,sincethis regionisneveraffectedbytheeventsofinsertion/deletion. Finally,ourresultsareconsistentwiththosefrom[ 22 ]wheresixoutofthetenpopulations herestudiedwereanalyzedforDNAprimarydamagethroughCometassay.Bothhaemocytes andbraincellstakenfromindividualsfromradon-pollutedcavesweretestedandcomparedto acontrolgroupofcavecricketsrearedinabsenceofradon.Resultsindicatedastatisticallysignificantdose-effectincreaseofDNAdamageinallcaves,especiallyforthebraincells.Inconclusion,wecaninferfrompresentdatathattheincreasedsequenceheterogeneityat pDo500 satDNAlevelcanbeexplainedasaneffectofthemutationpressureinducedbyradonincave. Furthermore,wediscoveredaspecificcategoryofnuclearDNA,thehighlyrepetitivesatDNA sequences,wheretheeffectsoftheexposureathighlevelsofradon-relatedionizingradiation aredetectable.FutureresearchescouldbeaddressedtoevaluateandinvestigateifsatDNA mightbeavaluabletooltorevealtheeffectsofradoninotherorganisms. Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 9/11

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AcknowledgmentsWeareindebtedtoMauroRampiniandGiorgioPintuswhohelpedoneofus(GA)incollectingsamplesinthedifferentcaves.SaraCocchihelpedusinthelabworking.Wealsothank GabrieleGentileforhissuggestionsincloningtechniquesandPaoloGrattonforhishelpin LMMstatisticalanalysis.StefanoDeFelicihelpedusintheimplementationof Fig1 .AuthorContributionsConceivedanddesignedtheexperiments:GAVSDC.Performedtheexperiments:GA.Analyzedthedata:GA.Contributedreagents/materials/analysistools:GA.Wrotethepaper:GA DCVS.References1. IyengarMAR.TheenvironmentalbehaviorofRadium.TechRepseries,1990:310 2. SperrinM,GillmoreG,DenmanTRadonconcentrationvariationsinaMendipcavecluster.Environ ManageHealth2001;12:476 – 482. 3. AlberingHJ,HagemanGJ,KleinjansJCS,EngelenJJM,KoulishcerL,HerensC.Indoorradonexposureandcytogeneticdamage.Lancet1992;340:739.PMID: 1355848 4. KhanMA,CrossFT,BuschbomRL,BrooksAL.Inhaledradon-inducedgenotoxicityinWistarrat,SyrianHamster,andChinesehamsterdeep-lungfibroblastsinvivo.MutatRes.1995;334:131 – 137. PMID: 7885364 5. JostesRF.Genetic,cytogenetic,andcarcinogeniceffectsofradon:areview.MutatRes.1996;340: 125 – 139.PMID: 8692177 6. BilbanM,VaupoticJ.Chromosomeaberrationsstudyofpupilsinhighradonlevelelementaryschool. HealthPhys.2001;80:157 – 163.PMID: 11197464 7. CristaldiM,IeradiLA,UdroiuI,ZilliR.Comparativeevaluationofbackgroundmicronucleousfrequenciesindomesticmammals.MutatRes.2004;559:1 – 9.PMID: 15066568 8. HaklJ,HunyadiI,VarhegyiA.Radonmonitoringincaves.In:DurraniSA,IlicR,editors.Radonmeasurementsbyetchedtrackdetectors.WorldScientific;1997.pp.261 – 283. 9. CignaAA.Thedistributionofradonconcentrationincaves.IntJSpeleol.2003;32:113 – 115. 10. BaubronJC,AllardP,SabrouxJC,TedescoD,ToutainJP.Soilgasemanationsasprecursoryindicatorsofvolcaniceruptions.JgeolSocLond.1991;148:571 – 576. 11. AytekinH,BaldikR,CelebiN,AtaksorB,TasdelenM,KopuzG.Radonmeasurementsinthecavesof Zonguldak(Turkey).RadiatProtDosimetry2006;118:117 – 121.PMID: 16120690 12. SbordoniV,DeMatthaeisE,CobolliSbordoniM.Phosphoglucomutasepolymorfismandnaturalselectioninpopulationsofthecavecricket Dolichopodageniculata .J.Zoolog.Sist.Evol.Res.1976;14: 292 – 299. 13. SbordoniV,AllegrucciG,CesaroniD,SbordoniCobolliM,DeMatthaeisE.Geneticstructureofpopulationsandspeciesof Dolichopoda cavecrickets:Evidenceofperipatricdivergence.Boll.Zool.1985; 52:139 – 156. 14. SbordoniV,AllegrucciG,CacconeA,CarchiniG,CesaroniD.MicroevolutionarystudiesinDolichopodinaecavecrickets.In:BaccettiBeditor.EvolutionaryBiologyofOrthopteroidInsects.HorwoodLtd. Publ,Chichester,U.K.1987.pp.514 – 540 15. AllegrucciG,CesaroniD,SbordoniV.Adaptationandspeciationof Dolichopoda cavecrickets(Orth. Rhaph.):geographicvariationofmorphometricindicesandallozymefrequencies.BiolJLinnSocLond. 1987;31:151 – 160. 16. DePasqualeL,CesaroniD,DiRussoC,SbordoniV.Trophicniche,agestructureandseasonalityin Dolichopoda cavecrickets.Ecography1995;18:217 – 224. 17. AllegrucciG,MinasiMG,SbordoniV.Patternsofgeneflowandgeneticstructureincave-dwellingcricketsoftheTuscanendemic, Dolichopodaschiavazzii (Orthoptera,Rhaphidophoridae).Heredity1997; 78:665 – 673. 18.CesaroniD,MatarazzoP,AllegrucciG,SbordoniV.Comparingpatternsofgeographicvariationin cavecricketsbycombininggeostatisticmethodsandManteltests.JBiogeogr.1997;24:419 – 431. Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 10/11

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19. AllegrucciG,TodiscoV,SbordoniV.Molecularphylogeographyof Dolichopoda cavecrickets(Orthoptera,Rhaphidophoridae):ascenariosuggestedbymitochondrialDNA.MolPhylogenetEvol.2005;37: 153 – 164.PMID: 15964214 20. AllegrucciG,RampiniM,GrattonP,TodiscoV,SbordoniV.Testingphylogenetichypothesesforreconstructingtheevolutionaryhistoryof Dolichopoda cavecricketsintheeasternMediterranean.JBiogeogr.2009;36:1785 – 1797. 21. AllegrucciG,TrucchiE,SbordoniV.Tempoandmodeofspeciesdiversificationin Dolichopoda cave crickets(Orthoptera,Rhaphidophoridae).MolPhylogenetEvol.2011;60:108 – 121.doi: 10.1016/j. ympev.2011.04.002 PMID: 21514393 22. GustavinoB,MeschiniR,FranzettiG,GrattonP,AllegrucciG,SbordoniV.Genotoxicitytestingfor radonexposure: Dolichopoda (Orthoptera,Rhaphidophoridae)aspotentialbio-indicatorofconfinedenvironments.CurrZool.2014;60:299 – 307. 23. BachmannL,VenanzettiF,SbordoniV.Characterizationofaspecies-specificsatelliteDNAfamilyof Dolichopodaschiavazzii (Orthoptera,Rhaphidophoridae)cavecrickets.JMolEvol.1994;39:274 – 281.PMID: 7932789 24. BachmannL,VenanzettiF,SbordoniV.TandemlyrepeatedsatelliteDNAof D . schiavazzii :atestfor modelsontheevolutionofhighlyrepetitiveDNA.JMolEvol.1996;43:135 – 144.PMID: 8660438 25. MartinsenL,VenanzettiF,JohnsenA,SbordoniV,BachmannL.Molecularevolutionofthe pDo500 familyin Dolichopoda cavecrickets(Orthoptera,Rhaphidophoridae).BMCEvolBiol.2009;9:301 – 314.doi: 10.1186/1471-2148-9-301 PMID: 20038292 26. RojasAA,Vazquez-TelloA,FerbeyreG,VenanzettiF,BachmannL,PaquinB,etal.Hammerhead-mediatedprocessingofsatellitepDo500familytranscriptsfrom Dolichopoda cavecrickets.NucleicAcids Res.2000;28:4037 – 4043.PMID: 11024185 27. MartinsenL,JohnsenA,VenanzettiF,BachmannL.Phylogeneticfootprintingofnon-codingRNA: hammerheadribozymesequencesinasatelliteDNAfamilyof Dolichopoda cavecrickets(Orthoptera, Rhaphidophoridae).BMCEvolBiol.2010;10:3 – 11.doi: 10.1186/1471-2148-10-3 PMID: 20047671 28. DePasqualeL.Misuredellaconcentrazionediradonevariabilit à geneticadipopolazionicavernicoledi Dolichopoda(Orthoptera,Rhaphidophoridae).TesididottoratoinScienzeAmbientali, “ Ambientee UomoinAppennino ” ,Universit à deglistudidell ’ Aquila.1998. 29. BellocchiE,BoifavaF,DalMolinL,MarchettoG.L ’indicedilanglierelaradioattivit à ambientaleindoor induegrottedell ’ altopianocarsicodelFaedo-Casaron(Vicenza).SpeleologiaVeneta2009;17:77 – 90. 30. ThompsonJD,GibsonTJ,PlewniakF,JeanmouginF,HigginsDG.TheClustalXwindowsinterface: flexiblestrategiesformultiplesequencealignmentaidedbyqualityanalysistools.NucleicAcidsRes. 1997;24:4876 – 4882.PMID: 9396791 31. LibradoP,RozasJ.DNAspv.5.AsoftwareforcomprehensiveanalysisofDNApolymorphismdata. Bioinformatics2009;25:1451 – 1452.doi: 10.1093/bioinformatics/btp187 PMID: 19346325 32. BenzécriJP.L ’ analysedesdonnées.II.L ’ analysedescorrespondenses.Dunod,Paris;1973 33. Hammer Ø ,HarperDAT,RyanPD.PAST:Paleontologicalstatisticssoftwarepackageforeducation anddataanalysis.PalaeontologiaElectronica.2001;4(1):9pp.Available: http://palaeo-electronica. org/2001_1/past/issue1_01.htm 34. AngelettiD,CarereC.Comparativeecogenotoxicology:MonitoringtheDNAofwildlife.CurrZool. 2014;60:252 – 25 35. DixonDR,PruskiAM,DixonLRJ,JhaAN.Marineinvertebrateeco-genotoxicology:Amethodological overview.Mutagenesis2002;17:495 – 507.PMID: 12435847 36. LewisC,GallowayT.Genotoxicdamageinpolychaetes:Astudyofspeciesandcell-typesensitivities. MutatRes.2008;654:69 – 75.doi: 10.1016/j.mrgentox.2008.05.008 PMID: 18579434 37. LacazeE,GeffardO,BonyS,DevauxA.Genotoxicityassessmentintheamphipod Gammarusfossarum byuseofthealkalineCometassay.MutatRes.2010;700:32 – 38.doi: 10.1016/j.mrgentox. 2010.04.025 PMID: 20451657 38. PalomequeT,LoriteP.SatelliteDNAininsects:areview.Heredity2008;100:564 – 573.doi: 10.1038/ hdy.2008.24 PMID: 18414505 Radon,SatelliteDNAandCaveCrickets PLOSONE|DOI:10.1371/journal.pone.0122456March30,2015 11/11


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