Phe362Tyr in AChE: A Major Factor Responsible for Azamethiphos Resistance in Lepeophtheirus ...

14 Pages·2016·1.26 MB·English

RESEARCHARTICLE
Phe362Tyr in AChE: A Major Factor
Responsible for Azamethiphos Resistance in
Lepeophtheirus salmonis in Norway
KiranpreetKaur1*,PederAndreasJansen2,VidarTeisAspehaug3,TorEinarHorsberg1
1 NMBUSchoolofVeterinaryScience,SeaLiceResearchCentre,POBox8146Dep.,NO-0033,Oslo,
Norway,2 NorwegianVeterinaryInstitute,Pb750,N-0106,Oslo,Norway,3 PatoGenAnalyseAS,Postboks
1527,6025,Ålesund,Norway
*[email protected]
Abstract
Organophosphates(OP)areoneofthemajortreatmentsusedagainstthesalmonlouse
(Lepeophtheriussalmonis)inNorwegiansalmonidaquaculture.TheuseofOPsincethe
OPENACCESS
late1970shasresultedinwidespreadresistantparasites.Recently,wereportedasingle
Citation:KaurK,JansenPA,AspehaugVT,
mutation(Phe362Tyr)inacetylcholinesterase(AChE)asthemajormechanismbehind
HorsbergTE(2016)Phe362TyrinAChE:AMajor
FactorResponsibleforAzamethiphosResistancein resistanceinsalmonlousetowardsOP.Thepresentstudywascarriedouttovalidatethis
LepeophtheirussalmonisinNorway.PLoSONE11 mechanismatthefieldlevel.Atotalof6658salmonlousesampleswereenrolledfrom56
(2):e0149264.doi:10.1371/journal.pone.0149264
differentfishfarmsacrosstheNorwegiancoast,fromVestAgderinthesouthtoFinnmarkin
Editor:XinghuiQiu,InstituteofZoology,Chinese thenorth.AllthesamplesweregenotypedusingaTaqManprobeassayforthePhe362Tyr
AcademyofSciences,CHINA
mutation.AstrongassociationwasobservedbetweenareaswithfrequentuseoftheOP
Received:July7,2015 (azamethiphos)andthePhe362Tyrmutation.Thiswasconfirmedat15siteswhereresults
Accepted:January30,2016 fromindependentlyconductedbioassaysandgenotypingofparasitescorrelatedwell.Fur-
thermore,genotypingofsurvivingandmoribundparasitesfromsixbioassayexperiments
Published:February16,2016
demonstratedahighlysignificantnegativecorrelationbetweenthefrequencyofresistance
Copyright:©2016Kauretal.Thisisanopen
allelesandtheprobabilityofdyingwhenexposedtoazamethiphosinabioassay.Basedon
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits theseobservations,wecouldstronglyconcludethatthePhe362Tyrmutationisamajorfac-
unrestricteduse,distribution,andreproductioninany torresponsibleforOPresistanceinsalmonlouseonNorwegianfishfarms.
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles.
Funding:Thestudywasfinanciallysupportedbythe Introduction
NorwegianResearchCouncilthroughthecentrefor
Norwegiangovernmentregulationsrequireareductionofthesalmonlouse(Lepeophtheirus
InnovationSeaLiceResearchCentre,grantnumber
NFR203513/O30.Thefundershadnoroleinstudy salmonis)burdeniftheabundanceexceeds0.5adultfemaleparasitesperfishonsalmonfarms.
design,datacollectionandanalysis,decisionto Duetothelimitednumberofchemotherapeutantsavailable,andtheshortageofothercontrol
publish,orpreparationofthemanuscript.This options,antiparasitictreatmentsthathavebeenusedtocontrolL.salmonis,andagainstwhich
fundingorganisationdidnotplayanyroleinthestudy theparasitehasdevelopedresistance,arestillinuse.Thelackofeffectivealternativeshas—and
design,datacollectionandanalysis,decisionto
iscontinuesto-agrowingresistanceproblem.Eventhoughsomesurveillanceprogramshave
publishorpreparationofthemanuscriptandprovided
beenconductedtoeducatefishfarmers,fishhealthservicesandgovernmentbodiesaboutthe
onlyfinancialsupportintheformofsalariesto
KiranpreetKaurandTorEinarHorsberg.Thespecific growingproblem[1,2],alotstillneedstobedone.
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Phe362TyrinNorway
rolesofalltheauthorsarearticulatedinthe'author Inordertocontroltheresistanceprobleminthesalmonlouse,itisimportanttodiagnoseit
contributions'section.Oneoftheauthorsofthestudy first.Iftheresistanceisdetectedatanearlystage,bettermanagementstrategiescouldbeused
isfromacommercialcompanycalledPatoGen
tocontrolitsspread.Unfortunately,theattemptssofarhavefailedtoattainthisgoal.
AnalyseAS,whocontributedinsamplecollection,
Organophosphates,inhibitorsoftheenzymeacetylcholineesterase(AChE)whichisrespon-
runningofTaqManProbeassayandcompilationof
dataforanalysis.Moreover,thisauthordidnotplay sibleforterminatingtheeffectofnerveimpulsesincholinergicsynapses,havebeenusedfor
anyroleinthestudydesign,statisticaldataanalysis treatmentagainstthemarineectoparasiticcopepodsalmonlouse(L.salmonis)inNorwegian
orthedecisiontopublish. salmonidaquaculturesincethelate1970s.Thefirstagentusedwasmetrifonate(Neguvon™),
CompetingInterests:Thecommercialcompany followedbydichlorvos(Nuvan™)in1986[3]andazamethiphos(Salmosan™)in1994[4].Inthe
PatoGenAnalyseASoffersthegenotypingserviceto early1990s,thefirstcasesofreducedefficacyoforganophosphatetreatmentswerenotedin
theircustomers.Thisdoesnotaltertheauthors' Mid-Norway[5].Thisreducedsensitivityledtotheterminationofazamethiphosusein1999.
adherencetoPLOSONEpoliciesonsharingdata
Eventhoughtheresistancetowardsazamethiphoswaswidespread,thecauseofresistancewas
andmaterials.Theauthorshavedeclaredthatno
unknown.Azamethiphoswasre-introducedasatreatmentagentagainstthesalmonlousein
competinginterestsexist.
2008[6].Newreportsofreducedefficacyofazamethiphostreatmentscamefromthefieldin
2009.In2013and2014,asurveillanceprogram,usingbioassaystotestforresistance,revealed
awidespreaddistributionofazamethiphosresistanceonNorwegianfishfarms[1,2].
Recently,twogenescodingforAChEinL.salmoniswereidentifiedandcharacterized[7].
ThetwoAChEsinL.salmonisarehighlysimilartoeachother(84%similarityattheprotein
level),anobservationquiteuniquetoL.salmonisandpreviouslyhasnotbeenobservedin
otherarthropods.ThetwoAChEsweredenotedasace1aandace1b.Ace1awaspredominantly
expressedinthedifferentdevelopmentalstagesofsalmonlousecomparedtoace1bandwas
activeinthecephalothorax,indicatingthatace1aplaysamajorroleinsynaptictransmission.
Inaddition,thescreeningofbothace1aandace1binresistantstrains(5strains)andsensitive
strains(2strains)ledtotheidentificationofasinglemissensemutationonlyinace1a,leading
toachangefromphenylalaninetotyrosineatcodon362[8].Thesignificantlyhigherfrequency
of362TyrinL.salmonissamplesresistanttoazamethiphosindicatedaclearassociationof
Phe362Tyrwithreducedsensitivitytowardsazamethiphos.Furthermore,3Dmodelling,small
scaletreatmenttrialsforgeneticcharacterizationwithazamethiphosandenzymaticinhibition
assaysprovidedastrongargumentinfavorofPhe362Tyrmutationbeingtheculpritbehind
azamethiphosresistanceinL.salmonis[8].
However,theaboveconclusionswerebasedonobservationsfromexperimentsconducted
onlaboratorycultivatedstrains.Theaimofthisstudywastoexplorethebiggerpicture:thesce-
narioonfishfarmsacrosstheentireNorwegiancoast.WhatroledoesPhe362TyrplayintheL.
salmonisresistancetowardsOPthroughoutthecountry?IsitamajorfactorresponsibleforOP
resistanceinNorway?Oristhereamorecomplexstorybehindit?Theseintriguingquestions
providedthebasisofthepresentstudywhereweattemptedtofindanswerstotheabove
questions.
MaterialsandMethods
Thisepidemiologicalstudyofgenotypesassociatedwithsensitivitytowardsazamethiphosin
salmonlousewasbasedonsalmonlousesamplessubmittedtothelaboratorybysalmon-pro-
ducingcompanies,andbioassaysconductedunderthenationalresistancemonitoringprogram
byvariousfishhealthservicesalongtheNorwegiancoast[2].
Samples
Atotalof6658salmonlousesamples(preadultandadultstages)weresubmittedtothelabora-
toryfrom56differentfishfarmsovertheperiodof2012–2014(onefarmwassampledtwice).
Mostsamples(4548)weresubmittedfrom41randomfarmswheretherewasnoinformation
aboutthetreatmenthistoryorsensitivitystatusoftheparasitestowardsazamethiphos.A
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secondsetofsamples(1696)wassubmittedfrom15sitesincludedinthenationalmonitoring
programforsalmonlouseresistance[1],howevernotatthesametimeasthesurvey.Thus,for
thesesamples,bioassayresultsbasedonatotalof1336sampleswerealsoavailable.Thesewere
two-dosebioassays,whichhadbeenconductedindependentlybylocalfishhealthservices.The
thirdsetofsamples(414)includedparasitesoriginatingfromsitesincorporatedintheresis-
tancemonitoringprogram(6sites)andweretheparasitesusedinthebioassaysinthissurvey.
Afterexposureto0,0.4or2ppb(μg/L)azamethiphosfor24hours[9],sampleswereseparated
intoaliveandmoribundparasites.Forthesesamples,boththegenotypingresultsandtheindi-
vidualphenotypicresponsetoazamethiphosexposure(aliveormoribund)wereavailable.
ThesalmonlicewerecollectedaspertheinstructionsprovidedbyPatoGenAnalyseASand
storedin70%ethanolorRNAlaterat-20°Cuntilanalysis.AllsamplesweregenotypedbyPato-
GenAnalyseAS.
Genotyping
AstandardTaqManassaywasdevelopedbyPatoGenAnalyseASfortherapidandhigh
throughputscreeningofthemutation(Phe362Tyr)identifiedinace1a[8].
Onbasisofthisassay,eachparasitecouldbeclassifiedashomozygotewildtype(SS;Phe362/
Phe362),heterozygote(RS;362Tyr/Phe362)orhomozygotemutated(RR;362Tyr/362Tyr).
Statisticalanalysesandmodeling
Thestatisticalanalysescomprisedofthreedifferentsetsofdatawithaccompanyinganalyses,
whichwetermedthespatial,bioassayandindividuallouseanalysis,respectively.
Spatialanalysis. Thedatasetaccompanyingthespatialanalysisconsistedofthe6658gen-
otypedsamplesofsalmonlouseoriginatingfrom56farmsalongthecoast(seeS1Table).The
questionsthatwereaddressedintheanalysisofthesedatawere1)whethertherewasapredict-
ablespatialpatterninthedistributionofmutated362Tyrgenotypes?and2)whetherthisdistri-
butionwasassociatedwithfarmtreatmentsorlocaltreatmentdensity?
Totestwhethertherewasapredictablespatialpatterninthedistributionofmutatedgeno-
types,wefirstassignedageographicalindex(geoindex)toeachsampledfarm.Thegeoindex
accountsforbothlongitudeandlatitudeusingthemethodpresentedinKristoffersenetal.
[10].Thisgeoindexordersthefarmsinasouthtonorthgradientandadditionallyaccountsfor
theireasttowestgradient.Thegeoindexwasconstructedusingalocalpolynomialregression
[11],wherethedependentvariablecomprisedoffarmlatitudesandtheindependentvariable
offarmlongitudes.Eachfarmwasthenprojectedontotheregressionlineandattributeda
geoindexaccordingtotheexpressiongiveninKristoffersenetal.[10].Thegeoindexregression
lineisshowninFig1.
Totestifthedistributionofmutated362Tyrgenotypeswasassociatedwithfarmtreatments
usingazamethiphos,orlocaldensitiesofsuchtreatments,reportedtreatmentdatawerecom-
piled.ReportingofbathtreatmentstotheNorwegianFoodSafetyAuthorityaccordingtocate-
goriesofsubstancesismandatory,includingazamethiphos,deltamethrin,andcypermethrin,
oras“other”.Herewesummarizethenumberofreportscategorizedasazamethiphosforevery
reportingsalmonfarmforeachoftheyears2012–2014.
Itismandatorytotreatallpensonthefarmwhenusingabathtreatment,[12].Thistakes
timeandtreatmentreportsfromgivenfarmsareoftenrepeatedoverconsecutiveweeks.To
summarizetheaccumulatednumberofbathtreatmentsperyear,weregardedsequencesof
treatmentsoverconsecutiveweekstocountasonetreatment.Thenumberoffarmsreporting
theuseofazamethiphos,thetotalnumberoftreatmentreportsandtheaccumulatednumber
oftreatmentsaresummarizedinTable1.Thenumberoftreatmentsforthefarmssampledfor
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Fig1.Relativequantitiesofthekerneldensityoftreatmentsalongwiththegeoindexline.Mapsshowingrelativequantitiesofthekerneldensityof
treatmentsalongwiththegeoindexline(leftpanel),andtheproportionoftheSS,RSandRRgenotypes(rightpanel,notethatlocationsoffarmsare
displacedinareaswithclustersoffarmstoavoidoverlappingsymbols).
doi:10.1371/journal.pone.0149264.g001
salmonlouseforthegivensamplingyearwasextractedfromthesummarynumberoftreat-
ments(Table1)andgiveninS1Table.
Thenumbersoftreatmentsperfarmperyearwereusedtocalculatelocaldensitiesoftreat-
ment.Foreachyearbetween2012–2014,alltreatmentreportingfarmswereprojectedontoa
mapinArcView(ESRI,Redlands,CA,USA)usingtheprojectionWGS84n33.Spatialkernel
densitiesoftreatmentwerethencalculatedforthenumberoftreatmentsperfarm,usingthe
KernelDensitytoolintheArcViewextensionSpatialAnalyst(ESRI,Redlands,CA,USA),a
gridsizeofonebyonekmandsearchradiusof25km.Thekerneldensityquantitiesforthe
farmssampledforsalmonlousewerethenextractedbythe“Extractvaluestopoints”toolin
SpatialAnalyst.Kerneldensitiesoftreatmentforsampledfarmsinthegivenyearofsampling
aregiveninS1Table.
Table1. Thenumberoffarmsreportingtheuseofazamethiphos,thetotalnumberoftreatment
reportsandtheaccumulatednumberoftreatments,fortheyears2012–2014.
Year Farms Totalnumberofreports Numberoftreatments
2012 244 601 422
2013 204 486 311
2014 290 568 479
doi:10.1371/journal.pone.0149264.t001
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Forthestatisticalanalysisofthespatialdataweusedtheproportionoflicewithinunique
farmsamplesingivenyearsthatharboredamutationforazamethiphosresistance,i.e.p=(RR
+RS)/(RR+RS+SS),asthedependentvariable.Sinceproportionsarenotnormallydistributed,
thedependentvariablewastransformedbytakingthearcsinofthesquarerootofthepropor-
tion[13].Fortheanalysis,weusedgeneraladditivemodels(GAM)oftheform:
pffiffiffi
arcsin p¼b þ b fX þ b X
0 1 1 n n
pffiffiffi
wherearcsin pisthedependentvariable,β istheintercept,f denotesasmoothfunctionof
0 1
thespatialvariableX withcoefficientβ ,andX representsanyotherco-variateenteredinthe
1 1 n
modelwithcoefficientsβ .GAMwasusedtoallowfornon-lineareffectsofthespatialvari-
n
ables.Sincethevariableexpressingthenumberoftreatmentsperfarmwashighlyskewed,it
wasenteredasafactorialvariablewiththreeclassesrepresenting0,1or>1treatments.Also
thekerneldensityoftreatmentswasskewed,soitwasenteredasasquareroottransformedvar-
iableintheanalyses,yieldingareasonablysymmetricdistribution.
Theanalysiswasdoneusingthe“mgcv”libraryinR[14].First,wetestedthevariablesin
univariatemodels.Thenweaddedvariablesinaforwardselectionprocedure,whilealsocheck-
ingforcorrelationbetweenvariables.
Bioassayanalysis
Thedatasetaccompanyingthebioassayanalysisconsistedof1336salmonlicefrom15different
salmonfarms(seeS1Table),whichweretestedinbioassayswiththeoutcomemoribundor
aliveatconcentrationsof0,0.4or2ppbazamethiphos,respectively.Thequestionthatwas
addressedintheanalysiswaswhetherthefarmproportionsoflicewithasusceptiblegenotype
(SS),andtheconcentrationofazamethiphosinthebioassaytests,contributedtopredictthe
testoutcome.Thecontrolexperimentswithconcentration=0gavelessthan4%mortality,and
hencethesewereomittedfromfurtherassessments.
Toassesswhetherfarmlevelsusceptibilityandazamethiphosconcentrationpredictedthe
outcomeofbioassaytestsforindividualsalmonlouse,amultivariablemixedeffectslogistic
modelwasdeveloped.Themodelhadtheform:
pffiffiffiffiffiffiffiffiffi
logitPðYi¼1jXÞ¼ b þb p:SSþb conc: þ oFarm
0 1 2
wherethedependentvariableexpressesthebinomialoutcome(moribundoralive)onalogit
pffiffiffiffiffiffiffiffiffi
scaleforindividualparasites,β istheintercept, p:SSdenotesthesquarerootofthepropor-
0
tionofSSgenotypesonfarmswithcoefficientβ ,conc.isatwolevelfactor(0.4and2ppb)
1
accountingfortheazamethiphosconcentrationwithcoefficientβ andFarmisarandomeffect
2
offarmwithcoefficientω.Theintra-classcorrelationcoefficient(ICC)wascalculatedaccord-
ingtoDohooetal.[13]as:
s2 =ðp2=3 þ s2 Þ
Farm Farm
whereσ2 representsthevariancebetweenfarmsadjustedfortheassumedvariancebetween
Farm
licewithinfarm(π2/3).
Individuallouseanalysis. Tofurtherassessthephenotypeofdifferentgenotypeswith
respecttoazamethiphossusceptibility,bioassayswereconductedwithlicesampledfromsix
differentfarmswheretheoutcome(moribundoralive)wasclassifiedforindividualparasites.
Alltheindividualsalmonliceweresubjectedto24-hourbioassaysateither0.4or2ppbaza-
methiphosconcentration,alongwithacontrolgroup(0ppb).Afterclassification,individual
parasites(n=441,seeS1Table)werecollectedandlatergenotypedforthePhe362Tyrmuta-
tion.Thequestionthatwasaddressedintheanalysiswashowdoindividualgenotypes,along
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Phe362TyrinNorway
withdifferentconcentrationsofazamethiphos,predicttheoutcome(moribundoralive)ofbio-
assaytests.
Thebioassaytestswithindividualgenotypingoflicepostexposurewerefirstanalyzedina
logisticmodelsimilartothatofthebioassayanalysis,withfarm(n=6)asrandomeffect.There
was,however,nosignificantvariancethatwasexplainedatthefarmlevelinthismodel.Hence,
thesedatawereanalyzedwithlogisticregressionwithonlyfixedeffects,inamodeloftheform:
logitPðYi¼1jXÞ¼ b þb Genotype þb conc:
0 1 2
whereGenotypeisanindividuallousefactorofthreelevels(SS,RSandRR)andconc.isathree
levelfactoraccountingforazamethiphosconcentration(0,0.4and2ppb).
Ethicalstatement
Thestudyonlyinvolvedthesalmonlouseandnofishwerebeingused.Alltheparasiteswere
collectedfromthefishfarms(locationsmentionedintheMaterialsandMethods)withthe
farmers'consent.
Afterfarmersconsent,nospecialpermissionswererequiredforthesamplecollection.The
studydidnotinvolveendangeredorprotectedspecies.
Results
Spatialanalysis
The6658salmonlousesamplesinthisstudywereenrolledfromfishfarmsdistributedthrough-
outtheNorwegiancoast,fromVestAgderinthesouthtoFinnmarkinthenorth(Fig1).These
areasrepresentalargepartofthesalmon-producingcoastinNorway.Allthe6658samples
weregenotypedforPhe362TyrmutationusingTaqManprobeanalyses.Screeningofthesam-
plesrevealedthatPhe362TyrwaswidespreadthroughouttheNorwegiancoast.Theproportion
ofdifferentgenotypes(SS,RSandRR)forthePhe362Tyrmutationindifferentareasisshown
inFig1,rightpanel.ForthewholeNorwegiancoastperse,theproportionofsalmonlousewith-
outPhe362Tyrmutation(i.e.theSSgenotype)wassignificantlylower(23.1%)thanthesalmon
licewithPhe362Tyrmutation(76.9%,ofwhich42.2%wereRSand34.7%RR,respectively).The
frequenciesofthedifferentgenotypespercountyaregiveninTable2.
Inthespatialanalysis,thefactoryearwasnotsignificantlyassociatedtotheproportionof
mutated362Tyrgenotypes,andwasthereforeexcludedfromfurthermodels.
Thevariablesexpressingfarmtreatmentsandlocaldensityoftreatment(Kerneldensity)
werebothsignificantlyassociatedwiththeproportionofmutated362Tyrgenotypesinuni-var-
iablemodels(Pearsonr=0.53).However,whenbothvariableswereenteredaslinearterms
intothestatisticalmodel,thefactorialfarmtreatmentswasnolongersignificant(F=0.71,
p=0.5)andwasthereforeexcludedfromfurthermodels.
Thegeoindexvariableenteredasanon-linearsmoothtermwasstronglyassociatedwiththe
proportionofPhe362Tyrmutation,accountingfor56%(adj.R2=0.561)ofthevarianceinthe
dependentvariable(p<0.001,AIC=-5.48).AsshowninFig2a,theproportionofPhe362Tyr
mutationwaslowestintheareaswiththelowestandthehighestgeoindexvalues.Theseareas
correspondtoVestAgder(lowestgeoindexvalue)andFinnmark(highestgeoindexvalue)
counties,respectively.
Whenthekerneldensityofazamethiphostreatmentswasenteredasanon-linearsmooth
terminthestatisticalmodel,itwasalsostronglyassociatedwiththeproportionofPhe362Tyr
mutation,accountingfor44%(adj.R2=0.444)ofthevarianceinthedependentvariable
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Table2. PercentagedistributionofthePhe362Tyrmutation(RSandRRgenotypes)versusthewildtype(SSgenotype)inthesamplescollected
fromfishfarmsalongtheNorwegiancoast.
County SS RS RR Totalnumber
Finnmark 86.4% 13.6% 0.0% 118
Nordland 20.9% 40.7% 38.4% 2320
NordTrøndelag 20.5% 48.0% 31.5% 337
SørTrøndelag 23.2% 44.2% 32.6% 362
Møre&Romsdal 27.9% 45.8% 26.3% 567
Sogn&Fjordane 21.4% 48.9% 29.7% 276
Hordaland 13.5% 43.3% 43.2% 2140
Rogaland 25.4% 59.2% 15.4% 240
VestAgder 77.5% 20.8% 1.7% 298
doi:10.1371/journal.pone.0149264.t002
(p<0.001,AIC=3.69).Thesquarerootofthekerneldensityoftreatmentsyieldedacloseto
linearrelationshipwiththedependentvariable(Fig2b).
Thefishfarmsintheareaswiththegeoindex~12thatcorrespondtolatitudeofabout65.0
decimaldegreesnorthhadthehighestproportionofPhe362Tyrmutation(Fig2aand2c).
TheseareascorrespondtoNordTrøndelagcounty.Predictionsfromtheunivariatemodel
yieldedminandmaxexpectedproportionsofmutatedgenotypesamountingto0.14and0.95,
respectively,alongthegeoindexline(givenbyminandmaxof:sin(modelprediction)2).
Whenboththegeoindexsmoothtermandalinearkerneldensityoftreatmenttermwere
enteredtogetherintheGAMmodel,thetreatmentvariablebecameslightlynon-significant
(p=0.055,Table3).Predictionsforthedependentvariableusingtheunivariategeoindexmodel,
however,correlatedcloselywiththekerneldensityoftreatments(correlationcoefficient=0.47),
indicatingthatthegeoindexeffectiscloselyassociatedwithlocaltreatmentdensities.
Bioassayanalysis
Boththetreatedgroups(0.4ppband2ppbazamethiphos)hadhighermortalityratesthanthe
control(0ppbazamethiphos)groupinthebioassaytests(Table4).Themortalityratewason
Fig2.Effectsofthegeoindexorthekerneldensityoftreatments(KD)ontheproportionofmutatedgenotypes.GAM-plotsshowingthenon-linear
relativeeffectsofthegeoindex(leftpanel)orkerneldensityoftreatments(KD)ontheproportionofmutatedgenotypes(RRorRS)infarmsampledsalmon
lice.Thetwoplotsrepresentunivariatefittingoftheproportionmodel.Therelationshipbetweenfarmgeoindexesandtheirnorthcoordinateisalsoshown
(rightpanel).
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Table3. Statisticsforamodelenteringanon-linearfunctionofgeoindexandkerneldensityoftreat-
ments(KD)aspredictorsforthedependentvariableexpressingtheproportionofmutatedgenotypes
insalmonlousesamplesfromfarms(adj.R2=.573,AIC(-6.51)). Coeffreferstoregressioncoefficient
andSEMtostandarderrorofmean.
Coefficient SEM Pvalue
Intercept 0.92 0.087 <0.001
KD 1.51 0.77 0.055
Smoothterm edf Ref.df.
GeoIndex 7.48 8.38 0.003
doi:10.1371/journal.pone.0149264.t003
average28%inthe0.4ppbgroups,and43%inthe2ppbgroups.Atboth0.4ppband2ppb,all
fullysensitiveparasites(SS)areexpectedtobeimmobilized[9].
Boththeproportionofsusceptibleliceonfarmsandthetwolevelsofazamethiphosconcen-
trationweresignificantlyassociatedwiththeoutcomeofthebioassaytests(Table5).
Theintra-classcorrelationcoefficient(ICC)was0.19(95%CI:0.09–0.28),indicatingthat
approximately19%ofthevariancewasatthefarmlevel.
Thepredictedprobabilityofsalmonlousedyinginthebioassaytestsincreasedfromcloseto
zeroto~0.9withincreasingproportionsofSS-genotypesinthefarmpopulationsofsalmon
louse(Fig3).Themaindifferencebetweenthelowandthehighconcentrationwasasteeper
increaseintheestimatedprobabilitiesofdyingwithincreasedproportionofSSgenotypesat
thehighconcentration.Fig3alsoshowsthefarmspecificpredictionsfortheprobabilityoflice
dyingwhenaccountingforthefarmrandomeffects,whichdeviatesmoderatelyfromthatof
thefixedeffectmodel.
Individuallouseanalysis
Theprobabilityofindividualsalmonlousedyingwassignificantlyassociatedtobothgenotype
andazamethiphosconcentrationinthebioassay-testsconductedfortheindividuallouseout-
come(Table6).ThestatisticalmodelpredictedthatlicewithoutthePhe362Tyrmutation(SS)
wouldpracticallyonlysurviveinthecontrolexperiments(0ppbazamethiphos),whereasthe
Table4. Asummaryofoutcomesofsalmonlouseviabilityinbioassaytestsatdifferentconcentrationsofazamethiphos. Theaverage%immobi-
lizedandaliveparasitesinthe15assays,andtheirstandarddeviation,aregiven.Thetotalnumberofparasitesarealsogiven.
Concentrationsppb(μg/L) ImmobilizedAvg.%(SD) AliveAvg.%(SD) Number(n)
0.0 4(5) 96(5) 430
0.4 28(27) 72(27) 460
2.0 43(29) 57(29) 446
doi:10.1371/journal.pone.0149264.t004
Table5. Finalmixedlogisticmodelforlice-levelmortalityinbioassaytests(CoeffreferstoregressioncoefficientandSEMtostandarderrorof
mean). AICinthefullmodelwas17889.
Parameters Level Coeff SEM p
Intercept(β ) -3.2676 0.6015 <0.001
0
Susceptibility(prop.)(β ) 4.8554 1.2496 <0.001
2
Concentration(β )(ppb) 0.4 0
3
Concentration(β )(ppb) 2 0.8074 0.1617 <0.001
3
Randomeffects(ω)Farm σ2
Farm
Randomeffects(ω)Farm 0.7968 0.2305
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Fig3.Relationshipbetweenthesusceptiblegenotype(SS)andthedeathofparasiteunderazamethiphostreatment.Theprobabilityofsalmonlouse
dyingasafunctionoftheproportionofsusceptiblesalmonlouseonfarms(squareroottransformed),whenexposedtolow(0.4ppb,leftpanel)orhigh
(2.0ppb,rightpanel)concentrationsofazamethiphos,respectively.Thelinesrepresentmodelpredictionsusingthefixedeffects(thedottedlinesaccountfor
the95%CIforthesusceptibility-coefficient).Thepointsrepresentfarm-specificmodelpredictionsaccountingfortherandomeffects.
doi:10.1371/journal.pone.0149264.g003
Table6. Generallinearmodel(GLM)forthebinomialoutcomeofsalmonlouseexposuretoazamethiphosinbioassayteststests(Coeffrefersto
regressioncoefficientandSEMtostandarderrorofmean).
Parameters Level Coeff SEM p
Intercept(β ) -7.8025 0.9026 <0.001
0
Genotype RR 0
Genotype RS 1.1044 0.4223 0.008
Genotype SS 5.0248 0.5472 <0.001
Concentration 0 0
Concentration 0.4 4.7769 0.8333 <0.001
Concentration 2.0 6.0988 0.8602 <0.001
doi:10.1371/journal.pone.0149264.t006
PLOSONE|DOI:10.1371/journal.pone.0149264 February16,2016 9/14
Phe362TyrinNorway
Fig4.Relationshipbetweendifferentgenotypesandtheprobabilityofdyingunderazamethiphos
treatment.Theestimatedprobabilityofsalmonlouseofvariousgenotypesdyinginbioassaytestswhen
exposedto0(control)ppb(upperpanel),0.4ppb(midpanel)and2.0ppb(lowerpanel)azamethiphos,
respectively.Theerror-barsaccountforthe95%CIforthegenotypecoefficient.
doi:10.1371/journal.pone.0149264.g004
PLOSONE|DOI:10.1371/journal.pone.0149264 February16,2016 10/14