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EffectsofOrganicAmendmentson MicrobiotaAssociatedwiththe Culex nigripalpus MosquitoVectoroftheSaint LouisEncephalitisandWestNileVirusesDagneDuguma,aMichaelW.Hall,bChelseaT.Smartt,a JoshD.NeufeldcFloridaMedicalEntomologyLaboratory,IFAS,UniversityofFlorida,VeroBeach,Florida,USAa;Facultyof ComputerScience,DalhousieUniversity,Halifax,NovaScotia,Canadab;DepartmentofBiology,Universityof Waterloo,Waterloo,Ontario,CanadacABSTRACTPollutionfromnutrientsinaquatichabitatshasbeenlinkedtoincreasesindiseasevectors,includingmosquitoesandotherpestiferousinsects.One possibilityisthatchangesinmosquitomicrobiomesareimpactedbynutrientenrichmentsandthatthesechangesaffectvarioustraits,includinglarvaldevelopment, susceptibilitytolarvalcontrolagents,andsusceptibilityoftheadultmosquitoesto pathogens.Wetestedthishypothesisusingeldmesocosmssupplementedwith low-andhigh-organic-nutrientregimensandthensampledmicrobialcommunities associatedwiththenaturallycolonizing Culexnigripalpus mosquitovector.Byhighthroughputsequencingof16SrRNAgenesequences,wefoundnosignicantdifferencesinoverallmicrobialcommunitiesassociatedwithsampledmosquitoes,despite detectingdiscernibledifferencesinenvironmentalvariables,includingpH,dissolved oxygen,andnutrientamendments.Nevertheless,indicatorspeciesanalysisrevealed thatmembersofthe Clostridiales weresignicantlyassociatedwithmosquitoesthat originatedfromhigh-nutrientenrichments.Incontrast,membersofthe Burkholderiales wereassociatedwithmosquitoesfromthelow-nutrientenrichment.Highbacterialvariabilityassociatedwiththelifestagesofthe C.nigripalpus waslargelyunaffectedbylevelsofnutrientenrichmentsthatimpactedlarvalmicrobialresources, includingbacteria,ciliates,andagellatesinthelarvalenvironments.IMPORTANCEMosquitomicrobiotaprovideimportantphysiologicalandecological attributestomosquitoes,includinganimpactontheirsusceptibilitytopathogens, tness,andsensitivitytomosquitocontrolagents. Culexnigripalpus mosquitopopulationstransmitvariouspathogens,includingtheSaintLouisandWestNileviruses, andproliferateinnutrient-richenvironments,suchasinwastewatertreatmentwetlands.Ourstudyexaminedwhetherincreasesinnutrientswithinlarvalmosquitodevelopmentalhabitatsimpactmicrobialcommunitiesassociatedwith C.nigripalpus mosquitoes.Wecharacterizedtheeffectsoforganicenrichmentsonmicrobiomesassociatedwith C.nigripalpus mosquitoesandidentiedpotentialbacterialmicrobiota thatwillbefurtherinvestigatedforwhethertheyaltermosquitolifehistorytraits andfortheirpotentialroleinthedevelopmentofmicrobial-basedcontrolstrategies.KEYWORDSaquaticchemistry,bacteria,diseasevectors,foodweb,lifestages, microbiome,mosquito,pollutionNutrientpollutionduetoexcessuseofnitrogenandphosphoruscanleadtoan increasedriskofvector-bornediseases( 1).Previouseldstudiesreportedan increase intheabundanceofmosquitovectorswithanincreaseinnutrientsin mosquitolarvaldevelopmentalsites( 6).Moreover,increasesinnutrientenrichments inmosquitolarvaldevelopmentalsiteshavebeenknowntoreducetheefcacyReceived 2January2017 Accepted 5January 2017 Published 1February2017 Citation DugumaD,HallMW,SmarttCT, NeufeldJD.2017.Effectsoforganic amendmentsonmicrobiotaassociatedwith the Culexnigripalpus mosquitovectorofthe SaintLouisencephalitisandWestNileviruses. mSphere2:e00387-16. https://doi.org/10.1128/ mSphere.00387-16. Editor Garret Suen,UniversityofWisconsin, Madison Copyright 2017Dugumaetal.Thisisan open-accessarticledistributedundertheterms ofthe CreativeCommonsAttribution4.0 International license. Address correspondencetoDagneDuguma, duguma@u.edu.RESEARCHARTICLE EcologicalandEvolutionarySciencecrossm January/February2017Volume2Issue1e00387-16 msphere.asm.org 1 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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andpersistenceoflarvalcontrolagents( 12).Higherdosesofmosquitolarvicidesare often requiredtohaveasignicantreductioninmosquitolarvalpopulationinorganicrichenvironments,suggestingahighereconomiccostofmosquitocontrolinpolluted environmentsthaninlesspollutedenvironments. Nutrientenrichmentsaregenerallythoughttocausechangesinmicrobialcommunities,includingbacteria,ciliates,agellates,microalgae,androtifersthatareconsideredessentialforlarvalmosquitodevelopment( 9, 10, 16),andcouldalteraquaticfood webs inanunpredictablemanner.Forexample,elevationofnutrientsinfreshwater streamsalteredinvertebratepredator-preyrelationshipsfromlineartocurvilinear( 17, 18).However,theunderlyingmechanismsofnutrientsandmosquitovectorinteractions arenotfullyunderstood. Culexnigripalpus TheobaldisamajorvectorofSaintLouisencephalitisvirusandis responsiblefortransmittingotherpathogensinthesoutheasternUnitedStates,includingWestNilevirus( 19, 20). Culexnigripalpus isamongthedominantmosquitospecies foundduringearlysuccessionstagesofnewlydevelopedaquatichabitats,including followingrainfallandinpollutedtreatmentwetlands( 21).Signicantgenetic variations areknowntoexistamongvariouspopulationsof C.nigripalpus (25).Variations inabundanceamonglarvaldevelopmentalsites( 21),susceptibilitytoinfection and transmissionofpathogensamonggeographicpopulations( 26),andsusceptibility to organophosphate-basedpesticides( 27)havealsobeendocumented.However,little is knownaboutwhethernutrient-mediatedchanges,includingwaterqualityvariables andmicrobialconsortiafoundinlarvaldevelopmentalhabitats,caninuencethe mosquito-associatedmicrobiomesfor C.nigripalpus developingindifferentenvironments. Bacteriaassociatedwithmosquitoesarefoundtobecrucialsourcesofnutritionfor successfullarvaldevelopment( 28),affectmosquitosusceptibilitybyvariouspathogens (32),impactresistancetopesticides(36, 37),andinuencemosquitooviposition (38).Asaresult,understandingtheeffectsofnutrientsonmicrobialcommunities associated withmosquitoesiscriticalfordisentanglingtheunderlyingcausesof variabilityindiseasetransmission,variationsinmosquitoproductionamongvarious aquatichabitats,andlackofsusceptibilitytopesticides.Inaddition,thisknowledgeis importantforthedevelopmentofnovelmicrobial(e.g., Wolbachia)mosquitocontrol strategies.Wehypothesizedthatdifferentnutrientregimensinlarvalhabitatsimpact microbialcommunitiesassociatedwithmosquitoesdevelopingduringthesuccession ofthesehabitats.Inordertotestthishypothesis,wecharacterizedmicrobiotaassociatedwith C.nigripalpus developingintwodifferentresource(nutrient)regimensunder naturaleldconditions.Inaddition,wecharacterizedmicrobialcommunitiesindifferentlifestagesof C.nigripalpus toidentifypotentialsymbiontsassociatedwithalllife stages. RESULTS Environmentalvariablesinthewatercolumn. The waterqualityindicatorsdifferedsignicantlybetweentwocontrastinglarvalenvironments(i.e.,highandlow nutrients)inoutdoorexperimentalmesocosms( Fig.1).SignicantlylowerpHvalues (Fig.1A [F1,4 55, P 0.002])anddissolvedoxygenconcentrations( Fig.1B [F1,4 25, P 0.007])werefoundinhigh-nutrientmesocosms.Incontrast,higherchemical oxygendemand(COD)wasfoundinthehigh-nutrientmesocosms(mean standard error[SE],239 45.8mg/liter)comparedtothelow-nutrientmesocosms(143 8mg/liter)onday7,althoughthedifferencewasnotstatisticallysignicant( P 0.107). Asimilartrendbutlowerconcentrationwasobservedonday9,with195 19.8 mg/literand150 18.6mg/literinthehigh-andlow-nutrienttreatments,respectively. Ahigherconcentrationoftotalnitrogen(signicancetestedafterBonferronicorrection)wasalsofoundinhigh-nutrienttreatmentsthaninthelow-nutrienttreatments andwasvariableacrosstime( Fig.1C [F1,4 5.8, P 0.07]).Similarly,ahighertotal phosphorusconcentrationwasfoundinthehigh-thaninthelow-nutrient-treated mesocosms( Fig.1D [F1,4 4.7, P 0.12]).TemperatureandlightintensityinthewaterDugumaetal. January/February2017Volume2Issue1e00387-16 msphere.asm.org 2 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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columnvariedtemporallybutwererelativelyuniformamongmesocosmsandwerenot signicantlyaffectedbynutrientenrichments(datanotshown). Theabundancesofsmall(0.2-to1.999-mequivalentsphericaldiameter[ESD])and large(2-to60-mESD)organicparticlesdifferedsignicantlybetweenthetwo treatmentsontherstdaythatthemesocosmswereexposedtoegg-layingfemale mosquitoes( Fig.2).Meantotalabundanceofsmallparticleswassignicantlygreaterin high-nutrient treatmentsthanlow-nutrienttreatments( Fig.2A and B [F1,15 10.5, P 0.005]).Similarly,themeanabundanceoflargeparticleswasapproximately5-fold greaterinthehigh-nutrienttreatmentsthaninthelow-nutrienttreatments( Fig.2C and D [F1,15 14.6, P 0.002]). Microeukaryoteabundance. Microscopicexaminationofwatersamplesformicroeukaryotes(i.e.,ciliates,agellates,androtifers)revealedasignicantdifferencein thecombinedabundanceofthesemicroorganismsbetweenthetwotreatments( Fig.3 FIG1 Waterqualityparameters.Mean SE(n 3)pH,dissolvedoxygen,totalnitrogen,andtotal phosphorusinwaterofthelow()-andhigh()-nutrienttreatmentsofdifferentiallytreatedlarval habitats(mesocosms).The x axisrepresentstimeindaysaftermesocosmswereuncoveredand Culex mosquitoeslaideggraftsonwater.The y axisdenotestheconcentrationorvaluesofwaterquality parameters.Forexample,themesocosmswereexposedtoegg-layingfemalemosquitoeson2November2015. FIG2 Heterotrophicandautotrophicparticle(cell)abundanceinwatercolumn.Small(0.2-to1.999-m equivalentsphericaldiameter[ESD])(A)andlarge(2-to60-mESD)(C)particlesizedistributioninhigh (solidlines)-andlow(dashedlines)-organic-nutrient-enrichedmesocosms,andmean SEtotalparticle abundance(n 3)ofsmall(B)andlarge(D)particlesizesinwateroflow-andhigh-nutrienttreatments onday0.Culex MicrobiomeandNutrientInteractions January/February2017Volume2Issue1e00387-16 msphere.asm.org 3 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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[F1,16 24.1, P 0.0002])andvariedacrosstime(F2,15 4.3, P 0.04).Signicantly greaternumbersofmicroeukaryoteswerefoundinhigh-nutrienttreatmentsondays0 and7,butthatdifferencedecreasedbyday9. Mosquitolarvaabundance. Microscopicexaminationofthedippersamplesrevealednosignicantdifferencesintotal Culex larvalabundancebetweenthetwo treatmentsateither5daysor7daysafterthemesocosmswereexposedtonaturally occurringmosquitoes(F1,4 0.006, P 0.9).Themean SEnumberof C.nigripalpus larvaefoundinthelow-nutrienttreatmentswas41 10,comparedwiththehighnutrienttreatments,with32 27larvaeperdipsample.Veryfewindividualsofthe southernhousemosquito Culexquinquefasciatus Saywereobservedonthissampling dateinthelow(average; 1larvae)-andhigh(~4larvaeperdippersample)-nutrient treatments.Asimilartrendwasobservedaweekafteruncoveringthemesocosms(data notshown). Diversityofbacterialcommunitiesinmosquitoesfromdifferentnutrienttreatments. Atotalof4,859,297sequencesin1,751operationaltaxonomicunits(OTUs) weregeneratedfrom48mosquitosamples,including6half-eggrafts,9earlyand12 lateinstarlarvae,7pupae,and14newly(1dayaftereclosion)emergednon-bloodfedfemaleadultsof C.nigripalpus.Assembledandquality-checkedsequenceshada meanlengthof416baseswithameanoverlapof49.8bases.Onaverage,83,408 sequencesfromeggrafts,106,809fromearlyinstarlarvae,and116,281fromlateinstar larvae,89,430frompupae,and83,107fromfemaleadultspersamplewereobtained. Thesewereclassiedinto28bacterialphylawith Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Actinobacteria,and Acidobacteria dominatingthebacterialphyla foundassociatedwiththismosquitospecies( Fig.4). Proteobacteria accounted for nearly70%ofsequences,followedby Firmicutes,with15%ofallsequences.Approximately56%ofthesequenceswereidentiedas240genera,with Arcobacter (Epsilonproteobacteria: Campylobacteraceae)beingthemostabundant(29%)genus,followed by Thorsellia (7%).Fewarchaealsequences(0.002%)wererecoveredfromthismosquito species,andthosewereprimarilyfromfemaleadults. Theprinciplecoordinate(PCoA)ordinationsbasedonweightedUniFracmeasures revealednosignicantdifferencesinbacterialcommunitycompositionfoundamong mosquitoesdevelopinginlow-andhigh-nutrienttreatments( Fig.5A [multiresponse permutation procedure[MRPP; A 0.005; P 0.081]).Thegreatestvariation(indicated byprincipalcoordinate1[PC1])detectedamongsampleswasattributedtothehigher abundanceofOTUscorrespondingto Arcobacter (OTU0)andanunidentiedspeciesof Comamonadaceae (OTU2). Arcobacter (Epsilonproteobacteria)wasmostlyassociated withlarvaeof C.nigripalpus,whereastheunidentiedspeciesin Comamonadaceae (Betaproteobacteria)wasfoundassociatedwithalllifestagesof C.nigripalpus,including eggrafts.Differencesinbacterialcommunitiesamonglarvalsamplesfromlow-and high-nutrienttreatmentswerenotsignicant(MRPP; A 0.018; P 0.084). FIG3 Microeukaryoteabundanceinwatercolumn.Mean SE(n 3)abundanceofciliateprotists, agellates,androtifersinthemesocosmswithhigh()-andlow()-nutrienttreatmentsondays0,4, and9afteregglayingbymosquitoes.Errorbarsnotseenarecontainedwithinthesymbols.Dugumaetal. January/February2017Volume2Issue1e00387-16 msphere.asm.org 4 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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Indicatorspeciesanalysisrevealedthatmembersof Clostridiales dominatedbacterial communitiesassociatedwithmosquitoesdevelopinginhigh-nutrientregimens(indicatorvalues 0.5to0.8; P 0.01),whereasmosquitoesfromlow-nutrienttreatments wereenrichedwith Burkholderiales (seeTableS1inthesupplementalmaterial[indicator value 0.48; P 0.01]).Althoughdifferencesamongstageswereapparent,indicator speciesanalysisbylifestagesrevealedthat Comamonadaceae OTUswerestrongly FIG4 Dominantbacterialphylafoundindifferentstages.Shownarebacterialphylaineggs,earlyandlatelarval instars,pupae,andnewlyemergedfemaleadultsof Culexnigripalpus developedundertheeldconditions.Only phylawithanaverageabundanceof 0.1%wereincluded.Otherunclassiedsequencesaccountedfor0.3%. Archaea andanadditional16phylaaccountedfor 0.1%.Eggsamples1,4,and5,earlyinstarsamples7,10,11, and18,lateinstarsamples14,18,20,41,42,46,and47,pupasamples20and38,andfemaleadultsamples32 to36weretakenfromhigh-nutrientregimens.Theremaining27sampleswerederivedfromlow-nutrient regimens. FIG5 PCoAordinationbasedontheweightedUniFracdistancemetric.Shownisordinationofmicrobialcommunitiesassociatedwith Culexnigripalpus mosquitoescoloredbyeithernutrienttreatmentlevel(A)orlifestage(B).Theeffectsofnutrientsonmicrobial communitiesassociatedwiththemosquitoeswerenotsubstantial(MRPP; A 0.005, P 0.08)betweenthetwotreatments.However, themicrobiotainthedifferentlifestagesdifferedweaklybutsignicantly(MRPP; A 0.08, P 0.001).Largeopencirclesindicatethe 10mostabundantbacterialtaxaassociatedwithsamples.Culex MicrobiomeandNutrientInteractions January/February2017Volume2Issue1e00387-16 msphere.asm.org 5 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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associatedwithalllifestages,exceptpupalsamples(seeTableS1andFig.S1inthe supplementalmaterial). Nonmetricmultidimensionalscaling(NMDS)analysisbasedonBray-Curtisdistance measuresrevealedsignicantdifferencesbetweenmicrobiotasamplesthatoriginated fromdifferentlifestagesofmosquitoes( Fig.5B;seeFig.S2inthesupplemental material [MRPP; A 0.08; P 0.001]).Microbialsamplesfromfemaleadultswere signicantlyseparatedfromtheimmaturestagesandeggraftsof C.nigripalpus. Ninecoretaxawerefoundinallsamplesofalllifestagesof C.nigripalpus,including Thorselliaanophelis OTU4, Oleomanas OTU7,twounidentiedspecies(OTUs1and2) of Comamonadaceae,two Hydrogenophaga species(OTUs18and1537),anunidentied species(OTU71)of Cyanobacteria,anunidentiedspecies(OTU8)of Firmicutes,andan unidentiedspecies(OTU3)of Tenericutes (seeFig.S3inthesupplementalmaterial). Bacterialcommunitiesineggraftsof Culexnigripalpus. Bacterialsequencesfrom eggraftsof C.nigripalpus groupedinto530OTUsthatweredominatedbyanunknown species(OTU2)of Comamondaceae (35%),followedby Agrobacterium OTU10(12%) (seeTableS2andFig.S3inthesupplementalmaterial). Bacterialcommunitiesinimmaturestagesof Culexnigripalpus. An Epsilonproteobacteria member, Arcobacter (31%),andtwoOTUscorrespondingtospeciesin Betaproteobacteria (anunknownspeciesin Comamondaceae [14%]and Vogesella [Neisseriaceae])dominatedbacterialcommunitiesassociatedwithearlylarvalinstars. Thorselliaanophelis wasalsorecoveredfromtheearlyinstarstagebutinamuchlower (1%)proportion(seeTableS3inthesupplementalmaterial).Bacteriainlateinstar larvaewerealsodominatedby Arcobacter (27%), Thorselliaanophelis (10.5%),andan unknowngenusof Mollicutes (10%). Hydrogenophaga (14%), Thorsellia (11%),andan unknownspeciesof Comamondaceae (10%)dominatedbacterialcommunitiesinpupal samples. Bacterialcommunitiesinnewlyemerged Culexnigripalpus femaleadults. Bacteriainnewlyemerged(12haftereclosion)non-blood-fedfemaleadultsreared fromeggtoadultsoutdoorswereenrichedwithanunknownspecies(OTU2)of Comamondaceae (20%), Oleomonas (7%),and Arcobacter (4.6%)(TableS3). Wolbachia wasalsofoundin2ofthe14adultsamples,constituting91%and 1%oftheir respectivesequencesbutwasabsentin12othersamples.Theseresultssuggestalikely inclusionof Culex quinquefasciatus,whichisaknownhostof Wolbachia inthose samplesduringDNAextraction.Othernotablespecieswererecoveredatlowerproportions(1%)andinclude Thorselliaanophelis. DISCUSSION Effectsoforganicenrichmentsonmicrobialcommunitiesassociatedwith Culex nigripalpus. We testedthehypothesisthatorganicnutrientenrichment,aprimary factorforeutrophicationandpollution,wouldalterthemicrobiallarvalresourcesand therebyimpactmicrobialcommunitiesassociatedwith Culex diseasevectorsinreplicatedoutdoormesocosmexperiments.Resultsofourstudyrevealedthatasignicant increaseinabundanceofsestonicparticlesandplanktonicmicroeukaryotes(i.e.,ciliates,agellates,androtifers)intreatmentswithhigh-nutrientenrichmentswerein agreementwiththebottom-upresourcehypothesis:i.e.,increasingnutrientswill increasetheabundanceofbothautotrophicandheterotrophicmicroorganisms( 39, 40).Thiswasfurthercorroboratedwiththeincreaseinchemicaloxygendemand,a predictor oftheamountoforganicmaterialavailableforoxidation,andmicrobial consumptioninthehigh-nutrienttreatmentscomparedtolow-nutrienttreatmentsin thisstudy.Totalconcentrationsofnutrientsincreasedimmediatelyfollowingthe uncoveringofthemesocosms,suggestingautotrophicandaerobicmicrobialcolonizationofthemesocosms. Despitetheapparentdifferencesinmicrobiotaandchemicalvariablesinthewater column( Fig.1to3 ),microbialcommunitiesassociatedwithmosquitoesdevelopingin these twolarvalenvironmentswerenotaffectedsignicantly.Thiscouldbeduetoa combinationoffactors,includingfoodweb-mediatedfactors,suchasdifferencesintheDugumaetal. January/February2017Volume2Issue1e00387-16 msphere.asm.org 6 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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abundanceofplanktonicbacteriovores(i.e.,agellates,ciliates,androtifers),andthe remarkablevariabilityinmicrobialcommunitiesamongsampleswithineachtreatment group.Predationofbacteriabyplanktonicbacteriovoreshasbeenknowntobeintense ( 41),andthesegroupsmighthaveaffectedthebacterialdiversityinthewatercolumn, a feedingzoneof Culex mosquitoes.Alternatively, Culex larvaeareconsideredomnivores,feedingonavarietyoflowertrophicmicroorganisms,includingbacteria( 16),and therefore theimpactonbacterialdiversityinthemosquitoesmaynotnecessarilybe affectedbyincreasesinbottom-upresources.Apreviousstudyofmicrobiotainother Culex mosquitoessampledfromdifferenthabitatswithdifferentnutrientconcentrations(inuencedbylarvalcontroltreatments)alsodidnotrevealsignicantdifferences inthebacterialcommunitiesinthelarvaesampledfromthedifferentlarvalhabitats ( 42). Increases innutrientssuchasnitrogenandphosphorusinaquatichabitats,asa resultofrunofffromagriculturalpracticesandothernonpointsources,havebeen reportedtoinuencetheabundanceofdiseasevectors,particularly Culex mosquitoes thataremoreadaptedtopollutedenvironments( 6, 8, 45, 46).Inaddition,organic enrichments havebeenshowntoinuencemosquitocontrolstrategies.Forexample, underhigh-organic-richenvironments,theefcacyofafungalbiologicalcontrolagent, Lagenidiumgiganteum,wassignicantlyreduced( 12).Otherstudiesreportedthat increase inorganicorinorganicmaterinthewatercolumnnegativelyinuencedthe efcacyofthecommonlyused Bacillus-basedlarvalcontrolagents( 13, 47).Itislikely that thediversemicrobialcommunitiesandsestonicparticlesingestedbymosquito larvaemightprovideimmunityorprotectionofmidgutepitheliumthatisconsidered aprimarytargetof Bacillusthuringiensis serovarisraelensis(Bti)toxinsinpolluted environments( 13). Mosquitoes, ingeneral,and Culex mosquitovectors,inparticular,areconsidered primarycolonizersofnewlycreatedfreshwateraquatichabitatsandarewelladapted topollutedenvironments( 42, 46, 48).Thedifferenceinabundancesof C. nigripalpus betweentreatmentswasnotsignicant,suggestingthatmosquitoabundancewasnot inuencedbywatercolumnnutrientconcentrations,especiallyduringtheinitialcolonizationofnewlyformedaquatichabitats.Thisstudyandothershaveshownthat C.nigripalpus mosquitoesprefertolaytheireggsanddevelopinhighlyeutrophic habitatsthantheir C.quinquefasciatus congeners( 21, 23). Culexquinquefasciatus was consideredtobethedominantspeciescolonizerofeutrophichabitats( 9, 24),butits abundance duringthesuccessioninourmesocosmswasnegligiblecomparedtothat of C.nigripalpus. Bacterialcommunitiesassociatedwithdifferentlifestagesof Culexnigripalpus. Microbialcommunitiesassociatedwith C.nigripalpus sampledduringtheautumn variedsignicantlyamonglifestagesfromthesamecohort.Bacterialcommunitiesfrom femaleadults,eggs,andpupaeweredominatedby Alphabacteria and Betaproteobacteria,whereasbacteriafromlarvaeweredominatedby Arcobacter ( Epsilonproteobacteria), Hydrogenophaga,and Agrobacterium ( Alphaproteobacteria), Thorsellia ( Gammaproteobacteria),and Clostridium ( Firmicutes). Arcobacter isubiquitousinaquaticenvironmentsand,as amemberof Epsilonproteobacteria,mightbeassociatedwithsulfurcycling(49, 50).The mesocosms usedinthisstudywerelledwithwellwater,whichcontainsarelatively highsulfurconcentration(~100mgsulfate/liter[D.Duguma,unpublisheddata]).Nearly 92% Arcobacter sequenceswerefromlarvae,whereasveryfewwerefoundassociated witheggs(0.4%),pupae(2.8%),andadults(4.6%),suggestingthat Arcobacter found associatedwiththemosquitoesinthisstudymightbewaterborneandthusingested bythemosquitolarvae.Several Arcobacter spp.areknowntobepathogenictohumans andanimals( 51)andassociatedwithpollutedenvironments( 52).Althoughwehave not ruledoutexperimentallythatthisbacteriumcanbetransstadiallytransmitted acrosslifestagesorisapathogen, Arcobacter foundassociatedwithpupaeandadult C.nigripalpus mosquitoeswaslikelyingestedbythelarvaefromthewaterandpassed downtopupaeandadults.ConsideringthatmembersofthisgenusofbacteriaareCulex MicrobiomeandNutrientInteractions January/February2017Volume2Issue1e00387-16 msphere.asm.org 7 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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recognizedasemergingpathogenstohumansandanimals( 51),therecoveryofa relatively smallproportion(e.g.,4.6%)of Arcobacter sequencesinfemaleadultssuggestsfurtherstudy,includingapossibilitythatthesebacteriamightbeharboredin salivaryglands,withadirectimplicationforpathogentransmission( 53).Salivaryglands have beenshowntoharbordiversebacterialcommunities( 53). Agrobacterium OTUs werefoundinallsamples,fromeggtoadultstagesof C.nigripalpus,withthehighestabundancefoundassociatedwiththeeggs.Membersofthis genusareknowntotransfergeneticmaterialsbetweenthemselvesandothereukaryotes,suchasplants( 54),andthisgenuswasamongthedominantgenerarecovered from Aedes mosquitoes(55).Althoughwerinsedtheeggraftsmultipletimeswith distilled water,itispossiblethatsomeofthecommunitiesfoundassociatedwitheggs mighthavebeenunintentionallycosampledfromthewatersurfaceduringthesamplingoftheeggrafts.Futurestudieswillinvestigatewhethersomeofthecommunities foundassociatedwitheggsareobligatesymbionts. Thorselliaanophelis wasalsofoundin C.nigripalpus,withthegreatestabundanceof thisbacteriumfoundinpupae(11.0%)andinlateinstarlarvae(10.5%).Theabundance ofthisspecieswasconsiderablylowerineggs(0.02%),earlyinstars(0.5%),andadults (0.14%),corroboratingpreviousstudiesthatthissymbiontislikelyingestedbylarvae andtransferredtothesubsequentdevelopmentalstages( 43, 56).Thedominance and persistenceof Thorsellia spp.inlifestagesof Culex mosquitoesinthisstudyandin previousstudies( 43, 44)and Anopheles (56)mosquitoessuggestastrongconsideration for thedevelopmentofparatransgenicmosquitocontrol( 59). In conclusion,differencesinenvironmentalhabitatvariationsmightnotaffectthe internalbacterialcommunitiesassociatedwith Culex mosquitovectors,whichinstead maybeinuencedbyseasonalvariations.Forthersttime,weidentiedmicrobial communitiesassociatedwith C.nigripalpus acrossdevelopmentalstagesandidentied potentialcandidatesthatwillbefurtherinvestigatedfortheirroleinbionomicsand controlofthismosquitospecies. MATERIALSANDMETHODSMesocosmexperiment. Ourexperimentaldesigninvolvedtwocontrastinglarvalenvironmentsin outdoorexperimentalmesocosmsduringautumn2015.Twodifferentlarvalenvironmentalconditions werecreatedon27October2015byaddingtwonutrientregimens:0.2and1%(wt/vol)(lowandhigh, respectively)rabbitfood(alfalfapellets)tothreereplicatedoutdoormesocosmslledwith378litersof wellwaterattheUniversityofFlorida,FloridaMedicalEntomologyLaboratory(seeFig.S4inthe supplementalmaterial).Thesurfaceareaanddepthofwaterwere0.85m2and0.5m,respectively. Outdoormesocosmscanbeusedtoexaminevariousecologicalhypotheses,includingtheeffectsof nutrientsandclimatechangeonaquaticfoodwebs( 18, 42, 43, 60, 61).Alfalfa-basedorganicmatteris commonly usedtoattractegg-layingfemalemosquitoesandsupportstheproductionof Culex mosquitoesforlongerperiodsoftime( 9, 38, 43, 62).Theorganicmatterwasallowedtofermentinthe mesocosms for~1weekwhilecoveredwithatarp.Naturalovipositionby Culex mosquitoesoccurredin allmesocosms 24hafteruncoveringthemesocosms(i.e.,on2November2015).Twoeggraftslikely laidbytwofemale Culexnigripalpus mosquitoesfromeachofthesixmesocosmsweresampledonday 1(3November2015).Oneeggraftlaidbyanindividualmosquitosampledfromeachofthemesocosms wasplacedinmodiedBioQuipmosquito-rearingchambers(seeFig.S5inthesupplementalmaterial) andthensubmergedineachofthesixmesocosmstoallowaccesstolarvalmicrobialfoodresourcesand developmentofthesemosquitoesundertheeldconditions.Thesubmergedportionofthedevicehas screenmeshes(300nylon)builtintoeachmosquitobreeder(BioQuip,Inc.,RanchoDominguez,CA,USA) toallowaccesstolarvalresourcesinthewatercolumn,whereastheabovewaterportionofthedevice capturesadultsemergingfromthesamecohortofeggs.Thesecondeggrafttakenfromeachofthe mesocosmswastakentothelaboratory,triplerinsedwithdistilledwater,andasepticallycutintotwo halves.One-halfoftheraftsfromeachofthecontainerswerepreservedin95%ethanolforDNA extraction,whiletheremaininghalveswereplacedin200mlofdistilledwaterinsterileplasticcupsand thenallowedtohatchinanenvironmentalchamberatatemperatureof27Cforpositivemorphological identicationofthelarvaeto Culexnigripalpus. Mosquitoandwatersampling. Samplesoftwo(earlyandlateinstars) Culexnigripalpus larvalstages, pupae,andadultmosquitoesdevelopedfromthesameeggraftsintheBioQuiprearingchamberswere takenondifferentdays(seeTableS4inthesupplementalmaterial),preservedin95%ethanol,andstored at 20CuntilDNAextraction.Inaddition,mosquitolarvalsamplesweretakeninve350-mlstandard dipsfromeachofthemesocosmsatdays7and9afterthemesocosmswereexposedtoegg-laying femalemosquitoestodeterminetheidentityandabundanceofmosquitoesfoundinthemesocosms. Watersamplesweretakenin250-mlamberplasticbottlesondays2,7,and9aftermosquitoes colonizedthemesocosmstodeterminetotalnitrogen,phosphorus,andchemicaloxygendemand(COD)Dugumaetal. January/February2017Volume2Issue1e00387-16 msphere.asm.org 8 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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inthewatercolumnusingaHachDR3900spectrophotometer(HachCompany,Loveland,CO).Thewater samplesintegratedboththesurfacewaterand~10cmbelowthesurfaceofthewaterandwerecollected fromthecenterofthemesocosm.DissolvedoxygenandpHinthewatercolumnweredetermined insitu usingaYSIProfessionalPlusmultiparameterinstrument(YSI,Inc.,YellowSpring,OH).Temperatureand lightintensityinthemesocosmsweremonitoredcontinuouslyusingaHoboPendanttemperature/light datalogger(OnsetComputerCorp.,Bourne,MA). Watersampleswerecollectedinduplicate50-mlsterilecentrifugetubesondays0,4,and9afterthe mesocosmswereopenedandpreservedwith1%Lugolsiodinesolutiontoquantifytheabundanceof microeukaryotes(i.e.,ciliates,agellates,androtifers)foundinthewatercolumn.Themicroeukaryotes werecountedbydirectmicroscopyonahemocytometerusingaLeicainvertedmicroscope(Leica Microsystems,Inc.,BuffaloGrove,IL).Particlesizedistributionsforsmall(0.2-to1.999-mESD)andlarge (2-to60-mESD)particlesthatincludebothheterotrophicandautotrophiccommunitiesweredeterminedusingaMultisizer4ECoulterCounterparticlesizeanalyzer(BeckmanCoulter,Inc.,Miami,FL)by apreviouslypublishedprocedure( 42). DNA extraction,PCR,andMiSeqIlluminalibrarypreparation. Thegeneralschemeofthisstudy followedproceduresdescribedinpreviousstudies( 43, 63).Briey,pooledDNAsamplesfrom1to3 individuals fromeachofthelifestagesof C.nigripalpus wereextractedusingtheDNeasybloodand tissuekitfollowingthemanufacturersprotocol(Qiagen,Valencia,CA)inalaminarowhood.Priorto DNAextraction,mosquitoesweresurfacesterilizedwith95%ethanolandrinsedthreetimesusing molecularbiology-gradeUltraPurewater(QualityBiological,Inc.,Gaithersburg,MD).Thesampleswere gentlyvortexedfor10sinbetweenrinsing.Themosquitoeswerelefttoairdryunderalaminarow hoodbeforeextraction.Poolingofindividualinsectsformicrobialanalyseshavebeenusedroutinelyin characterizationofcommunityprolesininsects( 43, 44, 64).Poolingofindividualsmayhaveseveral advantages, includingmaximizingthesequenceyieldpersample(abovenegativecontrols)todiscern microbialcommunitydifferencesbetweentreatmentsamplesininsects( 64).WealsoextractedDNAfrom egg raftstodetermineiftherewerematernallytransmittedsymbionts(e.g., Wolbachia)orunknown symbiontswerepresentinthisspecies.DNAfromadultmaleswasnotextractedinthisstudybecause maleshavenoknownsignicanceintransmittingpathogens. ThePCRprocedures,sequenceassembly,andanalysesfollowedpreviousproceduresdescribedin otherstudies( 65).Briey,~460-bpampliconsweregeneratedusingPCRfromtheV3andV4regions of 16SrRNAgenesusingPro341FandPro805R,whichtargetbothbacteriaandarchaea( 68).Amplicons from eachofthesamplesandreplicateno-templatecontrolsweretaggedwithunique6-basebarcodes, ampliedusingIllumina-specicprimers,andsequencedaccordingtoapreviouslyestablishedprotocol ( 67)withsomemodication.ThemodicationincludedasecondroundofPCRwith15cyclesforsamples with lowamplicationontherstroundofPCR.InbriefallPCRproductswerecombinedandsubjected to250-bpendsequencing(Reagentkitv2,500)onaMiSeq(Illumina,SanDiego,CA). Dataanalysis. UsingAXIOMEtomanagesequencesanalysis( 69),16SrRNAgenereadswere assembled byPANDAseqversion2.10(70),withaqualitythresholdof0.9(whichrejectssequenceswith low-quality scores),aminimumoverlapof10bases,andaminimumassembledlengthof100bases,and sequenceswithambiguousnucleotideswererejected.Operationaltaxonomicunits(OTUs)werepicked at97%identityusingtheUPARSEalgorithmUSEARCHversion7.0.1090( 71)with denovo chimera checking.TaxonomicclassicationwasperformedontherepresentativesequenceofeachOTUusing RDPversion2.2( 72)viaQIIME(73),trainedagainsttheGreengenes(August2013revision)(74)reference set withaminimumposteriorprobabilityof80%.Sequenceswererareedtothelowestnumberof sequencespersample(i.e.,43,611)foralphaandbetadiversityanalyses.Todeterminemicrobial communitydifferencesamongmosquitosamplesoriginatingfromhigh-andlow-nutrienttreatments, principalcoordinateanalysis(PCoA)andnonmetricmultidimensionalscaling(NMDS)ordinations,based ontheBray-Curtisdissimilaritymeasures,wereconductedusingtheveganRpackageversion2.2-0( 75). In addition,aPCoAordinationbasedonUniFracdistancemeasureswascarriedoutwithQIIMEto determinebacterialcommunitydifferencesamongsamplesofmultipletreatmentsandlifestages. Multiresponsepermutationprocedures(MRPP)wereusedtotestdifferencesamongsamplegroups basedondistancemeasures.CorebacterialtaxaweredeterminedbasedonOTUsrepresentedbyatleast onesequencepersampleinallsamples( 76). Repeated-measures analysisofvariance(ANOVA)usingJMP(77)wasconductedtoassessdifferences in environmentalvariables(e.g.,nutrients,pH,dissolvedoxygen,andmicroeukaryotes)andlarval mosquitoabundanceinthewatercolumnbetweenthetwotreatments.One-wayANOVAwasperformed toassessdifferencesinmeanabundanceoftotalcountsofsmallandlargesestonicparticles.Meanswere separatedbyTukeystestat P 0.05,afterperformingBonferronicorrectiononcalculated P values. Accessionnumber(s). AllsequencedataforthisstudyweresubmittedtotheEuropeanBioinformaticsInstituteunderaccessionno. PRJEB17885.SUPPLEMENTALMATERIAL Supplementalmaterialforthisarticlemaybefoundat https://doi.org/10.1128/ mSphere.00387-16. FIG S1, EPSle,0.3MB. FIGS2, PDFle,0.1MB. FIGS3, EPSle,0.3MB. FIGS4, PDFle,0.2MB.Culex MicrobiomeandNutrientInteractions January/February2017Volume2Issue1e00387-16 msphere.asm.org 9 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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FIGS5, PDFle,0.2MB. TABLES1, PDFle,0.1MB. TABLES2, PDFle,0.1MB. TABLES3, PDFle,0.1MB. TABLES4, PDFle,0.1MB. ACKNOWLEDGMENTS KatjaEngel,DanielVelez,SaraOrtiz,andArthurSimas-Domingosarethankedfor their technicalassistance.WethankJamesNewmanforsketchingschematicdiagrams ofthemesocosms,JamesMcNealyforsuggestionsonthedesignofthemodied BioQuipmosquitobreeder,andBarryAltoforvaluablecommentsonthemanuscript. D.D.conceivedanddesignedthestudy.D.D.performedtheexperiments.D.D., M.W.H.,andJ.D.N.analyzedthedata.D.D.,J.D.N.,andC.T.S.contributedreagents.D.D., M.W.H.,C.T.S.,andJ.D.N.wrotethemanuscript.Allauthorsreadandapprovedthenal versionofthemanuscript. D.D.wassupportedbyfundingfromFloridaDepartmentofAgricultureandConsumerServices(projectno.00123786).J.D.N.wassupportedbyaDiscoveryGrantfrom theNaturalSciencesandEngineeringResearchCouncilofCanada(NSERC).Publication ofthisarticlewasfundedinpartbytheUniversityofFloridaOpenAccessPublishing Fund.Thefundershadnoroleinstudydesign,datacollectionandinterpretation,nor thedecisiontosubmittheworkforpublication. REFERENCES1.JohnsonPT,TownsendAR,ClevelandCC,GlibertPM,HowarthRW, McKenzieVJ,RejmankovaE,WardMH.2010.Linkingenvironmental nutrientenrichmentanddiseaseemergenceinhumansandwildlife. EcolAppl20:16. https://doi.org/10.1890/08-0633.1. 2. CamargoJA,AlonsoA.2006.Ecologicalandtoxicologicaleffectsof inorganicnitrogenpollutioninaquaticecosystems:aglobalassessment. EnvironInt32:831849. https://doi.org/10.1016/j.envint.2006.05.002. 3. SanfordMR,ChanK,WaltonWE.2005.Effectsofinorganicnitrogen enrichmentonmosquitoes(Diptera:Culicidae)andtheassociated aquaticcommunityinconstructedtreatmentwetlands.JMedEntomol 42:766. https://doi.org/10.1093/jmedent/42.5.766. 4. SunishIP,ReubenR.2001.FactorsinuencingtheabundanceofJapaneseencephalitisvectorsinriceeldsinIndia.I.Abiotic.MedVetEntomol15:381. https://doi.org/10.1046/j.0269-283x.2001.00324.x. 5. VictorTJ,ReubenR.2000.Effectsoforganicandinorganicfertiliserson mosquitopopulationsinriceeldsofsouthernIndia.MedVetEntomol 14:361. https://doi.org/10.1046/j.1365-2915.2000.00255.x. 6. DugumaD,WaltonWE.2014.Effectsofnutrientsonmosquitoesandan emergentmacrophyte, Schoenoplectusmaritimus,foruseintreatment wetlands.JVectorEcol39:1. https://doi.org/10.1111/j.1948-7134 .2014.12063.x. 7. AwololaTS,OduolaAO,ObansaJB,ChukwurarNJ,UnyimaduJP.2007. Anophelesgambiae s.s.breedinginpollutedwaterbodiesinurban Lagos,southwesternNigeria.JVectorBorneDis44:241. 8.LundA,McMillanJ,KellyR,JabbarzadehS,MeadDG,BurkotTR,Kitron U,Vazquez-ProkopecGM.2014.Longtermimpactsofcombinedsewer overowremediationonwaterqualityandpopulationdynamicsof Culexquinquefasciatus,themainurbanWestNilevirusvectorinAtlanta, GA.EnvironRes129:20. https://doi.org/10.1016/j.envres.2013.12.008. 9. RodcharoenJ,MullaMS,ChaneyJD.1997.Organicenrichmentofbreedingsourcesforsustainedproductivityofmosquitoes(Diptera:Culicidae). JVectorEcol22:30. 10.NguyenTT,SuT,MullaMS.1999.Bacteriaandmosquitoabundancein microcosmsenrichedwithorganicmatterandtreatedwitha Bacillus thuringiensis subsp. israelensis formulation.JVectorEcol24:191. 11.ChavesLF,KeoghCL,Vazquez-ProkopecGM,KitronUD.2009.Combined sewageoverowenhancesovipositionofCulexquinquefasciatus (Diptera:Culicidae)inurbanareas.JMedEntomol46:220. https:// doi.org/10.1603/033.046.0206. 12. JaronskiST,AxtellRC.1982.Effectsoforganicwaterpollutiononthe infectivityofthefungus Lagenidiumgiganteum (Oomycetes:Lagenidiales)forlarvaeof Culexquinquefasciatus (Diptera:Culicidae):eldand laboratoryevaluation.JMedEntomol19:255. https://doi.org/ 10.1093/jmedent/19.3.255. 13. Ben-DovE,SaxenaD,WangQ,ManasherobR,BoussibaS,ZaritskyA. 2003.IngestedparticlesreducesusceptibilityofinsectlarvaetoBacillus thuringiensis.JApplEntomol127:146. https://doi.org/10.1046/ j.1439-0418.2003.00732.x. 14. MullaM,DarwazehHA,DavidsonEW,DulmageHT.1984.Efcacyand persistenceofthemicrobialagent Bacillussphaericus againstmosquito larvaeinorganicallyenrichedhabitats.MosqNews44:166. 15.MargalitJ,BobrogloH.1984.Theeffectoforganicmaterialsandsolids inwateronthepersistenceof Bacillusthuringiensis var. israelensis serotypeH-14.ZAngewEntomol97:516. 16.MerrittRW,DaddRH,WalkerED.1992.Feedingbehavior,naturalfood, andnutritionalrelationshipsoflarvalmosquitoes.AnnuRevEntomol 37:349. https://doi.org/10.1146/annurev.en.37.010192.002025. 17. DavisJM,RosemondAD,EggertSL,CrossWF,WallaceJB.2010.Longtermnutrientenrichmentdecouplespredatorandpreyproduction.Proc NatlAcadSciUSA107:121. https://doi.org/10.1073/pnas .0908497107. 18. HulotFD,LacroixG,Lescher-MoutouF,LoreauM.2000.Functional diversitygovernsecosystemresponsetonutrientenrichment.Nature 405:340. https://doi.org/10.1038/35012591. 19. DayJF,StarkLM.2000.FrequencyofSaintLouisencephalitisvirusin humansfromFlorida,USA:1990.JMedEntomol37:626633. https://doi.org/10.1603/0022-2585-37.4.626. 20. RutledgeCR,DayJF,LordCC,StarkLM,TabachnickWJ.2003.WestNile virusinfectionratesin Culexnigripalpus (Diptera:Culicidae)donot reecttransmissionratesinFlorida.JMedEntomol40:253. https:// doi.org/10.1603/0022-2585-40.3.253. 21. OMearaGF,Cutwa-FrancisM,ReyJR.2010.Seasonalvariationinthe abundanceof Culexnigripalpus and Culexquinquefasciatus inwastewaterpondsattwoFloridadairies.JAmMosqControlAssoc26:160. https://doi.org/10.2987/09-5971.1. 22. DayJF,CurtisGA.1989.Inuenceofrainfallon Culexnigripalpus (Diptera: Culicidae)blood-feedingbehaviorinIndianRiverCounty,Florida.Ann EntomolSocAm82:32. https://doi.org/10.1093/aesa/82.1.32. 23. ReyJR,OMearaGF,OConnellSM,Cutwa-FrancisMM.2006.Factors affectingmosquitoproductionfromstormwaterdrainsandcatchbasins intwoFloridacities.JVectorEcol31:334. https://doi.org/10.3376/ 1081-1710(2006)31[334:FAMPFS]2.0.CO;2. 24. WermelingerED,BenignoCV,MachadoRN,CabelloPH,MeiraAM, FerreiraAP,ZanuncioJC.2012.Mosquitopopulationdynamic(Diptera:Dugumaetal. January/February2017Volume2Issue1e00387-16 msphere.asm.org 10 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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Culicidae)inaeutrophiseddam.BrazJBiol72:795. https://doi.org/ 10.1590/S1519-69842012000500003. 25. NayarJK,KnightJW,MunstermannLE.2002.Temporalandgeographic geneticvariationin Culexnigripalpus Theobald(Culicidae:Diptera),a vectorofSt.Louisencephalitisvirus,fromFlorida.JMedEntomol 39:854860. https://doi.org/10.1603/0022-2585-39.6.854. 26. SardelisMR,TurellMJ,DohmDJ,OGuinnML.2001.Vectorcompetence ofselectedNorthAmerican Culex and Coquillettidia mosquitoesforWest Nilevirus.EmergInfectDis7:1018. https://doi.org/10.3201/ eid0706.010617. 27. BoikeAH,Jr,RathburnCB,Jr,FlooreTG,RodriguezHM,CoughlinJS. 1989.Insecticidetoleranceof Culexnigripalpus inFlorida.JAmMosq ControlAssoc5:522. 28.CoonKL,VogelKJ,BrownMR,StrandMR.2014.Mosquitoesrelyontheir gutmicrobiotafordevelopment.MolEcol23:2727. https:// doi.org/10.1111/mec.12771. 29. Daz-NietoLM,DAlessioC,PerottiMA,BernCM.2016. Culexpipiens developmentisgreatlyinuencedbynativebacteriaandexogenous yeast.PLoSOne11:e0153133. https://doi.org/10.1371/journal.pone .0153133. 30. MitrakaE,StathopoulosS,Siden-KiamosI,ChristophidesGK,LouisC. 2013. Asaia accelerateslarvaldevelopmentof Anophelesgambiae.PathogGlobHealth107:305. https://doi.org/10.1179/2047773213Y .0000000106. 31. CoonKL,BrownMR,StrandMR.2016.Gutbacteriadifferentiallyaffect eggproductionintheanautogenousmosquito Aedesaegypti andfacultativelyautogenousmosquito Aedesatropalpus (Diptera:Culicidae). ParasitVectors9:375. https://doi.org/10.1186/s13071-016-1660-9. 32. FinneyCA,KamhawiS,WasmuthJD.2015.Doesthearthropodmicrobiotaimpacttheestablishmentofvector-bornediseasesinmammalian hosts?PLoSPathog11:e1004646. https://doi.org/10.1371/ journal.ppat.1004646. 33. GlaserRL,MeolaMA.2010.Thenative Wolbachia endosymbiontsof Drosophilamelanogaster and Culexquinquefasciatus increasehostresistancetoWestNilevirusinfection.PLoSOne5:e11977. https://doi.org/ 10.1371/journal.pone.0011977. 34. ZinkSD,VanSlykeGA,PalumboMJ,KramerLD,CiotaAT.2015.Exposure toWestNilevirusincreasesbacterialdiversityandimmunegeneexpressionin Culexpipiens.Viruses7:5619. https://doi.org/10.3390/ v7102886. 35. DennisonNJ,JupatanakulN,DimopoulosG.2014.Themosquitomicrobiotainuencesvectorcompetenceforhumanpathogens.CurrOpin InsectSci3:6. https://doi.org/10.1016/j.cois.2014.07.004. 36. BerticatC,RoussetF,RaymondM,BerthomieuA,WeillM.2002.High Wolbachia densityininsecticide-resistantmosquitoes.ProcBiolSci269: 1413. https://doi.org/10.1098/rspb.2002.2022. 37. DuronO,LabbP,BerticatC,RoussetF,GuillotS,RaymondM,WeillM. 2006.High Wolbachia densitycorrelateswithcostofinfectionforinsecticideresistant Culexpipiens mosquitoes.Evolution60:303. https:// doi.org/10.1111/j.0014-3820.2006.tb01108.x. 38. HazardEI,MayerMS,SavageKE.1967.Attractionandovipositionstimulationofgravidfemalemosquitoesbybacteriaisolatedfromhay infusions.MosqNews27:133. 39.LiessA,DiehlS.2006.Effectsofenrichmentonprotistabundancesand bacterialcompositioninsimplemicrobialcommunities.Oikos114: 15. https://doi.org/10.1111/j.2006.0030-1299.14516.x. 40. HartmanWH,RichardsonCJ,VilgalysR,BrulandGL.2008.Environmental andanthropogeniccontrolsoverbacterialcommunitiesinwetlandsoils. ProcNatlAcadSciUSA105:17842. https://doi.org/10.1073/ pnas.0808254105. 41. PernthalerJ.2005.Predationonprokaryotesinthewatercolumnandits ecologicalimplications.NatRevMicrobiol3:537. https://doi.org/ 10.1038/nrmicro1180. 42. DugumaD,HallMW,Rugman-JonesP,StouthamerR,NeufeldJD,WaltonWE.2015.MicrobialcommunitiesandnutrientdynamicsinexperimentalmicrocosmsarealteredaftertheapplicationofahighdoseofBti. JApplEcol52:763. https://doi.org/10.1111/1365-2664.12422. 43. DugumaD,HallMW,Rugman-JonesP,StouthamerR,TereniusO, NeufeldJD,WaltonWE.2015.Developmentalsuccessionofthemicrobiomeof Culex mosquitoes.BMCMicrobiol15:140. https://doi.org/ 10.1186/s12866-015-0475-8. 44. DugumaD,Rugman-JonesP,KaufmanMG,HallMW,NeufeldJD, StouthamerR,WaltonWE.2013.Bacterialcommunitiesassociatedwith Culex mosquitolarvaeandtwoemergentaquaticplantsofbioremediationimportance.PLoSOne8:e72522. https://doi.org/10.1371/journal .pone.0072522. 45. WermelingerED,BenignoCV,MachadoRN,NascimentoTF,FerreiraAP, MeiraAM,SouzaMB,ZanuncioJC.2010.Occurrenceof Anopheles (Nyssorhynchus) rangeli (Gabaldonetal.)and Anopheles (Nyssorhynchus) evansae (Brethes)(Diptera:Culicidae)inaneutrophizeddam. NeotropEntomol39:449450. https://doi.org/10.1590/S1519 -566X2010000300022. 46. IshiiT,SohnSR.1987.Highlypollutedlarvalhabitatsofthe Culexpipiens complexincentralSweden.JAmMosqControlAssoc3:276. 47.AldemirA.2009.InitialandresidualactivityofVectoBac12AS,VectoBac WDG,andVectoLexWDGforcontrolofmosquitoesinAraratValley, Turkey.JAmMosqControlAssoc25:113. https://doi.org/10.2987/ 08-5836.1. 48. BatzerDP,WissingerSA.1996.Ecologyofinsectcommunitiesinnontidal wetlands.AnnuRevEntomol41:75. https://doi.org/10.1146/ annurev.en.41.010196.000451. 49. TalayF,MolvaC, AtabayHI.2016.Isolationandidenticationof Arcobacter speciesfromenvironmentalanddrinkingwatersamples.FoliaMicrobiol (Praha)61:479484 https://doi.org/10.1007/s12223-016-0460-0. 50. HnelI,TomasoH, NeubauerH.2016. Arcobacteranunderestimated zoonoticpathogen?BundesgesundheitsblattGesundheitsforschungGesundheitsschutz59:789. https://doi.org/10.1007/s00103-016-2350-7. 51. HsuTT,LeeJ.2015.Globaldistributionandprevalenceof Arcobacter in foodandwater.ZoonosesPublicHealth62:579. https://doi.org/ 10.1111/zph.12215. 52. GarrenM,RaymundoL,GuestJ,HarvellCD,AzamF.2009.Resilienceof coral-associatedbacterialcommunitiesexposedtoshfarmefuent. PLoSOne4:e7319. https://doi.org/10.1371/journal.pone.0007319. 53. SharmaP,SharmaS,MauryaRK,DasDeT,ThomasT,LataS,SinghN, PandeyKC,ValechaN,DixitR.2014.Salivaryglandsharbormorediverse microbialcommunitiesthangutin Anophelesculicifacies.ParasitVectors 7:235. https://doi.org/10.1186/1756-3305-7-235. 54. DunningHotoppJC.2011.Horizontalgenetransferbetweenbacteria andanimals.TrendsGenet27:157. https://doi.org/10.1016/j.tig.2011 .01.005. 55. ZouacheK,RaharimalalaFN,RaquinV,Tran-VanV,RavelosonLH,RavelonandroP,MavinguiP.2011.Bacterialdiversityofeld-caughtmosquitoes, Aedesalbopictus and Aedesaegypti,fromdifferentgeographic regionsofMadagascar.FEMSMicrobiolEcol75:377. https://doi.org/ 10.1111/j.1574-6941.2010.01012.x. 56. BrionesAM,ShililuJ,GithureJ,NovakR,RaskinL.2008. Thorsellia anophelis isthedominantbacteriuminaKenyanpopulationofadult Anophelesgambiaemosquitoes.ISMEJ2:7482. https://doi.org/ 10.1038/ismej.2007.95. 57. KmpferP,LindhJM,TereniusO,HaghdoostS,FalsenE,BusseHJ,Faye I.2006. Thorselliaanophelis gen.nov.,sp.nov.,anewmemberofthe Gammaproteobacteria.IntJSystEvolMicrobiol56:335. https:// doi.org/10.1099/ijs.0.63999-0. 58. LindhJM,TereniusO,FayeI.2005.16SrRNAgene-basedidentication ofmidgutbacteriafromeld-caught Anophelesgambiae sensulatoand A.funestus mosquitoesrevealsnewspeciesrelatedtoknowninsect symbionts.ApplEnvironMicrobiol71:7217. https://doi.org/ 10.1128/AEM.71.11.7217-7223.2005. 59. WilkeAB,MarrelliMT.2015.Paratransgenesis:apromisingnewstrategy formosquitovectorcontrol.ParasitVectors8:342. https://doi.org/ 10.1186/s13071-015-0959-2. 60. StewartRIA,DossenaM,BohanDA,JeppesenE,KordasRL,LedgerME, MeerhoffM,MossB,MulderC,ShurinJB,SuttleB,ThompsonR,Trimmer M,WoodwardG.2013.Mesocosmexperimentsasatoolforecological climate-changeresearch.AdvEcolRes48:71. https://doi.org/ 10.1016/B978-0-12-417199-2.00002-1. 61. KentAD,YannarellAC,RusakJA,TriplettEW,McMahonKD.2007. Synchronyinaquaticmicrobialcommunitydynamics.ISMEJ1:3847. https://doi.org/10.1038/ismej.2007.6. 62. LealWS,BarbosaRM,XuW,IshidaY,SyedZ,LatteN,ChenAM,Morgan TI,CornelAJ,FurtadoA.2008.Reverseandconventionalchemical ecologyapproachesforthedevelopmentofovipositionattractantsfor Culex mosquitoes.PLoSOne3:e3045. https://doi.org/10.1371/journal .pone.0003045 63. DadaN,Jumas-BilakE,ManguinS,SeiduR,StenstrmTA,OvergaardHJ. 2014.Comparativeassessmentofthebacterialcommunitiesassociated with Aedesaegypti larvaeandwaterfromdomesticwaterstoragecontainers.ParasitVectors7:391. https://doi.org/10.1186/1756-3305-7-391.Culex MicrobiomeandNutrientInteractions January/February2017Volume2Issue1e00387-16 msphere.asm.org 11 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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64.RubinBER,SandersJG,Hampton-MarcellJ,OwensSM,GilbertJA, MoreauCS.2014.DNAextractionprotocolscausedifferencesin16S rRNAampliconsequencingefciencybutnotincommunityprole compositionorstructure.Microbiologyopen3:910. https://doi.org/ 10.1002/mbo3.216. 6 5.BartramAK,LynchMDJ,StearnsJC,Moreno-HagelsiebG,NeufeldJD.2011. Generationofmultimillion-sequence16SrRNAgenelibrariesfromcomplex microbialcommunitiesbyassemblingpaired-endIlluminareads.ApplEnvironMicrobiol77:3846. https://doi.org/10.1128/AEM.02772-10. 66. KennedyK,HallMW,LynchMD,Moreno-HagelsiebG,NeufeldJD.2014. EvaluatingbiasofIllumina-basedbacterial16SrRNAgeneproles.Appl EnvironMicrobiol80:5717. https://doi.org/10.1128/AEM.01451-14. 67. RossAA,NeufeldJD.2015.Microbialbiogeographyofauniversity campus.Microbiome3:66. https://doi.org/10.1186/s40168-015-0135-0. 68. TakahashiS,TomitaJ,NishiokaK,HisadaT,NishijimaM.2014.Developmentofaprokaryoticuniversalprimerforsimultaneousanalysisof BacteriaandArchaeausingnext-generationsequencing.PLoSOne 9:e105592. https://doi.org/10.1371/journal.pone.0105592. 69. LynchMDj,MasellaAP,HallMW,BartramAK,NeufeldJD.2013.AXIOME: automatedexplorationofmicrobialdiversity.Gigascience2:3. https:// doi.org/10.1186/2047-217X-2-3. 70. MasellaAP,BartramAK,TruszkowskiJM,BrownDG,NeufeldJD.2012. PANDAseq:paired-endassemblerforIlluminasequences.BMCBioinformatics13:31. https://doi.org/10.1186/1471-2105-13-31. 71. EdgarRC.2013.UPARSE:highlyaccurateOTUsequencesfrommicrobial ampliconreads.NatMethods10:996. https://doi.org/10.1038/ nmeth.2604. 72. WangQ,GarrityGM,TiedjeJM,ColeJR.2007.NaiveBayesianclassier forrapidassignmentofrRNAsequencesintothenewbacterialtaxonomy.ApplEnvironMicrobiol73:5261. https://doi.org/10.1128/ AEM.00062-07. 73. CaporasoJG,KuczynskiJ,StombaughJ,BittingerK,BushmanFD, CostelloEK,FiererN,PeaAG,GoodrichJK,GordonJI,HuttleyGA,Kelley ST,KnightsD,KoenigJE,LeyRE,LozuponeCA,McDonaldD,MueggeBD, PirrungM,ReederJ,SevinskyJR,TurnbaughPJ,WaltersWA,WidmannJ, YatsunenkoT,ZaneveldJ,KnightR.2010.QIIMEallowsanalysisof high-throughputcommunitysequencingdata.NatMethods7:335. https://doi.org/10.1038/nmeth.f.303. 74. DeSantisTZ,HugenholtzP,LarsenN,RojasM,BrodieEL,KellerK,Huber T,DaleviD,HuP,AndersenGL.2006.Greengenes,achimera-checked 16SrRNAgenedatabaseandworkbenchcompatiblewithARB.Appl EnvironMicrobiol72:5069. https://doi.org/10.1128/AEM.03006-05. 75. OksanenJ,KindtR,LegendreP,OHaraB,StevensMHH,OksanenMJ, SuggestsM.2007.Theveganpackage.CommunityEcolpackage10. https://CRAN.R-project.org/packagevegan. 76. RivireD,DesvignesV,PelletierE,ChaussonnerieS,GuermaziS,WeissenbachJ,LiT,CamachoP,SghirA.2009.Towardsthedenitionofa coreofmicroorganismsinvolvedinanaerobicdigestionofsludge.ISME J3:700. https://doi.org/10.1038/ismej.2009.2. 77. SAS,Inc.2013.JMPrelease11.0.SASInstitute,Inc,Cary,NC.Dugumaetal. January/February2017Volume2Issue1e00387-16 msphere.asm.org 12 on February 1, 2017 by guest http://msphere.asm.org/ Downloaded from
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