Citation
Effects of organic amendments on microbiota associated with the Culex nigripalpus mosquito vector of the Saint Louis encephalitis and West Nile viruses

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Title:
Effects of organic amendments on microbiota associated with the Culex nigripalpus mosquito vector of the Saint Louis encephalitis and West Nile viruses
Series Title:
mSphere 2 (1): e00387-16. doi: 10.1128/mSphere.00387-16
Creator:
Duguma, Dagne
Hall, Michael W.
Smartt, Chelsea T.
Neufeld, Josh D.
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Washington, DC
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American Society for Microbiology
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English
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Journal Article

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Subjects / Keywords:
aquatic chemistry
bacteria
disease vectors
food web
life stages
microbiome
mosquito
pollen

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Abstract:
Pollution from nutrients in aquatic habitats has been linked to increases in disease vectors, including mosquitoes and other pestiferous insects. One possibility is that changes in mosquito microbiomes are impacted by nutrient enrichments and that these changes affect various traits including larval development, susceptibility to larval control agents, and susceptibility of the adult mosquitoes to pathogens. We tested this hypothesis using field mesocosms supplemented with low and high organic nutrient regimens, then sampled microbial communities associated with the naturally colonizing Culex nigripalpus mosquito vector. By high-throughput sequencing of 16S rRNA gene sequences, we found no significant differences in overall microbial communities associated with sampled mosquitoes, despite detecting discernable differences in environmental variables including pH, dissolved oxygen, and nutrient amendments. Nevertheless, indicator species analysis revealed that members of the Clostridiales were significantly associated with mosquitoes that originated from high nutrient enrichments. In contrast, members of the Burkholderiales were associated with mosquitoes from the low nutrient enrichment. High bacterial variability associated with the life stages of the Cx. nigripalpus were largely unaffected by levels of nutrient enrichments that impacted larval microbial resources, including bacteria, ciliates, and flagellates in the larval environments.
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Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Dagne Duguma.
Publication Status:
Published
Funding:
This article was published with assistance from the University of Florida's Open Access Publishing Fund.

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University of Florida Institutional Repository
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University of Florida
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This item is licensed with the Creative Commons Attribution License. This license lets others distribute, remix, tweak, and build upon this work, even commercially, as long as they credit the author for the original creation.

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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. 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