Author affiliation: Dagne Dagne Duguma affiliation: Author head: Running Josh D. Neufeld 32962, UnitedFL, States of America America of States This article is protected Allrights bycopyright. reserved. 10.1111/1365-2664.12422 of Record.versiondifferences and article Please between the cite this Version this as doi: through the copyediting, typesetting, pagination for publication accepted This articlehasbeen 3 2 1 of a application after high communitiesMicrobial and nutrient dynamics experimentalin microcosms are altered Wan : Shiqiang Editor type:Standard PaperArticle Accepted Date : 05-Mar-2015 :02-Mar-2015 Date Revised Received Date :04-Sep-2014 Accepted L Medical Florida Entomology affiliation: Current Department United CA92521, Riverside, California University Riverside, of of Entomology, Department Article

of Biology, N2L3G1,Ontario, Waterloo, University Canada of Waterloo, Bti 2 ,

William E. E. Walton William andaquatic community interactions microbial

dose of 1, 3 1 , Michael, Hall

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and undergone full peer review buthasnot been review undergone fullpeer and and proofreading process, which may lead to aboratory, University of Florida, Vero Beach, University of aboratory, Florida, 2 , PaulRugman-Jones , 1 , Richard Stouthamer , Richard 1 , This article is protected Allrights bycopyright. reserved. Summary 2009 Florida, Laboratory, University author: D. of Medical Entomology Duguma, Florida Corresponding Accepted Article longer than thelonger period during were which numbers larval warranted reduced. mosquito This significantly that application reduced abundancemosquito by more than 50% persisted 5 betaproteobacteria by microcosms subject to a dosehigh of physicochemical variables inthe water columnphysicochemical variables during the 44-day study.field significantlyreduced inthe high 3. control,untreated were assigned to 300 replicate L microcosms. dose of 16S of rRNA abundantgenes. most The in aquatic subject microcosms to a low communities Bacterial 4. in present the water column were assessed by sequencing Illumina 2. Two 2. of effects have beensuggested extendedfor vector control however,in some environments; the with andblackflies, ahistorymosquitoes of and high specificity efficacy. dosesof High 1 . . Bacillus thuringiensis Bacillus . Mucilaginibacter Synthesis and applications Culex Bti Bti Bti abundance, biomass, phytoplankton sestonic particulates, andnutrients were

Bti Bti (VectoBac th and untreated control, St SE, Vero Beach, FL 32962, USA, [email protected] Email: application application on the native microfauna inthe environment are poorly understood. ) and ) ), and), were diverse more otherthan in the treatments. Sphingomonas , Sediminibacterium

G) treatments (high = 48.1 kg ha 48.1 kg = (high treatments G) subsp. subsp. . The biotic and abiotic changes resulting from a biopesticide biopesticide a from resulting changes abioticand biotic The . israelensis Bti Bti (class treatment. These changes affected other Bti. Bti. (phylum (phylum Bacteria inthe alphaproteobacteria ( Bti ) is) widely the most against usedbiopesticide , -1 ; low; =0.6 kg ha

high Bti ) , , were suppressed in ) and Cyclobacteriaceae treatment Polaromonas -1 ), in addition inaddition an), to

were dominated were dominated (class (phylum (phylum Bti

control unintentionalmicrocosms, phytoplankton effects, Keywords: This article is protected Allrights bycopyright. reserved. processes associated with are habitats aquatic unexploredmosquito 2000). &Boisvert (Boisvert of possible interactions However, Roucaute mosquito operational (Lagadic, &Caquetfor control 2014; Lagadic &Caquet 2014). withintensively no studied, effects known detrimental found to date at dosages recommended pesticides.synthetic The effects of specificity, but alsoits ascomparedhigh highly to ecotoxicological profile other favourable Lacey 2003). & Merritt The widespread adoption of thuringiensis Bacillus Introduction Accepted agent for which dipteran insects, act asdisease vectors Article cycling couldaffectcycling the functioning of aquatic webs.food abundance anditsalgal on primaryeffect production, communities andnutrient microbial Bti habitats. Results innatural ofapplications our study suggest thethat adoption widespread of and regulations violaterecommendations country-specific biopesticide governing might cost of operational but application, repeated they could exceed manufacturer’s reduce abundancemosquito anfor extended period of time and therefore lessen the than control. Application rates mosquito for greater the labelrate a for into ecosystem-level effectsthe of investigation further application aboveapplication the recommended labelrate shouldbediscouraged where inhabitats nutrient dynamics, subsp. israelensis

bacteria communities Bti Bti with and microbial communities effects on ecosystem-level on nontarget invertebrates andvertebrates have been ( Bti ) is a commercially successful microbial control , biopesticide, vector, interactions, biopesticide, community Bti , Bti, Culex Bti, , isnot only due its effectiveness andto

(mainly mosquitoes andblack(mainly mosquitoes flies; Bti Bti application rates used routinely used routinely rates application mosquitoes, outdoor aquatic Bti Bti biopesticide can biopesticide Chlorella The suggested authors that the biomass wasAlgal expected increase asto a of result areported significant in reduction abundancethe of two species microalgal ( organic matter aswell (Merritt, Dadd & 1992).Walker micro-organisms protozoa,(i.e. bacteria, algaeand microcrustaceans) and onotherfeed responsible regulation“top–down” for of resources. Mosquito larvae are known of predators communities to themicrobial suggesting removal of mosquitoes, larvalthat are mosquitoes Xu 2008; & Walker aquatic ecosystemaquatic inhabitants &(Chen 2005; Folt Jackson comprise the base of many chains food might have direct or indirect effects on andfish other importancehas immense several for First,reasons. a reduction inautotrophs (e.g. algae) that habitats,physicochemistry inmosquito such as wetlandstreatment or other aquatic ecosystems hypothesis. this support indicatorsto water quality key other or measure communities However, suppression. phytoplankton Su&Mulla didnot (1999) characterize bacterial when resulted larval habitats in bacterial anincrease some Similarly, tocompared untreated wetlands control (Östman, &Persson Lundström 2008). Vinnersten important components of wetland food webs(e.g. Hershey wetlandof components food important are that insect groups of the andproduction abundance the are lower knownto production Ciliophora This article is protected Allrights bycopyright. reserved. Accepted Article A communities. increase in microbial 4.5-fold the abundance of protozoans (i.e. Contrary Contrary expectedto the of outcome such a top–down Su& hypothesis, Mulla (1999) Understanding effectsUnderstanding the of Several studies demonstrated positiveSeveral anindirect of studies effect sp.) in microcosms , Zoomastigophora et al et . In2008). eachof these studies, the changes attributed authors in

treated with doses high of ) was observed in Bti was usedagainst Bti Bti treatment applications water asa improved quality result of application on microbial communities and application on communities microbial water

(e.g. Flavobacteriaceae Bti -treated wetlands (15-treated kg VectoBac Gha Aedes triseriatus Bti Bti application, but application, opposite occurred.the tocontrol et al et et al Bti . 1998; &Paz Poulin, Lefebvre ) ) taxa intree-hole mosquito . Reductions 2013). inprimary application on application aquatic Say Culex

larvae Chen (Kaufman, mosquito larvae. mosquito Closterium Amoeba sp. and , -1 ) particulates, and nutrientsparticulates, were reduced significantly. and composition The bacterial diversity of abundance wasmosquito reduced by the high microcosms freshwater asingle following column physicochemistry. conditions in environments) havefield noeffect on community and structure microbial water This article is protected Allrights bycopyright. reserved. nutrients in wetlandsin recycling andtreatment algicidal potential andantibiotic effects of Lagadic, Roucaute2010; &Caquet Secondly, 2014). communities playroles important microbial Accepted than thehigher recommended labelrate VectoBacfor previously by and (1999) Su&Mulla tested the null lower rates or hypothesis application that communities and duplicated themicrobial high water variables. used rate quality application We VectoBac control, outdoor inreplicate G, microcosms freshwater onthe focusing primarily contributors essential into ecosystems. transformation nutrient aquatic onmicrobial application communities, which components important of are websfood and Su1998; 2000), about &Mulla &Boisvert islacking effects Boisvert the of 1999; knowledge Article studies suggestingDespite inaquaticchanges ecosystem communities and processes caused by control agents. mosquito applications less pesticide for athan and week mayto potentiallyrecognize fail important visual Most existing inspection. of the assessment protocols environments monitor following instead micro-organisms, typically consider fo scrutiny. increasing environmental assessmentThe current protocols biopesticides for donot wetland. impact on the operations treatment Thirdly, of water mosquito-control isunder quality &Mulla(Su Yudina 1999; We report anWe characterization in-depth in variables and microbiota of environmental multiple In study,In effects wethis the of acommercial investigated

et al Bti applications impact applications aquatic components Hershey(e.g. . 2003). could have adramatic impact on the efficacy a of Bti application to After control mosquitoes. cusing on a few physicochemical variables and Bti treatment, phytoplanktontreatment, sestonic biomass,

G (11.2-22.4 kg ha (11.2-22.4 kg G Bti formulation used for mosquito mosquito used for formulation -1 in polluted polluted in Culex et al et Bti .

Bti

environmental variables habitats insensitive aquatic environmental arethat subjected control.to mosquito of rates biopesticides significantlythat reduce application high suggest that was findings reduced. Our abundance mosquito that period the effectsThe on some of variablesthe inthe environmental high changesThese mosquitoes. alsoaffectedother physicochemical inthe watercolumn. variables controlsuntreated and those subject to avery low September 2012. September microcosmsThe were enriched 40 with sulphate [(NH g of ammonium column was 41.1 ( Su &Mulla & Duguma 2014). 1999; nitrogen ammonium concentration intheThe Walton water (17% crude Mill, protein) (Brookhurst Riverside, and Creek, Miller and CA)] Lilly Brands, 50 nitrogen 24%sulphur; pellets g of21% rabbit Walnut = 0.6 kg = wetlands(Popko inCalifornia treatment Station. Station. Twelve microcosms =(area 1 m Experiment Agricultural Riverside California of University the Facility of Research Control designandExperimental treatments ITU mg ITU of design. Amicrocosms usinga corncob experimental completely randomized formulation granule This article is protected Allrights bycopyright. reserved. andmethods Material communities in subjectmicrocosms highto (Su &Mulla (Su high The 1999). respectively)microcosm, were based selected on a previous study a using formulation similar

AcceptedBti Article This This study was conducted in outdoors fibreglass microcosms at the Aquatic andVector (VectoBac G; Valent Biosciences Corporation, Libertyville, IL, USA) a USA) (VectoBac Libertyville, IL, with Valent200 toxicity BiosciencesCorporation, G; of -1 was used. 12 (equivalent The rates to application 000 ITUs and962 000 ITUs per - ha -1 and high and high = 48.1kg ha ± 0.58 SE) mg L 0.58 SE) mg Bti

treatment used in presentthe study was approximately twice -1 24 nutrient hafter levels enrichment. in are Similar found -1 ) and) an untreated treatment control were to assigned the et al 2 ) were toflooded depth 0.3-m (300 L) on28 Bti Bti . 2006).. On 2 October 2012, two Culex treatment differedtreatment significantly in from those CA) to promote CA) mosquito colonization (Nguyen, Bti Bti mosquito abundancemosquito affectmight key treatment, which affect treatment, larval didnot the Bti Bti treatment persisted well after persistedtreatment well after Bti treatments treatments (low 4 ) 2 SO 4 ; concentration wasconcentration categorized 300 size into categories across dynamicthe range of each μ Blanks 100-consistedBlanks of unfiltered (for either APHA 10200H of (1995). MD) Columbia, method Scientific, spectrophotometer (Shimadzu 1601 UV-Visible following atcentrifugation 262 × wereand then pigments inextracted 90% alkalineacetone (Wetzel &Likens After 1991). laboratory andthe then at frozen -20 ºC. were Filters a using ground and mortar Teflon pestle were filtered through 45-mm membrane filters (0.8- 45-mmmembrane filters through were filtered filtered water wasfiltered in determined 50- three 10- a through aperturesusing of 100 inbulkparticles water samples were in quantified counts3–8 replicate of 0.5–2-mL samples using Coulter CA, (Beckman Analyzer Inc., USA). aMultisizerenumerated Brea, The 4Particle estimated asestimated the concentration of chlorophyll placedonice and immediately totransported laboratory.the Phytoplankton biomass was six during the dates 44days the experiment of reported S1). here (Table were Samples (2.8ha kg habitats, whereasenriched the low Phytoplankton andsestonicPhytoplankton particulates This article is protected Allrights bycopyright. reserved. concentrationthe maximum (22.4recommended ha kg

Accepted20- the size; for pore m Article Sestonic particle distributionsSestonic (equivalent A single 1-L wasA single sample below watertaken the surface inthe centre of each microcosmon -1 ) ) for incomparatively control habitats.mosquito less enriched μ m aperture m andmesh the particle (0.6–10 distribution g μ for 5 for pigmentmin, concentration was a determined using Biospec- m and 280 and m μ m and 30- Bti μ μ treatment was minimum rate recommended below the m. Approximately 10 mL of Approximatelym. bulk water 10mL was filtered m apertures) electrolyte (ISOTON II). Particle Particle (ISOTON II). electrolyte m apertures)

μ L replicates a using 20- spherical 0.6– diameter 224[ESD]: μ a m andm 280- in duplicate samples of 25–100-mL. induplicate Samples μ m pore size) under darkened conditions in -1 ) of VectoBac) in control G mosquito for μ m apertures) or filter-sterilized (0.2- or filter-sterilized m apertures) μ m or 30- μ m in0.1–m 0.2ESD) mL μ m aperture. μ m) were AquaCheck, Perstop Analytical, Wilsonville, OR, USA). OR, Analytical, Perstop Wilsonville, AquaCheck, h) of each datesampling usinganelectronic andsensor arrayh) afternoon (ICM (~15·30 and temperature dissolved oxygen concentration [DO])were inthemeasured (~08·30 morning pH, (i.e. each microcosm waterin parameters the of physicochemical Other information). Bourne, inoneComputer Inc., USA) of MA, the control (seemicrocosms S1inSupporting Fig. everyrecorded h 0.5 entire studythe for a using water temperature data logger (HOBO, Onset Plus tests, HachCo., USA). Loveland, water of Surface CO, temperature each microcosm was wereparameters measureda HachDR2800 using colorimetrically spectrophotometer (TNT asphosphate phosphorus, total chemical oxygen demand (COD) andsulphate. These nitrate–ni ofconcentration ammonium–nitrogen, parametersPhysicochemical (Table S1). S1). (Table The samplewas 24first taken h 1(on October 2012) before communities Bacterial centre of each microcosm. the and from corners two taken from wereSamples analysis. nutrient for were taken samples samples perdipper microcosm were taken just below water the 24surface hafter waterthe andotherMosquitoes macroinvertebrates This article is protected Allrights bycopyright. reserved. μ andaperture counts were then summed across broadthree size 0.600–1.000 categories (small,

Accepted Article1.001–10.000 medium, m; Two water 40-mL samples were in50-mL collected sterile, centrifuge tubes on datesfour enumerateTo andidentify mosquitoes immature invertebrates, and other 350-mLthree waterThe sample collected phytoplankton biomass alsowasfor used determine theto μ m; and large, 10.001–224 andlarge, m; trogen, nitrite–nitrogen, total nitrogen, total nitrogen, nitrite–nitrogen, trogen, μ m ESD) ESD) statistical analyses. for m Bti treatment, while treatment, the analysis to assureanalysis normality to the data. of One-way by ANOVA followed at Tukey’s test was 2011). out when11 (SAS transformation Logarithmic Inc. to carried necessary prior biomass, particulates and phytoplankton sestonic analysis Statistical werethat assigned to withQIIME e-value amaximum of parallel_assign_taxonomy_blast_py Sequences script 0.001. usingconducted BLAST the SILVAagainst 111 reference database (Quast and 4.2 version discarded (Edgar UCHIME out usingcarried cd-hit-est version 4.5.4 (Li & were Chimeras 2006). Godzik detected using of sequences similar Clustering (i.e. This article is protected Allrights bycopyright. reserved. NEXTflex (Duguma described andextracted, a oftarget region bacterialthe was 16Sgene via amplified PCRaspreviously samples were remaining 9, 16taken and44 days post-treatment. DNAwasTotal genomic Acceptedend sequences were assembled PANDAseq using version 2.5.0 (Masella out using PyNast using carried QIIME 1.2.0 version (1.7.0) (Caporaso Sequence analysis, and alignment assignment. Article CoreFacility. and Sequencing Genotyping subjected then 150-baseto sequencing paired-end ona at MiSeq UCLA (Illumina) the each normalized 10 to nm and combined to create two samples. multiplexed samplesThe were did not duringfield amplify steps. PCRor either Illumina preparation A total of 41samples were indices 6-base Scientific,(Bioo Inc., Austin, Seven TX, ofUSA). the samples collected from the Repeated-measures ANOVAsRepeated-measures were employed abundance, to mosquito compare Managed by 1.6.0 AXIOME(Lynch version

DNA sequencing (and protocols)kits and anidentifying NEXTflex with DNAbarcode et al Eukaryota . 2013). Illumina libraries 2013). were . generated Illumina eachfor sample using or provided noBLAST hit were discarded. ≥ 0.97 identity) to operational0.97 identity) to units (OTUs)taxonomic was et al et al . 2011). Taxonomy assignments were assignments . Taxonomy 2011). physiochemical variables using JMP using physiochemical variables version . 2013), analysis sequence of reads was et al et al et . Allpaired- 2010). et al . 2012). . 2013). via the P <0.05 ANOVA and ANOVA testTukey’s R. through Averagedepth. estimates were between compared were Alpha diversity calculated estimates QIIME 10 through subsamplingsfor at even sequence UniFrac di usingweighted APCoAordination 2010). whichClaude& the library2013), vegan inR(Paradis, 2004; leverages Strimmer Roberts Bti microcosms, but chlorophyllmicrocosms, microcosmstreatment control increase A (Fig. 1). was similar initially seeninthe high andsestonicPhytoplankton particulates This article is protected Allrights bycopyright. reserved. withcompared dark the colour observedgreen inthe low high in the study. the throughout Water differ significantly Chlorophyll =92; Results communities using in bacterial between samples AXIOME pipeline(Lynch 1.6.0 version torarefied 6,139 assembled sequences per sample), were carried out to determine differences based (NMDS)scaling on analysis, metric multidimensional randomly sequences sampled (i.e. withadjusted the before Bonferonni correction the Tukey’s was test carried out. was separate usedto significantly means different oneach sampling date. The among among treatments (Table 1). of number The particles inthe size medium range (1.001–10.000 after treatments (Fig. S2).

Accepted andcontrol treatments was higher 83-fold than that in found the high Article Phytoplankton biomass increased exponentially for exponentially biomass increased 30the Phytoplankton for first days inthe low Principal coordinate analysis and measures using coordinate Bray-Curtis dissimilarity Principal non- (PCoA) the Similarly, counts of sestonic counts Similarly, in particulates waterthe column were also significantly different P <0.001; 1; By Fig. dayTable1). 44of experiment, chlorophyllthe

a concentrations inthe microcosms of the low a concentration began to declineninedays after application (F

Bti stances was alsocarried out using QIIME. treatments at each time point using Bti Bti Bti Bti and control andcontrol 44treatments days treatment appeared clear, and control andcontrol didtreatments not a Bti concentration inlow concentration treatment (Fig. 1). P -values were Bti Bti and Bti et al et

2, 9 . This article is protected Allrights bycopyright. reserved. Bti 1).S4; AfternoonTable of measurements DO concentrationthe were similarly high inthe lower of inmicrocosms lower the high S5; 1). S4, AtTable 17days after butmicrocosms, those changes were only in evident measurements taken inthe afternoon (Figs physicochemical variablesOther were suppressedtreatment relative to those in the low treatments andcontrol S2).(Table 1). S3; Table (Fig. ammonium not but nitrates, and nitrites in a reduction from resulted 1). This 3; Table (Fig. treatments control in depleted that significantly microcosms received high 17daysfirst following nutrients inInorganic the water column buttime, without any clear pattern 1; Fig.(Table 2). (0.600–1.000 andparticles chlorophyll patternas the chlorophyll similar μ twice (at 38twice (at and44 days after Towards endthe of the study, sulphate concentrations in waterthe column were measured buttreatments, this for nutrient, differencethe was at apparent day 17 (Fig. 3; 1).Table m ESD) wasm in significantly reduced the microcosms treated with high

Accepted treatment 14days after Article The high phosphorus was inhigh Total depleted similarly allthree In treatments, decreased nitrogen total at asimilar rate (~43% per week) over the Bti μ dose also treatment inchangesresulted to the pHand DOcontent of the m ESD) m ESD) and large (>10.000 Bti Bti a application (Fig. However, 3). application by day 30, wastotal nitrogen concentration were positively ( correlated Bti application (Fig. S5; application 1). Table chemicalThe in oxygen demand Bti Bti application), and application), levels inmicrocosms that received high a Bti concentration (Fig. 2B). concentration (Fig. countsThe of medium-sized treatment relative the low to application, pH (measured at 15·30 h) was significantly pH (measured application, at 15·30 μ m) alsom) particles varied across treatments and/or Bti -treated microcosms-treated relative the other to Bti treatments, relativelowto Bti and control andcontrol (Fig. treatments r =0.75, Bti andshowed a P <0.05). Small Bti Bti and Bti

post-treatment). post-treatment). Overall, 45 were with recovered over 98% bacterial of times and three oncebefore, dates (i.e. sampling on four the microcosms from taken samples communities Bacterial ( andmicrocosms, abundance their didnot differ significantly among the three treatments the of enrichment and week flooding after first the during sampled were respectively, primary colonizers. Nearly colonizers. 90%and97%primary of the numbers total of chironomids in highand washigher the significantly 4C). Fig. abundanceThe late of instars early instars, and pupaevaried across time ( between days 3and30 ( high the P also initially significantly lower than in lower control than the significantly and lowalso initially days initial of andflooding the after microcosms, abundancein their the high This article is protected Allrights bycopyright. reserved. was intreatment significantlythan the low greater Culex quinquefasciatus andotherCulex mosquitoes invertebrates days after high the P =0.043; Fig.=0.043; 4A). contrast, theIn meannumber late and (3rd of instar 4th) larvae in mosquito

Accepted>0.05). Noother invertebrate were indipgroups collected samples these from microcosms. Article We generatedWe 4 095 139 in bacterial sequences 42 resulting 412 41from OTUs water Two other dipteran families (i.e. Ephydridae (i.e. dipteran Two other families After the the After Three Bti Bti Culex Bti treatment was significantly reduced by 54–74%relative to the other treatments treatment was also reduced (by ~75% compared to the control attreatment) 44 Bti application (Table S2). (Table application treatment, treatment, the abundanceof early &2nd) (1st instarsmosquito inthe high mosquito species( mosquito Say) were in found the duringmicrocosms the study. F 2, 33 2, = 4.2, = 4.2, Culex tarsalis tarsalis Culex P =0.023; Fig.=0.023; pupae Mosquito 4B). were collectedfirst 14 Bti ontreatment the last sampling date.

and also occurredChironomidae) among the Coquillett, Coquillett, Bti and untreated microcosms Bti application ( application Culex stigmatosoma Culex F 2, 33 2,

Bti and ephydrids, = 19.2; = 19.2; treatment was treatment

( Dyar and F 2,33 P <0.05), P = 3.5, = 3.5, <0.001; Bti

index) differed significantly whenindex) high differed the comparing This article is protected Allrights bycopyright. reserved. S8). (Fig. groups ( date between high and the low were treatment groups observed beginning at the second sampling betweendifferences any metric thefor sampling pre-treatment Significant date. differences Shannon index diversity andphylogenetic (PD). As expected, were there no significant ofcomparisons levels treatment Alpha diversity (MRPP: dates other the from separated datesignificantly were this sample. from per Samples sequences on recovered date werethis classified to eight just phyla (Fig. S6): 1 containing 892 941 sequences. Based on rarefied sequence sets, ~99.9% of sequencesthe communities bacterial Pre-treatment (1.5%), and (30.1%), classifiedsequences into only S6): seven phyla (Fig. dominated by dominated (0.7%), BD1-5 Bacteroidetes Accepted the sequencesthe per sample date. onthis sampling sequences. Article Three Three weremetrics chosen to analyse speciesrichness andevenness: observed species, samples 3Water taken days 26 initial generated flooding after 801 bacterial OTUs

Bacilli P <0.05; S8). Fig. By sampling date, the twofinal metrics (observed species andShannon Cyanobacteria and Bacteroidetes, (23.5%), (23.5%), Alphaproteobacteria A Clostridia, Clostridia, =0.307; (14.5%), T represented =-17.7; dominated by members of (0.2%) and (1.4%). Firmicutes Firmicutes P (19.7%), <0.001; S7). Fig. and

the third abundant phylummost with 9–23% of the Betaproteobacteria Cyanobacteria (7.5%), Firmicutes Firmicutes Actinobacteria Bti Bacteroidetes Flavobacteria, (0.1%). vs. the low (1.3%), ) accounted for 39–61% of accounted the ) for 39–61% mainly represented by members of Proteobacteria (7.2%), (35.4%), Bti accounted for 16–31% of and control treatment andcontrol Verrucomicrobia Proteobacteria Proteobacteria (primarily (53.6%), (53.6%), (0.8%),

abundance insome members of high to compared treatments Bacillales A differentiated from werethe untreated andlow significantly control high the communities from Bacterial 5). (Fig. applications treatment three the from date andby was sampling eventreatments more evident inanNMDS profiles bacterial of plot This article is protected Allrights bycopyright. reserved. low from significantly (MRPP: significantlywere samples differentiated taken ondaysfrom 9 and16 after application treatment by was treatment by explained PCoA2(Fig. S9). taken 44Samples days after of asseparation samples, shown by was PCoA1, by date. sampling of separation samplesThe revealed of aseparation bysamples treatment and date S9). sampling (Fig. The greatest of diversity post-treatment bacterialBeta application communities the high the last samplingthe date separately grouped the other from two dates (Fig. sampling whereas 6), from the separated from two treatments inordination other spaceand were characterized by OTUs species: as by SILVA classified #10, (OTU groupedtreatments together. samplesThese were by dominated of OTUs

Accepted=0.0652; Article PCoA of weighted UniFracPCoA of distances (Fig. 6) revealed samples that from low Principal coordinate analysis usingBray-Curtis coordinate similarity Principal (PCoA) distance measures Overall, members of Overall, Mucilaginibacter Leadbetterella A Bti , =0.1264; T Sphingomonadales Sphingomonadales

=-4.6 samples separated poorly by ordination. within UniFrac-based date this A; T P =-8.9; Bti Bti and =0.002). and Cyanobacteria and control treatmentand control samples (Fig. 5). separation of The the samples Sediminibacterium P Solitalea. Solitalea. <0.001). high samples from Similarly, Bti and treatment microcosms. observed increase in aslight We Betaproteobacteria Cyanobacteria Burkholderiales Samples from high Samples from , Sphingobacteriales species. and control The low ), dominated in dominated lowthe Cyclobacteriaceae , Gammaproteobacteria Bti , -treated microcosms became-treated microcosms Cytophagales Bti Bti Bti treatments (MRPP: and two treatments Bti Bti Proteomonas Proteomonas and Bti , and control samples taken on Bacteroidetes Bti Flavobacteria

Bti separated application application Bti and control andcontrol treatment sp.

,

significantly reduced in significantly the high abundance in water observedthe column inthis orstudy, previously (Su was& Mulla 1999), 1, 2B).(Figs. is It unknownwhether the suppression algal biomass or of sestonic particle andparticulate dynamicsPhytoplankton application high Bti following the high the diversity bacterial andcommunity changed increased control, significantly composition following microcosms inthe high asphotosynthesis in reductions well phosphorus andtotal as nitrogen total in occurred changes Concomitant inphysicochemicallarvae. associatedfactors with reduced (1.001–10.000 particles cause anenhancement phytoplankton andbacteria,of the abundance of chlorophyll-containing on pressure sestonic particles the reduction oflarvae by mosquito following would abiopesticide expectationsreduced grazing that concentrations.and to Contrary spectra nutrient size particle larvae abundanceandconcomitantly the altered micr reduced environments) polluted for rate recommended maximum the than higher two-fold microcosms. particles asparticles compared to the untreated control, of the ahigh application abundance of larvae mosquito andcause changes inthe nutrientsmicrobiota, and sestonic This article is protected Allrights bycopyright. reserved. Accepted Article averyWhereas low doseof Discussion Deltaproteobacteria Our study revealed that phytoplankton that study revealed Our Bti Bti treatment.

, Verrucomicrobiae

Bti Bti μ m ESD) was ESD) the reduced reduction greatly m of following mosquito treatment. Astreatment. compared to the low Bti Bti (0.6 kg VectoBac (0.6 Gha kg Bti treatments approximatelyweeks after two , Actinobacteria

and particles between 1and 10 obial community, algal -1 and ) didnot) significantly reduce the Frankiales Bti Bti treatment andtreatment the untreated inthe high Bti dose(48 kg ha μ biomass, sestonic biomass, m ESD were m ESDwere Bti application application Bti treated treated Culex -1 , significantly among the significantly treatments, DOthe concentration inthe high in the samplesphotosynthesis. Unlike morning where DO concentration did not differ Impacts of high Bti application rate on application ofrate Impacts highBti physicochemical variables reduced abundancemosquito high (i.e. (low (Kaufman when were present. mosquitoes of reduction mosquitoes. abundanceThe of edible- were pressure herbivores protists, larval released byrotifers) (i.e. small thefrom feeding high the in lower were effects. algicidal byprotozoans the removal larvae of or ofmosquito bacterial taxaproliferation may have that unknown whether the application of nearly three afternearlymonths three the and4) the 44days discussed here. Duguma (2013) found thesethat differences persisted for the treatments persisted beyond 30days the that abundancewasmosquito reduced after days three phosphorus wasTotal positively (Spearman’s correlated biomass declinedat aboutalgal samethe time (~two weeks) after phytoplanktonthe reduced biomass associated the high with This article is protected Allrights bycopyright. reserved. Accepted Articleof or recycling formulation G products of degradation toxins or to related directly Algal biomass isaAlgal source of organic andmatter oxygengenerates asa of result The reduction innutrientsThe resulting high from the numbersAlthough of size the large particles were comparatively (in constant fact, they Bti and control treatments), andcontrol algal biomass was as high compared to microcosms with et al et . but 2006), inour study where Bti application. Differences in application. the nutrient water-column concentrations among Bti treatment treatment than inthe other Fig. 2C), treatments, ispossible it that Bti Bti Bti Bti Anopheles in carcassesmosquito & Khawaled(Zaritsky is It 1986). also treatment. Bti indirectly favoured competitive release of heterotrophic indirectly release competitive favoured Bti treatment). larvae are depress biomass known to algal Culex Bti sized and increased particles algalbiomass Bti mosquitoes were mosquitoes abundant in microcosms application (Fig. 3) (Fig. might haveapplication beendue to , proprietary, of components VectoBacthe r = 0.68, Bti treatments. Phosphorus and P Bti <0.001) biomass with algal Bti- application (Figs. 1, (Figs. 3B). application treated microcosms

(cf. Figs. 3, This article is protected Allrights bycopyright. reserved. date 44 days(i.e. after larvae was among comparable the treatmentsthree on day 4B). 30 (Fig. At the last sampling hypothesis “top–down”on mosquitoesand the Btiof Effects 1995), oxidation (APHA reduced was inhigh to significantly andan oxygen of of utilization the organic amount indicator issusceptiblepotential that matter was its peakphytoplankton at likely Chemical (Fig. S5). oxygen which demand, isa ofmeasure was in other than lower the in thetreatments afternoon when photosynthesis by the Accepted ofconcentration bacteria-sized particles in small high suppressed asa of result high abundancein found the than abundance of the findings, as habitats a tree-hole ofresult 2008; Walker Xu hypothesistop–down regulation (e.g. Su& population Kaufman,Nguyen, Chen Mulla & 1999; bacterial taxato inaquatic agreeing with thehabitats removal of the predominantmosquitoes, date. on sampling this column water high in the (Fig.treatments 4). isunclearIt what caused this ofenhancement larval and pupalabundance was inthemosquitoes high higher significantly in instars late the control and low Article intotreatments) the microcosms declinedacross experiment 4A). the (Fig. abundanceThe of byrecruitment mosquitoes (which was inthe high relatively higher about oneafter month. Changes inthe abundance of The effectsThe of enrichmentthe that favoured of proliferation the Previous studies using using Previous studies Bti treatment it coincidedwithbut treatment bacterial the increased diversity observed inthe et al . 2008). Xu Bti application), the application), mean abundance of both larvae andpupae of Culex Bti Flavobacteriaceae for attributed exclusionmosquito changesfor experiments in larvae Moreover, reduction. mosquito we that found the Aedes et al et Bti Bti Bti treatments also declined; abundancetreatments alsodeclined; the of late-instar microcosms, suggesting that this group of bacteria was . (2008) of aproliferation reported mosquito removal by incontrol found microcosms was 3times greater Bti treatment than in lowthe andcontrol Culex Bti Bti microcosms was not significantly early indicate instars that Bti Bti -treated microcosms S2).(Table application. Contrary to their their Contrary to application. Culex Bti treatment than the other the treatment than Flavobacteriaceae mosquitoes waned Culex in

This article is protected Allrights bycopyright. reserved. Firmicutes the microcosms than onlater dates sampling (Figs S7, S8). application andwere were diverse among more incomposition treatment comparatively similar community dynamicsBacterial inBti treated microcosms eithersamples from the controluntreated or low high or westudy, didnot recover microcrustaceans or predators (e.g. ostracods)copepods, and odonates immature were collected indipsamples. In the current same the microcosms (e.g. Duguma and 2014), microcrustaceans (e.g.Walton cladocerans, withbiomass the abundance of Kroeger, Duquesnepredators. &Liess (2013) found chlorophyll apositive correlation of microcrustaceans daphinds)(e.g. are known to decrease phytoplankton in the absence of their withtreated explored inthe explicitly study.present of potentialThe influence protozoan communities onbacterial grazing however was not ofmajority the heterotrophic protozoans, didnot differ among themarkedly treatments (Fig. 2C). abundance of the large which ESD), sestonic particles includesthe 10–224 likely (i.e. µm are known to populations bacteria influence (e.g. predation).through In our study, the in populations protozoan wetlands with treated after 30 daysuntil treatments other the to relative increased Accepted Article Kaufman, Chen1999; & 2008; Walker Xu bytop–down regulation ashypothesizedmosquitoes by Su& (Nguyen, several studies Mulla The bacterialThe communities at days three after nutrient enrichment and 24 h before the DuquesneKroeger, &Liess demonstrated (2013) the reduction of chlorophyll inmicrocosms Lundström,Östman, &Persson Vinnersten reported (2008) a increase 4.5-fold of the dominated water dominated samples on sampling datethis and Bti (Vectobac 12 AS) in the absence of microcrustacean colonization. Filter-feeder colonization.Filter-feeder absence microcrustacean of in the AS) 12 (Vectobac Culex pipiens et al larvae. previousmosquito In studies conducted in . 2008) was not evident inour study. Bti compared with compared untreated habitats. Protozoa Bti treated microcosms. Proteobacteria Bti Chaoborus, Chaoborus, treatment (Fig. 2). Overall, the 2). (Fig. treatment Overall,

were probably early colonizers odonates) intheodonates) dip , Bacteroidetes

and ( S9). 5, high from communities taken bacterial separation from significant habitats.aquatic no negative effect of increased inhigh increased In contrast, of likely interaction not reveal the werestudies based onlaboratory bioassays with very cultivable microbial few This article is protected Allrights bycopyright. reserved. Contrary inlaboratoryspp.) studies. Yudina to (2003) demonstrated the activities antibacterial of the endotoxins of in what is ecosystems. to natural diversity relative found and the bacterial community inthe present source water supply might have enhanced microbial system matures. especially of addition enrichmentThe protocol, the carbon inthe pellets, rabbit and habitats declinethen asinteractions within the webfood become more complex as the expected to increase as succession and colonization occur after inundation of newly formed high dates S6). butsample bacterial diversity it declinedacross (Fig. wastime, inOverall, greater the organicof Asthe (rabbit thematter pellets). aged, habitat composition bacterial across changed species of & 2002; Yudina (Koskella Stotzky organisms AcceptedAlphaproteobacteria Article Ordination of samples Ordination controlfrom andlow Previous laboratory studiesPrevious laboratory showed contradictory ofresults the effects of Bti Cyanobacteria -treated microcosms microcosms in-treated than the other be Bacterial (Fig. S7). treatments diversity might Actinobacteria Polaromonas Bti treatments. ) were reduced in microcosms that received a high dose high receiveda in microcosms that were ) reduced Bti , Cytophagales, Cyclobacteriaceae on selected speciesof bacteria, algae and ofThe results fungi. these ( (i.e. three Betaproteobacteria Bti Micrococcus with the diverse microbial more et al et ) and) et al et Bti spp., . 2003; Revina. -treated microcosms demonstrated a . (2003), Koskella &Stotzky (2003), Koskella . (2002) reported Candidatus Nocardia calcaea ( Bacteroidetes Aquiluna ( et al et Bti Bti

-treated microcosms (Figs. communities found in found communities . 2005).. Yudina on six Gram-positive ), and two

Actinobacteria and Bti

of Sphingomonas species anddo

on micro- Bti Bti Steptomyces (Table S3). S3). (Table et al ) .

application when reduced wasapplication inthe it high significantly This article is protected Allrights bycopyright. reserved. Cyanobacteria and this and this taxon was significantly suppressed inthe high treatments than in samples from highdate (1.7±0.3%; sampling samples from last in onthe than treatments treatments n=4) average proportionsgreater per sample inboth low (47±6.9%; n=4) and control (26±8.3%; n=4) correct position ofcorrect taxonthis iscurrently unresolved. (Table S3). The fate of Bti formulations in the water column 1982; Danger (Cole growth algal whereas (e.g. nutrient-rich nitrogen, proliferation phosphorus) are thought media encourage to Carbon-rich 2012). Armbrust resources are considered bacterial generally favourable for absenceor of bacterialpredators 1982; Morin(Cole Hulot, 2001; &Loreau & Parker Amin, dependingon another theresources availability of (e.g. carbon:nutrient and ratios) the presence (Horner-Devine has beenassociated with increased bacterial abundance but reduced overall bacterial diversity belinkedmight treatments to the An inhibited ofgrowth algae. increase inprimary production for nearlyfor a after month Bti 16S specific ninedaysrRNA afterprimers gene application andwe didnot attempt to recover reduction of reduction food resources inthe high disappear disappear waterfrom the column soonafter application (Ohana, Margalit & 1987).Barak The

Accepted the substrate. from have, not therefore, out whether We ruled larval mosquito the mortality Article Cyanobacteria The suppressionThe of some bacterial taxa andincreased diversity occurred that inthe high In our study,In we didnot recover any Proteomonas sequences as were identified et al. et occurred about similarat abundances inalltreatments until day 44after 2003). andalgae Bacteria also are known one interactindirectly with to Bti is classified as application was application dueto the in toxins recycling the water column or et al . 2007). Bti Bti Cryptophytaceae treated microcosms. sequences the water column from bacteria- by using Proteomonas Bti Bti treatment, insignificantly occurring in database, the NCBI and the (OTU #10) by database the SILVA Nearly (Fig.treatment S6). 86% of Bti formulations rapidly formulations Bti Bti

observed at otherobserved at levels trophic web inthe food up tofor 44days. it However, iscurrently where other coexist with where organisms warrantsfilter other investigation. feeding mosquitoes, ofconsequences these biotic andabiotic changes to or natural agricultural ecosystems,aquatic and diversity, bacterial nutrient biomass andincreased reduced algal inthe column. water The unknown what aspect of the This article is protected Allrights bycopyright. reserved. Accepted acknowledges from andthe funding Ian HelenMoore Fund Aquatic Entomology, for B. MullensDr. andDr. Paineprovided constructiveT. on comments D.D. this manuscript. J. andGreer laboratories, Popko D. assistancefor inthe laboratory. Two anonymous reviewers, us in to useequipment allowing their Drs. andB.Federici Miller, thank Paine, for T. We T. Acknowledgments onemonthapproximately by avery high environment. practices cannot be recommended widespread for as adoption it may eventually the impact companion mosquito andprimary productionfauna isabsent, this deviation authorized from Article Farajollahi development resistance 2010;of low Rapleyrepeated (Ritchie, &Benjamin doseapplications (e.g. were rates controlrecommended mosquitoes previously of prolong container-dwelling shown to habitats. of granular-formulation of andsubsequent stirring the substrates Margalit &Barakfiltering (Ohana, 1987). spores withthe the soilor particulate substrates and toxicity was restored after three weeks by of toxicity the of loss the of cause primary Our studyOur demonstrates anda strong significant effect of a high application rateof a Aedes Bti et al spp.) in spp.) experimental studies and costs andthereby application reduce potential the (Vectobac WG (Vectobac and VectobacWG application higherrates thanWDG) the

In ourIn microcosm study, the abundance of . 2013).. Although or the in incontainers risk islimited situations where larval Bti

(VectoBac (VectoBac onG) inaquatic microbial communities microcosm Bti application impacted microbial community impacted microbial application composition, Bti Bti Bti application rate and rate application concomitant changes were was and dueto bindingof thesettling immediate Culex mosquitoes wasmosquitoes reduced for Cole, J.J. J.J. Cole, (1982) betweenInteractions bacteria and inaquatic algae ecosystems. Annual & C.Y. Chen, C.L. Folt, High(2005) plankton densities reduce biomagnification. mercury APHA. (1995) APHA. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., J.G.,Caporaso, J., E.K., F.D., Kuczynski, Costello, J., Stombaugh, Bushman, Bittinger, M.Boisvert, &Boisvert, J. (2000) Effects of This article is protected Allrights bycopyright. reserved. Accepted S.A., Parker, M.S. (2012) Amin, between E.V. & Armbrust, and diatoms Interactions bacteria. References Article are data uploadedasAll experimental information. onlinesupporting http://www.ebi.ac.uk/ena/data/view/ERP005772. PRJEB6267. dataAll sequence were Europeansubmitted Nucleotideto Archive with accessionnumbers AccessibilityData have authors The Riverside. noconflict interest. declared of acknowledges Counciloffunding thefrom AESatUC CanadaResearch (NSERC). W.E.W Engineering Sciences and Natural the from DiscoveryaGrant from funding acknowledges Year J.D.N Fellowship. and Dissertation Society of UCPresident’s America, Entomological Review of EcologyandReview Systematics ScienceandEnvironmental Technology 336. QIIME allows analysis of high-throughput community sequencing data. community high-throughput sequencing QIIME of analysis allows MD Baltimore, Association. Inc., Public Health USA. and Technology organisms: anontarget review laboratory and of experiments. field BiologyReview Molecular Microbiology

Standard Standard Methods ofthe for Examination and American Water Wastewater. , 10 , 517-561. , Bacillus thuringiensis Bacillus 13 , , 291-314., , 76 39 , 667-684., , 115-121.

var. var. israelensis Biocontrol Sciences Biocontrol Nature ontarget and

Methods et al , . (2010) 7 , 335-, Hulot, F.D., MorinHulot, & P.J. M.Loreau, between Interaction (2001) and algae loopin microbial Horner-Devine, M.C., Leibold, M.A., Smith, V.H. & Smith, V.H. M.A., diversity M.C., Bohannan, Bacterial Leibold, B.J.M. (2003) Horner-Devine, Farajollahi, A., Williams, G.M., Condon, G.C., Kesavaraju B., Unlu I. & Gaugler R. (2013) Unlu R.(2013) & B., I. Gaugler Kesavaraju Condon, G.C., G.M., A., Williams, Farajollahi, Hershey, A.E., Lima, A.R., Niemi, G.J., & Regal, R.R. & Effects of(1998) G.J., R.R. Regal, A.R., Niemi, A.E., Lima, Hershey, Edgar, R.C., Haas, B.J., J.C., R. &Knight, Edgar, Quince, C. Clemente, UCHIME(2011) improves Duguma, D. & D. Duguma, Effects (2014) W.E. Walton, of nutrients on andmosquitoes anemergent Duguma, D., Rugman-Jones, P., Kaufman, M.G., Hall, M.W., Neufeld, J.D., Stouthamer, R. Stouthamer, Neufeld, P.,J.D., Rugman-Jones, D., M.G., Kaufman, M.W., Duguma, Hall, D. Duguma, (2013) Influence of nutrients andintegrated mosquito onmanagement tactics This article is protected Allrights bycopyright. reserved. Accepted Article M.,Danger, Oumarou, D. Benest, C., &Lacroix, (2007) G. experimental microcosms. experimental patterns alongpatterns a ofgradient primary productivity. wetlands. israelensis Association, Control and immediate residualfor control of ofAssessment a application direct of two sensitivity andspeed of detection. chimera Ecology macrophyte, Riverside. Riverside. California and University of PhD habitat mosquitoes their microbiomes. Dissertation. two emergent aquatictwo plants emergent of PLoS bioremediation importance. ONE, communitiesWalton, Bacterial associated (2013) with W.E. and nutrient limitation of phytoplankton. of limitation phytoplankton. and nutrient , 39, Ecological Applications (Bti)and methoprene on inMinnesota nontarget macroinvertebrates 1-13. Scheonoplectus

29 , 385-388. 385-388. , Oikos,

maritimus,

, 95 8 , 41-60. , 231-238., Aedes albopictus Functional Ecology, useinfor treatment wetlands. Bacillus thuringiensis israelensis thuringiensis Bacillus Bioinformatics, Ecology Letters Bacteria . Journal of AmericanJournal Mosquito

27 can control stoichiometry cancontrol

Culex 21 , 2194-2200. , 202-210., Bacillus thuringiensis Bacillus thuringiensis , mosquito larvae and mosquito 6 , 613-622., Journal of 8 , e72522. , formulations formulations

Vector & Kaufman, M.G., S. & Chen, Kaufman, E.D. Leaf-associated bacterial (2008) and taxa shifts Walker, fungal J.M., Maknojia, M.G., Bayoh, (2006) S., & E.D. Vulule, M.N., Kaufman, E., Wanja, Walker, This article is protected Allrights bycopyright. reserved. J. &Stotzky, Koskella, G. Larvicidal(2002) toxins from & K.A. A., Roach, Adite, A.T., Jackson, Wine Lagadic, L. &Caquet,Lagadic, T. (2014) L.,Lagadic, M. Roucaute, &Caquet, (2014) T. Lacey, &Merritt, L.A. R.W. (2003) safetyThe of Microbial black bacterial agents for and fly I., Duquesne, S.Kroeger, &Liess, M. biodiversity asan Crustacean (2013) important factor for Accepted &Li, A. Godzik, W. Cd-hit:(2006) a program fast clusteringfor andcomparing large sets of Article in response to larvae of the mosquito, treehole of MedicalEntomology Journal ofImportance algal biomassto andgrowth development of African river. andstructure, yieldsfish inconstructed andnatural wetlands inthe anfloodplain of Ecology 51 invertebratesaquatic in Atlantic coastal wetlands.French pp.151–168. Dordrecht, Publishers. Hajek), Netherlands The Academic Kluwer need and insecticides: methods for risk assessment inaquatic control environments.mosquito larval control. mosquito Microbiology selected activity andbacteria, against fungi, invitro. algae morrisoni protein or nucleotide sequences.protein 3rd vol Elsevier edition Toxicology, 1. Inc., Academic Press, pp. 355–359. , 102-113., , (strain (strain 55 , 673-684. , Canadian Journal and of Fisheries Aquatic Sciences 48 , 262-267. tenebrionis Journal of Ecology Vector Bacillus thuringiensis Bacillus ), and), , 43 Bioinformatics israelensis , 669-676. miller, K.O. (2013) Primary production, food production, Primary web (2013) K.O. miller, Bti sprays donot adversely nontarget affect Environmental impactsEnvironmental of microbial have no or microbiostaticmicrobicidal . . P. In: (Ed.), Encyclopedia Wexler, of Onchlerotatus triseriatus , Bacillus thuringiensis Bacillus 22 , 38 , 1658-1659., H.M.T Hokkanen(eds & A.E. , 390-400., Journal of AppliedJournal Ecology, Canadian Journal of Canadian Journal of Anopheles gambiae , 70 subspp. , 543-553., . Microbial Microbial kurstaki larvae. larvae. , Nguyen, T.T.H., Su, Su, Nguyen, T.T.T.H., &Mulla M.S. (1999) R.W., Dadd, (1992) & E.D. R.H. behavior,Merritt, Feeding natural andnutritional food, Walker, Quast, C., Quast, Pruesse, E., Gerken P., Schweer,J., Yilmaz, Yarza, T., P., Lefebvre, B., Poulin, G. & Paz, L. Red (2010) flag spray:green for adverse effects trophic of This article is protected Allrights bycopyright. reserved. D.A., Han, & S.K., Popko, B. analysis Lanoil, Molecular of (2006) ecological Walton, W.E. E., J. & Claude, Paradis, K. APE: Strimmer, (2004) analyses of phylogenetics and evolution inR A. Phosphorus-chlorophyll (1994) underMazumder, contrasting herbivory relationships and & Hall, Bartram, K., M.W., Masella, J. D. AXIOME:A. Lynch, A.P., M.D., Neufeld, (2013) Accepted Lundström, & Östman, Ö., PerssonJ.O. Vinnersten, Effects (2008) T.Z. of larvae mosquito J. & Margalit, B., Ohana, Z.E. Barak, Fate (1987) of Article Nucleic Acids Research RNA ribosomal databasegene project: improved processing anddata web-based tools. birds.on breeding withenriched organic andmatter treated with of larval mosquitoes. relationships Aquatic Sciences Aquatic stratification: predictionsthermal andpatterns. of exploration diversity. automated microbial Culicidae) mosquitoes. mosquitoes. Culicidae) bacterial communities in planktonic constructed wetlands invaded by language. formulation. Hydrobiologia, removal with conditions simulated field under Bioinformatics Journal ofJournal Ecology Vector Bacillus thuringiensis Bacillus

607 , 51 Journal , 231-235. , 390-400. Journal of MedicalEntomology , , 41 20

of , D590-D596., , 289-290.,

Applied . AppliedandEnvironmental Microbiology .

Annual Reviewof Entomology israelensis Bacteria

Ecology , 2, 191-201. Bacillus thuringiensis Bacillus GigaScience and abundancemosquito in microcosms , Bacillus (

47 Canadian of Fisheries Journal and Bti , 884-889., ) on) protozoan natural communities.

thuringiensis , 43 , , 1153-1163. 2 , 3. , 3. et al. et , subsp. subsp. 37 subsp. (2013) (2013) SILVA The , 349-376. Culex , israelensis 53 israelensis (Diptera: , 828-831.,

Bti

Zaritsky, A. &Zaritsky, K. Khawaled, (1986) Toxicity incarcasses of Ritchie, S.A., Rapley, L.P. & Benjamin, S. &Benjamin, S.A., Rapley, L.P. (2010) Ritchie, Yudina, T.G., Konukhova, AV,Yudina, Revina, Zalunin,T.G., I.A. L.I., L.P., &Chestukhina, Kostina, G.G. This article is protected Allrights bycopyright. reserved. Chen, Y., S.,Xu, M.G., M. Bagdsarian, Kaufman, S., Maknojia, & Bacterial (2008) E.D. Walker, &Likens, R.G. (1991) G.E. Wetzel, Yudina, Chestukhina, G., Zalunin, I.A., L.I., M.A., Kostina, Dronina, T.G., Revina, L.P., Su, &Mulla,Su, T. Microbial M.S. (1999) agents Cary, Release10.0. SAS NC. Inc., JMPSAS (2011) Institute Software: Ecology. R and D.W. labdsv: Analysis(2010) for Multivariate Roberts, package Ordination Accepted Article bioassay. israelensis israelensis (2003) Antibacterial activity Cyt-proteins Antibacterial (2003) Cry-and of from subsp. subsp. of entomocidal Novel(2005) proteins crystals antibacterial from feeding. feeding. community in structure of habitats treehole mosquitoes in mosquitoes microcosms. Bacillus sphaericus http://CRAN.R-project.org/package=labdsv. version 1.4-1. MedicineandHygiene,Tropical control provides residual of israelensis Journal of Association American Control Mosquito Journal of Journal Mosquito Association Control the American -killed -killed . Canadian Journal of Microbiology Aedes aegypti . Canadian of Microbiology Journal suppress eutrophication, enhance and water control quality, Aedes aegypti Environmental Entomology Environmental Limnological analysesLimnological larvae against scavenging larvae: implications to larvae: larvae to against implications scavenging

82 , 1053-1057. Bacillus thuringiensis insmall containers. Bacillus thuringiensis Bacillus Ochlerotatus triseiatus , 49 , 37-44., . Springer-Verlag, Berlin. Bacillus thuringiensis thuringiensis Bacillus , 51 , 28 , 141-148., Bacillus thuringiensis thuringiensis Bacillus , , 761-767. subsp. 24 , 219-227., American JournalAmerican of subsp. Bacillus thuringeinsis , 2 : influences of larval israelensis , 555-558. israelensis var. var.

and subsp et al. ( Bti

.

) samples coloured by sample date B) of PCoAordination samples co ordination, PCoA the in placements sample associated with taxadominant 10 most of the biplot A) Panel treatments. three samples from water column in communities bacterial comparing matrix 6.UniFrac distanceFig. PCoA. based B) (Panel on aBray-Curtis distance matrix. of communities by date bacterial post-treatment sampling ordination (Panel and A) treatments Fig. 5. Nonmetric multidimensional scaling plot of post-treatment samples and larvicideanof treatments mosquito untreated control. rates application twoassigned to in microcosms C) pupae (Panel B), and (Panel instars fourth 4. Fig. larvicide andanuntreated control. in B) (Panel the watercolumn of microcosms toassigned of two rates a application mosquito This article is protected Allrights bycopyright. reserved. L N (mg concentration nitrogen 3.Total andFig. phosphorus total in the column. water mean ±chlorophyll: SE; 1.Phytoplankton biomassFig. in the column. water legends Figure Accepted Article(1.001–10.000 control.untreated The y-axis counts of represents small (0.600–1.000 in assignedSE) microcosms to two rates application larvicide of treatmentsand mosquito an 2 Fig. illustration. facilitate to horizontally and of anuntreated larviciderates treatments on control. Points each date sampling are offset . Culex Sestonic particulates in the columnwater mosquito abundance.

μ m, Panel andlargem, (10.001–224 B), -1 : mean ± SE) (Panel A) and total phosphorus PO (mg ±total and mean A) : SE) (Panel n = 4) inthe column water of assigned microcosms to two application A PCoA ordination basedon aweightedA PCoAordination distance UniFrac . Abundance and of secondfirst instars and A), (Panel third loured by andC) treatment of PCoA ordination . Sestonic particle. Sestonic concentration (mean ± μ Phytoplankton biomass Phytoplankton (total m, Panel C) particles. particles. PanelC) m, Total nitrogen nitrogen Total μ m, Panel A), medium PanelA), medium m, 4 L -1 . NMDS. : mean mean ±: SE) Table 1. Repeated-measures analysisTable of variance of water parameters quality S9 Fig. S8 Fig. S7 Fig. S6 Fig. S5 Fig. S4 Fig. S3 Fig. S2 Fig. S1 Fig. S3 Table This article is protected Allrights bycopyright. reserved. S2 Table S1 Table may information be in supporting found Additional the of onlineversion this article. Supporting Information

Particles (0.6–1.0 µm) µm) (0.6–1.0 Particles

Sources Treatments Time Treatment × Time Accepted mL chlorophyll µg Total Article . phyla bacteria Major in column water found . Dissolved concentration oxygen inwater column level . pH species . Bioavailable nitrogen of . water Colour Temperature . . Principal coordinate. analysis Principal . Alphadiversity comparisons plot scaling multidimensional Nonmetric . . Sequence. abundance (percentage per of sample) 11 bacterial . Sulphate and . oxygenchemical demand concentration L (mg Sampling schedule. of microcosms -1

P F P F 0.0022 <0.0001 <0.0001 0.0022 <0.0001 16.4 (2,9) 90.2 (5,45) 16.4 (10,45) 16.4 (5,45) 90.2 (2,9) 16.4 0.0001 <0.0001 <0.0001 0.0001 <0.0001 92 (2,9) 131.97 5,45) 38.9 38.9 (10,45) 5,45) 131.97 92 (2,9) -1 )

taxa taxa This article is protected Allrights bycopyright. reserved.

1 stated. Astated.

DO (time, 15·30 h) 15·30 (time, DO

DO (time, 08·30 h) 08·30 (time, DO

Total N (mg L pH (time, 15·30 h) pH (time, 15·30

Total P (mg L Particles (10–224 µm) (10–224 µm) Particles pH (time, 08·30 h) pH (time, 08·30 NH NO NO

Particles (1.001–10 µm) (1.001–10 µm) Particles AcceptedF Article values from within are values univari subjects from 4 2 3 -N (mg L -N (mg L -N (mg L P value <0.003 issignificant. Numbers inthe brackets are degrees of freedom. -1 -1 -1 -1 -1 ) ) ) ) ) P F P F P F P P F F P P P P P P F F F F F F 0.0017 <0.0001 <0.0001 0.0017 <0.0001 14.1 (2,9) 11.8 (5,45) 5.4 (10,45) 5.4 (5,45) 11.8 (2,9) 14.1 0.0017 <0.0001 <0.0001 0.0017 <0.0001 14.1 (2,9) 42.9 (6,54) 5.4 (10,45) 5.4 (6,54) 42.9 (2,9) 14.1 000 000 <0.0001 <0.0001 <0.0001 53.3 (2,9) 103.4 6,54) 7.9 (12,54) 7.9 6,54) 103.4 (2,9) 53.3 .66001 0.0127 0.6626 0.0011 0.0183 <0.0001 0.0852 0.0183 <0.0001 0.4 (2,9) 5.0 (5,45) 2.6 (10,45) 2.6 (5,45) 5.0 (2,9)0.4 6.4 (2,9) 52.2 (7,63) 1.7 (14,63) 1.7 (7,63) 52.2 (2,9)6.4 0.6917 <0.0001 0.7959 0.6917 <0.0001 0.0001 <0.0001 0.0205 0.0001 <0.0001 0.0004 <0.0001 <0.0001 0.0004 <0.0001 0.0012 <0.0001 <0.0001 0.0012 <0.0001 0.001 <0.0001 0.0687 0.0005 <0.0001 <0.0001 0.0005 <0.0001 . 29 1. 54) 2.2(10,45) (5,45) 15.9 (2,9)0.4 36.0 (2,9) 63.7 (5,45) 23.3 (10,45) 23.3 (5,45) 63.7 (2,9) 36.0 21.0 (2,9) 32.9 (6,54) 11.8 (12,54) 11.8 (6,54) 32.9 (2,9) 21.0 15.4 (2,9) 17.8 (5,45) 5.2 (10,45) 5.2 (5,45) 17.8 (2,9) 15.4 (10,45 1.9 (5,45) 13.5 (2,9) 16.6 19.8 (2,9) 41.8 (6,54) 4.9 (12,54) 4.9 (6,54) 41.8 (2,9) 19.8 ate unadjusted epsilonvaluesate unlessotherwise

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article

Supplemental Material is available at http://faculty.ucr.edu/~walton/pubs.htm