The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol chloroform isoamyl alcohol DNA extraction

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1 Moleculr Ecology Resources (215) 15, doi: / The room temperture preservtion of filtered environmentl DNA smples nd ssimiltion into phenol chloroform isomyl lcohol DNA extrction MARK A. RENSHAW,* BRETT P. OLDS,* CHRISTOPHER L. JERDE,* MARGARET M. MCVEIGH* nd DAVID M. LODGE* *Deprtment of Biologicl Sciences, University of Notre Dme, 1 Glvin Life Sciences Center, Notre Dme, IN 46556, USA, Unit 117, Environmentl Chnge Inititive, 14 Est Angel Boulevrd, South Bend, IN 46617, USA Astrct Current reserch trgeting filtered mcroil environmentl DNA (edna) often relies upon cold mient tempertures t vrious stges, including the trnsport of wter smples from the field to the lortory nd the storge of wter nd/or filtered smples in the lortory. This poses prcticl limittions for field collections in loctions where refrigertion nd frozen storge is difficult or where smples must e trnsported long distnces for further processing nd screening. This study demonstrtes the successful preservtion of edna t room temperture (2 C) in two lysis uffers, CTAB nd Longmire s, over 2-week period of time. Moreover, the preserved edna smples were semlessly integrted into phenol chloroform isomyl lcohol (PCI) DNA extrction protocol. The successful ppliction of the edna extrction to multiple filter memrne types suggests the methods evluted here my e rodly pplied in future edna reserch. Our results lso suggest tht for mny kinds of studies recently reported on mcroil edna, detection proilities could hve een incresed, nd t lower cost, y utilizing the Longmire s preservtion uffer with PCI DNA extrction. Keywords: edna extrction, edna preservtion, environmentl DNA, Lepomis mcrochirus Received 16 Mrch 214; revision received 9 My 214; ccepted 12 My 214 Introduction The detection of mcroil DNA in environmentl wter smples, herefter referred to s edna, is urgeoning field of reserch often involving the detection of rre species, including invsive species (Dejen et l. 212; Golderg et l. 213; Jerde et l. 213; Tkhr et l. 213; Piggio et l. 214) nd endngered species (Olson et l. 212; Thomsen et l. 212). Three importnt considertions for edna reserch re the cpture of the edna, the preservtion of the edna nd the successful extrction of the edna (Pilliod et l. 213). The filtrtion of wter smples is routine cpture mechnism of edna present in qutic environments s it is sclle to the environment nd llows for the concentrtion of rre edna frgments from lrge volumes of wter. Vrious filter memrne types nd filter pore sizes hve een utilized for the filtrtion of wter smples in edna studies (Minmoto et l. 212; Thomsen et l. 212; Golderg et l. 213; Jerde et l. 213; Piggio et l. 214). Correspondence: Mrk A. Renshw, Fx: ; E-mil: These choices cn impct the efficiency of the edna cpture (Ling & Keeley 213), ut re currently difficult to quntify etween studies s other spects (i.e. edna preservtion nd extrction) re not held constnt (Turner et l. 214). For studies utilizing filtrtion of wter smples, the preservtion of edna is often relint on cold mient tempertures (Mhon et l. 21; Tkhr et l. 212, 213; Jerde et l. 213; Wilcox et l. 213). This is the cse t multiple points of the smple collection process, including the use of ice in the trnsport of wter smples from collection sites to lortories, storge of wter smples in freezers for filtering t lter time points nd freezing of filters following the processing of wter smples (Mhon et l. 21; Thomsen et l. 212; Pilliod et l. 213; Tkhr et l. 213; Wilcox et l. 213; Piggio et l. 214). This relince on cold mient tempertures poses prcticl limittions for field collections in loctions where refrigertion nd frozen storge is difficult (i.e. ckcountry loctions ccessile only y foot) or where smples must e trnsported long distnces for further processing nd screening (i.e. interntionl trvel). The justifiction for such temperture control is to 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd. This is n open ccess rticle under the terms of the Cretive Commons Attriution License, which permits use, distriution nd reproduction in ny medium, provided the originl work is properly cited.

2 FILTERED MACROBIAL EDNA PRESERVATION/EXTRACTION 169 limit DNA degrdtion in edna smples, concern of prmount importnce in edna studies s the trgeted frgments re often degrded t the time of collection nd/or re present in vnishing mounts, due to the rrity of the orgnisms producing the edna. While in situ field filtrtion cn e overcome with portle pumps (Pilliod et l. 213; Wilcox et l. 213), hving method tht preserves filtered edna nd prevents further degrdtion would enefit field scientists tsked with collecting smples under trnsport or temperture limittions. The storge of filters in ethnol is current room temperture preservtion option (Golderg et l. 213; Pilliod et l. 213), ut the ethnol is not itself incorported into the DNA extrction process. A numer of preservtion uffers, in ddition to ethnol, hve shown to e effective with tissue smples t room temperture (Seutin et l. 1991; Longmire et l. 1997; Kilptrick 22; Rhodes et l. 23) nd should serve in similr cpcity with edna cptured on filters. Mny of these room temperture preservtion uffers simultneously fcilitte the lysis of cellulr memrnes, relesing intrcellulr components, such s DNA, into the preservtion uffer. For edna studies, the ssimiltion of room temperture preservtion uffer into DNA extrction protocol would increse the efficiency of the extrction (i.e. include edna tht wshes off the filter prior to extrction or edna relesed through lysis of cellulr memrnes) while voiding hssles relted to the storge of smples t cold mient temperture (Seutin et l. 1991; Kilptrick 22). The extrction of filtered edna is often ccomplished with commercil kit, such s MoBio s PowerWter â DNA Isoltion kit (Olson et l. 212; Jerde et l. 213; Wilcox et l. 213; Piggio et l. 214) or Qigen s DNesy â Blood nd Tissue kit (Minmoto et l. 212; Golderg et l. 213; Pilliod et l. 213; Kelly et l. 214). Phenol chloroform isomyl lcohol DNA extrctions (Smrook et l. 1989), herefter PCI, re populr extrction technique utilized in conjunction with room temperture preservtion uffers for tissue smples in vrious pplictions of conservtion genetics (Miller 26; Smith & Hughes 28; Wirgin et l. 21). Additionlly, this protocol hs een recently employed for the extrction of mcroil edna cptured on polycronte trck-etch filters, nylon filters nd glss fire filters (Brnes et l. 214; Deiner & Altermtt 214; Turner et l. 214). The PCI extrction protocol hs the potentil to drsticlly reduce per smple costs currently ssocited with edna reserch nd ssimilte room temperture preservtion uffer into the extrction process, integrting trnsport nd storge mechnism tht does not rely on cold mient tempertures. As edna projects currently employ vriety of cpture, preservtion nd extrction protocols (Lodge et l. 212; Pilliod et l. 213), some stndrdiztion could help with comprisons of other spects of the reserch tht my e hevily influenced y environmentl conditions, such s the use of vrious filter memrne types nd pore sizes in the cpture of trgeted edna frgments (Brnes et l. 214; Turner et l. 214). The room temperture preservtion would dditionlly llow for ppliction in conditions not suited for cold storge of smples. With these considertions in mind, we conducted set of four experiments to compre (i) preservtion with CTAB nd Longmire s uffers mong fresh smples, nd smples stored for 1 nd 2 weeks t 2, 2 nd 45 C, (ii) the ppliction of the PCI protocol for edna extrction from cellulose nitrte filters, polyethersulfone filters, polycronte trck-etch filters nd glss microfire filters (iii) the PCI DNA extrction protocol with two commercil DNA extrction kits currently fetured in edna reserch nd (iv) different pproches to the PCI DNA extrction protocol. Mterils nd methods Seprte rooms were used for fish husndry, pre-pcr lortory work nd post-pcr lortory work. The 7-gllon mesocosm, with pproximtely 1 juvenile luegill (Lepomis mcrochirus), ws monitored throughout the experiment following institutionl niml cre nd use protocols. For ll the four experiments, 25 ml wter smples were collected nd filtered immeditely through single filter; unless otherwise specified, DNA extrctions immeditely followed the terminus of smple filtrtion for ech experiment. Smples were filtered with 47-mm mgnetic filter funnels (Pll), nd ech filter funnel ws completely immersed in 1% lech for minimum of 1 min nd thoroughly rinsed with DI wter prior to ny susequent filtrtion. A single smple from ech experimentl tretment ws filtered efore filtering second smple from ech experimentl tretment nd then third nd so on, rndomly spreding the filtering effort cross ll experimentl tretments nd minimizing the potentil impct of contmintion on ny one experimentl tretment. Unless otherwise specified, the filters were plced in 2-mL microcentrifuge tues nd completely immersed in 9 ll of CTAB uffer (1.4 M NCl, 2% (w/v) cetyltrimethyl mmonium romide, 1 mm Tris, 2 mm EDTA nd.25 mm polyvinylpyrrolidone; Coyne et l. 25). The CTAB uffer ws chosen s it hs een used successfully for ongoing reserch (Brnes et l. 214; Turner et l. 214). Unless otherwise specified, DNA extrctions followed modified PCI extrction nd ethnol precipittion (Smrook et l. 1989): (i) the 2-mL microcentrifuge tues (filters nd preservtion uffer) were incuted in 65 C wter th for 1 min; (ii) 9 ll of PCI (one phse, 25:24:1, Amresco) ws dded to ech 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd.

3 17 M. A. RENSHAW ET AL. tue, nd smples were vortexed for 5 seconds. The ddition of chloroform disintegrtes polycronte trck-etch (herefter PCTE ) nd polyethersulfone (herefter PES ) filter memrnes (Strk et l. 1998; Turner et l. 214), nd s such, extr cre ws given to mix the liquid lyers for the cellulose nitrte (herefter CN ) nd glss microfire (herefter GMF ) filter memrne types, which remined intct; (iii) tues were centrifuged t 15 g for 5 min, nd 7 ll of the queous lyer ws trnsferred to fresh set of 2-mL microcentrifuge tues; (iv) 7 ll of chloroform isomyl lcohol, herefter CI (24:1, Amresco), ws dded to ech tue nd smples were vortexed for 5 seconds; (v) tues were centrifuged t 15 g for 5 min, nd 5 ll of the queous lyer ws trnsferred to fresh set of 2 ml tues; (vi) 1.25 ml of 1% ice-cold ethnol nd 2 ll of 5 M NCl were dded to ech tue, nd smples were precipitted t 2 C overnight; (vii) the precipitte ws pelleted y centrifugtion t 15 g for 1 min, nd the liquid ws decnted; (viii) pellets were dried in vcuufuge t 45 C for 15 min, followed y ir drying until no visile liquid remined; nd finlly, (ix) pellets were rehydrted with 2 ll of 19 TE Buffer, low EDTA (USB). All DNA extrctions were ssyed with qpcr Tq- Mn â primers nd proe trgeting 1-p frgment of the luegill cytochrome gene (Tkhr et l. 213) in the following 2 ll mixes: 1 ll of TqMn â Environmentl Mster Mix 2. (Life Technologies), 1.8 ll of ech primer (1 lm stock concentrtion),.25 ll of the Tq- Mn â proe (1 lm stock concentrtion), 4 ll of edna extrct nd 2.15 ll of sterile wter. The cycling prmeters were s follows: single step t 5 C for 2 min, single step t 95 C for 1 min nd 55 cycles t 95 C for 15 seconds followed y 6 C for 1 min. To quntify the DNA copy numer in ech edna extrct, stndrd ws creted (s follows) nd included on ech qpcr plte long with the edna extrcts. A DNA frgment ws synthesized y Integrted DNA Technologies sed on the sequence from GenBnk Accession no. JN strting t loction nd ending t loction The 5-p frgment included the 1-p region of the luegill cytochrome gene trgeted y the ssy flnked y n dditionl 2-p on either side. The copy numer of the synthesized stndrd ws determined y multiplying the numer of moles y Avogdro s numer. A seril dilution of the stndrd ws run on ech qpcr plte nd provided regression line from which the unknown copy numers of the edna extrcts could e estimted. All qpcr ssys were run on Mstercycler ep relplex rel-time PCR system (Eppendorf) nd nlysed with the ccompnying relplex 2.2 softwre. Two negtive controls were included on ech qpcr plte, oth contining the forementioned 2 ll mix except for dditionl sterile wter in plce of edna extrct. Filter preservtion experiment A pilot experiment evluted the use of four storge uffers: 2% DMSO uffer (2% DMSO,.25M EDTA, sturted with NCl; Seutin et l. 1991), RNAlter (Qigen #7616), CTAB uffer nd Longmire s uffer (.1 M Tris,.1 M EDTA, 1 mm NCl,.5% (w/v) SDS; Longmire et l. 1997). The 2% DMSO nd RNAlter were not comptile with the PCI DNA extrction protocol s they oth yielded sustntil precipitte tht ppered to completely inhiit qpcr mplifiction. The removl of the 2% DMSO nd RNAlter immeditely prior to the DNA extrction nd replcement with CTAB reduced the resulting precipitte, ut the qpcr ssys gin filed to mplify. As such, only the CTAB uffer nd Longmire s uffer were evluted in the filter preservtion experiment. For ech experimentl tretment, five 25 ml wter smples were ech immeditely filtered through single PCTE filter (1.2 lm; Millipore). Filters were plced in 2-mL microcentrifuge tues nd completely immersed in 9 ll of either CTAB uffer (35 smples totl) or Longmire s uffer (25 smples totl). For ech storge uffer, set of five filters ws extrcted immeditely, herefter fresh. For the CTAB, n dditionl 1 filters were kept t ech of the three temperture regimes: 2, 2 nd 45 C. For ech temperture regime, 5 of the filters were extrcted fter 1-week storge period, while the remining 5 were extrcted fter 2-week storge period. For the Longmire s uffer, the sme protocol ws pplied for only two of the temperture regimes, 2 nd 45 C. The 2 C regime ws only used with the CTAB storge uffer s this hs een used with success in the pst (Brnes et l. 214; Turner et l. 214) nd served s enchmrk ginst which the other temperture regimes nd storge uffer could e compred. All DNA extrcts from given storge uffer were ssyed once simultneously on the sme qpcr plte with seril dilution of the stndrd for the quntifiction of DNA copy numer. Ech plte ws then run second time to produce two qpcr replictes for ech smple. Filter memrne type experiment The fesiility of DNA extrction from different filter memrne types with the PCI extrction protocol ws evluted for four different filter memrne types:.8 lm CN (Whtmn),.8 lm PES (Pll), 1. lm PCTE (GE Osmonics) nd 1.5 lm GMF (Whtmn). For ech filter memrne type, ten 25 ml wter smples were ech filtered through single filter nd completely immersed with 9 ll of CTAB in 2-mL microcentrifuge tue. DNA ws extrcted nd ethnol precipitted with the previously descried PCI protocol. All of the 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd.

4 FILTERED MACROBIAL EDNA PRESERVATION/EXTRACTION 171 smples (in duplicte) were run simultneously on single qpcr plte with seril dilution of the stndrd for the quntifiction of DNA copy numer. duplicte) were run simultneously on single qpcr plte with seril dilution of the stndrd for the quntifiction of DNA copy numer. PCI kit comprison experiment Two historiclly populr comintions of mcroil edna filtrtion memrne types with commercil kit edna extrctions hve utilized.45-lm CN filters with Qigen s DNesy â Blood nd Tissue kit (Ahmed et l. 21, 213; Golderg et l. 211, 213; Pilliod et l. 213, 214) nd 1.5-lm GMF filters with MoBio s Power- Wter â DNA Isoltion kit (Olson et l. 212; Jerde et l. 213; Wilcox et l. 213; Piggio et l. 214). As such, comprisons etween the PCI DNA extrction protocol nd these two commercil DNA extrction kits focused on the sme pirings of filters nd extrction kits. A totl of forty 25 ml wter smples were collected, nd twenty of them were ech filtered immeditely through single.45-lm CN filter (Spectrum). Ten of the CN filters were completely immersed in 9 ll of CTAB, incuted in 65 C wter th for 1 h nd then put through the PCI extrction nd ethnol precipittion protocol s outlined previously. DNA ws extrcted from the remining 1 CN filters following Qigen s recommendtions for the DNesy â Blood nd Tissue kit, with some modifictions. Filters were completely immersed in 567 ll uffer ATL nd 63 ll Proteinse-K (rther thn the recommended 18 nd 2 ll, respectively) nd incuted in 65 C wter th for 1 h. Following the incution time, 63 ll uffer AL nd 63 ll 1% ethnol were dded to the 2-mL tue, insted of the recommended 2 ll of ech solution. A totl of three centrifugtion itertions were required to lod the entire contents of the 2-mL tue, minus the CN filter, onto the silic memrne, s compred to the single centrifugtion step normlly required. The reminder of the protocol followed the mnufcturer s recommendtions. The other 2 smples were ech filtered immeditely through single 1.5 lm GMF filter (Whtmn). Ten of the GMF filters were completely immersed in 9 ll of CTAB, incuted in 65 C wter th for 1 h nd then put through the PCI extrction nd ethnol precipittion protocol s outlined previously. DNA ws extrcted from the remining 1 GMF filters following MoBio s recommendtions for the PowerWter â DNA Isoltion kit, with the exception tht the ed-eting step ws performed until the filters ppered to e completely liquefied rther thn the 5 min recommended mximum. DNA extrctions from oth kits were eluted from their respective spin columns with the ddition of 2 ll of19 TE Buffer, low EDTA (USB), rther thn the recommended Buffer AE (Qigen) nd Solution PW6 (MoBio). All of the smples (in DNA extrction experiment Vritions on the PCI extrction protocol were evluted, utilizing totl of forty 25 ml wter smples ech filtered immeditely through single 1.2-lm PCTE filter (Millipore). Twenty of the filters were plced in 2-mL tues with 9 ll of CTAB, nd DNA ws extrcted with the PCI protocol. An lternte precipittion protocol ws evluted using isopropyl lcohol with 1 of the smples in conjunction with the previously mentioned ethnol precipittion on the remining 1 smples. The isopropyl lcohol precipittion ws conducted s follows: (i) 5 ll of ice-cold isopropyl lcohol nd 25 ll of 5M NCl were dded to the 5 ll recovered from the queous lyer (step 5 in the PCI protocol), nd tues were precipitted t 2 C overnight; (ii) the precipitte ws pelleted y centrifugtion t 15 g for 1 min, nd the liquid ws decnted; (iii) 15 ll of room temperture 7% ethnol ws dded to ech tue; (iv) tues were centrifuged t 15 g for 5 min, nd the liquid ws decnted; (v) 15 ll of room temperture 7% ethnol ws dded to ech tue second time; (vi) tues were centrifuged t 15 g for 5 min, nd the liquid ws decnted; (vii) pellets were dried in vcuufuge t 45 C for 15 min, followed y ir drying until no visile liquid remined; nd finlly, (viii) pellets were rehydrted with 2 ll of19 TE Buffer, low EDTA (USB). An lternte DNA extrction protocol eliminted the use of phenol (step 2 in the PCI protocol). The remining 2 filters were completely immersed with 7 ll of CTAB in 2-mL microcentrifuge tues nd incuted in 65 C wter th for 1 min. These smples were then extrcted strting with the ddition of 7 ll of CI (step 4 in the PCI protocol). The ethnol nd isopropnol precipittions were gin oth evluted, ech on 1 of the smples. All of the smples (in duplicte) were run simultneously on single qpcr plte with seril dilution of the stndrd for the quntifiction of DNA copy numer. Sttisticl nlyses ANOVA sttisticl tests were conducted individully for ech of the four experiments to test for differences etween men DNA copy numers. A two-sided t-test ws used to test differences in the verge mount of DNA recovered from fresh CTAB nd Longmire s extrctions within the filter preservtion experiment. Technicl replictes were verged for the nlysis, residuls from the ANOVAs nd t-test were checked for normlity 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd.

5 172 M. A. RENSHAW ET AL. using norml Q Q plots, nd pirwise comprisons in the ANOVA were performed using Tukey s post hoc test. All sttistics nd plots were conducted nd creted in Mthemtic (Wolfrm Reserch, Inc., Version 9..1., Chmpign, IL 213). All tests conformed to the normlity ssumptions unless otherwise indicted. Results For ll of the qpcr pltes run for this study, the qpcr stndrd curve slope rnged from to 3.498, the y-intercept rnged from to 39.26, the efficiency rnged from.93 to.99, nd the R 2 vlues rnged from.994 to 1.. All of the negtive controls filed to mplify throughout the entire experiment. Filter preservtion experiment All replictes mplified nd were incorported into the sttisticl nlyses for ll twelve of the experimentl tretments (Tle 1). For the CTAB preservtion uffer, reltive to fresh smples, Tukey s post hoc comprisons of the ANOVA results reveled significntly higher DNA copy numer in smples stored t ll the three tempertures ( 2, 2 nd 45 C) following the 2-week time intervl (Fig. 1 c). For the Longmire s preservtion uffer, the sme result ws oserved for the 45 C temperture (Fig. 1e), ut no significnt difference in copy numer existed etween fresh smples nd those stored t 2 C (Fig. 1d). A two-sided t-test of the fresh extrctions reveled significntly higher yield in DNA copy numer for the Longmire s preservtion uffer s compred to the CTAB preservtion uffer (P-vlue <.1; Fig. 1f). Filter memrne type experiment For three of the four filter memrne types (CN, PES nd PCTE), ll 1 smples mplified; one of the 1 smples for the GMF memrne type filed to mplify, nd s such, only nine of the smples were used in the sttisticl nlyses (Tle 1). Tukey s post hoc comprisons of the ANOVA results reveled tht the.8-lm CN nd.8-lm PES filters did not differ significntly nd oth yielded significntly more copies of DNA thn the 1.-lm PCTE nd 1.5-lm GMF filters; the 1.-lm PCTE filters yielded significntly more copies of DNA thn the 1.5-lm GMF filters (Fig. 2). PCI kit comprison experiment All 1 of the smples mplified nd were incorported into the sttisticl nlyses for ech of the four experimentl tretments (Tle 1). Tukey s post hoc comprisons of the ANOVA results reveled tht the CN filter with PCI Tle 1 Outline of ll four experiments, with the tretments evluted in ech experiment (Tretment) nd the numer of smples nlysed per experimentl tretment (N) Experiment Tretment N Filter preservtion CTAB; fresh 5 CTAB; 2 C; 1 week 5 CTAB; 2 C; 2 weeks 5 CTAB; 2 C; 1 week 5 CTAB; 2 C; 2 weeks 5 CTAB; 45 C; 1 week 5 CTAB; 45 C; 2 weeks 5 Longmire s; fresh 5 Longmire s; 2 C; 1 week 5 Longmire s; 2 C; 2 weeks 5 Longmire s; 45 C; 1 week 5 Longmire s; 45 C; 2 weeks 5 Filter memrne type.8 lm; cellulose nitrte (CN) 1.8 lm; polyethersulfone (PES) 1 1 lm; polycronte 1 trck-etch (PCTE) 1.5 lm; glss microfire 9 (GMF) PCI kit comprison.45 lm; cellulose nitrte 1 (CN); PCI.45 lm; cellulose nitrte 1 (CN); Qigen 1.5 lm; glss microfire 1 (GMF); PCI 1.5 lm; glss microfire 1 (GMF); MoBio DNA extrction PCI strt; ethnol precipittion 1 PCI strt; isopropnol precipittion 1 CI strt; ethnol precipittion 1 CI strt; isopropnol precipittion 1 extrction yielded significntly more copies of DNA thn the other three experimentl tretments; the GMF filter with the MoBio extrction yielded significntly more copies of DNA thn oth the GMF filter with PCI extrction nd the CN filter with Qigen extrction, which were not significntly different from one nother (Fig. 3). DNA extrction experiment All 1 of the smples mplified nd were incorported into the sttisticl nlyses for ech of the four experimentl tretments (Tle 1). Tukey s post hoc comprisons of the ANOVA results reveled no sttisticlly significnt differences mong the four experimentl tretments (Fig. 4). Discussion Both preservtion uffers, CTAB nd Longmire s, successfully preserved filtered edna t 2 C over 2-week period of time. The DNA copy numer incresed from 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd.

6 FILTERED MACROBIAL EDNA PRESERVATION/EXTRACTION 173 DNA copy numer (copies/4 μl) DNA copy numer (copies/4 μl) ANOVA: P <.1 DNA copy numer (copies/4 μl) DNA copy numer (copies/4 μl) ANOVA: P < () () (c) Fresh 1 week 2 weeks Fresh 1 week 2 weeks Fresh 1 week 2 weeks ANOVA: P =.16 ANOVA: P < (d) (e) (f) Fresh 1 week 2 weeks Fresh 1 week 2 weeks DNA copy numer (copies/4 μl) DNA copy numer (copies/4 μl) ANOVA: P <.1 c 2 2 sided T-Test: P <.1 35 CTAB Longmire s Fig. 1 Box nd whisker plots for the filter preservtion experiment. The top nd ottom of the whiskers represent the mximum nd minimum vlues, the top nd ottom of the oxes represent the 75% nd 25% qurtiles, nd the lines inside the oxes represent the medin vlues. Significnce in pirwise comprisons of tretments is noted y letters, nd c where different letters represent sttisticlly significnt differences. Two preservtion uffers, CTAB nd Longmire s, were evluted over 2-week intervl of time. () CTAB with 2 C storge, () CTAB with 2 C storge, (c) CTAB with 45 C storge, (d) Longmire s with 2 C storge, (e) Longmire s with 45 C storge nd (f) comprison etween CTAB nd Longmire s for fresh extrctions. DNA copy numer (copies/4 μl) 2 1 ANOVA: P <.1 CN PES PCTE GMF Fig. 2 Box nd whisker plots for the filter memrne type experiment. The top nd ottom of the whiskers represent the mximum nd minimum vlues, the top nd ottom of the oxes represent the 75% nd 25% qurtiles, nd the lines inside the oxes represent the medin vlues. Significnce in pirwise comprisons of tretments is noted y letters, nd c where different letters represent sttisticlly significnt differences etween experimentl tretments. The four tretments were.8- lm cellulose nitrte filters (CN),.8-lm polyethersulfone filters (PES), 1.-lm polycronte trck-etch filters (PCTE), nd 1.5- lm glss microfire filters (GMF). the initil time point to the 2-week time point, ssuming the results from the fresh tretment indicte the initil copy numer in ll corresponding experimentl tretments. This trend ws oserved for the filters in the 2 nd 45 C regimes s well. One possile explntion for the oserved trend is incresed cell lysis efficiency for oth uffers with n increse in time. This explntion indictes it is possily eneficil to leve the filters in the lysis uffer for n extended period of time prior to the c DNA copy numer (copies/4 μl) 15 1 ANOVA: P <.1 GMF, Moio GMF, PCI CN, Qigen CN, PCI Fig. 3 Box nd whisker plots for the PCI-kit comprison experiment. The top nd ottom of the whiskers represent the mximum nd minimum vlues, the top nd ottom of the oxes represent the 75% nd 25% qurtiles, nd the lines inside the oxes represent the medin vlues. Significnce in pirwise comprisons of tretments is noted y letters, nd c where different letters represent sttisticlly significnt differences etween experimentl tretments. The four tretments were 1.5- lm glss microfire filters (GMF) with MoBio extrction, 1.5-lm glss microfire filters (GMF) with PCI extrction,.45-lm cellulose nitrte filters (CN) with Qigen extrction nd.45-lm cellulose nitrte filters (CN) with PCI extrction. DNA extrction. Alterntively, longer incution times in the 65 C wter th could significntly increse the yield of extrcted DNA s het fcilittes cell lysis nd the relese of intrcellulr components, such s DNA (Lu et l. 25). In comprison etween the two uffers, the Longmire s uffer produced significntly higher DNA copy numer thn the CTAB uffer for the fresh c 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd.

7 174 M. A. RENSHAW ET AL. DNA copy numer (copies/4 μl) 7 2 PCI ethnol PCI isopropnol CI ethnol ANOVA: P =.242 CI isopropnol Fig. 4 Box nd whisker plots for the DNA extrction experiment. The top nd ottom of the whiskers represent the mximum nd minimum vlues, the top nd ottom of the oxes represent the 75% nd 25% qurtiles, nd the lines inside the oxes represent the medin vlues. There ws no sttisticl significnce in pirwise comprisons etween the four experimentl tretments: PCI extrction with ethnol precipittion, PCI extrction with isopropnol precipittion, CI extrction with ethnol precipittion nd CI extrction with isopropnol precipittion. extrctions. In the 45 C regime, the Longmire s uffer demonstrted copy numer increse from the initil time point to the 1-week time point to the 2-week time point; in comprison, significnt reduction in copy numer ws oserved in the CTAB uffer from the 1-week time point to the 2-week time point, possily representtive of DNA degrdtion in the elevted storge temperture. These results suggest tht while oth preservtion uffers re dequte for the room temperture preservtion of filtered mcroil edna, the Longmire s uffer outperformed the CTAB uffer. The relince of the PCI extrction pproch on chemicls tht re hrmful to humns is disdvntge ssocited with the hndling nd proper disposl of the phenol nd chloroform. When hndling these chemicls, certin mesures should e considered to reduce the risk of skin contct (i.e. lortory cot nd gloves), eye contct (i.e. sfety glsses) or inhltion (i.e. fume hood). It is lso recommended to equip lortories with eyewsh sttions nd sfety showers in the event of exposure. In ddition to sfety mesures for hndling, the hzrdous nture of oth chemicls requires them to e disposed of in mnner tht conforms to the sfety regultions s governed y the orgniztion with which ny lortory is ffilited. One vile lterntive for reserchers tht re interested in using either the CTAB or Longmire s preservtion uffers without relying on phenol nd chloroform for the DNA extrction is the integrtion of the room temperture preservtion uffer with Qigen s DNesy â Blood nd Tissue kit (Herth et l. 21; Miller et l. 21). On the other hnd, the PCI pproch provides severl dvntges. First, the PCI extrction protocol potentilly yields significntly more copies of trgeted edna frgments, s demonstrted y the use of.45-lm CN filters with oth Qigen s DNesy â Blood nd Tissue kit nd the PCI protocol. It is noteworthy tht numer of recommended modifictions of the DNesy â protocol exist for incresing the detection proility of trgeted species, including the ddition of Qigen s QIAshredder nd incresing incution times (Golderg et l. 211). These modifictions could potentilly provide more comprle results to those chieved in the current study with the CTAB preservtion uffer nd PCI DNA extrction. In the comprison etween MoBio s PowerWter â DNA Isoltion Kit nd the PCI protocol with the 1.5-lm GMF filters, however, the commercil kit yielded significntly more edna thn the PCI protocol. The MoBio kit, s implemented in the current study, shreds prt the GMF filters through ed-eting step, exposing the internlly cptured mteril (s ccomplished y GMF filters through tortuous pth) nd possily incresing lysis efficiency. These results suggest tht the ddition of ed-eting step should e considered for the use of the GMF filters with the preservtion uffer nd PCI extrction. Second, the PCI pproch is sustntilly cheper thn the commercil kits. Potentil per smple costs for edna extrction with MoBio s PowerWter â DNA Isoltion kit re over $8 (USD), for Qigen s DNesy â Blood nd Tissue kit re over $2 (USD) nd for the PCI extrction protocol re <$.2 (USD). These vlues re rough estimtes s numer of fctors cn impct their clcultion, ut the reltive difference in costs etween methods is well represented. And finlly, the regents used in the process re well understood, in the pulic domin, nd esily tested nd dpted to individul reserch projects. The lck of sttisticl significnce etween the pproches employed in the current study (DNA extrction with nd without the use of phenol nd DNA precipittion with oth ethnol nd isopropnol) suggests tht reserchers hve flexiility with the extrction protocols while producing comprle results. The PCI extrction protocol ws successful for ll four evluted filter memrne types: cellulose nitrte (CN), polyethersulfone (PES), polycronte trck-etch (PCTE) nd glss microfire (GMF). The discrepncy etween memrne types in the mount of DNA extrcted cn, in prt, e ttriuted to differences in pore size. A consistent reltionship etween pore size nd mount of DNA present in the extrct hs een previously demonstrted (Ling & Keeley 213), with mesurle decrese in DNA recovery with n increse in pore size (see Fig. S1, Supporting informtion for independent confirmtion of this reltionship). The one comprison etween memrne types with n equl stted pore size,.8 lm CN nd.8 lm PES, produced sttisticlly comprle copies of trgeted edna. This reltionship differs from 214 The Authors. Moleculr Ecology Resources Pulished y John Wiley & Sons Ltd.

8 FILTERED MACROBIAL EDNA PRESERVATION/EXTRACTION 175 previous oservtions y Ling & Keeley (213), where mixed cellulose ester filters recovered etween 2.6 nd 3.9 times more copies of plsmid DNA thn PES filters. In ddition to potentil differences etween the type of cellulose filter utilized (mixed cellulose esters comprises oth cellulose nitrte nd cellulose cette), the current study my lso highlight the complexity of n edna smple. Only singulr contriutor to edna yields (free-floting, extrcellulr DNA) ws evluted y Ling & Keeley (213). The increse in reltive cpture efficiency of the PES filters in the current study my highlight differences etween filter types in the potentil cpture efficiencies of other sources of edna, such s those tht re intrcellulr nd/or extrcellulr ut ound to other mteril in the environment (Siud & Gude 1996; Converse et l. 212; Thomsen et l. 212), nd more closely reflect mcroil edna cpture potentils in qutic systems where free-floting DNA is minority contriutor to totl edna (Turner et l. 214). The urgeoning field of mcroil edna reserch hs lredy produced noteworthy results in oth freshwter nd mrine environments, including the detection of fish (Thomsen et l. 212,; Jerde et l. 213; Kelly et l. 214), mphiins (Dejen et l. 212; Olson et l. 212; Thomsen et l. 212; Pilliod et l. 213, 214), reptiles (Piggio et l. 214), insect lrve nd crustcens (Thomsen et l. 212; Deiner & Altermtt 214), mmmls (Foote et l. 212; Thomsen et l. 212) nd molluscs (Golderg et l. 213; Deiner & Altermtt 214). And s the field continues to grow, the ppliction of next-genertion sequencing pltforms opens venues into iodiversity estimtes on lrge scle (Thomsen et l. 212) nd further integrtion into the conservtion nd mngement of nturl resources (Lodge et l. 212). The min gol of this study ws to evlute edna preservtion nd extrction for filtered mcroil environmentl DNA, with the potentil rod ppliction for studies in vriety of qutic environments. The Longmire s preservtion uffer provides reserchers with room temperture storge uffer tht dequtely hndles elevted tempertures (up to 45 C in the current study), nd the ssimiltion of the Longmire s preservtion uffer into PCI DNA extrction protocol hs the potentil to simultneously reduce per smple costs nd increse the recovery of trgeted edna frgments. The resulting increse in detection proilities for rre species will enefit future edna reserch for oth species-specific ssys nd lrge-scle iodiversity estimtes. Acknowledgements We would like to thnk K. Uy nd C. Gntz for ssistnce in the lortory; C. Gntz for ssistnce in the ongoing cre of the luegill; L. de Souz nd J. Godwin for stimulting ides for this study. We would lso like to thnk three nonymous reviewers who provided excellent insight nd feedck from which the mnuscript enefitted gretly. EPA Gret Lkes Restortion Inititive, Gret Lkes Fisheries Trust nd DoD SERDP provided funding. This is puliction of the Notre Dme Environmentl Chnge Inititive. References Ahmed W, Goonetilleke A, Grdner T (21) Humn nd ovine denoviruses for the detection of source-specific fecl pollution in costl wters in Austrli. Wter Reserch, 44, Ahmed W, Yusuf R, Hsn I et l. (213) Fecl indictors nd cteril pthogens in ottled wter from Dhk, Bngldesh. Brzilin Journl of Microiology, 44, Brnes MA, Turner CR, Jerde CL, Renshw MA, Chdderton WL, Lodge DM (214) Environmentl conditions influence edna persistence in qutic systems. 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