154 Journal of Vector Ecology June2007

scientificnote

PCR detection of Plasmodium and blood meal identification in a native New Zealand mosquito

B. Massey1,D. M. Gleeson1,D.slaney2,andD. M. Tompkins3

1Ecological Genetics Laboratory, Landcare Research, Private Bag 92170, Auckland, New Zealand 2Institute of Environmental Science and Research, P.O. Box 50 348, Porirua, New Zealand

3Landcare Research, Private Bag 1930, Dunedin, New Zealand

Received 1 December 2006; Accepted 20 March 2007

The application of molecular diagnostic techniquesbasedonDnAamplificationisrevolutionizingthestudyofwildlifediseaseecology (Leroyetal.2005,Lietal.2005).Wherepreviouslywehaveoftenlackedthenecessarytoolstounderstanddiseasedynamicsinnaturalhostcommunities,or have been limited to costly and time-consumingapproaches such as serology, Polymerase Chain Reaction(PCR) techniques (complemented by the sequencing ofamplifiedproductsorthedetectionofmolecularmarkers)now provide a relatively cheap and powerful tool fordocumenting and quantifying parasite transmissionthroughoftencomplexhost/vectorassociations.Theutilityof such knowledge for formulating disease managementstrategies,bothtocontrolcurrentoutbreaksandpre-emptfutureones,isclearlydemonstratedbyrecentworkonWestnile Virus in the United states (Kilpatrick et al. 2006),wheremoleculardiagnosticsprovedinvaluableintheirroleinparasitedetectionandidentification.

Although such techniques are now commonlyemployedinthedetectionofparasitesinvectors(e.g.,schallandsmith2006),theadditionalidentificationofhostbloodmeals(e.g.,Anselletal.2000,PriorandTorr2002,Molaeiet al. 2006) from the same vector individuals has, to thebestofourknowledge,onlybeenemployed in laboratoryexperimentalstudies(e.g.,Fabianetal.2004).However,thebenefitsofapplyingthisapproachtofieldsurveysareclear,withtheprovisionofdirectevidenceforlikelytransmissionpathways, as opposed to inference from multiple datasources.Herewereportsuchanapplication.OurfocuswasontheCulexmosquitovectorsofPlasmodium avianmalarialparasites in new Zealand. These protozoan parasites arebelieved to be introduced to new Zealand (Bennett et al.1993), emerging at higher prevalence in recent decades(Tompkins and Gleeson 2006), and potentially impactingonthenativebirdcommunity inasimilar fashionto thatobservedinHawaii(vanRiperetal.1986).Hence,inalsobeingthefirstapplicationofPCRforamplificationofavianmalariaparasitesfromamosquitovector,theworkreportedhereisanessentialtooldevelopmentfortheunderstandingand management of avian malarial disease dynamics andthe impactonnewZealand’shighlyendangeredendemicavifauna(sekerciogluetal.2004).

Mosquitoes were collected from eight pipe-traps

(nelson1980)andtenblack-paintedrestingboxes(Pletsch1970) placed out for five nights in early December 2005,atafarm10kmfromGisborne,newZealand(E2938126,n6272445). Previous surveys indicated a high prevalence(44%) of Plasmodium infection in birds in this locality(Tompkins and Gleeson 2006). Only one blood-engorgedfemale mosquito was collected during this period from aresting box and identified as the native Culex pervigilans usingthetaxonomickeysofsnell(2005).Collections,usingE67ModelPR101combinationlightandCO2 traps (http://www.entosupplies.com.au), of an average of 146 non-engorgedmosquitoespertrappernight(98%ofwhichwereidentified as Culex sp.), clearly indicated the low successrate of pipe-traps and resting boxes for collecting blood-engorgedfemalesinourstudy.

As previous studies have shown the head and thoraxtopossessinhibitorypropertiesforPCR(Arezetal.2000),onlytheabdomenof thesingleblood-engorgedmosquitowasusedforDnAextraction,performedusingamodifiedphenol–chloroform method (Kikuchi and Fukatsu 2003).The abdomen was ground by plastic pestle in a micro-centrifugetubecontaining120µlofwarm(65oC)extractionbuffer (0.5% sDs, 0.2 M naCl, 25 mM EDTA, and 10mM Tris pH 8.0). Proteinase K (0.02 mg/µl) was addedtothesample,whichwasthenincubatedfor1hat50°C.Following incubation, 120 µl phenol:chloroform:iso-amylalcohol(24:25:1)wasadded,andthesamplecentrifugedfor10minat14,000rpm.Thesamplewasthenprecipitatedin95%ethanolovernightina−20°Cfreezer,centrifugedforanother10minat14,000rpm,andwashedin70%ethanol.All the ethanol was carefully removed, and the air-driedpelletwasre-suspendedin100µlofhydrationbuffer(AquaPuregenomicDnAextractionkit,BIORAD).

ParasitedetectionwithinthemosquitobloodmealwascarriedoutusingPCRprimersL15368(5’AAAAATACCCTT CTA TCC AAA TCT 3’) and H15730 (5’CAT CCAATC CAT AAT AAA GCA T 3’), designed to amplify anapproximately355-base-pairfragmentofthemitochondrialcytochrome b gene of Plasmodium and Haemoproteusparasites as described in Ricklefs et al. (2004). The PCRreactionwascarriedoutina25-µlvolumecontaining10×PCRbufferand20mMMgCl2,2mMeachofdnTPs,0.2mMeachofprimerL15368andH15730and1.5U/reaction

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ofFaststartTaq (Roche), and5µlof extractedDnA.ThePCRwasperformedusingtheGeneAmpPCRsystem9700(AppliedBiosystems,FosterCity,CA,U.s.A.).Amplificationwasperformedusingaprogramof initialdenaturationof4minat95°C, followedby35cyclesof30sat94°C fordenaturation,60sat48°C,extensionfor1min30sat72°C,followedbyafinalextensionfor3minat72°C.

PCR was used to amplify host DnA fromthe mosquito blood meal using primers AvcytbF(5’GACTGTGACAAAATCCCnTTCCA 3’) and AvcytbR(5’GGTCTTCATCTYHGGYTTACAAGAC3’),designedasavian-specificprimerstoamplifya508-bpfragmentofthemitochondrialcytochromebgene(Molaeietal.2006).ThePCRreactionwascarriedoutina25-µlvolumecontaining10×PCRbufferand20mMMgCl2,2mMeachofdnTPs,0.2mMeachofprimerAvcytbFandAvcytbRand1.5U/reactionofFaststartTaq(Roche),and1µlofextractedDnA.Amplificationconsistedofinitialdenaturationfor4minat95oC,followedby36cyclesof30sat94oCfordenaturation,50sat60oC,extensionfor1minat72oC,followedbyafinalextensionfor3minat72°C.

FollowingbothPCRs,10µlofresultingproductswereanalyzed using 2% agarose gel in Tris acetate EDTA andvisualized under UV light following ethidium bromidestaining.ProductswerepurifiedusingtheHighPurePCRProduct Purification Kit (Roche Diagnostics), followingthe procedure outlined by the manufacturer, and directsequencing of purified products was achieved usingBigDyeTM Terminator Ready Reaction Cycle sequencingKit Version 3.1 (Applied Biosystems). Primers used inthe initial PCR reactions were also used for sequencing.sequenceswereanalyzedona3100–AvantgeneticAnalyzer(AppliedBiosystems,Hitachi),editedonsEQUEnCHERTMVersion 4.5 (Gene Codes Corporation), and identifiedusingtheBLAsTsearchalgorithmonthenCBIGenBanknucleotidedatabase.

Parasiteandblood-mealmtDnAwerebothsuccessfullyamplified from the one blood-engorged female Culex pervigilans. The sample was positive for the presence ofthe parasite cytochrome b fragment, with a closest (99%)matchtoPlasmodium relictum.Inaddition,ac. 508-base-pair amplicon fragment was produced from the sampleusingtheavian-specificprimers,withaclosest(99%)matchtotheintroducedblackbirdTurdus merula.AllresultswerereplicatedusingaduplicateroundofPCRandsequencing.Hence,wedemonstratethatthiscompletesetofinformationcanbeobtainedfromthesamevectorindividualssampledfrom the field, and that this approach can be usefullyemployed in field surveys designed to identify potentialparasite transmission pathways. This information, fromjust a single blood-engorged vector, has increased ourunderstandingofavianmalarialdiseasedynamics innewZealand. Whereas previously the role of the native C. pervigilans as a vector for malarial parasites was merelyhypothesized (Holder et al. 1999), we now have directevidenceforsucharolebeinglikely.Inaddition,withtheinferencethatthisnativemosquitohaspickedupmalarialparasites from an introduced bird (no other bird species

DnAmatcheswerefoundinthePCRproductamplified),apotentialtransmissionpathwayformalarialparasitesfromintroducedtonativebirdshasbeenidentified.

Acknowledgments

Our special thanks go to Rachel Paterson and JoseDerraikfortheirassistanceinthefieldandtoPeterHolderfor mosquito taxonomy. This research was funded bythe new Zealand Foundation for Research, science andTechnologycontractC09X0209.

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