Table Of Content©EntomologicaFennica.7September2018
Relative abundance and geographical variation
of Culex pipiens and Culex torrentium (Diptera: Culicidae)
in CO -baited traps in Denmark
2
DavidB.Byriel,BirgitKristensen,KirstineKlitgaard&RenéBødker
Byriel,D.B.,Kristensen,B.,Klitgaard,K.&Bødker,R.2018:Relativeabun-
danceandgeographicalvariationofCulexpipiensandCulextorrentium(Dipte-
ra:Culicidae)inCO-baitedtrapsinDenmark.—Entomol.Fennica29:112–
2
118.
EuropeanCulexpipiensandCulextorrentiumaremorphologicalfairlysimilar
mosquitospecieswithpotentiallydifferentvectorcompetencesforpathogenic
viruses. The relative abundance of the two species is therefore important for
quantifyingthepotentialfordiseasetransmissioninDenmark.Mosquitoeswere
sampledfrom74differentsitesinDenmarkwithCO andoctenol-baitedsuction
2
traps.Atotalof285Culexspecimenswereidentifiedtospeciesusingarestriction
enzymeassay.Culexpipienswasthedominatingspecieswith220(77%)speci-
menscaughtat22differentsites,while65(23%)specimenswereidentifiedasC.
torrentiumandonlycaughtat4sites.Theratioofthetwospeciesdifferedsignifi-
cantlybetweensiteswithC.torrentiumdominatinginjustasinglelocation.Both
mosquitospecieswerepredominantlycaughtlateintheDanishmosquitoseason,
frommid-Augustandonwards.
D. B. Byriel, DTU National Veterinary Institute, Bülowsvej 27, 1870 Frede-
riksbergC,Denmark;E-mail:[email protected]
B. Kristensen, DTU National Veterinary Institute, Bülowsvej 27, 1870 Frede-
riksbergC,Denmark
K. Klitgaard, DTU National Veterinary Institute, Bülowsvej 27, 1870 Frede-
riksbergC,Denmark
R.Bødker,DTUNationalVeterinaryInstitute,Bülowsvej27,1870Frederiksberg
C,Denmark
Received8February2017,accepted15September2017
1.Introduction whilstwingmorphologycanbeusedtodiscrimi-
natebetweenfemaleadultswithanaccuracy of
CulexpipiensLinnaeus,1758andCulextorren- morethan90%(Börstleretal.2014).Culexpi-
tiumMartini,1925(Diptera:Culicidae)aremor- piens and C. torrentium are both perennial spe-
phologicallyfairlysimilarspecieswithanover- ciesthathibernateasadults.Thelarvaeofthetwo
lappingrangeinnorthernEurope(Hessonetal. species often occur together, and are found in
2014).Accuratemorphologicalidentificationof semi-permanent waters such as edges of slow
thetwospeciescanonlybedoneonthebasisof runningstreams,invegetationatbordersoflakes,
male hypopygial characters (Service 1968), semi-permanentpools,marshyareas,man-made
ENTOMOL.FENNICAVol.29 (cid:127) CulexpipiensandC.torrentiuminDenmark 113
containers and reservoirs of sewage plants dant. Hessonetal. (2014) foundthatC. torren-
(Beckeretal.2010).Femalesofthetwospecies tium dominates in northern Europe whereas C.
are both anautogenous and ornithophilic and pipiens dominates south of the Alps. Contrary,
rarelybitehumans(Beckeretal.2010). anotherstudyfoundthatlessthan5%oftheCulex
CulexpipiensandC.torrentiumcanmaintain populationbelongedtoC.torrentiumand95%to
transmission of zoonotic viruses between birds. C.pipiensinthenorthernpartofGermanyclose
Culex pipiens can serve as a vector of several toDenmark(Rudolfetal.2013).
arboviruses including Japanese encephalitis, Exotic mosquitoes are spreading in Europe
Sindbis virus, Usutu virus and West Nile virus and recently thecompetentWNVbridgevector
(WNV) (Francy et al. 1989, Farajollahi et al. CulexmodestusFicalbi,1947wasdiscoveredin
2011).Lessisknownaboutthevectorcapability England and Denmark (Medlock et al. 2012,
of C. torrentium, probably due to it mistakenly Bødker et al. 2014). Denmark is located on the
being confused with C. pipiens, its holarctic migrationrouteofmanyPalaearctic-Africanmi-
distribution, and its late recognition (Harbach gratory birdsandantibodiesagainstWNVhave
1985).However,C.torrentiumisaknownvector beenfoundinmigratorybirdsinDenmark,with
ofSindbisvirus(Lundströmetal.1990)andwas 30seropositivesongbirdsamong1,056examined
recently also found susceptible to WNV from 2011 to 2015 and with positive birds de-
(Leggewieetal.2016).Duringthelast40years, tected in confirmatory tests of all five years
bothSindbisvirusandWNVhavebeenisolated (Lohse et al. 2016). In the same period of five
from wild-caught female mosquitoes in Europe years, 3,037 CulexmosquitoesfromtheDanish
(Lundström 1994, Nicolescu 1998, Jöst et al. vectorsurveillanceprogram(www.myggetal.dk)
2010, Hesson et al. 2015a) and outbreaks of were all tested negative for WNV (Lohse et al.
SindbisvirusandWNVinhumanshaveoccurred 2016).
(Lundström 1999, A Sirbu et al. 2011). Culex Herewepresentthefirststudyoftherelative
torrentiumappearstobeamoreefficientvectorof abundancesofadultC.pipiensandC.torrentium
SindbisvirusthanC.pipiens(Lundström1990), and their geographical variation in Denmark.
andrecentlyC.torrentiumwasfoundtobehighly Thisstudyistargetingadultmosquitoescollected
susceptible for dissemination of WNV even at inprivategardens,inpublicparks,andonanimal
lowtemperatures,andwithhigherinfectionrates productionfarms.Thetemporalandspatialabun-
thanC.pipiens(Leggewieetal.2016).Because danceoftheadultvectorsontheselocationsare
of the potential differences in vector compe- assumedtobethemaindriversofthepotentialfor
tences,researchrelatedtotheirdistribution,eco- diseasetransmissionfrombirdstohumansandto
logy and genetics has been conducted in Euro- productionanimalsinDenmark.
peancountries,anddifferentmolecularmethods
have been developed to separate females of C.
torrentiumfromC.pipiens(Hessonetal.2010, 2.Materialsandmethods
Rudolfetal.2013).
InDenmark,theprevalenceofC.pipiensand Mosquitoes were collected in 2012 using Mos-
C. torrentium is relatively unknown, because quito Magnet Independence CO-baited traps
2
they were previously considered one species. withoctenol(MosquitoMagnet,Litiz,PA,USA)
Culex torrentium was first discovered in Den- fromatotalof74sitesofrandomlygeneratedX–
markin1971(Iversen1971).Sincethen,littlere- Ycoordinatesfromthreetypesoflanduse.The
search on the distribution and occurrence of C. land use categories were urban gardens, recre-
torrentium has been done. Hesson et al. (2014) ational land use, and agricultural sites, which
conductedastudyonthelarvalprevalenceofthe were further divided into four categories, pig,
twospeciesthroughoutEuropeandincludedsix poultry, cattle and horse farms. At agricultural
artificial larval sampling sites in Denmark. The sites, the stable building or animal grazing fold
study found both species occurring together in nearest to the random coordinate was selected,
some artificial containers and separately in andthetrapwasplacedeitherfullyoutsideorin
others, but they were on average equally abun- theopeningofastable.Twoofthe74siteswere
114 Byrieletal. (cid:127) ENTOMOL.FENNICAVol.29
Fig.1.MapofthesamplingsitesinDenmark.
Sizeandcolourofthedotsrepresentthedaily
averagenumberofC.pipiens(black)andC.
torrentium(white).Crossesrepresentanab-
senceofCulex.Thetwoflagsrepresentthe
trapschosenassentinels.Onlythecatches
fromweeks33to39(272exx.)areincludedin
thismap(weeks19to32withonly13exx.).
selected as sentinel sites. The southern sentinel using the identification key by Becker et al.
sitewassampleddailyfromweek18to39andthe (2010).
northernoneweeklyfromweek21to39.These Species of the morphological fairly similar
sentinelsiteswerethereforesampledmorethan C.pipiensandC.torrentiumwereseparatedbya
theother72sitesandthesetwothereforerepre- restrictionenzymeassayaccordingtoHessonet
sented29%and26%ofallcollectionnights.Be- al. (2010) with modifications. DNA extraction
causeofalimitednumberoftrapsandtheaimto was performed with Qiagen DNeasy Blood &
samplealargenumberofdifferentareasofDen- Tissue kit (QIAGEN, Hilden, Germany) using
mark,thetrapsweremovedbetweentheremain- the manufacturer’s protocol for insect DNAex-
ingsiteseveryweek(temporarytraps).Thiswas traction.Mosquitolegsandthelowerpartoftho-
done in a total of 72 different temporary sites rax were used for DNA extraction, and a
fromweek21to37,wheretrapswereleftateach microtube pestle was used to homogenize the
site for three consecutive days, constituting the sample,asthisprocedureyieldedthebestresults.
remaning45%ofallcollectionsnights.Eachtem- ThePCRreactionsconsistedof:6µLDNA,5µL
porarycollectionsitewasonlysampledonce,and Buffer (10 x mM (NH)SO), 5 µLMgCl (2.0
4 2 4 2
between three and seven new collection sites mM),theprimerpair0.5µLTL2-N-3014and0.5
weresampledeachweek.Specieswereidentified µLC1-J-2183(Simonetal.1994),0.5µLDNTP
ENTOMOL.FENNICAVol.29 (cid:127) CulexpipiensandC.torrentiuminDenmark 115
Fig.2.BoxandwhiskerplotsofthedailycatchesoftwoCulexspeciesfromthesouthernsentinelCO-baitedcol-
2
lectionsite.Catchesarebasedonsevencollectionnightsperweek.Numberofspecimenswithminimum,maxi-
mum,median,25thand75thpercentilesareshownforeachweek.
(0.1 µM per sample), 0.20 µLTaq (0.02 U per Fromweek19to32atotalof10C.pipiensand3
µL),and32.3µLHO.Thermocyclerconditions C. torrentium were collected from 7 out of 50
2
were:94°Cfor3min,40cyclesof94°Cfor30s, sites. Whilethisrepresents2/3ofthecollection
46°Cfor30s,72°Cfor1min,andfinalexten- period, it only constitutes 4.6% of the collected
sionof72°Cfor7min.Restrictionenzymedi- Culexspecimens.Duringthelast1/3ofthecol-
gestion consisted of: 10 µL PCR reaction mix- lectionperiodfromweek33to39atotalof210
ture,18µLnuclease-freewater,1µ10XBuffer specimensofC.pipienswerecaughtfrom15out
tango, 0.1 µL FspBI (Thermo Scientific, Walt- of 24 different sites, and 62 specimens of C.
ham,MA,USA).Afteragentlemixing,itwasin- torrentiumwerecaughtfrom4outof24different
cubatedat37°Cfor3h,followedbyaninactiva- sites(Fig.1).Thespatialvariationintheratioof
tionofFspBIbyincubationat65°Cfor20min. the two species differed significantly with
Restrictions were run on a 2% agarose E-Gel® Fisher’sexacttestbetweenthecollectionsitesat
(Invitrogen,Carlsbad,CA,USA). week34(p=0.032,df=4),35(p=0.012,df=6),
Statisticalanalysesandboxandwhiskerplots 36(p=0.025,df=3)and37(p<0.0001,df=5),
weremadewithR-Studio(RCoreTeam2013). butnotatweek33(p=1.0,df=1)and38(p=1.0,
Statistical analyses were performed on weekly df=1).Atweek39,mosquitoeswereonlycap-
accumulatedmosquitocatches.Boxandwhisker turedinoneofthecollectionsites.Duringtheen-
plots were made as the daily catches of C. tirecollectionperiod,C.pipienswascaughtat22
torrentiumandC.pipiens(basedonsevennights differentsites,significantlymorethanC.torren-
forthesouthernsentineltrapandthreenightsfor tiumcaughtatjustfoursites(74sitesexamined;
thetemporarytraps)accumulatedforeachweek. Fisher’sexacttest:p=0.0001,df=1).
ThemapwasmadeinArcMap10.3(ESRI). In total, 95% of all C. torrentium were col-
lectedatthesouthernsentinelcollectionsite.This
trapconstituted29%ofallcollectionnightsofthe
3.Results 74traps.Thesouthernsentinelcollectionsitecol-
lected both species, with 61 specimens of C.
Atotalof285femaleCulexspecimensfromthe torrentiumand51ofC.pipiens.Thisallowedfor
entireseasonwereanalyzedusingtherestriction acomparisonofthephenology forthetwospe-
enzyme assay. Of these, 220 specimens were cies,whichshowedfairlyevenseasonaldynam-
identifiedasC.pipiensand65asC.torrentium. icsatthissitewithanabundancepeakfromweek
116 Byrieletal. (cid:127) ENTOMOL.FENNICAVol.29
Fig.3.BoxandwhiskerplotsoftheaveragedailycatchesoftwoCulexspeciesfromthreetosevenweeklytem-
porarytrapsites.Eachweeklycatchconsistof3consecutivecollectionnights.Numberofspecimenswithmini-
mum,maximum,median,25thand75thpercentileareshownforeachweek.
37to39(Fig.2).The210specimensofC.pipiens inclusion of an apparently rare C. torrentium
collected at the temporary sites were primarily hotspotinthesurveyasthesentinelsitewithrela-
foundinweeks34and36(Fig.3)butshoweda tively many collectionnightsincreasedthepro-
large geographical variation in abundance each portionofC.torrentiumto23%.Thetruenational
week. averageofC.torrentiuminDenmarkistherefore
likely to be lower than 23%; indeed, excluding
this sentinel site lowers the prevalence of C.
4.Discussion torrentiumtojust1.4%.Therefore,thetruenatio-
nalprevalencedependsonthefrequencyofsuch
Constituting 77% of all Culex specimens, this hotspotsinDenmark.
studyfoundC.pipienstobethedominantspecies Species ratios may also be strongly influ-
inDenmarkinoctenolandCO-baitedtraps.This encedbysamplingdesignwithC.pipiensbeing
2
isconsistentwiththefindingsfromthenorthern moreprevalentthanC.torrentiuminCO-baited
2
partofGermany,whereC.pipienswasfoundto traps compared to larval sampling and gravid
bethedominantspecies(Rudolfetal.2013),but traps(Weitzeletal.2011,Hessonetal.2015b).
not with C. torrentium being the dominant spe- Thereasonforthisremainsunknown,butithas
ciesinnorthernEuropeassuggestedfromalarger beenspeculatedthatC.torrentium,beingstrictly
European study (Hesson et al. 2014). While C. ornithophilic,spendsitstimearoundbirdnestsin
pipienswascollectedat22differentDanishsites, hightreesandonlyvisitsthegroundtooviposit
C.torrentiumwasonlycollectedatfoursites.At (Petri(cid:1) et al. 1999). Both C. pipiens and C.
the southern sentinel site C. torrentium was the torrentiumwerefoundtooccurinhighnumbers
dominantspecies with 54% during weeks 32 to late in the season. Culex pipiens hibernates as
39,andthissitecollected95%ofallspecimensof adultsfromAugustandslowlydepletesthefatre-
C.torrentium.Thus,whileC.pipiensonaverage servesandnevertakesbloodduringhibernation
dominatednationallyinbothaverageabundance (Sulaiman & Service 1983). While determining
and geographical distribution, one hot spot was Culex under a stereo microscope we found that
dominated by C. torrentium as expected for many of them had developed fat reserves. This
NorthernEurope(Hessonetal.2014).Ingeneral, observationtogetherwithinformationontheirhi-
the ratio of the two species varied significantly bernationbiology,suggeststhatthesemosquitoes
betweenthecollectionsitesandthecoincidental maynothavebeenattractedtoCO-baitedtraps
2
ENTOMOL.FENNICAVol.29 (cid:127) CulexpipiensandC.torrentiuminDenmark 117
insearchofbloodmeals,butratherasaresultof maleCulexpipiensandCulextorrentium.—Journal
exploringdesiredplacesforhibernation.Ifthisis ofVectorEcology39(1):204–212.
Bødker,R.,Klitgård,K.,Byriel,D.B.&Kristensen,B.
thecase,differencesintheirhibernationbiology
2014:EstablishmentoftheWestNilevirusvector,Cu-
and/orpossiblyphenologycouldalsoexplainthe
lex modestus, in a residential area in Denmark. —
low number of C. torrentium caught in CO- JournalofVectorEcology39(2):1–3.
2
baitedtraps. Farajollahi,A.,Fonseca,D.M.,Kramer,L.D.&Kilpa-
ThelateoccurrenceofC.pipiensandC.tor- trick,A.M.2011:“Birdbiting”mosquitoesandhu-
rentiumisaprobleminregardtothesamplingde- man disease: a review of the role of Culex pipiens
complex mosquitoes in epidemiology. — Infection,
sign used in this study, which was intended to
GeneticsandEvolution11(7):1577–1585.
catchabroadvarietyofmosquitoesfromasmany
Francy,D.B.,Jaenson,T.G.,Lundström,J.O.,Schildt,E.
differentsitesinDenmarkaspossible.Therefore, B., Espmark, A., Henriksson, B. & Niklasson, B.
many sites sampled early in the season that did 1989: Ecologic studies of mosquitoes and birds as
notcatchanyCulex,wouldmostlikelyhavebeen hostsofOckelbovirusinSwedenandisolationofIn-
kooandBataivirusesfrommosquitoes.—TheAme-
positiveforoneorbothofthestudiedspecies,if
ricanJournalofTropicalMedicineandHygiene41(3):
the sites had been sampled later in the season.
355–363.
Basedontheresultspresentedhere,wesuggest Harbach,R.E.,Dahl,C.&White,G.B.1985:Culex(Cu-
that studies investigating the ratio of C. pipiens lex)pipiensLinnaeus(Diptera:Culicidae):concepts,
andC.torrentiumshouldbecarriedoutlateinthe typedesignations,anddescription.—Proceedingsof
theEntomologicalSocietyofWashington87:1–24.
seasonandsupplementarytrapsnotbasedonCO
2 Hesson,J.C.,Lundström,J.O.,Halvarsson,P.,Erixon,P.
andoctanolshouldbeincludedinfuturesurveys.
&Collado,A.2010:Asensitiveandreliablerestric-
Inthepresentstudy,weusedarestrictionenzyme
tionenzymeassaytodistinguishbetweenthemosqui-
assay for species identification of 285 Culex toesCulextorrentiumandCulexpipiens.—Medical
specimens.Forfuturehighthroughputscreening andVeterinaryEntomology24(2):142–149.
ofCulexsamples,amoresuitablemethodcould Hesson,J.C.,Rettich,F.,Merdi(cid:1),E.,Vignjevi(cid:1),G.,Öst-
man,Ö.,Schäfer,M.&Scholte,E.J.2014:Thearbo-
betheqPCRassayfordifferentiationbetweenC.
virusvectorCulextorrentiumismoreprevalentthan
pipiensandC.torrentiumdevelopedbyRudolfet
CulexpipiensinnorthernandcentralEurope.—Me-
al.(2013). dicalandveterinaryentomology28(2):179–186.
Hesson,J.C.,Ignell,R.,Hill,S.R.,Östman,Ö.&Lund-
Acknowledgements.Themosquitosurveywasfundedby ström,J.O.2015b:TrappingbiasesofCulextorren-
the Danish Food and Veterinary Administration. Data tiumandCulexpipiensrevealedbycomparisonofcap-
analysiswasfundedbytheEMIDAERA-NETsupported turesinCDCtraps,ovitraps,andgravidtraps.—Jour-
project“VICERiskbasedSurveillanceforVectorBorne nalofVectorEcology40(1),158–163.
Diseases”.TheauthorsaregratefultoDr.JennyHesson Hesson,J.C.,Verner-Carlsson,J.,Larsson,A.,Ahmed,R.,
(UniversityofUppsala)forsendingreferencematerialto Lundkvist,Å.&Lundström,J.O.2015a:Culextor-
get started with the molecular identification, and to the rentiummosquitoroleasmajorenzooticvectordefi-
anonymous reviewers for their valuable comments and nedbyrateofSindbisvirusinfection,Sweden,2009.
suggestionstoimprovethispaper. —EmergingInfectiousDiseases21(5):875–878.
Iversen,T.M.1971:CulextorrentiumMartini(Dipt.Culi-
cidae)nyforDanmark,medenbeskrivelseaflarve-
biotopen. — Entomologiske Meddelelser 39: 235–
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