DTIC ADA534997: Prallethrin-Induced Excitation Increases Contact Between Sprayed Ultralow Volume Droplets and Flying Mosquitoes (Diptera: Culicidae) in a Wind Tunnel

0.73 MB·English

by  Defense Technical Information Center

VECTORCONTROL,PESTMANAGEMENT,RESISTANCE,REPELLENTS
Prallethrin-Induced Excitation Increases Contact Between Sprayed
Ultralow Volume Droplets and Flying Mosquitoes (Diptera: Culicidae)
in a Wind Tunnel
MIRIAMF.COOPERBAND,1,2FRANCESV.GOLDEN,2GARYG.CLARK,2
WILLIAMJANY,3ANDSANDRAA.ALLAN2
J.Med.Entomol.47(6):1099Ð1106(2010);DOI:10.1603/ME10021
ABSTRACT FemaleCulexquinquefasciatusSay(Diptera:Culicidae)mosquitoeswereexposedto
sublethalamountsofprallethrin,sumithrin,andpiperonylbutoxideappliedasultralowvolume(ULV)
dropletsinawindtunnel.Mosquitoeswerevideorecordedbefore,during,andaftertreatment,and
thenumberandsizeofdropletsontheirbodieswerelaterdeterminedusingacompoundmicroscope.
Apositivecorrelationwasfoundbetweenmosquitoesthatspentmoretimeßyingduringthetimeof
spraying and number of droplets on their bodies. Excitation, in the form of increased speed and
durationofßight,wasimmediateinmosquitoesexposedtoprallethrin,whereasexposuretosumithrin
didnotincreasetheirexposuretotheULVdroplets.Thelocationofdropletsonmosquitoes,theeffects
ofdropletvolume,andsubsequentmortalityarediscussed.
KEYWORDS ultralowvolumedroplets,excitation,mosquitobehavior,windtunnel,pyrethroids
Ultralowvolume(ULV)applicationofinsecticidesis showed a 2.5-fold reduction in droplets occurred in
a commonly accepted method for controlling or re- vegetated areas compared with open areas that re-
ducingpopulationsofadultmosquitoesandhasbeen sulted in a corresponding 10-fold reduction in mos-
extensively reviewed by Mount (1998). Such treat- quitomortality.RathburnandDukes(1989)further
mentsarecommonlyappliedusingbackpackortruck- stated that to achieve the same mortality in dense
mountedsprayers.Tobeeffectiveagainstßyingmos- vegetationasinopenareastheapplicationratewould
quitoes, and minimize nontarget effects, federal havetobeincreasedseveralfold(RathburnandDukes
requirements dictate the droplet spectrum of ULV 1989).
applicationsneedstohaveavolumemediandiameter Themovementoftinyobjects,suchasULVdrop-
(VMD)of(cid:1)30(cid:1)m,andatleast90%ofthespraymust lets,throughaßuid(e.g.,air)isdictatedbytheve-
be contained in droplets (cid:1)50 (cid:1)m for ground-based locityandlengthoftheobject,andtheviscosityofthe
applications(USEPA2006).Foreachproduct,droplet ßuiditismovingthrough.Whentinyobjectsapproach
parametersarespeciÞedbythemanufacturerwiththe largersurfacestheyencounterincreaseddragasthey
majorityofdropletsoccurringwithintheabove-men- entertheboundarylayer(thelayerofßuidadjacent
tioned range so that they will remain airborne for totheobjectwhichresistsmovingwithrespecttothat
extendeddurations,therebymaximizingthenumber object) (Loudon et al. 1994). Boundary layers in
ofdropletsthatmayimpingeonmosquitoestoopti- denselyvegetatedareasreduceairmovementandthe
mizemortality. movementofsmallerobjects(suchasULVdroplets)
Previous work by Curtis and Mason (1988) and amongvegetation(Pateletal.1985).Asaresult,mos-
Rathburn and Dukes (1989) revealed that the size, quitoesrestingonvegetation,orotherprotectedsur-
number, and volume of droplets that penetrate faces, are more protected from ULV droplets and
denselyvegetatedareasislowerthaninopenareas, morelikelytoreceiveasublethaldose,whereasair-
resulting in reduced efÞcacy of ULV insecticide ap- bornedropletsaremorelikelytoimpingeuponßying
plications. Indeed, Rathburn and Dukes (1989) mosquitoes.Ifthisassumptioniscorrect,itwouldbe
advantageousduringULVapplicationstousechem-
This article reports the results of research only. Mention of a icalsthatcauseexcitationandßushmosquitoesfrom
proprietaryproductdoesnotconstituteanendorsementorarecom- hidingplacesintotheaerosolapplication.
mendationbytheUSDAforitsuse.
The pyrethroid insecticide sumithrin is used as a
1CurrentafÞliation:OtisLaboratory,CenterforPlantHealthSci-
enceandTechnology,USDAÐAPHIS,1398W.TruckRd.,Buzzards mosquito adulticide in control programs worldwide
Bay,MA0254(e-mail:[email protected]). (Garciaetal.2009).Itisaneffectiveinsecticidebutis
2Center for Medical, Agricultural and Veterinary Entomology, not particularly known for its excitatory properties.
USDAÐARS,1600SW23rdDr.,Gainesville,FL,32608.
Prallethrinisarelativelyvolatilepyrethroidwithre-
3Clarke Mosquito Control, 159 Garden Ave., P.O. Box 72197,
Roselle,IL60172. pellentpropertiesagainstmosquitoes.Moreover,this
Report Documentation Page Form Approved
OMB No. 0704-0188
Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and
maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,
including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington
VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it

does not display a currently valid OMB control number.
1. REPORT DATE 3. DATES COVERED
2010 2. REPORT TYPE 00-00-2010 to 00-00-2010
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Prallethrin-Induced Excitation Increases Contact Between Sprayed
5b. GRANT NUMBER
Ultralow Volume Droplets and Flying Mosquitoes (Diptera: Culicidae) in
a Wind Tunnel 5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Center for Medical, Agricultural and Veterinary REPORT NUMBER
Entomology,USDA-ARS,1600 SW 23rd Dr,Gainesville,FL,32608
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release; distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
Female Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes were exposed to sublethal amounts of
prallethrin, sumithrin, and piperonyl butoxide applied as ultralow volume (ULV) droplets in a wind
tunnel. Mosquitoes were video recorded before, during, and after treatment, and the number and size of
droplets on their bodies were later determined using a compound microscope. A positive correlation was
found between mosquitoes that spent more time ?ing during the time of spraying and number of droplets
on their bodies. Excitation, in the form of increased speed and duration of ?ght, was immediate in
mosquitoes exposed to prallethrin, whereas exposure to sumithrin did not increase their exposure to
theULVdroplets. The location of droplets on mosquitoes, the effects of droplet volume, and subsequent
mortality are discussed.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE Same as 8
unclassified unclassified unclassified Report (SAR)
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
1100 JOURNALOFMEDICALENTOMOLOGY Vol.47,no.6
Fig.1. Schematicdiagramofthesideviewofthewindtunnel.
pyrethroidisthoughttohaveastrongexcitatorycom- openwhiletheplungerwasslowlypumped,allowing
ponent (Matsunaga et al. 1987, Groves et al. 1997, the spray cloud to enter the tunnel in a controlled
Emmrich et al. 2003). These properties make pral- manner.Eachtreatmenthaditsownspraychamberto
lethrinagoodcandidateasaßushingagent.Theob- minimizecontaminationbetweentreatments.
jectiveofthisstudywastotestproofofconceptthat Thewindtunnelwaslinedwithaluminumfoiland
femalemosquitoeswhenexposedtodropletsofpral- plasticwraptopreventcontaminationbetweeneach
lethrinresultedinincreasedexcitationandincreased treatment. Aluminum foil was used to line the ßoor
dropletcontactwithincreasedmortality. and back walls of the wind tunnel, whereas plastic
wrapwasusedalongthefrontandtopwalls.
Ahotwireanemometer(TSI,Inc.,Shoreview,MN)
MaterialsandMethods
wasusedtomeasuretheairßowinthewindtunneland
Insects. Mosquitoes used in this experiment were insidethescreenedcage.Thewindspeedinthetunnel
fromalaboratorycolonyofCx.quinquefasciatusfrom wasadjustedto100cm/stoattainwindspeedwithin
Gainesville,FL,maintainedsince1995byusingmeth- thescreenedcageof(cid:3)50cm/s,highenoughtouni-
ods described by Gerberg et al. (1994) and supple- formlycarrydropletsintothecage(Hoffmannetal.
mentedwithÞeld-collectedspecimensevery1Ð2yr. 2008). Under these conditions, the air in the tunnel
AdultsweremaintainedinBugDorm-1(30-cm3Mega- was completely replaced every 1.2 s. The average
ViewScienceCo.,Ltd.,Taichung,Taiwan)cagesinan temperature and humidity during experiments was
environmental chamber set at 27(cid:2)C, 70% RH, and a 24.8(cid:2)Cand69.2%,respectively,whereasthelightlevel
photoperiodof16:8(L:D)h.Beforebioassays,newly insidethescreenedcagewas106.8lux.
emergedadultswereheldfor4Ð8dtoallowformating Airbrush Calibration. A single action airbrush
and maturation and were provided with 10% sugar (model 350, Badger, Franklin Park, IL) was used to
watersolution.Abattery-operatedaspirator(Haush- produce the ULV droplets. The airbrush was tested
errÕs Machine Works, Toms River, NJ) was used to with DUET Dual-action Adulticide (hereafter re-
transferfemalemosquitoesintoascreenedpapercar- ferredtoasDuet)(ClarkeMosquitoControl,Roselle,
tonwheretheywereheldfor30Ð150minbeforebeing IL)withvariousnozzlesizesandpressuresettingsto
usedintests. determine which conÞguration produced droplets
Wind Tunnel. A push-pull Plexiglas wind tunnel thatconformedbesttothelabelinstructionsforDuet.
with a working section of 30 by 30 by 120 cm was Tests of settings were repeated two times. A plastic
designedforthisexperiment(Fig.1).Anupwindand settlingchamberwasconstructed(36.8cminwidthby
downwinddooronthetopofthewindtunnelallowed 30.5cmindepthby68.6cminheight)withaholefor
foraccess;theentiretopoftheworkingsectionalso insertionoftheairbrushnozzle57.2cmaboveaTe-
could be removed. The bottom of the wind tunnel ßon-coatedglassmicroscopeslide.Theairbrushwas
containedupwindanddownwindslotsforremovable sprayedfor0.5sintothesettlingchamber,anddrop-
attachmentpanelswhichcouldbesolid,haveahole lets were allowed to settle for (cid:3)10 s (Brown et al.
forinsertionofananemometer,oranattachedspray 1990). Droplets were measured under a compound
chamberforthecontrolledreleaseofinsecticideinto microscopeequippedwitha40(cid:4)objective,and10(cid:4)
thewindtunnel. ocularlenswitha10-mmreticulewith0.1-mmdivi-
Thespraychamberconsistedofacylinder(15.6cm sions.Astagemicrometerwasusedtodeterminethat
indiameterby16.2cminheight),withaplunger.The each eyepiece division was 2.5 (cid:1)m. During the be-
tipofanairbrushnozzle(seebelow)wasinsertedinto havioralbioassay,anozzleequippedwithaheavytip
a1.6-cmhole,located1.9cmfromthebottomofthe andÞneneedlewasused,andtheairpressurewasset
cylinder(adhesivetapecoveredtheholewhennotin to40psi.
use). The inner top surface of the spray chamber BioassayProcedures.Onequart(0.95liters)card-
containeda1.3-cmholewithaßapofacetatehanging boardmilkcartonsweremodiÞedforuseinthebio-
fromitwhichactedasavalve.Theacetatevalvesealed assays. The cartons had nylon tulle mesh (with
theholewhiletheairbrushwassprayedbutremained 0.075-mmÞberwidthand1-mm2openings)gluedto
November2010 COOPERBANDETAL.:MOSQUITOEXCITATIONANDULVDROPLETS 1101
bothends,sothataircouldenterthemeshandßow Table1. Componentsofthefourtreatmentsandcontroleval-
through the length of the carton. The side of the uated against Cx. quinquefasciatus in laboratory wind tunnel
bioassaysa
carton facing the video camera was cut out and re-
placed with a sheet of acetate to form a window.
Treatment(%)
Finally,aholewascutoutofthebottomofthecarton Content
A B C D(Duet) E(control)
(thebottomdeÞnedasthesidethatwouldlieonthe
ßoorofthewindtunnel),sothatanindividualmos- Prallethrin (cid:5) 1 1 1 (cid:5)
Sumithrin 5 (cid:5) (cid:5) 5 (cid:5)
quitocouldbeplacedinandremovedfromthecarton PBO 5 (cid:5) 5 5 (cid:5)
easily,andthroughwhichtheanemometercouldbe Inertingredients (cid:6) (cid:6) (cid:6) (cid:6) (cid:6)
inserted via a hole in the wind tunnelÕs replaceable
ßoorpanelformeasuringwindspeedwithinthecar- aDash((cid:5))indicatesthatingredientwasabsent;andplus((cid:6))
ton.Anewcartonwasusedforeachmosquitotoavoid indicatesthattheinertingredientsofDuet,aproprietarymixture
containinghydrotreatedparafÞnicoilandaromatichydrocarbons,
contamination.
makeupthebalanceofthesolution.
Onecarton,containingafemaleCx.quinquefascia-
tusmosquito(4Ð8dold),wasplacedatthedownwind
endofthewindtunnel.Apreliminarytestdetermined the same proprietary inert mixture of hydrotreated
that the test compounds would be sprayed into the parafÞnicoilandaromatichydrocarbonsusedinDuet
spraychamberfor0.5stoexposemosquitoestoa24-h andwereprovidedbyClarkeMosquitoControl.Ten
sublethal dose. A video camera (model WV-BP334, replicateswereconductedforeachoftheÞvetreat-
Panasonic, Sezhou, China) with automatic iris lens ments.Inbetweeneachreplicate,thespraychamber
(CCTV,1/3in.,3Ð8mm,F1.4,Rainbow,Irvine,CA) wasplungedseveraltimestoclearoutalldropletsthat
connected to a digital MPEG recorder (model mayhaveremainedinthechamber.Fivereplicatesof
EMR100, Canopus Co., Kobe, Japan) that was con- thesametreatmentwereconductedusinganewmos-
nected to a laptop computer captured the bioassay quitocartoneachtime,thentheentireliningofthe
videos in MPEG2 format at 30 frames per s. Power- wind tunnel was changed and Þve replicates of the
Director software version 6 (Cyberlink, Taipei, Tai- nexttreatmentwereconducted.Thenumberofrep-
wan)wasusedtopreparedigitalvideosforanalysis licatesandorderoftreatmentswereasfollows:5E,5D,
with Observer XT version 7 (Noldus Information 5C, 5B, 5A, and then this sequence of events was
Technologies, Wageningen, The Netherlands) and repeated. Between treatments the airbrush was dis-
Motusversion8.2(PeakPerformanceTechnologies, mantled and its parts rinsed in acetone Þve times.
Inc.,Centennial,CO)byreducingthemfrom30toÞve Before reusing the airbrush, clean acetone was
framespers,improvingcontrastandbrightness,and sprayed through it Þve times into a separate spray
cuttingÞlestomakeclipsofdesiredvideosegments. chamber.
Each recording had three segments: prespray, Mortality. As soon as a video recording was com-
spray,andpostspray.Theprespraysegmentservedas pleted,thecardboardcartonwasremovedfromthe
acontrolbaselineforeachmosquitoandconsistedof wind tunnel, and the mosquito was transferred by
themosquitobeingrecordedfor5minbeforespray- aspirationintoaclean,screenedpapercartonwithno
ing. A timer was used to time when the prespray foodorwater.Themosquitowasobservedevery30
segmentendedandthespraysegmentcommenced,at minfor8handonceafter24htoquantifymortality.
whichtimeapieceoffoilwaspassedinfrontofthe Knockdown(KD)wasdeÞnedasthemosquitolying
cameralenstosignalonthevideorecordthatspraying ontheßoorofthecarton,eitheronitssideorback,or
was about to begin. At the commencement of the uprightbutnotinastandingposture.Deathwasde-
spray segment, the airbrush trigger was pressed for Þnedasaknockeddownmosquitothatwasunrespon-
0.5 s, as measured using a digital stopwatch. Once siveifblownupongently.AbbottÕsformula(Abbott
sprayingwascomplete,workingquickly,oneperson 1925) was applied to compensate for any mortality
removedtheairbrushtipfromtheholewhileasecond foundincontrols.
personimmediatelycoveredtheholewithapieceof QuantificationofDroplets.Aftermortalitywasre-
tape.Thespraychamberwasthenslowlyplungedso corded, all mosquitoes were stored at (cid:5)12(cid:2)C. Then,
that the droplets of the treatment inside were dis- eachmosquitowasdismemberedandallappendages
placed into the wind tunnel. Each plunge took ap- andpartsweremountedontocleardoublestickytape
proximatelythirtysecondstocomplete,andthespray onamicroscopeslide.Eachwing,leg,antenna,andthe
chamberwasplungedÞvetimes,foratotalplunging head,thorax,andabdomenwasexaminedfordroplets
timeof2.5min.OncetheÞfthplungewascomplete, by using the compound microscope, as described
apieceoffoilwasagainpassedinfrontofthecamera above.Thelocationanddiameterofeachdropletwas
lens to signal on the video record that spraying and recorded, and the total droplet number and volume
plungingwerecomplete.Recordingcontinuedforan werelatercalculatedusingtheformulaforthevolume
additional5-minpostsprayperiod,bringingthetotal ofasphere,becausethespreadfactorisunknownfor
recordingtimeto(cid:3)12.5min. the surface of a mosquito, and droplets seemed to
Five treatments consisting of synergized pral- remainsomewhatspherical(Fig.2).
lethrin, sumithrin, and their mixture (a commercial DataAcquisitionFromVideos.Foreachvideore-
formulationoftheadulticideDuet)wereevaluatedin cording,aframe-by-frameanalysiswasconductedin
thisstudy(Table1).Allcompoundsweredilutedin whichthetwo-dimensional(2-D)positionwasdigi-
1102 JOURNALOFMEDICALENTOMOLOGY Vol.47,no.6
Fig.2. Dropletonthewingofamosquito.Whitescalebar(cid:8)50(cid:1)m.
tallyenteredintoMotus,whichthencalculatedx-and dropletnumber,aswellasvolumeandpercentageof
y-coordinatesforeachframe.Thesecoordinateswere timeßying.
exported to an Excel (Microsoft, Redmond, WA)
spreadsheet that was used to calculate 2-D vectors,
Results
distance traveled, velocity, and percentage of time
spent in motion (percent movement) in each seg- Droplet Spectra. The various airbrush conÞgura-
ment. tionsresultedindifferentdropletspectra(Table2).
Videoswereexaminedasecondtimetoquantifythe TheDuetlabelstatesthat“sprayequipmentmustbe
amount of time mosquitoes spent in each behavior adjusted so the volume median diameter (VMD) is
(e.g., walking, ßying, resting, knockdown) by using between8and30(cid:1)m(DV0.5(cid:1)30)andthat90%of
ObserverXTforeachofthethreevideosegmentsfor thesprayisindroplets(cid:1)50(cid:1)m(DV0.9(cid:1)50(cid:1)m).”
each mosquito. The percentage of time mosquitoes UsingaheavytipandaÞneneedle,andadjustingair
spentßyingdividedbythetotaltimewascalculated pressureto40psiproducedameanVMDof9.2(cid:1)m,
foreachvideosegment. arangeof1.6Ð37.2(cid:1)m,with3%ofdroplets(cid:7)30(cid:1)m.
StatisticalAnalysis.Thestudyusedadouble-blind These parameters were the closest to the label rec-
design.Meansofpercentageofmovement(i.e.,walk- ommendations for Duet and were used throughout
ing and ßying) were not normally distributed, how-
ever,whenmeansineachtimeperiodweresubtracted
from the other time periods, differences were nor- Table2. Airbrushconfigurationsandtheirresultingdroplet
mally distributed and could then be analyzed using spectra
proc GLM and Tukey means separation test (SAS
version9.2,SASInstitute,Cary,NC),withthe(cid:2)re- Tip Needle Air(pprseis)sure (V(cid:1)MmD) R((cid:1)anmg)e %(cid:7)30(cid:1)m
jectionregionof0.025ratherthan0.05,duetousing
each data set in two paired comparisons and subse- Fine Fine 30 21.9 1.6Ð76.0 14.0
Fine Fine 35 21.3 1.6Ð86.8 13.0
quentBonferronicorrectionofthecriticalalpha.In
Fine Fine 40 16.5 1.6Ð51.2 7.3
each time segment, the DunnettÕs test was used to Fine Fine 45 16.7 1.6Ð54.3 5.8
compareeachformulationtothecontroltreatmentfor Fine Fine 50 12.7 1.6Ð45.0 4.5
averagevelocityandpercentageoftimespentßying. Fine Medium 50 14.9 1.6Ð49.6 5.0
Medium Fine 50 13.3 1.6Ð62.0 4.3
For each of the three time segments, PearsonÕs cor-
Medium Medium 30 25.6 3.1Ð76.0 18.8
relation coefÞcients were calculated by plotting the Heavy Fine 30 13.6 1.6Ð46.5 2.0
numberofdropletsagainstthepercentoftimespent Heavy Fine 40 9.2 1.6Ð37.2 3.0
ßyingusingPROCCORR(SASInstitute).Correlation Heavy Fine 50 9.0 1.6Ð32.6 0.5
Heavy Large 30 22.3 1.6Ð103.9 15.0
analyses were also conducted between volume and
November2010 COOPERBANDETAL.:MOSQUITOEXCITATIONANDULVDROPLETS 1103
Fig.3. TotalpercentageofKDanddeadmosquitoesineachtreatmentovertime.A,Sumithrin,PBO,andinertingredients;
B,prallethrinandinertingredients;C,prallethrin,PBO,andinertingredients;D,sumithrin,prallethrin,PBO,andinert
ingredients(Duet);andE,onlyinertingredients(control).
the experiment. To simulate spraying a mosquito quitoesexposedtotreatmentA(sumithrinandPBO)
through a cage, a Teßon-coated slide in the settling thatdied,showedmoremovementaftersprayingwas
chamberwasplacedinsideameshcageandthesame overthanthosethatsurvived.
airbrush settings were used (n (cid:8) 5), resulting in a Mosquitoes in treatments D (Duet) and B (pral-
VMDof4.9,witharangeof1.6Ð15.9(cid:1)m,and0%of lethrin)spentsigniÞcantlymoretimeinßightduring
droplets(cid:7)30(cid:1)m.Sprayingcagedmosquitoesinthe thesprayperiodthanthecontrolmosquitoes,whereas
settlingchamberwiththesesettingsfor0.5s,andthen mosquitoesintreatmentsA(sumithrinplusPBO)and
removingthemafter10sresultedinasublethaldose C(prallethrinplusPBO)didnotspendmoretimein
ofDuetafter24h. ßightduringthesprayperiodcomparedwithcontrol
Mortality. More KD and recovery was seen with mosquitoes(Fig.7).
treatments B and C (prallethrin only, and pralleth- Duringthesprayperiod,velocityofmosquitoeswas
rin plus piperonyl butoxide [PBO], respectively), greaterwhenexposedtoformulationscontainingpral-
whereas the greatest mortality with least recovery lethrin (treatments BÐD) than the control. Mosqui-
fromKDwastreatmentD(Duet)(Fig.3). toes exposed to the sumithrin and PBO formulation
Droplets. The majority of the observed droplets (treatmentA)didnotmovesigniÞcantlyfasterthan
werefoundonthelegsandwingsofmosquitoes(Fig. controlmosquitoesduringthesprayperiod(Fig.8).
4),andmostofthedropletspermosquitomeasured Forallfourtreatmentsmosquitoesmovedfasterthan
2.5Ð5 (cid:1)m in diameter (Fig. 5). No differences were control mosquitoes during the postspray period.
foundbetweentreatmentsinnumberandsizeofdrop- Therewerenodifferencesbeforesprayingbetween
lets. any of the treatments and controls, indicating the
Movement. Mosquitoes exposed to treatment D bioassaywasfreeofcontamination(Figs.7and8).
(Duet) that died, showed more movement during Correlations.TherewasnosigniÞcantcorrelation
spraythanthosethatsurvived(Table3;Fig.6).Mos- between droplet number and droplet volume, nor
between droplet volume and percent of time spent
ßying in the three video segments. There was a sig-
Fig.4. Meannumberofdropletscountedondifferent Fig. 5. Average number of droplets of different sizes
bodypartsofmosquitoes. (micrometers)foundpermosquito.
1104 JOURNALOFMEDICALENTOMOLOGY Vol.47,no.6
Table3. Analysisofvariancetablecomparingdifferencesin
the percentage of time spent moving (sum of both walking and
flying),fromonetimesegmenttoanother,ofmosquitoesthatlived
tothosethatdied
Betweentimesegments Treatmenta df F Pb
Pretospray A 1 4.54 0.0657
B 1 4.29 0.0720
C 1 2.83 0.1308
D 1 17.35 0.0031
E 0
Pretopost A 1 9.16 0.0164
B 1 3.34 0.1050
C 1 0.53 0.4862
D 1 0.46 0.5167
E 0
Spraytopost A 1 0.19 0.6782
Fig.7. Percentageoftimemosquitoesspentßyingbefore
B 1 5.88 0.0415
(pre),during(spray),andafter(post)beingsprayed.As-
C 1 2.87 0.1287
D 1 15.72 0.0041 terisks(*)overbarsindicatethemeanofthattreatmentwas
E 0 signiÞcantlydifferentfromthecontrol(E)(P(cid:8)0.05;Dun-
nettÕstest);n.s.indicatesthatnosigniÞcantdifferenceswere
aA,Sumithrin,PBO,andinertingredients;B,prallethrinandinert detected.TheÞvetreatmentsconsistedofinertingredients
ingredients;C,prallethrin,PBO,andinertingredients;D,sumithrin, andthefollowing:A,Sumithrin,PBO,andinertingredients;
prallethrin, PBO, and inert ingredients (Duet); and E, only inert B,prallethrinandinertingredients;C,prallethrin,PBO,and
ingredients(control). inertingredients;D,sumithrin,prallethrin,PBO,andinert
bPvalueof(cid:1)0.025indicatesthattherewasasigniÞcantdifference
ingredients(Duet);andE,onlyinertingredients(control).
betweenthebehaviorofmosquitoesthatlivedandthosethatdiedin
8haftertheexperiment.
We found no correlation between volume (i.e.,
dose)anddropletnumberonmosquitoesbecausea
niÞcantcorrelation,however,betweenthenumberof singlelargedropletcanhaveamuchgreatervolume
dropletsandthepercentoftimespentßyingduring than several small droplets. Also, if an increase in
thesprayperiod(r(cid:8)0.536,P(cid:1)0.0001)butnotduring locomotion was a result of an increase in dose, we
the prespray or postspray periods (Table 4). There wouldexpecttoseeacorrelationbetweenvolumeand
wasnosigniÞcantrelationshipbetweenwalkingand movement,butsucharelationshipdidnotexist.In-
dropletnumberorvolume,norwasthereasigniÞcant stead,asigniÞcantpositivecorrelationwasfoundbe-
correlation between speed and droplet number for tweenmovementandnumberofdroplets,suggesting
anyofthetimesegments. that mosquitoes picked up additional droplets as a
result of being more active, rather than activity in-
creasingasaresultofincreaseddose.Thissuggestsif
Discussion
more ßight can be induced during the period when
Thisstudyprovidesexperimentalevidencethatfe- droplets are suspended in the air, the greater the
male Cx. quinquefasciatus exposed to ULV droplets likelihoodthatmosquitoeswillcomeintocontactwith
containing 1% prallethrin causes an immediate in- ULV droplets. Separating behavioral data between
crease in ßight activity and speed. Moreover, in- prespray,spray,andpostsprayintervals,wasintegral
creasedtimespentßyingwhiledropletsareairborne indetectingthesepatterns.
correlatespositivelywiththenumberofULVdroplets Although relationships between treatment and
thatimpingeonmosquitoes. ßight and ßight and droplet number were found, a
Fig.6. Amountofchange,fromonetimesegmenttoanother,ofthepercentageoftimemosquitoesspentinmotion(i.e.,
ßyingandwalking).Asterisks(*)representsigniÞcantdifferencesbetweenmosquitoesthatultimatelydiedversusthosethat
livedinaparticulartreatment(P(cid:1)0.025;TukeyÕstest).TheÞvetreatmentsconsistedofinertingredientsandthefollowing:
A,Sumithrin,PBO,andinertingredients;B,prallethrinandinertingredients;C,prallethrin,PBO,andinertingredients;D,
sumithrin,prallethrin,PBO,andinertingredients(Duet);andE,onlyinertingredients(control).
November2010 COOPERBANDETAL.:MOSQUITOEXCITATIONANDULVDROPLETS 1105
quefasciatus; however, excitation caused by pral-
lethrin was more immediate than sumithrin. More
workisneededtodeterminehowtheexcitatoryef-
fectsofprallethrintranslateintomortalityintheÞeld.
Becausedifferentspeciesmaynothavethesamebe-
havioralresponsestoagiveninsecticide(Cooperband
and Allan 2009), similar research on other species
shouldbeconductedtodetermineiftheeffectsob-
servedinourstudyarebroadlyapplicable.
Acknowledgments
Fig.8. DunnettÕstestineachtimesegmentcomparing WethankErinVrzalforassistingwithexperimentsand
themeanvelocity((cid:7)1cm/s)ofmosquitoesexposedtoeach EricPaulsenandLarryPittsforworkonthewindtunnel.This
formulationtothemeanvelocityofcontrolmosquitoes.As- studywasfundedbytheDeployedWar-FighterProtection
terisks(*)overbarsindicateasigniÞcantdifferencefromthe Program.Theresearchwasconductedunderanonfunded
control(P(cid:7)0.05);n.s.indicatesthatnosigniÞcantdiffer- cooperativeagreementwithClarkeMosquitoControl,Inc.
encesweredetected.TheÞvetreatmentsconsistedofinert
ingredientsandthefollowing:A,Sumithrin,PBO,andinert
ingredients; B, prallethrin and inert ingredients; C, pral- ReferencesCited
lethrin,PBO,andinertingredients;D,sumithrin,prallethrin,
PBO,andinertingredients(Duet);E,onlyinertingredients Abbott,W.S. 1925. Amethodofcomputingtheeffective-
(control). nessofaninsecticide.J.Econ.Entomol.18:265Ð267.
Brown,J.R.,T.P.Breaud,andV.Chew. 1990. ULVdroplet
spectra comparative analysis of six droplet collection
directrelationshipbetweentreatmentandnumberof methods.J.Am.Mosq.ControlAssoc.6:713Ð715.
dropletsonmosquitoeswasnotfound.Thisisprobably Cooperband,M.F.,andS.A.Allan. 2009. Effectsofdifferent
duetothecombinationoftherelativelysmallsample pyrethroidsonlandingbehavioroffemaleAedesaegypti,
size,variationsinindividualinsectbehavior,theirlo- Anopheles quadrimaculatus, and Culex quinquefasciatus
cationswithinthecarton,andinteractionswithvari- mosquitoes (Diptera: Culicidae). J. Med. Entomol. 46:
292Ð306.
ations in size, movement and location of droplets
Curtis,G.A.,andJ.Mason. 1988. Evaluationofequipment
withinthecartonthatwerenotmeasured,tracked,or
modiÞcationsanddosageratesofgroundULVapplica-
able to be controlled. Although considerable efforts
tionsofnaledagainstAedestaeniorhynchusinaFlorida
weremadetostandardizethespraycloud,variations USAcitrusgrove.J.Am.Mosq.ControlAssoc.4:345Ð350.
within the droplet clouds due to the formation of Dethier,V.G.,L.B.Browne,andC.N.Smith. 1960. The
turbulenteddiesastheytraveleddownwind,andthe designationofchemicalsintermsoftheresponsesthey
positionofthemosquitowithinthoseeddies,would elicitfrominsects.J.Econ.Entomol.53:134Ð136.
account for some degree of variation between indi- Emmrich,R.R.,J.W.Mikkonen,andT.A.Lajiness,inven-
viduals. tors;S.C.Johnson&Son,Inc.,assignee. 2003June24.
Article for insert control by passive evaporation of an
Sizeandnumberofdropletsfoundonmosquitoes
activeingredient.USPatent6,582,714B1.
were similar to results obtained by Lofgren et al.
Garcia,G.P.,A.E.Flores,I.Fernandez-Salas,K.Saaverdra-
(1973),however,inourstudydropletswereprimarily
Rodriguez,G.Reyes-Solis,S.Lozano-Fuentes,J.G.Bond,
foundonthelegsandwings,andrarelyontheanten- M.Casas-Martinez,J.M.Ramsey,J.Garcia-Rejon,etal.
nae. The fact that few droplets were found on the 2009. Recent rapid rise of a permethrin knock down
head,thorax,andabdomenmaybeduetothemanner resistancealleleinAedesaegyptiinMexico.PLoSNegl.
inwhichspecimenswereexaminedusingacompound Trop.Dis.3:e531:1Ð10.
microscope,providingonlyasilhouetteofthickbody Gerberg,E.J.,D.R.Barnard,andR.A.Ward. 1994. Manual
parts, so that only droplets located precisely on the formosquitorearingandexperimentaltechniques.Amer-
icanMosquitoControlAssociation,Inc.,LakeCharles,
edgewereabletobeobserved.
LA.
OurresultsconÞrmthatthetwopyrethroidstested,
Groves,R.L.,D.A.Dame,C.L.Meek,andM.V.Meisch.
prallethrinandsumithrin,arebothlocomotorstimu-
1997. EfÞcacy of three synthetic pyrethroids against
lantsasdeÞnedbyDethieretal.(1960)forCx.Quin- threemosquitospeciesinArkansasandLouisiana.J.Am.
Mosq.ControlAssoc.13:184Ð188.
Hoffmann,W.C.,B.K.Fritz,M.Farooq,andM.F.Cooper-
Table 4. Pearson correlation coefficients and P values for
band. 2008. Effectsofwindspeedonaerosolspraypen-
percentageoftimespentflyinginthethreevideosegmentscom-
paredwithnumberofdropletsanddropletvolumepermosquito etrationinadultmosquitobioassaycages.J.Am.Mosq.
afterexposure(n(cid:1)48) ControlAssoc.24:419Ð426.
Lofgren,C.S.,D.W.Anthony,andG.A.Mount. 1973. Size
Behavior No.droplets Dropletvol. of aerosol droplets impinging on mosquitoes as deter-
segment Correlation P Correlation P mined with a scanning electron microscope. J. Econ.
Entomol.66:1085Ð1088.
Prespray 0.05183 0.7265 (cid:5)0.09725 0.5108 Loudon,C.,B.A.Best,andM.A.R.Koehl. 1994. Whendoes
Spray 0.53671 (cid:1)0.0001 0.13302 0.3674
motion relative to neighboring surfaces alter the ßow-
Postspray (cid:5)0.10675 0.4702 0.06139 0.6785
througharraysofhairs.J.Exp.Biol.193:233Ð254.
1106 JOURNALOFMEDICALENTOMOLOGY Vol.47,no.6
Matsunaga, T., M. Makita, A. Higo, and I. Nishibe. 1987. Rathburn,C.B.,Jr.,andJ.C.Dukes. 1989. Acomparisonof
Studies on prallethrin, a new synthetic pyrethroid, for themortalityofcagedadultmosquitoestothesize,num-
indoorapplications.I.Theinsecticidalactivitiesofpral- berandvolumeofULVspraydropletssampledinanopen
lethrinisomers.Jpn.J.Sanit.Zool.38:219Ð223. and a vegetated area. J. Am. Mosq. Control Assoc. 5:
Mount, G. A. 1998. A critical review of ultralow-volume 173Ð175.
aerosolsofinsecticideappliedwithvehicle-mountedgen- [USEPA] U.S. Environmental Protection Agency. 2006.
eratorsforadultmosquitocontrols.J.Am.Mosq.Control Reregistrationeligibilitydecisionforpyrethrins.Caseno.
Assoc.14:305Ð334. 2580.U.S.EnvironmentalProtectionAgency,Washing-
Patel,V.C.,W.Rodi,andG.Scheuerer. 1985. Turbulence ton,DC.
modelsfornear-wallandlowReynoldsnumberßows:a
review.Am.Inst.Aero.Astro.Stud.J.23:1308Ð1319. Received29January2010;accepted3August2010.