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1 /pns /pns /pns Reserch Archive Cittion for published version: M. K. Rust, et l, Susceptibility of Ct Fles (Siphonpter: Pulicide) to Fipronil nd Imidcloprid Using Adult nd Lrvl Biossys, Journl of Medicl Entomology, Vol. 51 (3): , My DOI: Document Version: This is the Published Version. Copyright nd Reuse: 2014 The Author(s). This is n Open Access rticle distributed in ccordnce with the Cretive Commons Attribution Non Commercil ( )license, which permits non-commercil reuse, distribution, nd reproduction in ny medium, provided the originl work is properly cited. For commercil re-use, plese contct journls.permissions@oup.com Enquiries If you believe this document infringes copyright, plese contct the Reserch & Scholrly Communictions Tem t rsc@herts.c.uk

2 VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS Susceptibility of Ct Fles (Siphonpter: Pulicide) to Fipronil nd Imidcloprid Using Adult nd Lrvl Biossys M. K. RUST, 1,2 R. VETTER, 1 I. DENHOLM, 3 B. BLAGBURN, 4 M. S. WILLIAMSON, 5 S. KOPP, 6 G. COLEMAN, 6 J. HOSTETLER, 7 W. DAVIS, 7 N. MENCKE, 8 R. REES, 9 S. FOIT, 8 AND K. TETZNER 8 J. Med. Entomol. 51(3): 638Ð643 (2014); DOI: ABSTRACT The monitoring of the susceptibility of ßes to insecticides hs typiclly been conducted by exposing dults on treted surfces. Other methods such s topicl pplictions of insecticides to dults nd lrvl biossys on treted rering medi hve been developed. Unfortuntely, bseline responses of susceptible strins of ct ße, Ctenocephlides felis (Bouchè), except for imidcloprid, hve not been determined for ll on-niml therpies nd new clsses of chemistry now being used. However, the reltionship between dult nd lrvl biossys of ßes hs not been previously investigted. The dult nd lrvl biossys of Þpronil nd imidcloprid were compred for both Þeld-collected isoltes nd lbortory strins. Adult topicl biossys of Þpronil nd imidcloprid to lbortory strins nd Þeld-collected isoltes demonstrted tht LD 50 s of Þpronil nd imidcloprid rnged from 0.11 to 0.40 nnogrms per ße nd 0.02 to 0.18 nnogrms per ße, respectively. Resistnce rtios for Þpronil nd imidcloprid rnged from 0.11 to Bsed on the lrvl biossy published for imidcloprid, lrvl biossy ws estblished for Þpronil nd reported in this rticle. The rnges of the LC 50 s of Þpronil nd imidcloprid in the lrvl rering medi were 0.07Ð0.16 nd 0.11Ð0.21 ppm, respectively. Resistnce rtios for dult nd lrvl biossys rnged from 0.11 to 2.2 nd 0.58 to 1.75, respectively. Both dult nd lrvl biossys provided similr ptterns for Þpronil nd imidcloprid. Although the dult biossys permitted more precise dosge pplied, the lrvl biossys llowed for testing isoltes without the need to mintin on synthetic or nturl hosts. KEY WORDS Ctenocephlides felis, insecticide resistnce, resistnce monitoring The monitoring of insecticide resistnce in ct ßes, Ctenocephlides felis (Bouchè), hs been typiclly conducted with tests similr to World Helth Orgniztion (WHO) procedure tht exposes dults on treted Þlter pper (WHO 1970). Alterntive methods of determining the susceptibility of ct ßes to insecticides hve been conducted. Moyses (1995) found ssys with topicl pplictions of insecticides to dult ßes to be more sensitive nd less susceptible to interctions between substrtes nd insecticides thn the WHO Þlter pper method. A comprehensive review of insecticide resistnce in ct ßes by Bossrd et l. (1998) summrized the dt collected on crbmte, orgnophosphte, nd pyrethroid insecticides. Further studies reveled multiple cross-resistnce to 1 Deprtment of Entomology, University of Cliforni, Riverside, Riverside, CA Corresponding uthor, e-mil: michel.rust@ucr.edu. 3 Humn nd Environmentl Science Deprtment, University of Hertfordshire, United Kingdom. 4 Deprtment of Pthobiology, College of Veterinry Medicine, Auburn University, Auburn, AL Rothmsted Reserch, Hertfordshire, United Kingdom. 6 School of Veterinry Science, University of Queenslnd, Gtton, QLD, Austrli. 7 Byer Helth Cre, Animl Helth, Shwnee Mission, KS. 8 Byer Animl Helth GMBH, Leverkusen, Germny. 9 Byer Helth Cre, Brisbne, QLD, Austrli. mny of these insecticides (Bossrd et l. 2002). Moyses nd Gfeller (2001) reported the use of topicl pplictions to determine susceptibility of single lbortory strin to 13 different insecticides. To monitor the susceptibility of Þeld-collected isoltes to imidcloprid, lrvl biossy using n insecticide-impregnted food medium ws developed by Rust et l. (2002). This method llows for the worldwide shipment of ße eggs, permits the development of dignostic dose, nd minimizes the need for mintining ße isoltes in the lbortory (Rust et l. 2005). The reltionship between dult nd lrvl biossys of ßes hs not been previously investigted. The objective of this study ws to compre dult nd lrvl biossys of Þpronil nd imidcloprid ginst both lbortory strins nd Þeld-collected isoltes. The utility of ech biossy is discussed. Mterils nd Methods Mintennce of Ct Fles. The isoltes of C. felis used for this work were collected s prt of lrger study involving the monitoring of ct ße susceptibility to imidcloprid (Kopp et l. 2013). Fles were mintined on individul cts ccording to procedure modiþed from Metzger nd Rust (1996). The cts were housed in double cges nd segregted s much /14/0638Ð0643$04.00/ Entomologicl Society of Americ

3 My 2014 RUST ET AL.: CAT FLEA SUSCEPTIBILITY TO FIPRONIL AND IMIDACLOPRID 639 Tble 1. Informtion regrding the field-collected isoltes nd the lbortory strins tested Strin/isolte Loclity Collection dte Ct or dog Previous tretments Auburn Soquel, CA 1985 Ct Unknown Monheim Hnnover, Germny 1992 Ct Unknown UCR Plo Alto, CA 1978 Ct Unknown Clncy Queenslnd, Austrli 14 Aug 2007 Dog Severl unknown Inky Jefferson City, MO 30 Aug 2010 Ct Frontline combo, frontline plus Jeepers Chrleston, SC 3 June 2009 Dog Frontline Kiki Jcksonville, FL 24 My 2006 Ct Unknown Monster Rochester, NY 2 Nov 2009 Ct None Oliver Jcksonville, FL 6 Jn 2009 Ct Advntge multi Sssy Mount Dor, FL 2 Dec 2009 Dog Advntge Tweedle Dee Houston, TX 9 Jn 2009 Ct None s possible to minimize the chnce of cross-contmintion between isoltes. Three different rooms were used to mintin the cts nd ße isoltes. The mintennce of the cts nd rering of ct ßes were conducted under protocols pproved by the Institutionl Animl Cre nd Use Committee of the University of Cliforni, Riverside. Ct ße eggs were collected from trys underneth cts supporting ech Þeld-collected or lbortory isolte. The eggs nd debris were pssed through series of four sieves (10, 16, 20, nd 60 mesh), with the eggs being retined on the 60-mesh screen. The eggs were plced on lrvl ße-rering medium (1 prt nutritive medium [0.15 dried beef blood; AmericÕs Lbortories, # NK SD Hemoglobin Powder, Omh, NE] to 0.75 ground dog chow by weight to 0.1 inctive bkerõs yest [Red Str Bio Products-Nuttrex 55, Milwukee, WI] to three prts 30-mesh silic snd by volume) nd held t 26 1 C nd 80% reltive humidity (RH). Lrve completed development within 11Ð13 d, nd the cocoons nd lrvl medium were pssed through 16-mesh sieve to seprte cocoons. Adults emerged 16Ð18 d fter egg collection. To mintin the isoltes on ech ct, 30 mle nd 30 femle dult ßes were plced on ech ct every 2 wk. Typiclly, three to four genertions were required before sufþcient numbers of dult ßes were present for testing. Dt on Fle Strins nd Isoltes. Tble 1 provides collecting informtion regrding the strins nd isoltes tested. Not ll isoltes were tested with both imidcloprid nd Þpronil. Unfortuntely, there hs never been stndrd susceptible strin of C. felis used consistently for insecticide testing nd resistnce monitoring in different lbortories. Consequently, s much informtion s possible hs been collected bout the lbortory strins nd Þeld-collected isoltes used in this study. The Auburn strin ws originlly estblished t the University of Cliforni, Riverside (UCR) in The Monheim strin ws originlly obtined from the veterinry school in Hnnover, Germny, nd estblished t UCR in 2000.The UCR strin ws originlly obtined from Stnford Reserch Institute in Plo Alto, CA, in It hs since then been mintined on cts t UCR without ny exposure to insecticides. Comprisons were lso mde with dt collected previously for the Dnish Pest Infesttion Lbortory (DPIL) strin. DPIL ws originlly collected in Copenhgen, Denmrk, in 1981, trnsferred to Switzerlnd in 1984, nd rered on n rtiþcil system in 1990 (Moyses 1995, Moyses nd Gfeller 2001). Insecticides. Acetone solutions of technicl grde Þpronil (97.8%, Pestnl D-30926, Sigm-Aldrich, Seetze, Germny) nd imidcloprid (Byer Animl Helth, Monheim, Germny) were pplied to the lrvl-rering medium or directly to the dult ct ße. Adult Topicl Biossys. A 0.1- l droplet of insecticide ws deposited on the cuticle of ech ße using 27-guge needle in glss tuberculin syringe (Becton, Dickinson nd Co, Rutherford, NJ) held in n Isco model M Micropplictor (Instrumenttion Specilties, Sewrd, NE). The tip of the needle ws removed so tht the opening ws level insted of tpered s is common with hypodermic needles. This llowed the solvent to bed up t the end of the syringe. Adult ßes were tested when they were 18Ð20 d from the egg collection dte. Fles were plced into two or three test tubes (195 by 23 mm in dimeter) in groups of 60Ð100 ßes per tube by inverting the rering jr nd pouring them down glss funnel. They were plced in refrigertor t 3 C. Fles were immobile fter 40 min when the Þrst test tube ws removed. The ßes were lightly nesthetized with CO 2 nd poured into plstic petri dish (85 mm in dimeter by 14 mm in depth) sitting on chill plte (#1429, Bioquip, Rncho Dominguez, CA) nd covered with the plstic petri dish lid. Covering the petri dishes ws importnt becuse, despite the chilling, some ßes retined enough mobility to kick nd propel themselves out of the petri dish. The ßes were kept under gentle ßow of CO 2. The chilling tble ws mintined t round 0.4 C. The second nd third test tubes were removed from the refrigertor s needed. We detected no detrimentl effect of dditionl refrigertion nd CO 2 on survivl of the lterdosed ßes. A smll droplet of cetone nd insecticide (or cetone lone in the controls) ws initilly forced out of the syringe to ensure ßow; despite the smll perture of the syringe opening, some evportion occurred inside the needle shft between pplictions. After the test droplet ws forced out nd wiped off with Kimwipe tissue, second droplet ws forced out. An immobilized ße ws removed from the covered petri dish with Þne forceps nd ws brought to the tip of the

4 640 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3 syringe. The ße ws then plced in clen test tube to recover. Ten ßes were dosed nd plced per test tube fter which strip of Þlter pper (Whtmn #1, 18-mm wide by 90-mm long, tpered t one end) ws plced inside the test tube, tpered end down, to give the recovering ßes verticl substrte onto which they could crwl. The test tube ws then covered with smll squre of prþlm to conþne the ßes. The test tubes nd ßes were plced inside n environmentl chmber held t 27 1 C nd 56% RH. Fles were checked 24 h lter for survivl. A ße ws considered ded (or irreversibly ffected) if it ws either immobile on the bottom of the vil or, if moving, could not right itself nd crwl upwrd on the piece of Þlter pper. A series of doses ws dministered from lowest to highest concentrtion with corresponding control btch before nd fter ech insecticide series. All insecticide doses were mde up fresh from stock solution within n hour of use, nd ech group of 10 ßes of the sme dose received insecticide from seprte vil (i.e., three groups of 10 required three vils of prepred insecticide). The stock solutions were refrigerted for storge nd llowed to rech room temperture before the mking of new dilutions for testing. Tests were performed in three simultneous trils such tht the precontrol tretments were pplied Þrst nd in succession, followed by the lowest insecticide doses, then the next highest doses, nd so on. After pplying the highest dose, the syringe (glss plunger, glss body, nd metl needle) ws dismntled nd thoroughly clened with cetone nd llowed to sok 3Ð5 min in cetone to remove insecticide. After this soking, the syringe ws ressembled, rinsed few more times, nd the postcontrol tretments were pplied. Typiclly, the postcontrols showed no discernible increse in mortlity, such tht the Þnl clening process ws deemed successful in removing remnnt insecticide. However, imidcloprid ppered to dhere more tenciously to the syringe thn Þpronil (i.e., higher mortlity in the postcontrol tretments). Subsequently, fter the highest dose of imidcloprid, the syringe received severl dditionl ctive rinsing nd ws soked in the cetone for 10 min. This ws sufþcient to reduce the postcontrol mortlity to norml levels (i.e., 0Ð10%). In ddition, to further decrese potentil contmintion, one syringe ws speciþclly dedicted to use only with imidcloprid. Lrvl Biossys. To determine the ctivity of Þpronil nd imidcloprid ginst lrvl ct ßes, immture ßes were exposed to lrvl-rering medium treted with seril dilutions of ech insecticide. The medium ws plced in the bottom of plstic Srstedt vil (2 grms per vil) nd treted with 2 ml of nine seril dilutions of technicl Þpronil or imidcloprid in cetone (0.003, , 0.001, , , , , , nd %), resulting in concentrtions in the medium from 30 to 0.05 ppm (Rust et l. 2002). The mixture ws stirred, nd the medium ws llowed to dry for t lest 2 h. The treted mixture ws trnsferred to glss petri dish (60 by 15 mm). To permit n ccurte count of the number of eggs tht htched, ct ße eggs were glued to the underneth surfce of the petri dish lids. The petri dish lids were plced over the medi nd plced into incubtors mintined t 26 2 C nd 80% RH. The number of htched eggs ws counted on dy 5. The medium nd cocoons were pssed through 16-mesh screen on dy 12, nd the number of cocoons ws counted. The cocoons were plced in 2.5 cm in dimeter by 4.5 cm in length plstic snp cp vil, nd 5.5-cm-dimeter disk of Whtmn Þlter pper (Whtmn, Hillsboro, OR) ws plced over the top nd secured with snp cp lid. The vils nd cocoons were returned to chmber mintined t 26 2 C nd 80% RH. The number of dults tht emerged ws counted on dy 28. Sttisticl Anlyses. DoseÐmortlity lines for ech strin nd insecticide were determined using Polo softwre (LeOr Softwre, Menlo Prk, CA: Robertson nd Preisler 1992). Dt obtined for DPIL lbortory strin by other uthors, but using comprble topicl ppliction method, were used s bseline for determining resistnce rtios (RR 50 ) for the dults (Moyses nd Gfeller 2001). Becuse lrvl biossy dt were not vilble for DPIL, the Auburn strin ws selected s the bseline reference strin becuse of its high frequency of susceptible lleles for the Rdl (Bss et l. 2004b) nd knockdown resistnce mechnisms (Bss et l. 2004). Results Adult Topicl Biossys. Fipronil ws toxic to dult ct ßes, giving LD 50 vlues ginst the three lbortory strins (Auburn, Monheim, nd UCR) rnging Tble 2. The minimum lethl dose (nnogrms per fle) required to kill dult C. felis by topicl pplictions of fipronil Strin/isolte n Slope SE LD 50 (95% CL) LD 95 (95% CL) Rtio LD 50 Auburn (0.059Ð0.149) 0.42 (0.298Ð0.800) 0.61 Monheim (0.228Ð0.343) 0.75 (0.586Ð1.097) 1.56 UCR (0.229Ð0.330) 0.72 (0.557Ð0.992) 1.56 Clncy (0.043Ð0.213) 1.55 (0.845Ð6.897) 0.7 Jeepers (0.167Ð0.530) 1.56 (0.907Ð9.283) 1.94 Monster (0.229Ð0.514) 1.43 (1.008Ð3.791) 2.22 Oliver (0.170Ð0.330) 1.15 (0.760Ð2.520) 1.39 Sssy (0.154Ð0.382) 2.14 (1.031Ð31.781) 1.56 DPIL b (0.12Ð0.27) 0.69 (0.40Ð2.3) Rtio of isolte or strin/dpil. b DPIL Dnish Pest Infesttion Lbortory strin (Moyses nd Gfeller 2001).

5 My 2014 RUST ET AL.: CAT FLEA SUSCEPTIBILITY TO FIPRONIL AND IMIDACLOPRID 641 Tble 3. The minimum lethl dose (nnogrms per fle) required to kill dult C. felis by topicl pplictions of imidcloprid Strin/isolte n Slope SE LD 50 (95% CL) LD 95 (95% CL) Rtio LD 50 Auburn (0.052Ð0.071) 0.12 (0.105Ð0.178) 0.32 Monheim (0.067Ð0.094) 0.15 (0.118Ð0.262) 0.42 UCR (0.070Ð0.142) 0.55 (0.298Ð2.084) 0.56 Clncy (0.044Ð0.084) 0.20 (0.129Ð1.277) 0.37 Inky (0.042Ð0.060) 0.08 (0.069Ð0.147) 0.26 Kiki (0.071Ð0.291) 0.67 (0.415Ð1.981) 0.95 Monster (0.045Ð0.086) 0.14 (0.106Ð0.553) 0.37 Sssy (0.015Ð0.031) 0.13 (0.078Ð0.312) 0.11 Tweedle Dee (0.034Ð0.064) 0.10 (0.076Ð0.224) 0.26 DPIL b (0.15Ð0.22) 0.70 (0.54Ð1.1) Rtio of isolte or strin/dpil. b DPIL, Dnish Pest Infesttion Lbortory strin (Moyses nd Gfeller 2001). from 0.11 to 0.28 nnogrms per insect (Tble 2). These vlues were close to tht (0.18 nnogrms per insect) obtined previously with DPIL (Moyses nd Gfeller 2001). The LD 50 s of the Þeld-collected isoltes rnged from 0.13 to 0.40 nnogrms per ße, yielding RR 50 rnging from 0.61 to 2.22 compred with DPIL. Imidcloprid proved similrly toxic to Þpronil ginst dult C. felis (Tble 3). The LD 50 s rnged from 0.02 to 0.18, nd the RR 50, compred with the DPIL strin, rnged from 0.11 to Lrvl Biossys. Lrvl medium treted with Þpronil ws extremely toxic with concentrtion of 0.07 ppm, providing 50% kill of lrve (Tble 4). RR 50, compred with Auburn, rnged from 0.58 to Imidcloprid ws similrly toxic ginst lrve, with LC 50 s rnging from 0.09 to 0.21 ppm nd RR 50 rnging from 0.5 to 1.75 (Tble 5). Discussion Spot-on niml therpies hve been widely used to control dult ct ßes since the registrtion of imidcloprid (Advntge) nd Þpronil (Frontline) in the mid-1990s. Despite their widespred use for nerly 15 yr, there hve been no scientiþclly documented reports of reduced susceptibility of ßes to either of these compounds. A long-term progrm to monitor the susceptibility of imidcloprid hs not reveled ny reduced susceptibility of lrve in 1,347 isoltes collected from North Americ, Europe, nd Austrli (Blgburn et l. 2006, Kopp et l. 2013). There hs not been comprble monitoring progrm for other currently used spot-on niml therpies, including Þpronil. Unfortuntely, there re no fully insecticide-susceptible strins of C. felis in culture. Moleculr studies hve shown tht even existing long-stnding lbortory strins such s Auburn, UCR, nd Monheim contin lleles conferring resistnce to pyrethroids nd cyclodiene insecticides (Bss et l. 2004,b). Brunet et l. (2009) reported tht six strins thought to be susceptible to Þpronil were in fct homozygous for the Rdl muttion conferring resistnce to cyclodiene insecticides nd cross-resistnce to Þpronil. Consequently, we included in this study the topicl ppliction dt for dults collected by Moyses nd Gfeller (2001) becuse the DPIL strin hd been cultured since the 1980s nd ws one of the Þrst tested with Þpronil nd imidcloprid. Unfortuntely, this strin is no longer in existence. None of our lbortory strins or Þeld-collected isoltes of C. felis were signiþcntly less susceptible to Þpronil thn results obtined by Moyses nd Gfeller (2001) for DPIL. Interestingly, most of our strins nd isoltes were signiþcntly more susceptible to imidcloprid compred with the DPIL strin. In our study, smll residues of imidcloprid ppered to remin even fter norml wshing nd rinsing of syringes. The dult ßes were lso lightly nesthetized with CO 2 nd then conþned on cold plte before treting them. These procedurl chnges my hve contributed to incresed susceptibility nd lower LD 50 vlues. When treting holometbolous insects such s ßes, the insecticide pplictions my trget speciþc developmentl stges. For exmple, insect growth regultors such s methoprene nd pyriproxyfen typiclly ffect eggs, lrve, or both, wheres most other insecticides trget the dult ßes becuse they re pplied Tble 4. The lethl concentrtion of fipronil in lrvl-rering medium (ppm) required to kill lrvl C. felis Strin/isolte n Slope SE LC 50 (95% CL) LC 95 (95% CL) Rtio LC 50 Auburn (0.076Ð0.142) 0.33 (0.255Ð0.5567) Monheim (0.025Ð0.120) 0.72 (0.423Ð2.063) 0.58 UCR (0.049Ð0.206) 0.69 (0.438Ð1.952) 1.08 Clncy (0.014Ð0.331) 2.11 (0.783Ð49.141) 1.17 Jeepers (0.080Ð0.157) 0.46 (0.332Ð0.771) 1.00 Monster (0.006Ð0.282) 8.19 (2.297Ð ) 0.83 Oliver (0.070Ð0.270) 1.12 (0.654Ð3.013) 1.33 Sssy (0.010Ð0.279) 3.40 (1.291Ð57.499) 0.92 Rtio of strin or isolte/auburn.

6 642 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3 Tble 5. The lethl concentrtion of imidcloprid in the lrvl-rering medium (ppm) required to kill lrvl C. felis Strin/isolte n Slope SE LC 50 (95% CL) LC 95 (95% CL) Rtio LC 50 Auburn (0.094Ð0.158) 0.28 (0.212Ð0.518) Monheim (0.070Ð0.154) 0.34 (0.239Ð0.673) 0.92 UCR (0.039Ð0.214) 0.48 (0.260Ð2.957) 1.00 Clncy (0.059Ð0.120) 0.30 (0.207Ð0.608) 1.33 Inky (0.051Ð0.272) 0.52 (0.280Ð3.228) 1.25 Kiki (0.071Ð0.291) 0.67 (0.415Ð1.981) 1.50 Monster (0.133Ð0.272) 0.62 (0.473Ð0.968) 1.75 Sssy (0.100Ð0.198) 0.58 (0.419Ð0.964) 1.25 Tweedle Dee (0.141Ð0.216) 0.42 (0.341Ð0.560) 1.50 Rtio of strin or isolte/auburn. to the host. The developmentl stge nd ge of the insect cn ffect susceptibility to insecticides. Neonte codling moth lrve, Cydi pommonell (L.), were more sensitive thn Þfth instrs to zinphosmethyl mixed in rering medi. The ge of the dult moths ffected the sensitivity to topicl pplictions (Reuveny nd Cohen 2007). In two strins of tobcco whiteßy, Bemisi tbci Genndius, prepupl nymphs were more susceptible to imidcloprid thn dults, nd resistnce to imidcloprid conferred by overexpression of monooxygense enzyme ws much more potent in dults nd lrve (Nuen et l. 2008). In western ßower thrips, Frnkliniell occidentlis (Pergnde), lrve were more susceptible thn the dults to crinthrin, formetnte, nd methiocrb (Contrers et l. 2010). Higher resistnce levels were found in dults, nd this crried over to lrve t lower levels. Clerly, fctors such s feeding ctivity, developmentl processes, nd the route of exposure ffect the susceptibility of vrious life stges to insecticides. However, our studies suggest lrvl biossy to be the right lbortory test system for both dulticides, Þpronil nd imidcloprid. This is becuse of the mjor limittion of topicl biossys of dults, s the test is tht they require rtiþcil or nturl hosts to generte the lrge numbers of dult ßes. However, if 60 ße eggs re collected, it is possible to test for susceptibility using the lrvl biossy technique, provided dignostic dose hs been developed (Rust et l. 2005). To dte 1,347 ße isoltes from the Þeld hve been tested in lrvl biossy ginst imidcloprid, with no reports of reduced efþccy with spot-on pplictions (Rust et l. 2011, Kopp et l. 2013). In contrst, only few isoltes hve been tested with Þpronil. However, with the reported lrvl biossy, relible study could be implemented for further investigtion. Acknowledgments This reserch ws, in prt, funded by Byer Animl Helth. We thnk Lucio Rodriquez (UCR) for rering nd mintennce of ße isoltes. References Cited Bss, C., I. Schroeder, A. Turberg, L. M. Field, nd M. S. Willimson IdentiÞction of muttions ssocited with pyrethroid resistnce in the pr-type sodium chnnel of the ct ße, Ctenocephlides felis. Insect Biochem. Mol. Biol. 34: 1305Ð1313. Bss, C., I. Schroeder, A. Turberg, L. M. Field, nd M. S. Willimson. 2004b. IdentiÞction of the Rdl muttion in lbortory nd Þeld strins of the ct ße, Ctenocephlides felis (Siphonpter: Pulicide). Pest Mng. Sci. 60: 1157Ð1162. Blgburn, B. L., M. W. Dryden, P. Pyne, M. K. Rust, D. E. Jcobs, R. Bond, M. J. Hutchinson, I. Denholm, H. Melhorn, B. Vughn, et l New methods nd strtegies for monitoring susceptibility of ßes to current ße control products. Vet. Ther. 7: 86Ð98. Bossrd, R. L., N. C. Hinkle, nd M. K. Rust Review of insecticide resistnce in ct ßes (Siphonpter: Pulicide). J. Med. Entomol. 35: 415Ð422. Bossrd, R. L., M. W. Dryden, nd A. B. Broce Insecticide susceptibilities of ct ßes (Siphonpter: Pulicide) from severl regions of the United Sttes. J. Med. Entomol. 39: 742Ð749. Brunet, S., C. L. Meter, M. Murry, M. Soll, nd J. C. Audonnet Rdl polymorphism nd sequence nlysis nd reltion to in vivo Þpronil susceptibility in strins of the ct ße. J. Econ. Entomol. 102: 366Ð372. Contrers, J., P. J. Espinos, V. Quinto, J. Abellán, C. Grávlos, E. Fernández, nd P. Bielz Life-stge vrition in insecticide resistnce of the western ßower thrips (Thysnopter: Thripide). J. Econ. Entomol. 103: 2164Ð2168. Kopp, S., B. Blgburn, G. Colemn, W. Dvis, I. Denholm, C. Field, J. Hostetler, N. Mencke, R. Rees, M. Rust, et l Monitoring Þeld susceptibility to imidcloprid in the ct ße: world-þrst inititive twelve yers on. Prsitol. Res. 112: S47ÐS56. Metzger, M. E., nd M. K. Rust Egg production nd emergence of dult ct ßes (Siphonpter: Pulicide) exposed to different photoperiods. J. Med. Entomol. 33: 651Ð655. Moyses, E. W Mesurement of insecticide resistnce in the dult ct ße, pp. 21Ð34. In R. W. Meol (ed.), The Third Interntionl Symposium on Ectoprsites of Pets, April 2Ð4, College Sttion, TX. Moyses, E. W., nd F. J. Gfeller Topicl ppliction s method for compring the effectiveness of insecticides ginst ct ße (Siphonpter: Pulicide). J. Med. Entomol. 38: 193Ð196. Nuen, R., P. Bielz, I. Denholm, nd K. Gormn Age-speciÞc expression of resistnce to neonicotinoid insecticide in the whiteßy Bemisi tbci. Pest Mng. Sci. 64: 1106Ð1110. Reuveny, H., nd E. Cohen Toxicity of zinphosmethyl to vrious development stges of the codling moth Cydi pomonell (Lepidopter: Tortricide). Pest Mng. Sci. 63: 129Ð133.

7 My 2014 RUST ET AL.: CAT FLEA SUSCEPTIBILITY TO FIPRONIL AND IMIDACLOPRID 643 Robertson, J. L., nd H. K. Preisler Pesticide biossys with rthropods. CRC, Boc Rton, FL. Rust, M. K., M. Wggoner, N. C. Hinkle, N. Mencke, O. Hnsen, M. Vughn, M. W. Dryden, P. Pyne, B. Blgburn, D. E. Jcobs, et l Development of lrvl biossy for susceptibility to imidcloprid. J. Med. Entomol. 39: 671Ð674. Rust, M. K., I. Denholm, M. W. Dryden, P. Pyne, M. Willimson, B. Blgburn, D. E. Jcobs, N. Mencke, I. Schroeder, M. B. Vughn, et l Determining dignostic dose for imidcloprid susceptibility testing of Þeld-collected isoltes of ct ßes (Siphonpter: Pulicide). J. Med. Entomol. 42: 631Ð635. Rust, M. K., I. Denholm, M. W. Dryden, P. Pyne, B. L. Blgburn, D. E. Jcobs, R. Bond, N. Mencke, I. Schroeder, S. Weston, et l Lrge-scle monitoring of imidcloprid susceptibility in the ct ße, Ctenocephlides felis. Med. Vet. Entomol. 25: 1Ð6. (WHO) World Helth Orgniztion Instructions for determining the susceptibility or resistnce of ßes to insecticides, pp. 100Ð105. In Insecticide resistnce nd vector control. Technicl Report Series No WHO, Genev, Switzerlnd. Received 11 December 2013; ccepted 8 Februry 2014.