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Efficacy of Spinosad Against Acaricide-Resistant and -SusceptibleRhipicephalus (Boophilus) microplus and Acaricide-SusceptibleAmblyomma americanum and Dermacentor variabilisAuthor(s): Robert J. Miller, W. Hunter White, Ronald B. Davey, John E. George, andAdalberto Perez De LeonSource: Journal of Medical Entomology, 48(2):358-365. 2011.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/ME08222URL: http://www.bioone.org/doi/full/10.1603/ME08222

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VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Efficacy of Spinosad Against Acaricide-Resistant and -SusceptibleRhipicephalus (Boophilus) microplus and Acaricide-Susceptible

Amblyomma americanum and Dermacentor variabilis

ROBERT J. MILLER,1,2 W. HUNTER WHITE,3 RONALD B. DAVEY,1 JOHN E. GEORGE,4

AND ADALBERTO PEREZ DE LEON4

J. Med. Entomol. 48(2): 358Ð365 (2011); DOI: 10.1603/ME08222

ABSTRACT Various acaricide-resistant strains of Rhipicephalus (Boophilus) microplus, representa-tive of the major resistance mechanisms found in Mexico and Brazil, were exposed to spinosad usingthe Food and Agriculture Organization of the United Nations larval packet test and adult immersiontest (AIT). Larvae of all strains tested were found to be susceptible to spinosad. Conversely, spinosaddid not show toxic activity toward engorged females used in the AIT. In vitro tests against larvae,nymphs, and adults of acaricide-susceptible Amblyomma americanum and Dermacentor variabilisrevealed differences in activity that were dependent on tick species and parasitic life stage. Spinosadseemed to be a viable alternative to current acaricides available for tick control in the species tested.The larval packet test should be used for future monitoring of resistance, as the AIT did not provideuseful information with this chemical. The potential beneÞt for the use of spinosad in integrated pestmanagement or eradication programs is discussed.

KEY WORDS Rhipicephalus (Boophilus) microplus, Amblyomma americanum, Dermacentor varia-bilis, resistance

Rhipicephalus (Boophilus) microplus (Canestrini) in-fest cattle throughout the tropical and subtropicalregions of the world, causing billions of dollars inlosses directly by feeding on the blood of the host andindirectly through the transmission of the causativeagents of babesiosis and anaplasmosis (FAO 2004).Generally, cattle ticks are controlled through the useof acaricides and, because of many environmental andeconomic factors, the use of acaricides is especiallyhigh in tropical regions of the world. As a result, a highlevel of resistance to acaricides in R. microplus hasoccurred in every tropical region of the world where

chemical control has been used to control ticks oncattle (FAO 2004).

Initially, acaricide resistance in cattle ticks was di-rected to only a single class of acaricides. For example,the Þrst case of resistance to the organophosphate(OP), coumaphos, in Mexico was described in 1982(Aguirre et al. 1986) and came after heavy use of thisacaricide in a national tick eradication program in theprior decade. In reaction to this discovery, pyrethoid(PY) acaricides were approved for use in Mexico in1985 (Aguirre et al. 1986). By 1993 the Þrst report ofpyrethroid resistance was made in Mexico and 14 yrlater within the United States (Miller et al. 2007, San-tamaria and Fragoso 1994). After the discovery ofpyrethroid resistance in Mexico, many ranchers reliedon amitraz (AM), a formadine acaricide, to controlticks on cattle. Unfortunately, amitraz resistance wasreported in Mexico in 2002 (Li et al. 2004, Soberanes-Cespedes et al. 2002). Reports of probable resistanceto the macrocyclic lactone, ivermectin, in Brazil, werepublished �9 yr ago by Martins and Furlong (2001)and were conÞrmed by Klafke et al. (2006). Recently,Perez-Cogollo et al. (2010) described the Þrst case ofR. microplus resistance to ivermectin in Mexico. Fi-nally, resistance to the phenylpyrazole, Þpronil, hasbeen reported in Uruguay and Mexico (Castro-Janeret al. 2010, Miller et al. 2008b).

Currently, resistance has progressed to the extentthat it is common for resistant tick populations to besimultaneously resistant to multiple classes of acari-

In conducting the research described in this report, the investiga-tors adhered to the “Guide for the Care and Use of LaboratoryAnimals,” as promulgated by the Committee on Care and Use ofLaboratory Animals of the Institute of Laboratory Animal Resources,National Research Council. The facilities are fully accredited by theAmerican Association of Laboratory Animal Care. This article reportsthe results of research only. Mention of a proprietary product does notconstitute an endorsement or a recommendation by the USDA for itsuse.

1 United States Department of AgricultureÐAgricultural ResearchService, Cattle Fever Tick Research Laboratory, 22675 North Moore-Þeld Road, Edinburg, TX 78541.

2 Corresponding author: United States Department of AgricultureÐAgricultural Research Service, Cattle Fever Tick Research Labora-tory, Moore Air Base, Building 6419, 22675 North MooreÞeld Road,Edinburg, TX 78541 (e-mail: robert.miller@ars.usda.gov).

3 Elanco Animal Health Research and Development, A Division ofEli Lilly and Company, 2001 West Main Street, GreenÞeld, IN 46140.

4 United States Department of AgricultureÐAgricultural ResearchService, Knipling-Bushland U.S. Livestock Insects Research Labora-tory, 2700 Federicksburg Road, Kerrville, TX 78028.

cide, and it is anticipated that in the near future,resistance will develop to other macrocyclic lactonesas their use has increased because of their long activ-ity, ease of administration, and lack of other viablealternatives (Rodriguez-Vivas et al. 2007, Rosario-Cruz et al. 2008).

The development of tick populations resistant tomultiple classes of acaricide is a real threat to theworld cattle industry. In the past, adequate control ofacaricide-resistant ticks could be attained simply byswitching to a different class of acaricide. The currentsituation is more challenging because there is a highlikelihood that the ticks to be controlled will be re-sistant to several classes of acaricide necessitatingsome degree of acaricide resistance testing to identifyan efÞcacious active ingredient. Unfortunately, manycountries do not have the means to adequately test forresistance before selecting the proper chemical to use.In some cases, there simply will not be a candidatechemical to use because the population to be con-trolled is resistant to every class of chemical on themarket. This will likely be the trend in the future ifnew chemicals are not quickly introduced.

One relatively new chemical available for whichthere has not been any reported resistance in ticks isspinosad. Spinosad is a fermentation metabolite of theactinomycete, Saccharopolyspora spinosa, found in thesoil. It has long-acting contact and systemic activityagainst lepidopteron larvae, leaf miners, thrips, andtermites. The mechanism of action of spinosad is thedisruption in binding of acetylcholine in nicotinic ace-tylcholine receptors at the postsynaptic cell (Salgado1997). This study describes the susceptibility of sev-eral strains of acaricide-resistant R. microplus, col-lected from Mexico and Brazil to spinosad, as well asthe activity proÞle comparing spinosad with otheracaricides against multiple parasitic life stages of twoadditional ixodid ticks, Amblyomma americanum (L.)and Dermacentor variabilis (Say).

Materials and Methods

Ticks. Six strains of R. microplus were used in thisstudy (Table 1). The AM-, OP-, -and PY-resistant SanAlfonso strain was collected near Emiliano Zapata,Tabasco, Mexico, in 2001. Engorged females wereoriginally colonized at the National Center of Parasi-tology Laboratory, Jiutepec, Morales, Mexico, beforebeing transferred to the United States Department ofAgriculture (USDA), Agricultural Research Service,Cattle Fever Tick Research Laboratory (CFTRL, Ed-

inburg, TX) in 2002. The AM-, OP-, and PY-resistantSanta Luiza strain was collected from a ranch in Brazil,and was maintained at the Mexican National Parasi-tology Laboratory (Jiutepec, Morelos, Mexico) beforebeing established at CFTRL (Mission, TX) in 2000.The PY-resistant B&H Ranch strain was detected onnative cattle in Hidalgo County, Texas, located 1 milesouth of Mercedes, Texas, on the B&H Ranch in 2004.The OP- and PY-resistant San Roman strain was col-lected from a ranch in Champoton, Campeche, Mex-ico, and was established at the CFTRL in 1998. TheOP-resistant strain, Pesqueria, was collected in 2000 atthe port-of-entry in Reynosa, Tamaulipas, Mexico, oncattle that had originated from Pesqueria, NuevoLeon, Mexico. The susceptible strain, Las Palmas, wascollected in 2005 from an outbreak in Zapata County,Texas, 22 miles south of the town of Zapata, Texas. Ithas been tested and found susceptible to all classes ofacaricides commonly used to control R. microplus inMexico and is maintained in colony without acaricideselection (R.J.M., unpublished data). The San Alfonsoand Santa Luiza strains were challenged with amitraz,the B&H Ranch strain with permethrin, the San Ro-man strain with coumaphos, and the Pesqueria strainwith diazinon to increase or maintain resistance to therespective acaricides during their laboratory coloni-zation and maintenance (Li et al. 2003, 2004; Miller etal. 1999, 2008a; Davey et al. 2008). The procedures forrearing ticks on cattle, maintaining nonparasitic stagesin the laboratory, and challenging larvae with acari-cides were similar to those previously described(Davey and George 1998, Davey et al. 1980).

One strain each of A. americanum (Oklahoma StateUniversity, Department of Entomology and Plant Pa-thology, Stillwater, OK) and D. variabilis (Elward IILaboratories, Soquel, CA) were used in this study.Both strains were maintained by the vendors withoutacaricide selection and had previously been deter-mined to be susceptible to permethrin amitraz, spi-nosad, and chlorpyrifos (W.H.W., unpublished data).Larval Packet Test (LPT).The level of resistance to

spinosad in strains of R. microplus was determined bystandard bioassay using the Food and Agriculture Or-ganization of the United Nations (FAO) LPT (Stoneand Haydock 1962). Bioassays were conducted usingtechnical spinosad (89.7% AI, Elanco Animal Health,GreenÞeld, IN). Technical grade acaricide was addedto trichloroethylene (Sigma-Aldrich, St. Louis, MO)to make a stock solution. This stock was then dilutedthree separate times to generate a speciÞc concentra-tion of acaricide in two parts trichloroethylene andone part olive oil (Sigma-Aldrich). These formulationswere serially diluted at a 20 or 30% rate generating thedoses for testing (Fig. 1). One milliliter of each dilu-tion was applied evenly to a 7.5 � 9-cm piece of Þlterpaper (Whatman 1, Whatman, Maidstone, Kent,United Kingdom). The trichloroethylene was allowedto evaporate from the Þlter paper for 2 h under a fumehood. The treated papers were then folded in half andsealed on the sides with bulldog clips. This formed apacket into which �100, 14-d-old larvae were placed,and then the top of the packet was sealed with another

Table 1. Presence of resistance in Mexican and Brazilianstrains of Rhipicephalus (Boophilus) microplus to various classes ofacaricide

Strain Pyrethoids Organophosphates Amidines Reference

San Alfonso � � � Davey et al. 2008Santa Luiza � � � Li et al. 2004B&H Ranch � � � Miller et al. 2007San Roman � � � Li et al. 2005Pesqueria � � � Li et al. 2003Las Palmas � � � Miller et al. 2008a

March 2011 MILLER ET AL.: SPINOSAD EFFICACY AGAINST TICKS 359

bulldog clip. The packets containing larvae were heldinanenvironmental chamberat 27 �2�C,92%RH,andat a photoperiod of 12:12 liters:D for 24 h. After thistime, the packets were removed from the environ-mental chamber and opened, and the number of liveand dead larvae was counted. Larvae were considereddead if they were not able to walk. Acaricide concen-trations were calculated in relation to volume of oliveoil because the trichloroethylene was allowed to evap-orate.Parasitic Life State Packet Tests. Packet tests were

also used to determine the activity proÞle of acaricidesagainst larval, nymphal, and adult stages of A. ameri-canum and D. variabilis. Bioassays were conductedusing technical spinosad (89.7% AI, Elanco AnimalHealth) and Pestanal analytical standard (�98% AI,amitraz, permethrin, or chlorpyrifos, Chem Service,West Chester, PA). Acaricides were formulated in

trichloroethylene and olive oil (Sigma-Aldrich) in afashion analogous to the FAO LPT method and seriallydiluted to obtain a 4-log concentration range corre-sponding to 5, 0.5, 0.05, and 0.005% active ingredient.Acaricide concentrations were calculated in relationto volume of olive oil because the trichloroethylenewas allowed to evaporate. A volume of 0.2 ml of eachdilution was applied evenly to each 5.0 � 6.0-cm tissuebiopsy bag (Fisher, Pittburgh, PA), and the trichlo-roethylene was allowed to evaporate for 1 h under afume hood. Approximately 100 larvae, 10 nymphs, or10 adults were then placed into each packet, and theopen end of each packet was sealed with a 6-cm plasticdialysis clip. A total of three replicates (n � 3) wastested for each acaricide at each concentration andparasitic life stage. The packets containing ticks wereinverted and placed in an environmental chamber at27 � 2�C, 92% RH, and at a photoperiod of 12:12

Fig. 1. Bioassay results of various acaricide-resistant strains of R. (Boophilus) microplus exposed to spinosad usingthe FAO LPT.

360 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 2

liters:D for 24 h. After this time, the packets wereremoved and opened, and the number of live and deadlarvae, nymphs, and adults was counted. Death wasmeasured when individual ticks could not walk.Adult Immersion Test (AIT). The AIT was de-

scribed by Drummond et al. (1973) and a slightlymodiÞed method was used to determine the efÞcacyof commercial preparation of spinosad (2.46% AI,Elector, Elanco Animal Health, GreenÞeld, IN)against the tick strains used in this study. A serialdilution from the stock formulation was made at a rateof 50% using water as the diluting compound (Fig. 2).Final volumes of diluted acaricide were 50 ml. En-gorged female ticks (�24 h after repletion) wererinsed in tap water to remove feces and other debris.Washed ticks were dried by placing them on papertowels. Ten ticks were randomly assigned to groupsand weighed to ensure equal weights for each group.For the control solution and each of the acaricide testsolutions, ticks were placed directly into a containercontaining 25 ml of the treatment solution shakengently for 30 s. After 30 s, the acaricide was poured offthrough a wire mesh sieve and the ticks were retained.The ticks were then placed onto a clean paper towelto dry. All ticks of each group were then deposited intopetri dishes (9 cm diameter) sealed with tape. Theticks were stored at 27Ð28�C and 80Ð95% RH for ovi-position. After 14Ð20 d, the ticks were discarded, andthe eggs produced by the ticks in each replicate wereweighed. After weighing, the eggs from each replicatewere mixed thoroughly and arraigned into a single

rectangle (1 cm � 1 cm). Five small subsamples weretaken from this rectangle at regular intervals, andthese eggs were combined in a shell vial (one dram)sealed with a cotton plug. Each combination of Þvesubsamples weighed �0.05 g. The remaining eggswere discarded. For replicates containing 0.05 g ofeggs, all of the eggs were kept. The eggs were held inan incubator and checked daily for hatching. Fourteendays after the Þrst larva was observed, the vials werefrozen for at least 24 h. Percentage hatch of larvae wasestimated by spreading larvae into a single layer in aglass petri dish with a grid (1 cm � 1 cm) printed onthe bottom. Five squares were selected at random, andthe numbers of hatched and unhatched eggs werecounted. Three replicates were made of each acari-cide concentration. Estimation of acaricide efÞcacywas made by determining the estimated reproduction(ER) of each group of treated ticks with that of thecontrol ticks.

First, the ER was calculated as follows: ER � (g eggslaid/number of females) � (% hatch) � (number ofeggs/g). ER is an estimate of the number larvae pro-duced per female at each concentration of acaricideused in the bioassay.

Secondly, the ER of each group of treated ticks wascompared with that of its control group. The percent-age control was calculated as follows: % control ofER � (ER control ticks � ER-treated ticks)/ER con-trol ticks) � 100.Statistical Analysis. Probit analysis was conducted

on R. microplus bioassay results using PoloPlus (Le

Fig. 2. Regression analysis of the percentage of control of ER on acaricide-resistant strains of R. (Boophilus) microplusexposed to increasing concentrations of spinosad (Elector EC). Arrow indicates the labeled rate of Elector if used as a wholebody spray.

March 2011 MILLER ET AL.: SPINOSAD EFFICACY AGAINST TICKS 361

Ora Software 2004). This analysis included probittransformation of percentage mortality and naturallogarithm transformation of dose. Resistance ratiosand synergism ratios were calculated by taking intoaccount the variance and covariance of the slope andintercept of each regression line at the lethal concen-tration estimated at 50% mortality (LC50) for the com-parison in question using the method of Robertson andPreisler (1991). Resistance ratios were calculated rel-ative to the susceptible Las Palmas strain, and syner-gism ratios were calculated relative to the formulationthat contained acaricide only. SigniÞcance of eachcomparison was determined only if one was not in-cluded in the conÞdence interval (Robertson and Pre-isler 1991). Goodness-of-Þt of data for all probit re-gressions was assessed by the examination of plots ofstandardized residuals. A standardized residual wasdetermined by taking the difference of each observedvalue and its expected value, and then dividing by thestandard error of the difference. For good Þt, residualsplotted against dose were randomly scattered around0 and within a band between �2 and 2 (Robertson andPreisler 1991). Linear regression was performed onAIT data using a natural log transformation of thepercentage of control of ER versus spinosad concen-tration (Kuo et al. 1992). Linear regression was per-formed on parasitic life stage packet test data usingGraph Pad Prism 4.00 (GraphPad Software, San Diego,CA) to obtain slope, LC50, 95% conÞdence limits (CL)of the LC50, and goodness-of-Þt parameters (r2).

Results

LPT. None of the strains tested showed resistanceto spinosad (Table 2, Fig. 1). Resistance ratios at theLC50 estimate were only 0.3 (0.28Ð0.33), 1.41 (1.30Ð1.53), 0.74 (0.69Ð0.78), 1.73 (1.60Ð1.87), and 1.18(1.11Ð1.26) for the San Alfonso, Santa Luiza, B&HRanch, San Roman, and Pesqueria, respectively, whencompared with the susceptible Las Palmas strain. TheLPT bioassay technique was adequate for establishingthe susceptibility of R. microplus larvae to spinosad.Graphs of standardized residuals from each bioassayshowed that �85% of the observed data points werebetween 2 and �2 standardized residuals with nopatterned deviation from the predicted lethal concen-tration estimated from the probit model (Fig. 3).

AITs. Spinosad was not efÞcacious against engorgedfemale R. microplus. There was no observable corre-lation in the calculated percentage of control of ERbecause of increased spinosad concentration (r2 �0.028, 0.21, 0.54, and 0.0002 for the Las Palms, SanRoman, San Alfonso, and B&H Ranch strains, respec-tively; Fig. 2) even after immersion in concentrationsof spinosad as high as 10%.Parasitic Life Stage Tests. Spinosad, amitraz, per-

methrin, and chlorpyrifos were all highly effectiveagainst larvae, nymphs, and adultA. americanum,withspinsod having the lowest LC50 values against all threelife stages (Table 3). Spinosad was highly efÞcaciousagainst larval and nymphalD.variabilis,but along withamitraz was inactive against the adult stage of this tick,as reßected in the high LC50 and large 95% CL esti-mates (Table 3). In contrast, permethrin was efÞca-cious against all three life stages, with chlorpyrifosbeing the most efÞcacious.

Discussion

As long as ticks are controlled with acaricides, thedevelopment of resistance to the compounds used willcontinue. Populations of multi-resistant R. microplusexist today throughout the tropical regions of theworld and, as selection for resistance occurs, newtechnologies and chemistries must be developed andused in concert with integrated pest managementstrategies to control resistant ticks (Jonsson and Piper2007). Spinosad represents one of the new chemicaltechnologies very much needed to control cattle ticksand, as it is relativelynewto themarket, therehasbeenno documented resistance as of yet in cattle ticks.Spinosad has been shown to be 100% efÞcaciousagainst cattle ticks. Davey et al. (2001) showed thatspinosad applied as a whole body spray under shel-tered experimental conditions (stall trial) provided100% kill of larvae for 14 d posttreatment. However,when infested cattle were treated in a Þeld situation(pastured cattle), spinosad failed to eradicate theseticks. The authors indicated that the failure to eradi-cate ticks in the Þeld was likely the result of too longof an interval between spinosad treatments (21 d) andtoo low of a concentration used (0.05Ð0.08% activeingredient [AI]). Therefore, it is possible that spi-nosad could be used to eradicate R. microplus with

Table 2. Acaricide-resistant Rhipicephalus (Boophilus) microplus treated with spinosad using the FAO larval packet test

Strain n Slope (SE) LC50 (95% CL) �2 RRa (95% CI)

San Alfonso 1,387 5.23 (0.25) 0.30 (0.27Ð0.33) 51.7* 0.30 (0.28Ð0.33)Santa Luiza 2,451 2.04 (0.07) 0.70 (0.60Ð0.83) 127.2* 1.41 (1.30Ð1.53)B&H Ranch 2,370 4.16 (0.15) 0.37 (0.34Ð0.40) 54.4* 0.74 (0.69Ð0.78)San Roman 1,792 2.89 (0.11) 0.86 (0.74Ð1.01) 106.2* 1.73 (1.60Ð1.87)Pesqueria 2,688 4.00 (0.14) 0.59 (0.55Ð0.64) 69.4* 1.18 (1.11Ð1.26)Las Palmasb 2,457 4.48 (0.74) 0.50 (0.47Ð0.53) 51.4* - -

All values are % AI spinosad. *, Indicates signiÞcance (P � 0.05).a Resistance ratio, generated by determining the ratio between the LC50 estimate of the susceptible strain with the LC50 estimate of the

resistant strain under consideration. The 95% CL is also taken into consideration for the calculation of the RR and 95% conÞdence interval(CI) for each RR estimate (Robertson and Preisler 1991).b Las Palmas is a susceptible strain used as a reference to generate resistance ratios (RR).

362 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 2

repeated systematic treatments if the treatment in-terval was shortened and the spinosad concentra-tion increased. Further increases in efÞcacy arelikely to be obtained with the use of a spray race orspray dip machine rather than a hand-held sprayapparatus (Davey et al. 1997).

In this study, we demonstrated that spinosad wasefÞcacious against the larvae of many strains of single-and multi-resistant R. microplus originally collectedfrom Brazil, Mexico, and the United States, represent-ing almost all known resistance mechanisms nowknown in R. microplus. However, the reproductivecycle of engorged female adult R. microplus ticks wasnot affected by treatment with spinosad. These in vitroresults against R. microplus correlate well with the invivo results of Davey et al. (2001), in which some adult

ticks present on cattle at the time of treatment withspinosad survived to repletion, but a high degree ofresidual activity was observed against repeated infes-tations with larvae on spinosad-treated cattle. To rec-oncile these observations and further evaluate poten-tial differences in efÞcacy that may have beendependent on parasitic life stage, spinosad was eval-uated and compared with amitraz, permethrin, andchlorpyrifos against all three parasitic life stages of twomulti-host ticks that readily infest cattle in the UnitedStates, A. americanum andD. variabilis (USDA 1976).Whereas spinosad was highly effective against imma-ture stages of both ticks, activity was much loweragainst adult D. variabilis, as was the activity of ami-traz. The data clearly illustrated differences in activityproÞles for spinosad and the other acaricides that were

Fig. 3. Standardized residuals of R. (Boophilus) microplus exposed to spinosad using the FAO LPT.

March 2011 MILLER ET AL.: SPINOSAD EFFICACY AGAINST TICKS 363

dependent both upon parasitic life stage and tick spe-cies.

Although not yet proven in Þeld studies, in principlespinosad should be able to eradicate acaricide-resis-tant R. microplus with a 1- to 2-wk treatment intervalat a concentration �0.08% AI. Given the high level ofefÞcacy against immature life stages, it is also possiblethat spinosad would be useful as an adjuvant to exist-ing acaricides in longer term treatment programs de-signed to disrupt the life cycle of emerging outbreaksand minimize development of resistance to amitraz orcoumaphos. Additionally, future resistance testingshould be accomplished as part of integrated pestmanagement programs using larvae as the test sub-jects, as we found no mortality in the engorged femaleticks after exposure to spinosad. The FAO LPT orlarval immersion tests (not used in this research)could be used for this purpose.

Acknowledgments

We thank Drs. G. M. Klafke and A. Y. Li for critical reviewof this manuscript; D. Krska and M. Moses for technicalsupport; and the members of the USDA, Animal and PlantHealth Inspection Service, Veterinary Services, Cattle FeverTick Research Laboratory, and the USDA, Agricultural Re-search Service, CFTRL who made this research possible.

References Cited

Aguirre, J., A. Sobrino, M. Santamarıa, A. Aburto, S. Roman,M. Hernandez, M. Ortiz, and Y. A. Ortiz. 1986. Resis-tancia de garrapatas en Mexico, pp. 282Ð306. In A. H.Cavazzani and Z. Garcia (eds.), Seminario Internacionalde Parasitologia Animal, Cuernavaca, Morelos, Mexico.

Castro-Janer, E., L. Rifran, P.Gonzalez, J. Piaggio, A.Gil, andT.T.S. Schumaker. 2010. Rhipicephalus (Boophilus) mi-croplus(Acari: Ixodidae) resistance to Þpronil in Uruguayevaluated by in vitro bioassays. Vet. Parasitol. 169: 172Ð177.

Davey, R. B., and J. E. George. 1998. In vitro and in vivoevaluation of a strain of Boophilus microplus (Acari:

Ixodidae) selected for resistance to permethrin. J. Med.Entomol. 35: 1013Ð1019.

Davey, R. B., E. H. Ahrens, and J. E. George. 1997. Com-parative effectiveness of coumaphos treatments appliedby different methods for the control of Boophilus micro-plus (Acari: Ixodidae). J. Agric. Entomol. 14: 45Ð54.

Davey, R. B., J. Garza, Jr., G. D. Thompson, and R. O.Drummond. 1980. Ovipositional biology of the cattletick, Boophilus annulatus (Acari: Ixodidae), in the labo-ratory. J. Med. Entomol. 17: 287Ð289.

Davey, R. B., J. E. George, and D. E. Snyder. 2001. EfÞcacyof a single whole-body spray treatment of spinosad,against Boophilus microplus (Acari: Ixodidae) on cattle.Vet. Parasitol. 99: 41Ð52.

Davey, R. B., R. J. Miller, and J. E. George. 2008. EfÞcacy ofamitraz applied as a dip against an amitraz-resistant strainofRhipicephalus (Boophilus)microplus (Acari: Ixodidae)infested on cattle. Vet. Parasitol. 152: 127Ð135.

Drummond, R. O, S. E. Ernst, J. L. Trevino, W. J. Gladney,and O. H. Graham. 1973. Boophilus annulatus andBoophilus microplus: laboratory test of insecticides. J.Econ. Entomol. 66: 130Ð133.

[FAO] Food and Agriculture Organization of the UnitedNations. 2004. Guidelines for Resistance Managementand Integrated Parasite Control in Ruminants. FAO,Rome, Italy.

Jonsson, N. N., and E. K. Piper. 2007. Integrated ControlPrograms for Ticks on Cattle. School of Veterinary Sci-ence, The University of Queensland, Queensland, Aus-tralia.

Klafke, G. M., G. A. Sabatini, T. A. De Albuquerque, J. R.Martins, D. H. Kemp, R. J. Miller, and T. T. Schumaker.2006. Larval immersion tests with ivermectin in popula-tions of the cattle tick Rhipicephalus (Boophilus) micro-plus (Acari: Ixodidae) from State of Sao Paulo, Brazil. Vet.Parasitol. 142: 386Ð390.

Kuo, J., E. Fox, and S.McDonald. 1992. SigmaStat StatisticalSoftware for Working Scientists. Jandel ScientiÞc, SanRafael, CA.

Le Ora Software. 2004. A UserÕs Guide to Probit or LogitAnalysis. Le Ora Software, Berkeley, CA.

Li, A. Y., R. B. Davey, R. J. Miller, and J. E. George. 2003.Resistance to coumaphos and diazinon in Boophilus mi-croplus (Acari: Ixodidae) and evidence for the involve-

Table 3. Acaricide-susceptible life stages of Amblyomma americanum and Dermacentor variabilis treated with spinosad, amitraz,permethrin, or chlorpyrifos using packet tests

A. americanum D. variabilis

Slope (SE) LC50 (95% CL) r2 Slope (SE) LC50 (95% CL) r2

SpinosadLarva 30.85 (6.89) 0.03a (0.001Ð0.27) 0.67 35.29 (4.07) 0.07a (0.02Ð0.23) 0.88Nymph 40.00 (6.31) 0.16a (0.030Ð0.84) 0.80 40.00 (6.31) 0.16a (0.03Ð0.84) 0.80Adult 38.01 (5.65) 0.19a (0.041Ð0.95) 0.82 12.33 (2.32) 126.77b (9.90Ð28180) 0.74

AmitrazLarva 36.90 (3.47) 0.17a (0.07Ð0.44) 0.92 27.24 (5.82) 0.02a (0.001Ð0.15) 0.69Nymph 26.88 (5.73) 0.34a (0.04Ð5.03) 0.69 24.37 (5.97) 2.46b (0.21Ð293.09) 0.62Adult 35.34 (4.02) 0.34a (0.11Ð1.17) 0.89 9.67 (2.93) 2642b (26.73Ð3.24E 12) 0.52

PermethrinLarva 31.94 (5.85) 0.04a (0.003Ð0.23) 0.75 39.38 (5.74) 0.17a (0.04Ð0.81) 0.83Nymph 36.23 (4.50) 0.23a (0.07Ð0.87) 0.87 35.00 (6.34) 0.36a (0.06Ð3.14) 0.75Adult 35.42 (4.02) 0.29a (0.09Ð0.98) 0.89 22.33 (5.42) 3.79a (0.33Ð0.002) 0.63

ChlorpyrifosLarva 30.51 (7.20) 0.03a (0.001Ð0.28) 0.64 2.02 (0.58) 0.00000001a 0.55Nymph 31.64 (6.36) 0.03a (0.002Ð0.26) 0.71 20.09 (5.09) 0.004b (0.0001Ð0.05) 0.61Adult 30.33 (7.43) 0.99a (0.08Ð54.70) 0.63 30.40 (6.17) 0.86c (0.11Ð15.45) 0.71

LC50 values are % AI. For each acaricide and species within a column, LC50 values with a common superscript were not signiÞcantly differentby virtue of overlapping 95% CL. n � �1,200, 120, and 120 for bioassays involving larvae, nymphs, and adults, respectively.

364 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 2

ment of an oxidative detoxiÞcation mechanism. J. Med.Entomol. 40: 482Ð490.

Li, A. Y., R. B. Davey, R. J. Miller, and J. E. George. 2004.Detection and characterization of amitraz resistance inthe southern cattle tick, Boophilus microplus (Acari:Ixodidae). J. Med. Entomol. 41: 193Ð200.

Li, A. Y., J. H. Pruett, R. B. Davey, and J. E. George. 2005.Toxicological and biochemical characterization of cou-maphos resistance in the San Roman strain of Boophilusmicroplus (Acari: Ixodidae). Pestic. Biochem. Physiol. 81:145Ð153.

Martins, J. R., and J. Furlong. 2001. Avermectin resistanceof the cattle tick Boophilus microplus in Brazil. Vet. Rec.149: 64.

Miller, R. J., R. B. Davey, and J. E. George. 1999. Charac-terization of pyrethroid resistance and susceptibility tocoumaphos in Mexican Boophilus microplus (Acari: Ixo-didae). J. Med. Entomol. 36: 533Ð538.

Miller, R. J., R. B.Davey, and J. E.George. 2007. First reportof permethrin-resistant Boophilus microplus (Acari: Ixo-didae) collected within the United States. J. Med. Ento-mol. 44: 308Ð315.

Miller, R. J., A. Y. Li, M. A. Tijerina, R. B. Davey, and J. E.George. 2008a. Differential response to diazinon andcoumaphos in a strain of Boophilus microplus (Acari:Ixodidae) collected in Mexico. J. Med. Entomol. 45: 905Ð911.

Miller, R. J., G. C. Almazan, O. M. Estrada, R. B. Davey, andJ. E. George. 2008b. A survey for Þpronil-and-ivermec-tin-resistant Rhipicephalus (Boophilus) microplus col-lected in northern Mexico and the options for the man-agement of acaricide-resistant ticks with pesticides. InV. Z. Garcõa (ed.), VI Seminario Internacional deParasitologõa. Impacto de las enfermedades parasitariassobre la ganaderõa globalizada. INIFAP-INFARVET AM-PAVE-CNG-UV, 3Ð5 September 2008, Boca del Rõo, Ve-racruz, Mexico. JasonÕs Editores, S. A. de C. V., MexicoD.F., Mexico.

Perez-Cogollo, L. C., R. I. Rodriguez-Vivas, G. T. Ramirez-Cruz, andR. J.Miller. 2010. First report of the cattle tickRhipicephalus microplus resistant to ivermectin in Mex-ico. Vet. Parasitol. 168: 165Ð169.

Robertson, J. L., and H. K. Preisler. 1991. Pesticide Bioas-says with Arthropods. CRC, Boca Raton, FL.

Rodriguez-Vivas, R. I., M. A. Alonso-Diaz, F. Rodriguez-Arevalo, H. Fragoso-Sanchez, V. M. Santamaria, and R.Rosario-Cruz. 2007. Prevalence and potential risk fac-tors for organophosphate and pyrethroid resistance inBoophilus microplus tick on cattle ranches from the Stateof Yucatan, Mexico. Vet. Parasitol. 136: 335Ð342.

Rosario-Cruz, R., C. Almazan-Garcıa, R. J. Miller, D. I.Domınguez-Garcıa, R. Hernandez-Ortiz, and J. de la Fu-ente. 2008. Genetic basis and impact of tick acaricideresistance. Front. Biosci. 14: 2657Ð2665.

Salgado, V. L. 1997. The modes of action of spinosad andother insect control products. Down Earth 52: 35Ð43.

Santamaria, E. M., and S. H. Fragoso. 1994. Resistencia engarrapatas Boophilus microplus, a los ixodicidas en Mex-ico, pp. 473Ð474. InProceedings, XIV Pan American Con-gress of Veterinary Science, 9Ð15 October 1994, Aca-pulco, Mexico. Patrocinio Casa Autrey, S. A. de C. V.,Mexico D.F., Mexico.

Soberanes-Cespedes, N., M. Santamarıa-Vargas, H. Fragoso-Sanchez, and Z. Garcıa-Vazquez. 2002. Primer caso deresistencia al amitraz en la garrapata del Ganado Boophi-lus microplus en Mexico. (First case reported of Amitrazresistance in the cattle tick Boophilus microplus in Mex-ico.) Tec Pecu. Mex. 40: 81Ð92.

Stone, B. F., and K. P. Haydock. 1962. A method for mea-suring the acaricide-susceptibility of the cattle tickBoophilus microplus (Canestrini). Bull. Entomol. Res. 53:563Ð578.

[USDA] United States Department of Agriculture. 1976.Ticks of Veterinary Importance. United States Depart-ment of Agriculture, Washington, DC.

Received 26 September 2008; accepted 10 November 2010.

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