Survival of larvae of Boophilus annulatus, Boophilus microplus, and Boophilus hybrids (Acari: Ixodidae) in different temperature and humidity regimes in the laboratory.

The survival period for larvae of Boophilus annulatus (Say), Boophilus microplus (Canestrini) and hybridized Boophilus ticks was determined by exposure to various combinations of temperature (20, 25, 30 and 35°C) and relative humidity (32, 63, 75, 84 and 97% RH) in the laboratory. Results indicated that within a given temperature and RH regime, there was no difference (P> 0.05) in larval survival among the three species tested, indicating that these ticks respond similarly over a wide range of temperature and RH combinations. Larval survival in all three species was longest (P < 0.05) at 20°C and either 84 or 97% RH. With each increase in temperature at the 84 and 97% RH treatment levels, there was a corresponding significant (P < 0.05) decrease in larval survival. When the temperature reached 35°C at all humidities or when the RH was 63% or less at all temperatures, the mean larval survival period was 43 days or less in all cases and little difference (P> 0.05) was observed among the treatment regimes included. Results suggest that at a RH of 75% and more, the temperature is the determining factor in larval survival, whereas at a RH of 63% and less the RH is the determining factor in larval survival, regardless of temperature.     

Eprinomectin pour-on for control of Boophilus microplus (Canestrini) ticks (Acari: Ixodidae) on cattle

The efficacy of a commercial pour-on formulation of eprinomectin, a macrocyclic lactone, against experimental infestations of Boophilus microplus (Canestrini) ticks was evaluated in two trials involving 27 Bos taurus calves. The first trial was designed to evaluate the effects of a single treatment at a dose of 0.5 mg/kg of body weight against standard size B. microplus females (4.5-8.0 mm long). A significant reduction in tick numbers (P<0.05, Wilcoxon test) was observed between treated calves as compared to untreated ones from Day 3 (44% efficacy) after treatment to the end of the trial on Day 28 (96.9%), with a peak efficacy of 97.1% on Day 21. In the second trial the effect of eprinomectin on standard size tick numbers, engorgement weight and fertility of female ticks from calves with a single treatment dose of 1 mg/kg on Day 0 and calves treated twice at a dose of 0.5 mg/kg on Days 0 and 4 was evaluated. An efficacy >93% was obtained from Day 2 to Day 28 after treatment in calves treated twice at 0.5 mg/kg, and to the end of the trial (Day 35) in calves treated once with 1 mg/kg. The 1mg/kg treatment provided >98% residual efficacy for at least 7 days. During the first part of the second trial the efficacy of eprinomectin resulted from a dramatic adverse effect on engorgement weight and fertility of female ticks, with 100% control on Day 5 (dosage of 1 mg/kg) and on Days 6 and 7 (two doses of 0.5 mg/kg). Following Day 7, most of the effect was due to reduction in the number of standard size female ticks.     

Comparison of the reproductive biology between acaricide-resistant and acaricide-susceptible Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The reproductive fitness of Rhipicephalus (Boophilus) microplus (Canestrini) strains resistant to organophosphate (OP), pyrethroid (P), or formamidine (F) acaricides was compared to an acaricide-susceptible (SUS) strain to determine whether the acquisition of resistance affected reproductive fitness in the resistant strains. The SUS strain females had a 3.0 days preoviposition period, a 12.1 days oviposition period, a 22.5 days egg incubation period, a mean of 3670 eggs per female, and a mean percentage egg hatch of 78.1%, which were all remarkably similar to these same parameters reported for this species throughout the world. The reproductive biology of the P-resistant strain (PYR) and the F-resistant strain (FOR) were, for the most part, similar to those of the SUS strain. In the few instances where statistical differences did occur there was little evidence that the variation had any biological basis that could be attributed to a reduction in fitness related to resistance to P or F acaricides. Although the comparison of reproductive parameters of the OP-resistant strain (OPR) and the SUS strain identified statistical differences between the mean egg incubation and oviposition periods, the magnitude of the differences was not sufficient to conclude that the OPR strain was biologically less fit than the SUS strain. However, the OPR strain produced 30% fewer eggs (2562 eggs per female) than the SUS strain (3670 eggs per female) indicating the acquisition of resistance placed the OPR at a selective disadvantage relative to the SUS strain. This coupled with a lower, though non-significant, egg hatch was used to predict there would be a reduction of at least 34.1% in larval numbers available to potentially re-infest subsequent cattle than were available from the SUS strain. These data may aid the development of management strategies that can be used to control OP-resistant ticks.     

Susceptibility to two pyrethroids in Boophilus microplus (Acari: Ixodidae) populations of northwest Argentina. Preliminary results

Cattle are treated 6-12 times yearly to control Boophilus microplus ticks in the east zone of the Argentinean infested region, while 1-4 treatments are applied for tick control in the west zone. In the 1970s resistance to organo-phosphate acaricides was found in the east zone, but not in the west zone. However, a shift to synthetic pyrethroids (SP) was made through all infested regions. Currently, indications of resistance to SP in the east zone, but not in the west zone, are provoking to a switch to formamidine acaricides. During 1998 a total of 147 B. microplus engorged females were collected from 20 beef cattle ranches from the west zone of the Argentinean infested region. Individual progenies of these ticks were tested ('larval packet test') with cypermethrin and deltamethrin, and their LC 50 and LC 90 were compared to those estimated for the Milagro susceptible strain. No evidence of resistance to these SP was found. Due to sampling restraints the results are presented as preliminary. Nevertheless, a shift away from use of SP for control of B. microplus in the west zone appears to be unjustified and should be independent of the resistance circumstances observed in the east zone of the Argentinean tick infested region.     

Kinetics of energy source utilization in Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) embryonic development

The present work evaluates the kinetics of utilization of the main potential energy sources throughout the embryonic developmental stages of Boophilus microplus. The embryonic development of this arthropod is completed in 21 days. Cellularization of the blastoderm occurs on the 6th day and is rapidly followed by germ band extension and segmentation, whose first signs are visible on the 7th day. Cellularization is typically a maternal-driven process, carried out by molecular determinants deposited in the oocyte during oogenesis. On the other hand, segmentation is of zygotic nature, being the consequence of the synthesis of various components by the growing embryo. The enhancement in total B. microplus RNA was observed after cellularization, corroborating the replacement of maternal-driven processes by embryonic zygotic expression. An abrupt increase in oxygen consumption was observed from cellularization until the 8th day of development. The reduction in dry weight at the same period and the susceptibility of oxygen consumption to KCN suggest that the respiration process is activated during early embryonic development. A marked decrease in total lipid content occurred between the 5th and 7th days of development, suggesting this is the main energy source for cellularization. A major reduction in carbohydrate content occurred later, between the 7th and 9th days, and it could be assigned to the morphological segmentation of the embryo. Although the total amount of proteins remains unchanged from oviposition to hatching, a 15% reduction in vitellin (VT) content was observed before cellularization, up to the 4th day after egglaying. This observation was correlated to the synthesis of new proteins needed to support early embryo development. Additional 20% of VT was consumed thereafter, mainly at the end of embryogenesis, and in this case VT is probably used as energy source to the older embryo. Altogether, these data indicate different energy sources for maternal and zygotic driven processes.     

Accumulation of acaricide resistance mechanisms in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) populations from New Caledonia Island

Rhipicephalus (Boophilus) microplus has been pesticide-controlled for several decades in the pacific island of New Caledonia. Since 1996, pesticide-control has been based on either deltamethrin (Butox) or amitraz (Taktic) in herds harbouring deltamethrin-resistant ticks. In this island, the first R. microplus deltamethrin- and amitraz-resistances were detected in 1992 and 2003, respectively. Using LPT bioassays, we have undertaken to update data regarding the geographical distribution and the physiological diversity likely to be involved in these resistances. We confirmed that after 17 years of intensive use of deltamethrin, several resistances of moderate levels (<30-fold) have evolved and/or diffused in any part of the island. We also evidenced that amitraz-resistant phenotypes have recently evolved in diverse western tick populations, although none has reached fixation in any tick population yet. According to synergists bioassays, the physiological changes involved in amitraz-resistance in New Caledonia would involve target modification and detoxifying P450 cytochrom oxydase(s). It may also involve detoxifying esterase(s) although this later point will need confirmation on samples bearing higher frequency of resistant phenotypes. Results are discussed with regard to the local evolutionary dynamics of resistance.     

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 μg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 μg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.     

Efficacy of a single whole-body spray treatment of spinosad, against Boophilus microplus (Acari: Ixodidae) on cattle.

The efficacy of a single whole-body spray of spinosad, a naturally derived control agent, applied at three concentrations was evaluated against cattle infested three separate times prior to treatment and at four weekly intervals following treatment with Boophilus microplus (Canestrini). At 0.0167% active ingredient (AI) both tick numbers (1894 ticks per calf) and index of fecundity (IF) of females (258.3) were no different than that of the control group. However, spinosad treatment at both 0.05 and 0.15% AI resulted in fewer ticks per calf (600 and 935, respectively) with lower IF values for females (43.4 and 38.4, respectively). The percent control of ticks on the animals at the time of treatment (acute efficacy) was dramatically lower at 0.0167% AI (21.4%) than at 0.05 (86.3%) and 0.15% AI (87.9%). Spinosad treatments appeared to be more effective against immature stages (nymphs and larvae) than against adult ticks that were on the animals at the time of treatment. The mean weight of females that survived to repletion was similar (322-348 mg) in all groups. By contrast, the mean weight of egg masses produced by females was highest in the control group (155 mg), whereas each increase in spinosad concentration resulted in a substantial decrease in egg mass weight, with the 0.15% AI group averaging only 73 mg. The hatch rate of eggs derived from females ranged from 93.4% in control females down to 53.9% hatch for females treated at 0.15% AI spinosad. The residual efficacy of spinosad at 0.0167% AI was poor even at 1 week following treatment, resulting in 101 ticks per calf and a level of control of only 66.4%. At 0.05% AI, protection against successful reinfestation was high at 1-week post-treatment where only five ticks per calf reached repletion, and control of the IF of these females was 99.3%. The 0.15% AI treatment provided almost complete protection against reinfestation for 2 weeks following treatment (< or =5 ticks per calf), and control of the IF of these ticks was >99.9%. Thus, the use of spinosad at US ports-of-entry would be unacceptable because of the critical necessity of achieving 100% control with a single treatment to prevent the reintroduction of ticks. However, it is likely ticks could be eradicated using spinosad in tick infested areas of the US if repeated (systematic) treatments were applied to cattle maintained on the premises.     

Molecular analysis of Boophilus spp. (Acari: Ixodidae) tick strains

Boophilus spp. (Acari: Ixodidae) parasitize cattle and other farm and wild animals in tropical and subtropical regions of the world. Ticks belonging to the genus Boophilus have undergone evolutionary processes associated with habitat adaptation following biogeographical separation, resulting in strains with marked morphological differences. We have characterized at the molecular level B. microplus strains from Latin America and Australia, employing sequences derived from the bm86 coding region, an intron located within the bm86 gene, and DNA short tandem repeats (STR). A B. annulatus strain was employed for comparison. The results indicated that variation within the bm86 coding region is higher between B. microplus strains than between some B. microplus strains and B. annulatus. The sequence of the intron was not informative for phylogenetic analysis, varying among individuals of the same strain. Two STRs were identified in B. microplus (STRs BmM1 and BmM2) and one in B. annulatus (STR Ba1). Southern hybridization experiments with STRs BmM1 and BmM2 as a probe revealed the prevalence of dispersed moderately repeated DNA in the genome of B. microplus. The analysis of polymorphism at STR locus BmM1 evidenced differences within and between populations of B. microplus. These results support at the molecular level the existing differences between B. microplus strains and suggest tools to characterize these populations.     

Seasonal Variation of Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) in Cattle in Minas Gerais State,Brazil

The seasonal variation of the tick Boophilus microplus in the `Regiaõ Metalúrgica' region of Minas Gerais state was studied using 26 Bos taurus (Holstein)×Bos indicus (Gir) calves, of which 16 animals were 3/4 Holstein×1/4 Gir and 10 were 3/4 Gir×1/4 Holstein. Monthly tick counts were made between March 1996 and March 1998 and, although B. microplus infestations occurred throughout the year, they were greater during the rainy season. Rainfall was the climatic factor that most affected the seasonal variation. The animals with a preponderance of Holstein blood presented infestations that were significantly (p<0.05) greater than those of the other cross. Five of the animals (19.2%) maintained 45.5% of the parasite load of the entire group.     

Identification of point mutations in a putative carboxylesterase and their association with acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Chemical control based on the use of pyrethroid and organophosphate compounds has selected resistant genotypes in populations of Rhipicephalus (Boophilus) microplus. Point mutations in esterase-encoding genes represent one of the main resistance mechanisms in this species. In this study, the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) technique was used to investigate the presence of mutations in a fragment of a putative carboxylesterase in a population of ticks with a history of resistance. The digestion of a fragment of 372 pb with EcoRI revealed three genotypes: W, H and M, observed in different frequencies. The homozygous wild-type genotype (W) was detected only in sensitive strains, with high frequency. The heterozygous genotype (H) was observed in all the strains, albeit with higher frequency in the strains with a moderate resistance, while the homozygous mutant genotype (M) was found only in the moderate resistant strain and resistant strains, with higher frequency in the resistant strains. A comparison of the sequences indicated the presence of other mutations, besides EcoRI polymorphism in the moderate resistant and resistant strains. Also found was the presence of stop codons generating truncated proteins in the sensitive and moderate resistant strains. A domain analysis revealed the presence of additional domains in the resistant strain. These findings suggest that different point mutations, as well as the influence of post-translational modification mechanisms, are altering the activity of the translated proteins and may be associated with resistance.     

Evaluation of Metarhizium anisopliae (Hyphomycetes) for the control of Boophilus microplus (Acari: Ixodidae) on naturally infested cattle in the Mexican tropics

The efficacy of Metarhizium anisopliae on the control of Boophilus microplus in cattle infested naturally in the Mexican tropics was evaluated. The study was carried out on a ranch in Veracruz, Mexico. Twenty steers were randomly allocated into two groups of 10 cattle. Animals were naturally infested with B. microplus. Animals in the treated group were sprayed with M. anisopliae (strain Ma34) at a concentration of 1x10(8)conidia/ml every 15 days (four treatments). The other group remained as untreated control. Standard engorged female ticks were recorded on days 0, 1, 3, 5, 7 and 14 post-treatment. From the second application treatment (day 7) to the end of the experiment, animals in the treated group had lower tick infestation (P<0.05) with an efficacy of 40.0-91.2%. The results demonstrate the efficacy of repeated treatment with M. anisopiae (Ma34 strain) to control natural infestation of engorged female B. microplus on cattle in the Mexican tropics.     

Genetic incompatibility between Boophilus decoloratus (Koch, 1844) and Boophilus microplus (Canestrini, 1888) and hybrid sterility of Australian and South African Boophilus microplus (Acarina: Ixodidae)

Virgin females of both Boophilus decoloratus and Boophilus microplus, when mated with males of the other species, subsequently produced sterile eggs. Counts of spermiophore capsules in female seminal receptacles showed that the males of both species will mate with the females of both species and that B. microplus males show a slightly greater, but statistically insignificant, mating capacity than B. decoloratus males. South African B. microplus females, when mated with an Australian strain of B. microplus males, produced a 62% yield of viable hybrid progeny while the reciprocal cross produced only a 1,82% hatch of non-viable larvae. The hybrids were sterile when interbred and no hatch resulted when the Fl males were backcrossed with parent females. The reciprocal backcross of hybrid Fl females to parent males resulted in a low percentage hatch of non-viable larvae.     

In vitro and in vivo evaluation of deltamethrin and amitraz mixtures for the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in New Caledonia

Acaricide resistance is a major problem that hinders the control of the tropical cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), in many parts of the world where cattle production continues to suffer severe economic losses to tick infestation. Deltamethrin and amitraz have been used alone to control R. microplus in New Caledonia for the past decade, and tick populations have developed resistance to both acaricides. A study was conducted to evaluate the effectiveness of deltamethrin and amitraz mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant R. microplus on cattle at two dairy farms in New Caledonia. Results of laboratory bioassays using modified larval packet tests (LPT) revealed up to 16.59-fold resistance to deltamethrin, and up to 5.86-fold resistance to amitraz. Significant synergism was observed when amitraz was used as a synergist in deltamethrin bioassays. Amitraz significantly increased deltamethrin toxicity to tick larvae, while deltamethrin was much less effective on amitraz toxicity. Synergism of amitraz by deltamethrin only occurred when the deltamethrin concentration was relatively high. Results of on animal efficacy trials of deltamethrin and amitraz alone and mixtures of both at different concentrations revealed a similar pattern of synergism. Adding amitraz to a deltamethrin formulation led to dramatic increases of percent reduction of both immature and adult ticks. In contrast, adding deltamethrin to an amitraz formulation did not increase control efficacy. Results from this study may lead to the adoption of an acaricide mixture strategy for the control of pyrethroid-resistant R. microplus in New Caledonia and elsewhere.     

Factors affecting the detachment rhythm of engorged Boophilus microplus female ticks (Acari: Ixodidae) from Charolais steers in New Caledonia

As in most parts of the world where the cattle tick Boophilus microplus is established, resistance of ticks to acaricides occurs in New Caledonia. In order to implement laboratory resistance tests on larvae, engorged females collected in suspected farms are necessary. Investigations on the detachment schedule of the engorged females were conducted to explain certain field situations such as the lack or scarcity of engorged females on highly infested cattle driven from the pasture to the pen in the morning. Three experiments on Charolais steers naturally infested on pastures showed that: (1) engorged female burdens at sunrise are similar whether the steers spend the night in pasture or in a pen; (2) compared with steers maintained in a pen, morning detachment of females increases when the steers stay on the pasture or move from the pasture to the pen; (3) detachment rhythm of engorged females on steers staying the morning in a pen, is not influenced by feeding activity or exposition of steers to sun; (4) detachment occurs earlier for females attached on anatomical sites exposed to sun, and earlier from these sites for the steers in pasture or walking than for steers in a pen.     

Development of amitraz resistance in field populations of Boophilus microplus (Acari: Ixodidae) undergoing typical amitraz exposure in the Mexican tropics

The purpose of this study was to determine the effect of amitraz selection pressure on the development of resistance in field populations of Boophilus microplus in the Mexican tropics. Three farms (FA(1), FA(2) and FA(3)) in Yucatan, Mexico, were selected in this study. Amitraz was applied as a whole body sprays to all cattle on each farm for tick control once monthly for 15 months. From each farm, 20-30 B. microplus engorged females were collected every 3 months. The modified larval immersion test was used to test susceptibility of B. microplus to amitraz. Larvae were exposed to serial dilutions of amitraz. Probit analysis was used to determine lethal dose at 50% mortality and associated 95% confidence limits. The resistance factors found in the three farms during the 15 months of amitraz selection pressure were FA(1) (1, 2, 4, 4 and 13), FA(2) (1, 6, 23, 21 and 22) and FA(3) (2, 13, 2, 6 and 6). It is concluded that amitraz selection pressure on field populations of B. microplus increased the resistance level in all populations studied in the Mexican tropics.     

Morphological, histological, and ultrastructural studies of the ovary of the cattle-tick Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae)

This study presents the morphology of the ovary, as well as the dynamics of the vitellogenesis process in oocytes of the cattle-tick Boophilus microplus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a lumen delimitated by a wall of small epithelial cells with rounded nuclei. In this tick species, the oocytes were classified into six stages varying from I to VI and according to: cytoplasm appearance and presence of the germ vesicle, yolk granules, and chorion. Oocytes of various sizes and at different developmental stages remain attached to the ovary through a cellular pedicel until completing stage V. Afterwards, they are liberated into the lumen and from there to the exterior. Some oocytes (classified as type VI) showed an atypical appearance indicating that some of the cellular components would be undergoing a degenerative process and/or reabsorption.     

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50)<2) and resistant (differences and RR(50)≥2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases ≥2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay.     

Efficacy of amitraz applied as a dip against an amitraz-resistant strain of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infested on cattle

Selection pressure with the acaricide, amitraz was applied to a strain of Rhipicephalus (Boophilus) microplus (Canestrini) in 13 out of 18 generations, resulting in a 28.7-fold increase in resistance, indicating a shift in the phenotypic composition of the ticks, from a heterogenous mixture of both susceptible and resistant ticks when selection began to a homogenous composition of predominantly resistant ticks when selection ended. Resistant larvae of the last selected generation were infested on cattle three separate times at 20, 13, and 6 d (7 d intervals) before being dipped in a total immersion vat charged at 0.02, 0.044, and 0.096% active ingredient (AI) amitraz. There was a dose related, though not always significant, response to amitraz whereby each increase in concentration produced increased adverse effects. The number of ticks and index of fecundity (IF) of females recovered from cattle treated at 0.02 and 0.044% AI were not different from that of untreated ticks. However, female weight and egg mass weight of females recovered from cattle treated at 0.044% were lower than untreated females or females treated at 0.02% AI. All measured parameters obtained from ticks recovered from cattle treated at 0.096% AI were significantly lower than the untreated control group, but tick numbers and IF were not different from the other treated groups. At 0.02% AI the level of control was significantly lower (34.6%) than treatment at 0.096% AI (81.2%), while the level of control at 0.044% AI (57.6%) was intermediate between the other treated groups. Therefore, none of the amitraz treatments provided the required 99% control necessary for use in the U.S. Cattle Fever Tick Eradication Program, and the estimated line derived from regression analysis showed that a concentration of ca. 0.2% AI amitraz would be needed to achieve 99% control of the resistant ticks.