(Acari: Ixodidae) Cell Lines - BioOne

ARTICLE

In Vitro Generation of Organophosphate Resistant Boophilus microplus (Acari: Ixodidae) Cell Lines RAQUEL COSSIO-BAYUGAR,1 G. GALE WAGNER,

AND

PATRICIA J. HOLMAN2

College of Veterinary Medicine, Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843Ð 4467

J. Med. Entomol. 39(2): 278Ð284 (2002)

ABSTRACT Three organophosphate resistant Boophilus microplus Canestrini cell lines were generated by exposing B. microplus VIII-SCC cell line to incrementally increased toxic concentrations of the acaricide coumaphos. The development of resistance was evidenced by LC50 values elevated over those of control cells. The resistant cell lines selected in higher concentrations of organophosphate, designated C44 and C54, also had signiÞcantly slower duplication rates than a resistant cell line selected in lower concentrations of coumaphos (C34) and the nonresistant control cells. Resistant cell lines C44 and C54 also had signiÞcantly higher levels of esterase after exposure to coumaphos than resistant cell line C34 and the nonresistant controls. These in vitro results agree with reports of increased esterase activity associated with organophosphate resistance in B. microplus ticks in vivo. KEY WORDS Boophilus microplus, tick cell lines, organophosphate resistance, coumaphos, in vitro culture, esterases

THE CATTLE TICK, Boophilus microplus Canestrini, is the most important tick parasite of cattle in the world in terms of the economic losses it causes (Angus 1996). It was eradicated from the United States after a 55-yr campaign that began in 1906 (Graham and Hourrigan 1977). In Mexico, increases in B. microplus acaricide resistance to pyrethroids and organophosphate (OP) compounds (Aguirre et al. 1986, Ortiz et al. 1994) emphasize the need for an understanding of the fundamental mechanisms of resistance. Most pesticide resistance mechanisms involve either a change in sensitivity of the site of action or an increased capacity for detoxiÞcation (Oppenoorth 1984). The target site of the OP and carbamate insecticides is acetylcholinesterase (AChE), a serine esterase that hydrolyzes the neurotransmitter acetylcholine. Once AChE is inhibited by these insecticides, paralysis or death of the insect occurs (Hemingway and Karunartne 1998). A modiÞed AChE with reduced sensitivity to OP has been reported in OP resistant B. microplus in Australia (Roulston et al. 1967). In Mexican B. microplus tick strains, resistance to the OP acaricide coumaphos is caused by a multifactorial system involving an altered AChE target site (Wright and Ahrens 1987) and enhanced metabolic detoxiÞcation (Bull and Ahrens 1988). A myriad of detoxifying enzymes have been implicated in pesticide resistance. Cytochrome P450 mono 1 Cenid-Parasitologia Veterinaria INIFAP-SAGAR, Apartado Postal 206 Civac, Morelos, CP 62500, Mexico. 2 E-mail: [email protected].

oxygenase mediated resistance to organophosphate insecticides, as well as to organochlorine, carbamate, pyrethroid, and insect-organ growth regulator insecticides, has developed in numerous important pests (Oppenoorth 1985). Increased microsomal phosphatase and mixed function oxidase enzyme activities are associated with OP resistance in Lucilia cuprina (Wied), the sheep blowßy (Hughes and Devonshire 1982). Elevated glutathione S-transferase (GST) levels are reported in OP resistant Musca domesticus (L.), house ßies (Wang et al. 1991). However, increased detoxiÞcation by esterases is probably the most common mechanism of resistance to OP insecticides (Oppenoorth 1984) and may be important in OP acaricide resistance. In B. microplus ticks, nonspeciÞc esterases have been reported associated with resistance toward pyrethroid acaricides (De Jersey et al. 1985) and elevated levels of these enzymes are found in OP resistant B. microplus strains as well (Miranda et al. 1995, Rosario-Cruz et al. 1997, Jamroz et al. 2000). In vitro assays are attractive alternatives to traditional animal tests not only because they limit the use of animals, but also because they provide a cost effective approach for the rapid screening of large numbers of environmental xenobiotics and new drugs in development. In vitro assays allow experimental parameters to be rigorously controlled, reducing the variability between experiments (Guzzie 1994). Selection of cancer cell lines in tissue culture for resistance to cytotoxic drugs has been a key element in searching for genetic alterations that are responsible for drug resistance development in cells. Analysis

0022-2585/02/0278Ð0284$02.00/0 䉷 2002 Entomological Society of America

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COSSIO-BAYUGAR ET AL.: COUMAPHOS RESISTANT TICK CELL LINES

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of the phenotype of these drug-resistant cancer cells has allowed classiÞcation into two groups: cells demonstrating single agent resistance or resistance to a single class of anti-cancer drugs with the same mechanism of action, and cells broadly resistant to many chemically diverse anti-cancer drugs with different mechanisms of action (Gottesman et al. 1996). We hypothesized that a cell culture based system would help identify the mechanisms of detoxiÞcation and resistance to coumaphos. The current study reports the in vitro generation of B. microplus OP resistant cell lines. Materials and Methods Boophilus microplus VIII-SCC Cells. Cryopreserved B. microplus VIII-SCC cells (Holman 1981) were cultivated at 32⬚C, 5% CO2 in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 20% heat inactivated fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA) and 1% HB101 (reconstituted according to manufacturerÕs recommendations; Irvine ScientiÞc, Santa Ana, CA). The cultures were considered passage one at the time of recovery for the purpose of this study. Generation of Resistant Cell Lines. Preliminary analysis of coumaphos toxicity was based on measurements where viability was reduced 48 h after exposure to the OP compound. Boophilus microplus cells were seeded at a density of 2 ⫻ 105 cells in Nunclon delta ßat-sided tubes (Nalge Nunc International, Naperville, IL) in culture medium and maintained at 32⬚C, 5% CO2 for 120 h. The culture medium was removed and the cells dosed with serial two-fold dilutions of coumaphos from 0.086 to 22 ␮M in serum- and phenol red-free RPMI medium (BioWhittaker, Walkersville, MD) diluted from a stock solution of 22 mM coumaphos in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO). Control cells were exposed to medium only. After 48 h exposure, the cells were counted and percentage viability determined by trypan blue exclusion as follows: the cells were resuspended with a Pasteur pipet and trypan blue solution (Sigma-Aldrich, St. Louis, MO) was added to a Þnal concentration of 0.004%. After 5 min, viable and nonviable cells were separately counted using a hemocytometer. The coumaphos LC50 was then determined using doses based on the results of the toxicity study. Boophilus microplus cells were seeded at a density of 8 ⫻ 105 cells per Nunclon delta ßat-sided tube in culture medium and maintained at 32⬚C, 5% CO2 for 72 h. All culture medium was removed and replaced with test doses of 0, 11, 15, 18, or 22 ␮M coumaphos in serumand phenol red-free medium containing 0.1% DMSO. A 77.18 mM coumaphos in DMSO stock solution was used for dilutions. The cultures were incubated at 32⬚C, 5% CO2 for 48 h. The cells were counted and percentage viability determined as described above. The percentage viability means were calculated and plotted according to coumaphos concentrations. The LC50 (dose at which 50% of the cells were nonviable) was estimated by curve Þtting regression analysis

Fig. 1. Schematic of the generation of coumaphos resistant B. microplus cell lines.

(CA-Cricket Graph III, version 1.01, Computer Associated International, Islandia, NY). Exposure of the cells to coumaphos was done as follows: the cells were grown in 3 ml culture medium in 25-cm2 culture ßasks (Corning Costar, Cambridge, MA). At 80% conßuency the cells were exposed to 27 ␮M coumaphos medium (initial LC50) in serum- and phenol red-free medium for 48 h. The medium was then replaced with 3 ml of fresh culture medium and the cells were fed new medium every 4 d until they recovered to 80% conßuency. The cells were exposed in this manner to 27 ␮M coumaphos two more times at intervals of 59 and 36 d, respectively. These treated cells were designated 4C27 (Fig. 1). The coumaphos LC50 for the 4C27 cells was determined as described above, except the cells were dosed with 0, 11, 22 and 27 ␮M coumaphos. The cells were then exposed to the new LC50 level, 34 ␮M coumaphos (1.25 times the original LC50), three times, following the above protocol, and then designated C34. As controls, cells were treated similarly with 0.1% DMSO in serum- and phenol red-free medium only. A second batch of cells was exposed to 27 ␮M coumaphos under the same conditions as described above for 4C27 cells, but 80% conßuency was achieved in 20 d after each exposure. These cells were designated 5C27. The coumaphos LC50 for these cells was determined as above. The cells were then exposed three times to 44 ␮M coumaphos (1.6 times the original LC50) according to the protocol above. These cells were designated C44. One-half of these cells was exposed three times to 54 ␮M coumaphos (two times the original LC50) following the protocol above. These cells were designated C54. Due to the inability

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to maintain higher concentrations of coumaphos in solution, this was the highest concentration that could be used to expose the cells. As controls, cells were treated similarly with 0.1% DMSO in serum- or with phenol red-free medium only. To determine the effect of DMSO concentration on the B. microplus cells treated with coumaphos, cells were dosed with 0, 6.75, 13.5, 27 ␮M coumaphos in serum- and phenol red-free medium containing 0.24% DMSO as described above. Similarly, to determine the effect of DMSO alone on the B. microplus cells, the cells were dosed with increasing concentrations of DMSO (0, 0.1, 0.2, 0.4%) in serum- and phenol red-free medium as described above. This study was done in duplicate. Throughout the above experiments, as 80% conßuency was achieved after each exposure to either coumaphos or DMSO, the cells were subcultured and a portion was cryopreserved in RPMI 1640 supplemented with 20% heat inactivated FBS and 7.5% DMSO. Untreated and DMSO control cells were maintained at passage levels equivalent to the coumaphos exposed cells throughout the series of experiments. The generated cell populations will be referred to as resistant (coumaphos-exposed), nonresistant controls (no coumaphos exposure) and DMSO controls (DMSO treated only). Duplication rate determination. Boophilus microplus cells were seeded at a density of 4 ⫻ 105 cells in 12 Nunclon delta ßat-sided tubes in culture medium and maintained at 32⬚C, 5% CO2 for 24 h. At day 0, 2, four and six the concentrations of viable cells in triplicate tubes were determined as described above. Esterase assay. Esterase activity levels were determined in the B. microplus cells using a previously described esterase assay protocol (Dary et al. 1990). Boophilus microplus cells were seeded at a density of 2 ⫻ 105 cells/well in 96 well clear bottom black plates (Corning Costar) and maintained in culture medium at 32⬚C, 5% CO2 for 24 h. All culture medium was removed and replaced with 0 or 11 ␮M coumaphos in 0.1% DMSO in serum- and phenol red-free RPMI 1640 medium. After 24 h at 32⬚C in 5% CO2 the cells were resuspended and centrifuged at 5220 RCF for 5 min, then resuspended in 100 ␮l of PBS with 1% Triton X-100. The suspension was sonicated using a Vibra Cell (Sonics and Materials, Danbury, CT) with 10 pulses. The supernatant was transferred to a microplate and serial two-fold dilutions (1:1, 1:2, 1:4) were made to a Þnal volume of 50 ␮l. A solution of 3 mM ␣-naphthyl acetate was prepared for use by adding 250 ␮l of ␣-naphthyl acetate stock (120 mM in ethanol) to 10 ml PBS with 1% Triton X100. The samples were incubated for 20 min with 100 ␮l of ␣-naphthyl acetate followed by the addition of 100 ␮l of 0.0008% Fast Garnet in PBS with 1%Triton X-100. The reaction was incubated for another 10 min and read at 540 nm with a MRX Microplate Reader (Dynatech, Chantilly, VA). Statistical Analysis. For all assays, resistant cells were compared with nonresistant and DMSO control cells developing a complete randomized design using

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Fig. 2. Preliminary analysis of coumaphos toxicity in B. microplus cells. Viability of tick cell cultures was determined after exposure to coumaphos for 48 h. Percentage viability versus coumaphos concentration are plotted. Means ⫾ SE are shown, n ⫽ 2.

the analysis of variance (ANOVA) procedure from the Statistical Analysis System software (SAS Institute 1988) to test for signiÞcant effects (P ⬍ 0.05) of coumaphos treatment. For those effects that were significant, tests for multiple comparisons of the means were developed to detect the means that differed using TukeyÕs procedure (Lentner and Bishop 1993). Before statistical analysis involving multiple comparisons, the percentage data were converted by arcsine square-root transformation (Lentner and Bishop 1993). Results The cultured B. microplus cells showed reduced viability when exposed for 48 h to coumaphos concentrations between one and 22 ␮M (Fig. 2). For the LC50 determination cells were exposed to increasing concentrations of coumaphos dissolved in 0.1 and 0.24% DMSO. The LC50 values for these cells were 27 ␮M coumaphos in 0.1% DMSO (Fig. 3A) and 14.8 ␮M in 0.24% DMSO (Fig. 3B). DMSO alone had no effect on cell viability with concentrations as high as 0.4% (Fig. 4). Two different batches of cells were exposed three times to 27 ␮M coumaphos in 0.1% DMSO: one at intervals of 59 and 36 d (4C27) and the second at intervals of 20 d (5C27) (Fig. 1). The LC50 values were 34 ␮M for 4C27 and 41.4 ␮M for 5C27 (Fig. 5). The cells were exposed three consecutive times to higher concentrations of coumaphos, 34 ␮M for 4C27 (C34) and 44 ␮M for 5C27 (C44) (Fig. 1). Finally, the C44 cells were exposed to 54 ␮M coumaphos three times and designated C54 (Fig. 1). The exposure level was limited to 54 ␮M due to the coumaphos solubility constraints imposed by using a DMSO concentration of 0.1%. The coumaphos LC50 results showed that nonresistant control cells and DMSO control cells (0.1% DMSO), lost viability in the presence of 27 ␮M coumaphos, a concentration tolerated by the coumaphos exposed cells C34, C44, and C54 (at 27 ␮M, F ⫽ 29.37; df ⫽ 4, 10; P ⬍ 0.001). The coumaphos LC50 results

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Fig. 5. Coumaphos LC50 curve for B. microplus cells after three sequential exposures to 27 ␮M coumaphos. Curve Þtting regression analysis was used to determine the LC50. LC50 of 27 ␮M for control, untreated cells (r ⫽ 0.999); LC50 for 34 ␮M for 4C27 cells (long intervals between doses) (r ⫽ 0.982) LC50 of 41 ␮M for 5C27 cells (short intervals between doses) (r ⫽ 0.998). Adjusted percentage viability (surviving cells at 0 ␮M coumaphos ⫽ 100%) versus coumaphos concentration are plotted. Means ⫾ SE are shown, n ⫽ 3.

Fig. 3. Determination of the initial coumaphos LC50 for B. microplus cells. Viability of tick cell cultures was determined after exposure to coumaphos for 48 h. Curve Þtting linear regression analysis was used to determine the LC50. (A) LC50 of 27 ␮M was obtained when cells were exposed to coumaphos in 0.1% DMSO, r ⫽ 0.990. (B) LC50 of 14.8 ␮M was obtained when cells were exposed to coumaphos in 0.24% DMSO, r ⫽ 0.971. Percentage viability versus coumaphos concentration are plotted. Means ⫾ SE are shown, n ⫽ 4.

P ⬍ 0.001 and at 22 ␮M, F ⫽ 14.53; df ⫽ 4, 10; P ⬍ 0.001) (Fig. 6). All cell lines were at similar passage levels when these viability curves were performed (C34, passage 50; C44, passage 49; C54, passage 58; nonresistant controls, passage 57; DMSO controls, passage 63). Exposure to coumaphos resulted in changes in the morphological appearance of the B. microplus cells. Upon initial exposure to 11 ␮M coumaphos, the control cells became smaller, darker in appearance and vacuolated. Cells that remained alive at higher coumaphos concentrations appeared larger with cytoplasmic vacuoles. Vacuolated cells and cells of different size appeared in the culture designated C34 at concentrations of 22 ␮M coumaphos in 0.1% DMSO, but

also showed that DMSO control cells (0.1% DMSO) lost viability in the presence of 11 and 22 ␮M coumaphos, concentrations that were tolerated by the coumaphos exposed cells C34, C44, and C54 and nonresistant control cells (at 11 ␮M, F ⫽ 12.88; df ⫽ 4, 10;

Fig. 4. Effect of different concentrations of DMSO on the viability of B. microplus cells after exposure to DMSO for 48 h. Percentage viability versus DMSO concentration are plotted. Means ⫾ SE are shown, n ⫽ 2.

Fig. 6. Coumaphos tolerance levels of B. microplus cells. All cultures were exposed to various levels of coumaphos for 48 h before viability determinations. Nonresistant control cells (solid squares); DMSO treated control cells (solid circles); C34 resistant cells (open circles); C44 resistant cells (open squares); C54 resistant cells (open triangles). Adjusted percentage viability (surviving cells at 0 ␮M coumaphos ⫽ 100%) versus coumaphos concentration are plotted. Means ⫾ SE are shown, n ⫽ 3.

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Fig. 7. Duplication rates in B. microplus cells. Cell concentrations over time were determined in nonresistant control cells (solid squares); DMSO treated control cells (solid circles); C34 resistant cells (open circles); C44 resistant cells (open squares); C54 resistant cells (open triangles). Adjusted cell concentrations (cell concentration at day 0 ⫽ 1) versus time in days are plotted. Means ⫾ SE are shown, n ⫽ 3.

unexpectedly these cultures regained normal morphology with the 27 ␮M coumaphos in 0.1% DMSO exposures. The C44 cultures contained cells with cytoplasmic vacuoles at 27 ␮M. However, the C54 cultures appeared normal at this concentration. Since all the cell lines were maintained at roughly equivalent passage levels, the passage level of the cell line was unlikely to be responsible for the differences observed in viability and morphology. The duplication rates of cultures of each cell treatment regimen were measured. The nonresistant controls, DMSO controls, and C34 cells, treated with lower coumaphos levels, had a duplication rate of 5 d. In contrast, the cells treated with higher coumaphos concentrations had a slower duplication rate of seven and 8 d for C44 and C54, respectively (Fig. 7). Esterase levels were evaluated in C34, C44 and C54 resistant, nonresistant and DMSO control cells after 24 h exposure to 0 or 11 ␮M coumaphos. Basal levels of esterase activity were signiÞcantly less in coumaphos resistant cells when compared with nonresistant cells (F ⫽ 502.95; df ⫽ 4, 15; P ⬍ 0.001). C34 was 91% lower and C44 was 76% lower than both the nonresistant and DMSO controls (Fig. 8). C54 was 76% and 74% lower than nonresistant and DMSO controls, respectively (Fig. 8). When the cells were exposed to coumaphos for 24 h before assaying for esterases, all of the cell lines had decreased esterase levels (F ⫽ 125.71; df ⫽ 4, 15; P ⬍ 0.001). Nonresistant cells were 96% lower, DMSO control cells were 98% lower, C34 cells were 100% lower, C44 cells were 12% lower, and C54 cells were 11% lower compared with their basal values. However, signiÞcantly higher esterase levels were present in C44 and C54 resistant cells compared with C34, nonresistant and DMSO control cells (P ⬍ 0.05)

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Fig. 8. Boophilus microplus cell basal esterase levels (dotted bars) and esterase levels after coumaphos exposure (diamond hatch). Esterase levels (relative absobance) was measured in nonresistant control cells, DMSO treated control cells, and C34, C44, and C54 resistant cells. Relative absorbance at 540 nm versus dilutions of cell extracts are plotted. Means ⫾ SE are shown, n ⫽ 4.

(Fig. 8). C44 cells exhibited esterase levels 76% and 89% greater than nonresistant and DMSO control cells, respectively. C54 cells exhibited esterase levels 78% and 89% greater than nonresistant and DMSO control cells, respectively. Discussion Since one of the earliest reports of acaricide resistance in B. microplus ticks in Australia (Lee and Batham 1966), OP resistance has been reported in Boophilus spp. all over the world (Fluck and Rufenacht 1969, Matthewson et al. 1976, Harris et al. 1988). Coumaphos resistance in B. microplus most likely is multifactorial as suggested by Bull and Ahrens (1988). Understanding the molecular and cellular processes underlying the development of acaricide resistance in ticks is key to improving the detection and prevention of acaricide resistant tick populations. Our approach is to develop acaricide resistant cell populations to study at the cellular level the processes involved in the resistance phenomenon. Previously, comparative mechanistic analyses of insecticides on insect and mammalian cell lines have been useful in evaluating insecticide toxicity in vitro (Meola et al. 1997). Permethrin is a synthetic pyrethroid insecticide with low toxicity to mammals used extensively worldwide to control agricultural and medically important insect pests. It acts primarily on insect nerve cells by modulating voltage-activated sodium channels so that the time they remain open is prolonged (Narahashi 1992). Meola et al. (1997) showed a sensitivity to permethrin by epithelial cell lines from Anopholes gambiae (Giles) and Aedes albopictus (Skuse) mosquito larvae and a differential sensitivity by insect and mammalian nonexcitable cells, comparable to in vivo observations. Similarly, we found that the OP acaricide coumaphos, which inhibits acetylcholinesterases in insects (Hemingway and Karunartne 1998), induced cellular toxicity in embryonic B. microplus VIIISCC tick cell lines. Moreover, we found that after

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repeated exposure, cells showed an adaptation to acaricide assault and became resistant to increasingly higher doses. Since the B. microplus VIII-SCC tick cell line is not a cloned population, further studies will be needed to determine the actual mechanism of selection that is in play. It is possible that the selection for resistance is a selection of a resistant phenotype that was present in the cells from the onset of the experiment. Hence an LC50 of 27 ␮M coumaphos was observed in the original cell population. It is also possible that the acaricide exposure induced mutations in the cells, some of which were adaptive for survival in the presence of the OP, thus these cells continued to proliferate and express a resistant phenotype. The initial LC50 for B. microplus cells was 27 ␮M coumaphos in 0.1% DMSO and 14.8 ␮M in 0.24% DMSO. DMSO-enhanced penetration of substances across biological membranes has been reported (Brayton 1986) and may potentiate the effect of OP. Thus, the observed difference in sensitivity was probably due to enhanced penetration of the acaricide into the cell in the presence of higher concentrations of DMSO. DMSO alone in concentrations up to 0.4% showed little, if any, toxicity to the cell culture. To lessen the possible effects of DMSO on the cell cultures, all experiments were done with 0.1% DMSO, which was the lowest concentration that would maintain the solubility of coumaphos in the culture medium used. Boophilus microplus VIII-SCC OP resistant cell lines developed from incrementally increased exposure to coumaphos. Repeated exposure of B. microplus cells to coumaphos increased the tolerance of the cells, and the frequency of exposure was an important factor in the development of the OP tolerance. The shorter interval between exposures resulted in cells with a higher LC50. The elucidation of the biochemical basis for drug resistance which has been facilitated by in vitro development of mammalian cell lines that exhibit altered hormone and drug responsiveness (Moore et al. 1994, Simon and Shindler 1994, Chang et al. 1997) supports the potential of acaricide resistant cell lines as in vitro models to detect cellular adaptations to the acaricide. It has been suggested that tumors are extra-sensitive to drugs because their replication rates are higher than those of normal cells (Simon and Schindler 1994). This may explain their increased sensitivity to drugs that affect DNA replication and the cytoskeleton of the cell, which are the primary targets. However, the higher sensitivity may simply reßect higher intracellular drug concentration, since chemotherapeutic drugs accumulate in tumor cells at higher concentrations than in normal cells. Drug resistance may subvert the same mechanisms that make tumor cells hypersensitive. If tumor cells are more sensitive because of changes in their cell cycle program, then multiple drug resistance may result from cells remaining longer in a particular stage of the cell cycle (Simon and Schindler 1994). These points may explain the observation that replication rates in cells resistant to higher concentrations of coumaphos were slower than those of the

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untreated and DMSO control cells and the cells treated with reduced OP concentrations. In vivo, nonspeciÞc esterase activity has been found to be increased in OP resistant B. microplus (Miranda et al. 1995, Rosario-Cruz et al. 1997), and these enzymes also have been associated with resistance toward pyrethroid acaricides (De Jersey et al. 1985). Unexpectedly, basal esterase levels were signiÞcantly decreased in the resistant cell lines compared with the controls. When the cells were exposed to coumaphos immediately before the assay, decreased esterase levels compared with the basal levels resulted in all the cell lines, suggesting the presence of type B esterases, which are known to be sensitive to inhibition by organophosphates. However, resistant cell lines C44 and C54 exhibited signiÞcantly higher levels of esterases after OP exposure than C34 or the control cells, indicating induction of sufÞcient type B esterases in C44 and C54 to remain at detectable levels despite the presence of coumaphos. Alternatively, C44 and C54 may produce other esterases that use the ␣-naphthyl acetate but are not inhibited by OP. The increased levels of nonspeciÞc esterases found in the resistant cells after OP exposure suggest that similar resistance mechanisms may be induced both in vitro and in vivo, since elevated levels of nonspeciÞc esterases are found in OP resistant B. microplus strains as well (Miranda et al. 1995, Rosario-Cruz et al. 1997). The Þnding of similar resistance mechanisms in vitro and in vivo validates the use of acaricide resistant cell lines to study the phenomena of resistance development in the tick. Acknowledgments We are grateful for the extensive assistance provided by Andres Ducoing in the statistical analyses in this work. The senior author was supported for the duration of this study by a scholarship provided by the Consejo Nacional De Ciencia y Technologia. This research was supported by the Texas A&M University Texas Agricultural Experiment Station (project H6261) and by USDA Formula Animal Health Grant 1433.

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