Genetics and Mechanisms of Permethrin Resistance in the Santa Luiza Strain of Boophilus Microplus (Acari: Ixodidae)

VECTOR CONTROL,PEST MANAGEMENT,RESISTANCE,REPELLENTS Genetics and Mechanisms of Permethrin Resistance in the Santa Luiza Strain of Boophilus microplus (Acari: Ixodidae)

ANDREW Y. LI,1,2 RONALD B. DAVEY,3 ROBERT J. MILLER,3 1 1 FELIX D. GUERRERO, AND JOHN E. GEORGE

J. Med. Entomol. 45(3): 427Ð438 (2008) ABSTRACT The Santa Luiza strain of the southern cattle tick, Boophilus microplus (Canestrini) (Acari: Ixodidae), is resistant to both permethrin and amitraz. A study was conducted at the USDA Cattle Fever Tick Research Laboratory in Texas to investigate the genetic basis of permethrin resistance with cross-mating experiments, and to determine the mechanisms of permethrin resistance through synergist bioassays and biochemical analysis of esterase proÞles. The Mun˜ oz strain, an acaricide-susceptible reference strain, was used as the susceptible parent and the Santa Luiza strain, originating in Brazil, was used as the resistant parent. The Food and Agriculture Organization larval

packet test was used to measure the levels of susceptibility of larvae of the parental strains, F1, backcross, F2, and F3 generations to permethrin. Results of reciprocal crossing experiments suggested that permethrin resistance was inherited as an incomplete recessive trait. There was no signiÞcant

maternal effect on larval progenyÕs susceptibility to permethrin in the F1 and subsequent generations. Ϫ Ϫ The values of the degree of dominance were estimated at 0.700 and 0.522 for the F1 larvae with resistant and susceptible female parents, respectively. Results of bioassays on larval progeny of the F1 backcrossed with the resistant parent strain and of the F2 generations suggested that one major gene was responsible for permethrin resistance in the Santa Luiza strain. Selection of F3 larvae with either permethrin or amitraz led to signiÞcantly increased resistance to both permethrin and amitraz, indicating a close linkage between genes responsible for permethrin and amitraz resistance. The possible involvement of metabolic enzymes in permethrin resistance in the Santa Luiza strain of B. microplus was dismissed by the lack of enhanced synergism by TPP or PBO, as observed in synergist bioassays, as well as by the lack of enhanced esterase activity in the Santa Luiza strain relative to the susceptible strain. The results of this study suggest that other mechanisms, including a possible new sodium channel mutation that is different from the one currently known, may be responsible for permethrin resistance in the Santa Luiza strain of B. microplus.

KEY WORDS acaricide, inheritance, southern cattle tick, Boophilus microplus

Resistance to chemical acaricides remains a world- may be infested with B. microplus (Graham and Hour- wide problem for the control of the southern cattle rigan 1977, George 1996). In addition to coumaphos, tick, Boophilus microplus (Canestrini) (Acari: Ixodi- an organophosphate (OP) acaricide, which is used dae), an important ectoparasite of cattle and a major exclusively for the dipping treatment of all Mexican vector of Babesia spp., which causes bovine babesiosis cattle at the ports of entry, other acaricides, including or cattle fever disease (Bram and George 2000, George pyrethroids and amitraz, also have been used to elim- et al. 2004). Although B. microplus was eradicated inate outbreaks of ticks in the quarantine zone along from the United States in the 1940s after several de- the U.S.ÐMexican border in southern Texas. Due to the cades of intensive efforts, the USDA has since main- heavy use of chemical acaricides, B. microplus has tained an active Cattle Fever Tick Eradication Pro- developed resistance to all major classes of acaricides gram (CFTEP) to prevent the reestablishment of this that have been used in Mexico (Miller et al. 1999; Li pest into the United States from Mexico through cattle et al. 2003, 2004). Resistance to chemical acaricides in importation, stray cattle, or other wild animals that Mexican populations of B. microplus poses a serious threat to the continued success of the CFTEP (George et al. 2004). This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recom- In Mexico, B. microplus Þrst developed resistance to mendation by the USDA for its use. OP acaricides in the 1980s (Aguirre et al. 1986). Py- 1 USDAÐARS, Knipling-Bushland U.S. Livestock Insects Research rethroid acaricides and amitraz were introduced to Laboratory, 2700 Fredericksburg Rd., Kerrville, TX 78028. control OP-resistant ticks in the late 1980s. Resistance 2 Corresponding author, e-mail: [email protected]. 3 USDAÐARS, Cattle Fever Tick Research Laboratory, 22675 N. to pyrethroid acaricides emerged in the early 1990s MooreÞeld Rd., Edinburg, TX 78541. (Fragoso et al. 1995), and resistance to amitraz also 428 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 3 was detected for the Þrst time in 2001 (Soberanes et this tick strain. We describe here the genetic basis and al. 2002). Many of the Mexican tick populations be- mechanisms of permethrin resistance, as well as the came resistance to multiple acaricides (Santamarõ´aet linkage relationship between permethrin and amitraz al. 1999, Rodriguez-Vivas 2003, Li et al. 2004). Signif- resistance in the Santa Luiza strain of B. microplus. icant progress has been made in determination of mechanisms of resistance to OPs and pyrethroids in Materials and Methods the past decade (Li 2004). Enhanced metabolic de- toxiÞcation and insensitive target site are two major Tick Strains. Four strains of B. microplus were used mechanisms of resistance to pyrethroid acaricides in for investigating permethrin resistance mechanisms B. microplus (Pruett 2002; Guerrero et al. 2001, 2002; and genetics in this study. The Santa Luiza strain is a Li 2004). Toxicological and biochemical studies have resistant tick strain collected from a ranch in Brazil, led to the isolation and characterization of a pyre- and it was maintained at the Mexican National Para- throid-detoxifying esterase, CzEst9, which is respon- sitology Laboratory, Jiutepec, Morelos, Mexico, be- sible for moderate resistance in some of the tick strains fore being established at the USDA Cattle Fever Tick originating from Mexico (Miller et al. 1999, Pruett Research Laboratory (CFTRL) in Edinburg, TX, in 2002). Molecular studies have led to the identiÞcation 2000. The Santa Luiza strain is resistant to both per- of a sodium channel mutation that is responsible for methrin and amitraz, and it was used as a resistant high-level pyrethroid resistance in Mexican strains of parent strain in this study. The Mun˜ oz strain is a B. microplus (He et al. 1999; Guerrero et al. 2001, susceptible laboratory strain that was established at 2002). the CFTRL in 1999 from an outbreak of B. microplus Successful management of pesticide resistance re- ticks in Zapata County, TX. The Mun˜ oz strain was quires better understanding of the genetic, biological, susceptible to all major classes of acaricides; conse- and operational factors that inßuence the evolution of quently, it was used as the susceptible parental strain pesticide resistance in pest populations (Georghiou to cross with the resistant Santa Luiza strain in this and Taylor 1986). The genetic components of resis- study. The other strains of B. microplus used in the tance include the number and initial frequency of synergist bioassays were the Pesqueria strain and the resistance alleles, dominance of resistance alleles, in- San Felipe strain that originated in Mexico, and they tensity of selection, and relative Þtness of genotypes are maintained at the USDAÕs CFTRL. The Pesqueria (Georghiou and Taylor 1986). The genetic basis of B. strain is resistant to multiple acaricides and the San microplus resistance to earlier acaricides, such as or- Felipe strain is highly resistant to pyrethroids (Miller ganochlorine and organophosphate acaricides, has et al. 1999; Li et al. 2003, 2004). been well studied (Stone 1962, Wilson et al. 1971, Cross-Mating Experiments. Details of the proce- Stone et al. 1973, Stone and Youlton 1982). Resistance dures for tick rearing and cross-mating experiments to organophosphate compounds was found to be con- were described previously (Li et al. 2005). The indi- ferred by one or several closely related genes. These vidually tagged Hereford heifer calves had no prior resistant genes were found to be autosomal with in- exposure to Boophilus ticks, and they were randomly complete dominance in these studies. Similarly, resis- assigned to be infested with one of the tick strains or tance to organochlorine was found to be inherited as genotypes at a particular time throughout the course a single, near-complete dominant gene in two other of this study. The heifers were individually stan- tick species (Lourens 1979, 1980). A recent study on chioned in a covered, open-sided barn with walls sep- resistance to ßumethrin (a pyrethroid) in a Mexican arating each calf to prevent engorged ticks from es- strain of B. microplus demonstrated that resistance to caping. ßumethrin was controlled by more than one gene, and Parental Strains. Two heifers were Þrst infested expressed as a recessive or dominant trait, depending with Ϸ10,000 larvae derived from 0.5 g of eggs each on the ßumethrin concentration exposed (Tapia- from the Mun˜ oz (genotype, SS) and Santa Luiza Perez et al. 2003). strains (genotype, RR), respectively. Approximately We have previously characterized the mode of in- 250 metanymphs were removed from each host at heritance of amitraz resistance in the Santa Luiza 13Ð14 d postinfestation. The metanymphs of each strain of B. microplus (Li et al. 2005). As the Santa strain were placed collectively in separate vials placed Luiza strain also demonstrated a relatively high level in an incubator at 30 Ϯ 2ЊC and 92.5% RH to allow of resistance to permethrin, we also conducted per- molting to adults. The adults were separated by sex methrin bioassays for the parental, F1 from reciprocal within 24 h of molting, and they were used in recip- crosses of the parental strains, backcross, F2 and sub- rocal crosses. The ticks left on the animals were al- sequent generations, to determine the genetic basis of lowed to develop to repletion. Engorged females were permethrin resistance. The linkage relationship be- collected and placed in individual vials for laying eggs tween permethrin and amitraz resistance in this re- in a separate incubator. A portion of eggs from indi- sistant tick strain also was tested through selection of vidual females were mixed together, and larvae

F3 generation larvae pressured with either permethrin hatched from mixed eggs were used for bioassays or amitraz, and subsequent bioassays using the F4 when they reached 14Ð16 d old. generation larvae. Synergist bioassays and biochemi- Reciprocal Crosses. Eighty pair of Mun˜ oz males and cal analysis of esterases were also conducted to de- Santa Luiza females (type-I cross) were placed on one termine the mechanisms of permethrin resistance in heifer with four stockinette sleeves, each containing May 2008 LIETAL.: PERMETHRIN RESISTANCE IN B. microplus 429

20 mating pair. A second heifer was similarly infested [AI]) used in this study was a product of NOR-AM with 80 pair of Santa Luiza males and Mun˜ oz females Chemical Company (Wilmington, DE). The syner- (type-II cross). Engorged females were collected from gists used in this study include triphenylphosphate each crossing type, and individual females were (TPP, an inhibitor of esterases), piperonyl butoxide placed in a vial for laying eggs in an incubator. A (PBO, an inhibitor of cytochrome P450 monooxygen- portion of the egg mass from each female was added ases), and diethyl maleate (DEM, an inhibitor of glu- to a vial of mixed eggs of the same crossing type and tathione transferases), which were purchased from the resulting F1 larvae were used for bioassays. Aldrich Chemical (Milwaukee, WI). Backcrosses. Two heifers were each infested with Bioassay Techniques. A modiÞed version of the lar- Ϸ 5,000 F1 larvae derived from 0.25 g of mixed eggs val packet test (LPT) recommended by FAO (1971) from engorged females from one of the two crossing was used to determine permethrin toxicity to tick types. A third heifer was infested with Ϸ5,000 Santa larvae, levels of permethrin resistance, as well as the Luiza strain larvae derived from 0.25 g of mixed eggs. effect of synergists on permethrin toxicity (Miller et Approximately 250 metanymphs were removed from al. 1999). Larvae used for all bioassays were 12Ð16 d the heifer infested with one of the two F1 larval types, old. A stock solution of permethrin was made by dis- and Ϸ500 metanymphs were removed from the heifer solving technical grade permethrin in trichloroethyl- infested with the Santa Luiza strain larvae. The ene (Sigma-Aldrich, St. Louis, MO). The top concen- metamymphs of each type were collectively placed in tration (10%) was prepared by adding a volume of the separate vials in an incubator to allow molting to stock solution to a mixture of trichloroethylene and adults. Only the F1 type-II was used for reciprocal olive oil (Sigma-Aldrich), with a Þnal 2:1 ratio. Serial backcrosses with the Santa Luiza strain. The backcross dilutions from the top concentration were made using between F1 type-II males and Santa Luiza females was a diluent of two parts trichloroethylene and one part designated as type-A (genotype, SR), and the back- oil. Between nine and 12 concentrations of permethrin cross between Santa Luiza males and F1 type-II fe- were tested. When the effect of a synergist on per- males was designated as type-B (genotype, RS). Two methrin toxicity was evaluated, the synergist was additional heifers were each infested with one of the added into the diluent at a constant rate of 1%, the backcrossing types in four sleeves, each containing 20 highest rate at which no larval mortality in B. microplus pairs of males and females. The engorged females from was observed when applied alone. A volume of 0.7 ml each of the backcrossing types were collected and of each dilution was applied to a Whatman No. 1 Þlter placed in individual vials for laying eggs. Larvae from paper (7.5 by 8.5 cm, Whatman, Maidstone, Kent, mixed eggs of each backcrossing type were obtained United Kingdom). Three Þlter paper replicates were for bioassays as described above. prepared for each dilution. Treated Þlter papers were F and F Generations. The metanymphs of both F 2 3 1 placed in a fume hood for2htoallow trichloroeth- types that were left on the heifers (see above) were allowed to molt, inbreed and complete development ylene to evaporate before being folded in half and on two separate heifers. The engorged females were sealed with bulldog clips on both sides. Approximately collected and placed in an incubator for laying eggs. 100 larvae were placed into each packet, and the top was sealed immediately with another bulldog clip. The F2 larvae from mixed eggs oviposited by engorged females of each of the F crosses were tested for Packets were then held in an environmental chamber 1 Ϯ Њ susceptibility to permethrin as described below in 2.4. at 27 2 C, 90% RH for 24 h. Packets were removed Similarly, the larvae of the F type-II cross were reared from the environmental chamber, and mortality was 2 determined by counting live and dead larvae. to the F3 generation and tested for susceptibility to permethrin. A modiÞed FAO LPT was used for all amitraz bioassays in this study (Miller et al. 2002; Li et al. 2004, Acaricide Selection and the F4 Generation. A subset Ϸ 2005). Pieces (7.5 by 8.5 cm) of nylon fabric (Type ( 15,000) of the F3 generation larvae was challenged with 1.25% permethrin by exposing larvae in treated 2320, Cerex Advanced Fabrics, Pensacola, FL), in- Þlter paper packets (50 papers, Ϸ300 larvae each) for stead of the Whatman Þlter papers, were used as the 24 h in an incubator. The mortality was counted in 10 substrate. randomly chosen packets after 24 h, and survivors Larvae of four different genotypes, backcross type-I from all packets were placed on a heifer to complete and type-II; F2 type-I and type-II, were subjected to a discriminating concentration (0.5%) of permethrin to feeding and development. The F4 generation larvae from engorged females were used for bioassays to determine mortality. In total, 30 treated Þlter packets, determine their susceptibility to both permethrin and with Ϸ100 larvae each, were prepared for each geno- Ϸ amitraz. A second subset ( 15,000) of F3 generation type. The mortality was determined after 24 h, and larvae was similarly challenged with 0.1% amitraz, compared with the expected mortality based on the mortality determined, and survivors reared on another genotype composition of the tick samples tested. heifer. The F4 generation larvae from the engorged Gel Electrophoresis. Native esterase activity gel females also were used for bioassays to determine their electrophoresis was performed using extracts from susceptibility to both permethrin and amitraz. pools of 80 tick larvae extracted in 100 ␮l of extraction Chemicals. Technical-grade permethrin [92.2% ac- buffer and detecting ␣- and ␤-esterase activity as de- tive ingredient (AI)] was obtained from FMC (Phil- scribed in Jamroz et al. (2000). Protein concentration adelphia, PA). The formulated amitraz (Taktic, 12.5% was determined using the DC Protein Assay kit (Bio- 430 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 3

Table 1. Permethrin concentration–mortality responses of the parental strains, F1, backcross, F2, and F3 generations of B. microplus

Tick strain/crossing Genotypes of Slope Bioassay results n RF type larval progenya (SE) ␹2 (df) LC50 (95% CI) Muno˜z SS 1,639 3.22 (0.16) 32.80 (19) 0.0447 (0.0401Ð0.0496) 1.0 Santa Luiza RR 1,681 5.44 (0.22) 98.16 (16) 4.1538 (3.7793Ð4.5708) 92.9 F1 type-I SR 2,673 3.69 (0.31) 196.61 (25) 0.0883 (0.0605Ð0.1075) 2.0 F1 type-II RS 1,761 3.34 (0.20) 131.10 (25) 0.1320 (0.1027Ð0.1645) 3.0 Backcross type-II (A) SR, RR 3,107 1.43 (0.06) 147.84 (40) 0.7347 (0.5838Ð0.9073) 16.4 Backcross type-II (B) RS, RR 2,598 1.22 (0.05) 184.76 (40) 0.4437 (0.3234Ð0.5902) 9.9 F2 type-I SS, SR, RS, RR 4,145 1.41 (0.04) 219.71 (40) 0.2171 (0.1727Ð0.2683) 4.9 F2 type-II SS, SR, RS, RR 4,189 1.27 (0.03) 250.36 (43) 0.1763 (0.1375Ð0.2225) 3.9 F3 type-II SS, SR, RS, RR 3,283 1.15 (0.04) 193.75 (34) 0.1252 (0.0921Ð0.1684) 2.8

a Genotype designation: SS, susceptible homozygote; RR, resistant homozygote; SR, heterozygote with susceptible male and resistant female parents; RS, heterozygote with resistant male and susceptible female parents.

Rad, Hercules, CA), and 8.8 ␮g of total protein was inbred of the type-I cross are summarized in Table 1. loaded into each lane of the gel. The LC50 values of the susceptible parental strain Data Analysis. The concentrationÐmortality re- (Mun˜ oz) and the resistant parental strain (Santa sponses of all bioassays were analyzed using the Luiza) were measured at 0.0447 and 4.1538%, respec- POLO-PC program (LeOra Software 1987). Mortality tively. In comparison with the Mun˜ oz strain, the Santa data of all three replicates of each concentration were Luiza strain had a RF of 92.9 to permethrin, with a included in probit analysis. Resistance factor (RF) was relatively steep slope (5.44), indicating a homogenous calculated by dividing the LC50 of the Santa Luiza resistant strain. strain, F1, backcrosses, F2,F3, and F4 generations with The LC50 of F1 larvae from the reciprocal cross the LC50 of the reference Mun˜ oz strain. The same type-I and -II were 0.0883 and 0.1320%, with a resis- method also was used to calculate the RF for other tick tance factor of 2.0 and 3.0, respectively, which were strains. The synergism ratio (SR) of a synergist was signiÞcantly higher than that of the susceptible Mun˜ oz calculated by dividing the LC50 of the bioassay using strain and lower than that of the resistant strain. Both permethrin alone with the LC50 using both per- LC50 values of the F1 generations were closer to that methrin and the synergist. Differences between LC50 of the Mun˜ oz strain than to the Santa Luiza strain estimates were designated as signiÞcant when their (Table 1; Fig. 1). The value of the degree of domi- Ϫ Ϫ 95% conÞdence intervals (CI) did not overlap. nance was 0.700 and 0.522 for the F1 larvae from The degree of dominance (D) of permethrin resis- type-I and type-II crosses, respectively, suggesting tance trait in the F1 larvae from both reciprocal crosses that resistance to permethrin was inherited as an in- ϭ Ϫ Ϫ was estimated using the formula D (2X2 X1 complete recessive trait. Ϫ X3)/(X1 X3), where X1 is the log of the LC50 of the No signiÞcant difference was found between LC50 resistant strain, X2 is the log of the LC50 of the F1, and values of F1 larvae from the reciprocal crosses (type-I, X3 is the log of the LC50 of the susceptible strain and -II). Although the LC50 value of backcross type-II (Stone 1962). The expected mortality at each amitraz A was higher than that of the type-II B, the difference concentration for larvae resulted from reciprocal was not signiÞcant. Similarly, no signiÞcant difference backcrosses between F1 type-II and the resistant strain was found between LC50 values of the F2 type-I and (Santa Luiza), estimated on the basis of a single major F2 type-II larvae, and also between F2 type-II and F3 gene, was calculated using the formula X ϭ type-II larvae (Table 1). The data suggest that resis- ϩ (0.5)W(F1) (0.5)W(R strain), where X is the expected tance to permethrin in the Santa Luiza strain is con- larval mortality at the given concentration and W is trolled by an autosomal gene that is not sex-related. the mortality derived from their respective response Figure 2 illustrates the observed mortalities of lar- lines of the parental types at the given concentration vae from reciprocal backcrosses between males and

(Stone 1962, 1984). The relationships between the females of the F1 type-II and the resistant parent observed and expected mortality in both backcross (Santa Luiza strain) and the expected mortalities, were analyzed by chi-square goodness-of-Þt analysis which were calculated assuming monogenic inheri- (Tabashnik 1991). tance. The backcross type-A resulted from mating

between males of the F1 type-II and females of the Santa Luiza strain, and the backcross type-B resulted Results from mating between males of the Santa Luiza strain

Cross-Mating Experiments. The genotypes and and females of the F1 type-II. No signiÞcant difference concentrationÐmortality responses of larvae from the was detected between the observed and expected susceptible and resistant parent strains, the F1 from mortalities obtained in larval progeny from backcross reciprocal crosses (type-I and -II) between the pa- type-A (P ϭ 0.31Ð0.98) at the concentration of 1.681% rental strains, reciprocal backcrosses between the F1 and lower, except at 0.078% where the observed mor- Ͻ type-II and the resistant Santa Luiza strain, the F2 and tality was signiÞcantly lower than expected (P 0.01). F3 inbred generations of the type-II cross, and the F2 The observed mortality was signiÞcantly higher than May 2008 LIETAL.: PERMETHRIN RESISTANCE IN B. microplus 431

SS SR RS RR 99 Santa Luiza (RR) Munoz (SS)

90 F1 type I (SR) F1 type-II (RS)

30 Mortality (%)

1 0.01 0.1 1 10 Permethrin Concentration (% a.i.) Fig. 1. Permethrin concentration-mortality responses of the parental (Mun˜ oz and Santa Luiza) strains and two types of F1 generation resulting from reciprocal crosses between the parental strains. expected at the concentration of 2.5% and higher (P Ͻ values in larval progenies of backcross type-A and 0.01). For the larval progeny of backcross type-B, the type-B at some of the concentrations tested suggests observed mortality was signiÞcantly higher than ex- the involvement of other minor modifying genes in pected (P Ͻ 0.01), except at the concentrations of permethrin resistance in the Santa Luiza strain. Ͼ 0.626 and 1.25% permethrin (P 0.05). Table 2 shows F2 and F3 Generations. Fig. 3A shows the probit the results of a discriminating bioassay using a per- lines of the parent generations, the F1 hybrids (type-I methrin concentration (0.5%) that would kill all SR or and type-II) and the observed mortality of bioassays

RS heterozygotes. The mean mortalities of bioassays for F2 generation type-I and type-II. Very similar with larval progenies of both backcross type-A and doseÐmortality responses were observed for both type-B were not signiÞcantly different from the ex- types of the F2 generation, and there was no signiÞcant pected 50% mortality. The results suggest that one difference between the LC50 values (Table 1). Mor- major recessive gene is involved in permethrin resis- tality reached a plateau near 75% at permethrin con- tance in the Santa Luiza strain of B. microplus. The centrations between 0.2 and 2%, which would likely deviation of observed mortalities from the expected kill all SS, SR, and RS genotypes, but not the RR

SS RS RR 99

predicted observed BC-A 90 observed BC-B

30 Mortality ($)

1 0.01 0.1 1 10 Permethrin Concentration (% a.i.) Fig. 2. Comparisons between the observed and predicted mortalities in larval progenies of reciprocal backcrosses between the F1 type-II (RS) and the resistant parent strain (Santa Luiza, RR). BC-A represents backcross between the F1 type-II males and the resistant parent (Santa Luiza) females. BC-B represents backcross between the resistant parent (Santa

Luiza) females and the F1 type-II males. The arrow indicates the permethrin concentration (0.5%) used for discriminating RR from SS, SR or RS genotypes (see Table 2). 432 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 3

Table 2. Bioassays with discriminating concentration of per- F2 generation discriminating concentration bioassays methrin in the backcross and F2 generations provided further support for the involvement of single major recessive gene in permethrin resistance. Larval Permethrin Mean mortality Generation n genotypea concn (%) % (SD) Increase of Resistance in F4 Larvae as a Result of Selections of F Type-II Larvae. F type-II had a sim- Backcross SR, RR 0.5 30 54.0 (6.7) 3 3 type-II (A) ilar permethrin concentrationÐmortality response to Backcross RS, RR 0.5 29 50.3 (7.0) that of F2 type-II, and the LC50 value was 0.1763 and type-II (B) 0.1252% for F2 and F3, respectively (Fig. 3B; Table 1). F2 type-I SS, SR, RS, RR 0.5 30 76.5 (5.1) Selection of F type-II larvae with 1.25% permethrin F type-II SS, SR, RS, RR 0.5 30 75.9 (6.8) 3 2 resulted in a mean mortality of 77.9%. The larval prog- a Genotype designation: SS, susceptible homozygote; RR, resistant enies, designated as F4 type-II permethrin-selected, of homozygote; SR, heterozygote with susceptible male and resistant the F3 engorged females that developed on the host female parents; RS, heterozygote with resistant male and susceptible from larvae that survived permethrin selection had a female parents. signiÞcantly increased permethrin resistance level ϭ (RF 82.2, Table 3). Selection of F3 type-II larvae genotype (Fig. 3A). The expected mortality for per- with 0.1% amitraz resulted in a mean mortality of methrin concentrations was 75%. A discriminating 83.6%. The larval progenies, designated as F4 type-II concentration (0.5%) of permethrin led to a mean amitraz-selected, of the F3 engorged females that de- mortality of 76.5 and 75.9% in the F2 type-I and F2 veloped on a host from larvae that survived amitraz type-II larvae, respectively (Table 2). The results from selection also had a signiÞcantly increased permethrin

SS SR RS RR 99 A

30 Mortality (%) Mortality

F2 type-I F2 type-II 1 0.01 0.1 1 10

SS RSF4 RR 99 B

30 Mortality (%)

10 F3 type-II F4 permethrin-selected F4 amitraz-selected 1 0.01 0.1 1 10 Permethrin Concentration (% a.i.)

Fig. 3. Observed mortalities in the larval progenies of F2,F3 and F4 generations. (A) Observed mortalities of F2 type I and type II, resulting from inbreeding of F1 type-I and F1 type-II. The arrow indicates the permethrin concentration (0.5%) used for discriminating RR from SS, SR or RS genotypes (see Table 2). (B) F3 type II and F4 generations resulted from selection with discriminating concentrations of permethrin (1.25% [AI]) and amitraz (0.1% [AI]). May 2008 LIETAL.: PERMETHRIN RESISTANCE IN B. microplus 433

Table 3. Summary of concentration–responses to permethrin and amitraz in the parental strains and the F4 type-II larvae reared from selection of the F3 type-II larvae that were selected with either permethrin or amitraz

Bioassay results Acaricide Permethrin Amitraz Tick strain/generation n Slope LC50 (95% CI) tested ␹2 (df) RF RF (SE) Muno˜z(SS)a Permethrin 1639 3.22 (0.16) 32.80 (19) 0.0447 (0.0401Ð0.0496) 1 Amitraz 2005 1.57 (0.07) 144.25 (19) 0.0024 (0.0015Ð0.0036) 1 Santa Luiza (RR)a Permethrin 1681 5.44 (0.22) 98.16 (16) 4.1538 (3.7793Ð4.5708) 92.9 Amitraz 1001 4.60 (0.40) 50.62 (22) 0.4519 (0.3928Ð0.5076) 188.0 F4 type-II Permethrin-selectedb Permethrin 3651 2.91 (0.09) 179.9 (25) 3.6746 (3.1029Ð4.5784) 82.2 Amitraz-selectedb Permethrin 2158 3.47 (0.13) 145.9 (25) 3.2163 (2.7146Ð4.0361) 71.9 Permethrin-selectedb Amitraz 2311 0.77 (0.08) 81.9 (25) 0.3060 (0.1759Ð0.9346) 127.5 Amitraz-selectedb Amitraz 2362 1.17 (0.08) 96.0 (18) 0.3971 (0.2916Ð0.6215) 165.0

a Genotype designation: SS, susceptible homozygote; RR, resistant homozygote. b Selection of F3 type-II larvae with either permethrin or amitraz.

ϭ resistance level (RF 71.9, Table 3). The LC50 values somal, multigenic, and incompletely recessive trait in were indeed not signiÞcantly different from each the house ßy (Liu and Yue 2001, Shono et al. 2002), other, and the probit lines were almost identical (Fig. whereas a single, incompletely recessive, sex-linked 3B). A similar increase in the level of resistance to gene was involved in the horn ßy (McDonald and amitraz was also observed in the F4 larvae from the F3 Schmidt 1987). Resistance to permethrin was similarly engorged females developed from permethrin- or ami- inherited as an autosomal, multigenic, and incom- traz selected F3 type-II larvae (Table 3). Again, the pletely recessive trait in a predatory mite species, difference in the amitraz LC50 value between the Amblyseius fallacies (Garman) (Thistlewood et al. permethrin- and amitraz-selected F4 larvae was not 1995). Results of a more recent study in Mexico indi- signiÞcantly different (Table 3). Although being cate resistance to ßumethrin in a Mexican strain of B. slightly lower, the permethrin and amitraz LC50s and microplus also was autosomal and controlled by mul- RFs observed in either permethrin- or amitraz se- tiple genes (Tapia-Perez et al. 2003). It was suggested lected F4 generation were not signiÞcantly different that ßumethrin resistance could be expressed as a from those of the resistant parent (Santa Luiza strain, recessive or dominant trait, depending on the ßu- Table 3). methrin concentration to which ticks were exposed Levels of Permethrin Resistance and Synergism Ra- (Tapia-Perez et al. 2003). The results of the current tios in Different Tick Strains. Compared with the study revealed a different mode of inheritance for susceptible reference strain (Mun˜ oz), the RF in the permethrin resistance in B. microplus. Pesqueria and San Felipe strains was 48 and 285, re- In this study, it was determined that resistance to spectively (Fig. 4A). TPP (1%) signiÞcantly syner- permethrin in the Santa Luiza strain of B. microplus gized permethrin toxicity in all four tick strains tested. was inherited as an incomplete recessive trait involv- The SR ranged from 4.1 to 9.1 (Fig. 4B). PBO (1%) also ing a single major autosomal gene. Other minor genes signiÞcantly synergized permethrin toxicity, with SR might also exist that have some modifying effects on ratios ranging from 3.4 to 6 (Fig. 4C). DEM (1%) had the expression of the major resistance gene, as dem- no signiÞcant effect on permethrin toxicity (Fig. 4D). onstrated by the deviations of observed mortalities

Esterase Proﬁles in the Parents, Backcross, and F2 from the predicted values in the larval progenies of the Generations. No difference in the intensity of esterase backcross at certain concentrations (Fig. 2). Differ- bands was observed between the susceptible (Mu- ences in the results of genetic analysis of pyrethroid n˜ oz) and resistant (Santa Luiza) parental strains (data resistance in the Brazilian strain (Santa Luiza) we not shown). There were also no differences in esterase studied and the Aldama strain of B. microplus in Mex- band intensities between the parent strain larvae and ico (Tapia-Perez et al. 2003) may be caused by dif- the larval progenies of backcrosses or of the F2 gen- ferent mechanisms of resistance. Both insensitive so- erations (data not shown). dium channel and enhanced metabolic detoxiÞcation mechanisms have been found to play important roles in pyrethroid resistance in B. microplus (Miller et al. Discussion 1999, He et al. 1999, Jamroz et al. 2000, Guerrero et al. The genetics of resistance to permethrin and other 2001). An overexpressed esterase, CzEst9, which hy- pyrethroid pesticides have been investigated in vari- drolyzes pyrethroids, led to moderate resistance, ous pest species. In general, resistance to pyrethroids whereas the sodium channel mutation was responsible was found to be inherited as an autosomal and incom- for high levels of pyrethroid resistance in B. microplus pletely recessive trait (Roush et al. 1986, Ru et al. 1998, (Miller et al. 1999, Pruett 2002, Guerrero et al. 2002). Liu and Yue 2001, Shono et al. 2002). Differences exist It is entirely possible that some of the tick populations, in the number of genes involved and sex linkage in such as the Aldama strain reported by Tapia-Perez et pyrethroid resistance. For example, resistance to per- al. (2003) may possess both the target site and meta- methrin was determined to be inherited as an auto- bolic detoxiÞcation mechanisms of resistance. 434 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 3

300 A

250

100

50 Resistance Factor (RF)

0 10 B 8

2 Synergism Ratio (SR) - TPP - (SR) Ratio Synergism

0 10 C 8

4 2D Graph 2

2 Synergism Ratio (SR) - PBO

0 10 D 8

2 Synergism Ratio (SR) - DEM 0 Munoz Pesqueria Santa Luiza San Felipe Tick Strain Fig. 4. Comparisons of the resistance factors (RF, A) between four different tick strains (Mun˜ oz, Pesqueria, Santa Luiza and San Felipe) and synergism ratios of TPP, PBO and DEM (B, C, and D) in all tick strains tested. Dashed line indicates SR ϭ 1, where the synergist has no effect on toxicity of permethrin.

The polymerase chain reaction (PCR) assays from study failed to demonstrate the involvement of en- a recent study indicated there was no sodium channel hanced activity of detoxifying enzymes in permethrin mutation in larvae of the Santa Luisa strain (Li et al. resistance in the same tick strain. The results of gel 2007). Results of the synergist bioassays of the current electrophoresis showed similar levels of esterase May 2008 LIETAL.: PERMETHRIN RESISTANCE IN B. microplus 435 staining in the resistant (Santa Luiza) and suscep- The sodium channel PCR assay was developed us- tible (Mun˜ oz) parental strains, as well as the back- ing pyrethroid resistant populations of R. microplus cross and F2 generations. These data provided further from various ranches throughout Mexico (Guerrero et support to the observations from synergist bioassays al. 2001). The assay is designed to detect a nucleotide that dismiss the involvement of over-expressed ester- substitution in the S6 transmembrane segment of do- ases in permethrin resistance in this particular tick main III. This substitution leads to a Phe-to-Ile amino strain. Although the study revealed the genetic basis acid substitution in the sodium channel protein, and it of permethrin resistance in the Santa Luiza strain of B. has been associated with pyrethroid resistance in microplus, the actual mechanisms of resistance remain many Mexican R. microplus populations (Guerrero et unknown. One possibility is that the Brazilian tick al. 2001, 2002; Rosario-Cruz et al. 2005). Pyrethroid strain might have a different sodium channel muta- resistance-associated sodium channel gene mutations tion, which is likely to be responsible for the per- most commonly occur in the region containing the methrin resistance that was observed. S5-S6 transmembrane segment of domain II (Soder- The target of the pyrethroid pesticide action is the lund and Knipple 2003), although a survey of a mu- voltage-sensitive sodium channels. Pyrethroid mole- tagenized laboratory strain of Drosophila melanogaster cules that bind to the sodium channels disrupt the detected pyrethroid resistance-conferring point mu- channel inactivation kinetics, leading to repetitive tations in domain III (Martin et al. 2000). Additionally, neural discharge and eventual death of target pests two alternative exons in the Blattella germanica S3 (Soderlund and Bloomquist 1989). In insects, an in- transmembrane segment of domain III play a role in sensitive target site (sodium channel), known as pyrethroid resistance (Du et al. 2006), although, like knockdown resistance (kdr), is one of the major the mutagenized laboratory D. melanogaster strain, it mechanisms of resistance to pyrethroid insecticides. It is not clear whether these domain III variants occur has been demonstrated in insect species that kdr-type frequently in natural populations like the domain III resistance is caused by point mutations in the para S6 variant of R. microplus. It is possible that the Bra- family of sodium channel genes (Amichot et al. 1992, zilian strain of R. microplus used in this study possesses Williamson et al. 1993, Taylor et al. 1993, Guerrero et a target site gene mutation in a location other than that al. 1997, Liu et al. 2000). A second point mutation, detected by the target site PCR assay designed for super-kdr that confers a much higher level resistance Mexican ticks. This possibility could be addressed by has been identiÞed in both the house ßy and the horn sequence analysis of the Santa Luiza sodium channel, ßy (Williamson et al. 1996, Guerrero et al. 1997). segregating the most resistant and the most suscepti- Three sodium channel mutations were identiÞed in ble individuals using bioassay techniques selecting for pyrethroid-resistant German cockroach populations pyrethroid resistance and correlating any amino acid (Liu et al. 2000, Tan et al. 2002). When expressed in variants with pyrethroid resistance. The most obvious Xenopus oocytes, the primary mutation reduced so- location to sequence would be the S5-S6 region of dium channel sensitivity to deltamethrin by Þve-fold. domain II. However, cDNA clones containing this None of the two secondary mutations alone decrease region were sequenced in several highly pyrethroid sodium channel sensitivity. Combination of the pri- resistant strains of Mexican ticks and nucleotide sub- mary mutation with either one of the secondary mu- stitutions were not detected in the “classic” kdr and tation decreased sodium channel sensitivity to delta- super-kdr sites in domain II (Jamroz et al. 2000, He et methrin by 100-fold. When all three mutations were al. 1999). Another possible explanation for the puz- combined, a 500-fold decrease in sensitivity to delta- zling results from the PCR assay of the Brazilian ticks methrin was observed (Tan et al. 2002). None of the is alternative splicing of the sodium channel gene kdr or super-kdr mutations present in pyrethroid-re- leading to target site insensitivity. Alternative splicing sistant insects were detected in pyrethroid-resistant has been shown to lead to pyrethroid resistant forms populations of a mite species, Varroa destructor (Wang of the sodium channel in B. germanica (Tan et al. et al. 2002). Instead, four new point mutations were 2002), Musca domestica (Lee et al. 2002) and Plutella identiÞed and correlated with ßuvalinate resistance in xylostella (Sonoda et al. 2006). Further research will mites from Florida. However, only two of these resis- be required to investigate these possible target site- tance-related mutations were present in a resistant based resistance mechanisms in the Santa Luiza strain mite population from Michigan. Although the de- from Brazil. duced amino acid sequence from the southern cattle Should the new sodium channel mutation be tick B. microplus cDNA shares 71% identity with the proven to be responsible for permethrin resistance in corresponding region of the para-homologous protein the Santa Luiza strain of B. microplus, the new sodium of the Varroa mites, the sodium channel mutation channel mutation may be equivalent to kdr described detected in resistant B. microplus was not detected in in insects, such as the horn ßy (Guerrero et al. 1997, resistant mites (Wang et al. 2002). Similarly, none of Jamroz et al. 1998), as kdr alone confers low to medium the mutations present in resistant Varroa mites was levels of resistance. The highest permethrin resistance detected in B. microplus. The data from insect and mite has been reported in two Mexican strains of B. micro- species suggest that distinct sodium channel mutations plus, the San Felipe and San Roman strains, with a RF may be selected in the same pest species from differ- at 638 or higher (Miller et al. 1999, Li et al. 2007). ent regions possibly as a result of different history of Earlier studies provided a much higher RF (1840) for pesticide use and selection pressure. the San Felipe strain when a much lower LC50 436 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 45, no. 3 was used from another susceptible reference strain References Cited (Gonzalez) (Miller et al. 1999, Guerrero et al. 2001). Aguirre, J., A. Sobrino, M. Santamarı´a, A. Aburto, S. Roman, The allelic sodium channel mutation frequency was M. Hernandez, M. Ortiz, and Y. A. Ortiz. 1986. Resis- 80.2 and 100% for the San Felipe and San Roman tancia de garrapatas en Mexico, pp. 282Ð306. In A. H. strains, respectively. Given the high level of per- Cavazzani and Z. Garcia [eds.], Seminario Internacional methrin resistance and high frequency of the sodium de Parasitologia Animal, Cuernavaca, Morelos, Mexico. channel mutation, the currently known sodium chan- Amichot, M., C. Castella, A. Cuany, J. B. Berge, and D. nel mutation in the Mexican strains of B. microplus Pauron. 1992. Target modiÞcation as a molecular mech- could be equivalent to the super-kdr found in the ßy anism of pyrethroid resistance in Drosophila melano- species (Williamson et al. 1996, Guerrero et al. 1997). gaster. Pestic. Biochem. Physiol. 44: 183Ð190. It is also possible that the kdr-type of sodium channel Bram, R. A., and J. E. George. 2000. Introduction of nonin- mutation, which may potentially be found in the Bra- digenous arthropod pests of animals. J. Med. Entomol. 37: 1Ð8. zilian strain of B. microplus, also may coexist with the Chen, A. C., H. He, and R. B. Davey. 2007. Mutations in a super-kdr type in the Mexican resistant strains. putative octopamine receptor gene in amitraz-resistant A sharp increase of permethrin resistance levels in cattle ticks. Vet. Parasitol. 148: 379Ð383. the F4 larvae resulting from the F3 larvae selected with Du, Y., Z. Lui, Y. Nomura, B. Khambay, and K. Dong. 2006. permethrin at a concentration (1.25% [AI]) that re- An alanine in segment 3 of domain III (IIIS3) of the sulted in 77.9% mortality was expected. As the geno- cockroach sodium channel contributes to the low pyrethroid sensitivity of an alternative splice variant. Insect type composition of the F3 larvae was one fourth each of SS, RS, SR, and RR genotypes, the permethrin con- Biochem. Mol. Biol. 36: 161Ð168. centration (1.25% [AI]) used for selection eliminated [FAO] Food and Agriculture Organization. 1971. Recom- mended methods for the detection and measurement of all SS, SR, and RS genotype as expected (Fig. 3B). It resistance of agricultural pests to pesticidesÐtentative was surprising that selection of the F3 larvae with method for larvae of cattle ticks, Boophilus microplus spp. amitraz resulted in a similar increase of permethrin FAO method no. 7. FAO Plant Prot. Bull. 19: 15Ð18. resistance in the F4 larvae (Table 3; Fig. 3B). Similarly, Fragoso, H., N. Soberanes, M. Ortiz, M. Santamarı´a, and A. selection of F3 larvae with 0.1% amitraz drastically Ortiz. 1995. Epidemiologia de la resistencia a ixodicidas piretroides en garrapatas Boophilus microplus en la Re- increased amitraz resistance in the F4 larvae. This was expected because the amitraz concentration (0.1% publica Mexicana, pp. 45Ð57. In S. Rodriquez and H. [AI]) used for selection should theoretically eliminate Fragoso [eds.], Seminario internacional de parasitologia all susceptible homozygotes and heterozygotes (Li et animalÐResistncia y Control en Garrapatas y Moscas de Importancia Veterinaria. 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Factors inßuenc- ing the evolution of resistance, pp. 157Ð169. In National resistance to permethrin and amitraz in the Santa Research Council [ed.], Pesticide Resistance-Strategies Luiza strain of B. microplus are tightly linked. Results and Tactics for Management. National Academies Press, of a recent study revealed strong synergism between Washington, DC. permethrin and amitraz in this Brazilian tick strain and Graham, O. H., and J. L. Hourrigan. 1977. Eradication pro- other Mexican tick strains (Li et al. 2007). At the grams for the arthropod parasites of livestock. J. Med. molecular level, it is unknown how permethrin or Entomol. 6: 629Ð658. amitraz molecules interact with their own receptors Guerrero, F. D., R. C. Jamroz, D. Kammlah, and S. E. Kunz. and receptors for the other acaricide molecule. The 1997. 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