Comparison of the reproductive biology between

acaricide-resistant and acaricide-susceptible

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Ronald B. Davey a,*, John E. George b, Robert J. Miller a

a USDA, ARS, Cattle Fever Tick Research Lab., Moore Air Base, Bldg. 6419, 22675 N. Moorefield Rd., Edinburgh, TX 78541, USAb USDA, ARS, Knipling-Bushland U.S. Livestock Insects Lab., 2700 Fredricksburg Rd., Kerrville, TX 78029, USA

Received 2 December 2005; received in revised form 17 February 2006; accepted 21 February 2006

Abstract

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.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Rhipicephalus; Boophilus; Reproduction; Resistance

www.elsevier.com/locate/vetpar

Veterinary Parasitology 139 (2006) 211–220

* Corresponding author at: USDA, ARS, SPA, Cattle Fever Tick

Research Lab., Moore Air Base, Bldg. 6419, 22675 N. Moorefield

Rd., Edinburgh, TX 78541, USA. Tel.: +1 956 580 7262;

fax: +1 956 580 7261.

E-mail address: Ronald.Davey@ars.usda.gov (R.B. Davey).

0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved

doi:10.1016/j.vetpar.2006.02.027

1. Introduction

The United States Cattle Fever Tick Eradication

Program (CFTEP) has faced many challenges during

its 100-year history, but perhaps the greatest challenge

.

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220212

has been the development of widespread acaricide

resistance to the major classes of pesticides that have

been used to control Rhipicephalus (Boophilus) spp. in

Mexico during the past 30 years. Considerable

research in the U.S. in recent years has focused on

control technologies, characterization of resistance

mechanisms, and development of molecular assay

techniques associated with acaricide-resistant R. (B.)

microplus (Canestrini) ticks (Davey and George, 1998,

1999; He et al., 1999a,b,c, 2002; Miller et al., 1999,

2002; Guerrero et al., 2001; Davey et al., 2003, 2004;

Li et al., 2003, 2004, 2005a,b; Temeyer et al., 2004).

However, few studies conducted anywhere in the

world have specifically investigated the impact that

resistance has on biological factors, such as reproduc-

tion. The reproductive biology of R. (B.) microplus,

consisting of the preoviposition and oviposition

periods and fecundity of engorged females, along

with the incubation period and fertility of eggs was

documented several decades ago in widely divergent

parts of the world, such as Australia, Cuba, and the

USA, using acaricide-susceptible ticks (Hitchcock,

1955; Cerny and de la Cruz, 1971; Bennett, 1974a;

Davey et al., 1980a). But, with the exception of a

single Australian study (Bennett, 1974b) there appears

to be little specific information on the effect of

resistance on the reproductive processes of this

species, even though acaricide resistance seriously

threatens to undermine chemical control strategies

used against the species. Although it has been reported

that fitness reduction in pesticide-resistant arthropods

is likely to occur in the absence of pesticide pressure

(Roush and Daly, 1990), it is difficult to associate

fitness disadvantages specifically with resistance.

The purpose of this study was to compare the

reproductive fitness, as measured by oviposition,

fecundity, and fertility, of acaricide-resistant strains

of R. (B.) microplus with those of a tick strain that was

susceptible to the major classes of acaricides used to

eradicate or control the species. The rationale for the

study was based on the assumption that any selective

disadvantages in reproductive capacity of acaricide-

resistant ticks that could be demonstrated under

laboratory conditions could potentially explain the

occurrence of unusual reproductive patterns, such as

lower egg production or reduced egg viability that

might occur in naturally occurring resistant tick

populations. In addition, demonstration of selective

disadvantages associated with acaricide-resistant ticks

might provide insight for the development of strategies

that could be used to manage resistant tick populations.

2. Materials and methods

2.1. Tick strains

One strain of R. (B.) microplus that was susceptible

to acaricides and three strains that were resistant to

either organophosphate (OP), pyrethroid (P), or

formamidine (F) acaricides were evaluated in the

study. Each of the four strains had been maintained in

the laboratory for multiple generations using standard

rearing techniques (Davey et al., 1982). The acaricide-

susceptible strain (SUS) used in the study, to which all

of the resistant strains were compared, was originally

collected from an outbreak of ticks discovered in

Zapata Co., TX in 1999. No acaricidal pressure has

ever been applied to the ticks since its laboratory

colonization. However, larvae from most generations

were subjected to laboratory bioassay tests with OP, P,

and F acaricides using the larval packet test method

described by FAO (Anonymous, 1971) to track the

susceptibility level of the strain. The OP-resistant

strain (OPR) used in the study was originally obtained

from a ranch located in Champoton, Campeche, MX

in 1998. The strain was selectively pressured during

most generations of laboratory colonization with the

OP acaricide coumaphos to maintain or increase the

level of OP resistance. The P-resistant strain (PYR)

was collected from a ranch located near Soto la

Marina, Tamaulipas, MX in 1995 and colonized at our

laboratory in 1996. The strain was selectively

pressured during many generations of colonization

with the P acaricide permethrin to maintain or increase

the level of P resistance, and was subjected to

laboratory bioassay tests (FAO method) to track the

level of P resistance. The F-resistant strain (FOR) was

originally collected from a ranch in Tabasco, MX in

2001, and was colonized in our laboratory in 2002.

The strain was selectively pressured during numerous

generations since colonization with the formamidine

acaricide amitraz to maintain or increase the level of

resistance. Laboratory bioassays (FAO method) were

conducted on numerous generations to track the level

of F resistance in the strain.

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220 213

2.2. In vitro laboratory bioassays

Although some of the resistant strains had lower

levels of resistance to other acaricides than the one to

which they were assigned (i.e. coumaphos for the OPR

strain; permethrin for the PYR strain; and amitraz for

the FOR strain), the resistance to other chemicals was

not evaluated for this study. At the time the study was

conducted, laboratory bioassays with coumaphos

(OP), permethrin (P), and amitraz (F) were conducted

using the FAO larval packet test method (Anonymous,

1971) to establish the level of resistance of the four

tick strains to the acaricide to which they were

assigned. Briefly, the FAO technique is as follows:

technical grade acaricide is dissolved in trichlor-

oethylene to make a stock solution which is then

diluted to a top dose in diluent containing two parts

trichloroethylene and one part olive oil. The top dose

is serially diluted to make test dosages. AWhatman #1

filter paper (7.5 cm � 9.0 cm; Whatman, Maidstone,

Kent, UK) is treated with 1 ml of each test solution,

and the solvent is allowed to evaporate before being

folded into a packet into which ca. 100 larvae are

placed. The packets are placed in an incubator (27 8C,

92.5% RH, photoperiod 12:12 L:D) for 24 h after

which the numbers of live and dead larvae are counted.

The LC50 (lethal concentration for 50% of the ticks

tested) of each acaricide (coumaphos, permethrin, and

amitraz) was estimated for the SUS strain, whereas for

each resistant strain (OPR, PYR, and FOR) only the

LC50 of the acaricide to which the strain was assigned

was estimated. A resistance ratio (RR) value for each

appropriate acaricide (coumaphos, permethrin, and

amitraz) for each resistant strain (OPR, PYR, and

FOR) was calculated in comparison to the SUS strain

by dividing the LC50 of each resistant strain by the

LC50 of the SUS strain for the appropriate acaricide.

2.3. Evaluation design

Due to logistical limitations the four tick strains

evaluated in the study were tested sequentially rather

than simultaneously. The SUS strain was evaluated first,

followed by the PYR, OPR, and finally the FOR strain.

Because the study was designed to compare reproduc-

tive biology of each of the resistant strains (PYR, OPR,

and FOR) with that of the SUS strain, it was necessary to

determine the mean engorgement weight of females

from each strain to see whether there were differences,

since it is well known that engorgement weight is

strongly correlated with the number of eggs produced

by females (Drummond et al., 1969a,b; Bennett, 1974a;

Iwuala and Okpala, 1977; Davey et al., 1980a,b).

Unless engorgement weights of females in each of the

strains were similar a direct comparison between strains

would have been impossible because of the positive

correlation between female weight and numbers of eggs

deposited. Therefore, prior to the initiation of the study

50 randomly selected females from each of the four

strains were statistically analyzed to determine whether

there were differences in female engorgement weight

between any of the resistant strains and the SUS strain to

which they were each compared. Analysis showed that

the mean engorgement weight of SUS females was

significantly greater (SUS versus PYR: t = 6.0, df = 98,

P < 0.001; SUS versus OPR: t = 5.5, df = 98,

P < 0.001; and SUS versus FOR: t = 3.7, df = 98,

P < 0.001) than each of the resistant strains used in the

evaluation (data not presented). As a result of the

indicated differences in female engorgement weight

between the SUS strain and each of the resistant strains,

when the study was initiated, a procedure was devised to

standardize the female weights of each strain used in the

evaluation, so comparisons could be made on

reproductive parameters that were influenced by the

weight of the female, such as number of eggs laid by

each female. Since the SUS strain was tested first, the

engorged female ticks used in the evaluation were

randomly selected. However, prior to obtaining each

sample of females for each resistant strain (PYR, OPR,

and FOR), a subset of randomly selected engorged

females was created from the female ticks that detached

by using only females that had engorgement weights

that fell within the same weight range as the SUS strain.

While this weight standardization procedure prevented

the use of this parameter as a means of determining

differences in reproductive biology among the strains, it

was critical in providing the means for comparing other

reproductive parameters that were affected by female

weight.

Each tick strain was sequentially evaluated, as

previously stated, by infesting a naı̈ve Hereford heifer

calf weighing ca. 200 kg with ca. 5000 larvae that

were 14–21 days old. Each calf was monitored daily

until replete females began to detach. Engorged

females that detached on the 23rd day post-infestation

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220214

(day of maximum detachment of engorged females)

were used to obtain a random sample of 25 individuals

from each strain that were used in the evaluation. Each

of the 25 engorged females selected for evaluation was

weighed using a top loading PC 440 Mettler1 balance.

Each weighed female was placed individually in a

coded 9-cm diameter plastic petri dish and transferred

to an incubator calibrated to 27 � 2 8C, 92% RH, and

a 12:12 photophase (L:D) to allow for oviposition.

Each female was monitored at 24 h intervals until

oviposition began to determine the preovipositon

period. Once oviposition commenced, the eggs

deposited by each female during each 24 h time

period throughout the oviposition cycle were carefully

removed and placed in an empty, coded

17 mm � 60 mm (2-dram) shell vial fitted with a

cotton stopper, and the female and eggs were returned

to the incubator. This procedure allowed for the

determination of the duration of the oviposition period

and daily egg output of each female. The collection of

eggs at 24 h intervals from each female continued until

the female failed to oviposit three consecutive days, at

which time oviposition was considered to have ceased

and the female was discarded. The eggs collected from

each female on each day of the oviposition period

were monitored at 24 h intervals until the first larva

enclosed from the egg to determine the incubation

period of eggs collected on each day of oviposition.

After egg hatch began, all eggs remained in the

incubator for 4 weeks after deposition to insure

complete hatch of all viable eggs. After 4 weeks, vials

containing the eggs and larvae from each female on

each day of the oviposition period were removed from

the incubator and the larvae and unhatched eggs from

each sample were carefully counted to determine the

Table 1

In vitro laboratory bioassay results obtained from strains of Rhipicephalus (

resistant to organophosphate (OPR), pyrethroid (PYR), or formamidine (

Strain Acaricide Larvae, n Slope (S.E

SUS Coumaphos 2584 4.2 (0.1) a

OPR Coumaphos 1270 5.7 (0.3) b

SUS Permethrin 1413 4.2 (0.2) a

PYR Permethrin 938 2.3 (0.2) b

SUS Amitraz 1999 1.7 (0.07)

FOR Amitraz 1597 1.2 (0.05)

Slopes within the same acaricide followed by a different letter are significa

ticks; presented as % active ingredient (AI). RR = resistance ratio value

percentage egg hatch on each day of the oviposition

cycle.

2.4. Data analysis

Data obtained from the in vitro laboratory

bioassays of the four strains against the different

acaricides (OP, P, and F) were subjected to probit

analysis (LeOra Software, 1987) to determine if dose–

response estimates between the SUS strain and each

resistant strain were significantly different for the

appropriate acaricide and to establish the LC50 and RR

values of each strain to each appropriate acaricide. The

other measured variables (female engorgement

weight, preoviposition period, oviposition period,

number of eggs laid, incubation period, and percen-

tage egg hatch) were analyzed by general linear model

(GLM), one-way analysis of variance (ANOVA), and

differences among means in all analyses were

determined by Tukey’s test method (SAS, 1999).

3. Results

3.1. Laboratory bioassays

Analysis showed that the slope of the estimated

dose-mortality line of each acaricide-resistant strain

(OPR, PYR, and FOR) was significantly different

(P < 0.05) than the corresponding dose-mortality line

of the SUS strain for each acaricide (OP, P, and F,

respectively) (Table 1). The OPR strain was 8.4-fold

more resistant to coumaphos (OP) than the SUS strain,

the PYR strain was 39.4-fold more resistant to

permethrin (P) than the SUS strain, and the FOR

Boophilus) microplus that were either acaricide-susceptible (SUS) or

FOR)

.) x2 LC50 (95% CL) RR

212 0.034 (0.03–0.04) –

36 0.29 (0.27–0.31) 8.4

45 0.027 (0.025–0.03) –

19 1.08 (0.94–1.24) 39.4

a 94 0.002 (0.002–0.003) –

b 122 0.26 (0.18–0.39) 107.0

ntly different (P < 0.05). LC50 = lethal concentration for 50% of the

relative to the SUS strain for the indicated acaricide.

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220 215

Fig. 1. Accumulative oviposition curves for Rhipicephalus (Boo-

philus) microplus females that were either acaricide-susceptible

(SUS) or resistant to organophosphate (OPR), pyrethroid (PYR),

or formamidine (FOR) acaricides.

strain was 107 times more resistant to amitraz (F) than

the SUS strain.

3.2. Female engorgement weight

There was no significant difference (F = 0.18;

df = 3,96; P > 0.9) in engorgement weight among

females of the four strains that were evaluated in the

study (Table 2). In fact, there was a difference of only

9 mg between the mean weight of the strain with the

heaviest females (PYR strain) and that of the strain

with the lightest females (OPR strain). The results

indicated that the procedures used to standardize the

female engorgement weight of each of the four strains

were successful.

3.3. Preoviposition period

The mean preoviposition period for each of the four

strains showed that there was no significant difference

(P > 0.05) between the SUS strain and that of either

the OPR or the PYR strain. However, the preoviposi-

tion period of the FOR strain was significantly shorter

(F = 5.4; df = 3,96; P < 0.002) than all of the other

strains (Table 2), even though the difference between

the other means was �0.6 day.

3.4. Oviposition period

The mean oviposition period of the SUS strain

(12.1 days) was significantly longer (F = 13.4;

df = 3,96; P < 0.001) in duration than either the

OPR (8.4 days) or the PYR (9.9 days) strain, but was

not significantly different (P > 0.05) from the FOR

(11.0 days) strain (Table 2). The range of the

oviposition period for SUS and FOR females was

8–18 and 8–16 days, respectively, whereas the range

Table 2

Mean engorgement weight, preoviposition period, oviposition period, and n

(Boophilus) microplus females that were either acaricide-susceptible (S

formamidine (FOR) acaricides

Strain Engorgement weight (mg) Preoviposition period (da

SUS 362 � 56 a 3.0 � 0.2 a

OPR 359 � 23 a 3.2 � 0.5 a

PYR 368 � 60 a 3.0 � 0.6 a

FOR 365 � 45 a 2.6 � 0.5 b

Means within the same column followed by a different letter are significan

way analysis of variance (ANOVA). Differences among means were dete

of the oviposition period for PYR and OPR females

was considerably shorter at 8–13 and 4–13 days,

respectively. The cumulative percentages of the total

number of eggs that were produced on each day of the

ovipositional cycle by females of each strain were very

similar, although because of its significantly shorter

ovipositon period, the OPR strain deposited a slightly

higher percentage of the total egg mass on each day of

oviposition than the other strains (Fig. 1). Never-

theless, results showed that >93% of all eggs laid by a

female were deposited during the first 8 days of

oviposition, regardless of the strain.

3.5. Egg output

Although females of the SUS strain produced more

eggs than all other strains, there was no significant

difference (P > 0.05) in the egg output produced by

the SUS females, as compared to the PYR and FOR

females, which produced 8.9 and 7.7% fewer eggs,

umber of eggs oviposited (�S.D.) from four strains of Rhipicephalus

US) or resistant to organophosphate (OPR), pyrethroid (PYR), or

ys) Oviposition period (days) No. of eggs oviposited

12.1 � 2.7 a 3670 � 651 a

8.4 � 2.2 c 2562 � 816 b

9.9 � 1.4 bc 3344 � 797 a

11.0 � 2.2 ab 3388 � 626 a

tly different (P < 0.05) tested by general linear model (GLM), one-

rmined by Tukey’s method.

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220216

respectively, than the SUS strain females (Table 2).

However, females of the OPR strain produced

significantly fewer eggs (F = 10.7; df = 3,96;

P < 0.001) than all other strains tested. The OPR

strain females produced an average of 1108 fewer eggs

per female than the SUS strain females, a 30.2%

reduction in egg production.

3.6. Egg incubation period

The incubation period of eggs derived from females

of the four strains showed no significant difference

(P > 0.05) in duration time between the SUS strain

and either the OPR or FOR strains, either on any

specific day or over the entire ovipositional cycle

(Table 3). Conversely, PYR eggs had a significantly

longer incubation period (F = 15.8; df = 3,50;

P < 0.0001), on each day of oviposition, as well as

over the entire ovipositional cycle. Sequentially

deposited eggs laid during the first 6 days of the

oviposition period showed a slight, but consistent

tendency toward shorter incubation periods, regard-

less of strain. Incubation periods of eggs deposited on

Days 7–10 of the oviposition cycle were comparable

to those laid on Day 6, regardless of strain.

Subsequently, eggs of the SUS and FOR strains laid

Table 3

Mean daily incubation period of eggs (�S.D.) derived from four strains

acaricide-susceptible (SUS) or resistant to organophosphate (OPR), pyret

Day eggs were laid Mean incubation period � S.D. (days) fo

SUS OPR

1 22.6 � 0.6 a (25) 22.1 � 0.8

2 21.6 � 0.7 a (24) 21.1 � 0.6

3 21.3 � 0.8 a (24) 20.9 � 0.6

4 20.8 � 0.5 a (24) 20.4 � 0.6

5 20.7 � 0.8 a (24) 20.4 � 0.6

6 20.5 � 0.7 a (24) 20.5 � 1.2

7 20.8 � 0.9 a (23) 20.5 � 1.7

8 20.5 � 0.7 a (23) 20.1 � 1.2

9 21.2 � 1.2 a (18) 20.0 � 1.3

10 21.0 � 1.3 a (16) 20.0 � 0.7

11 20.7 � 1.0 a (11) 20.0 � 1.0

12 21.1 � 1.0 a (8) 19.0 a (1)

13 21.2 � 0.8 a (5) –

14 21.3 � 0.6 a (3) –

15 23.0 a (2) –

All days 21.2 � 0.7 a (15) 20.4 � 0.8

Numbers in parenthesis indicate the n value. Means within the same row

tested by general linear model (GLM), one-way analysis of variance (AN

on Days 11–15 tended to have an increased incubation

period.

3.7. Egg hatchability

Although eggs from the PYR strain produced the

highest overall hatch, followed by eggs from the

FOR strain, the SUS strain, and the OPR strain,

analysis showed no significant difference (F = 1.2;

df = 3,96; P > 0.3) in the overall mean egg hatch-

ability among the four strains (Table 4). On each

individual day of the ovipositional cycle, the only

significant differences (P < 0.05) in egg hatch

between the SUS strain and the three resistant

strains were observed in eggs deposited by SUS

females on Days 1 and 2 of the oviposition cycle,

which had significantly higher (P < 0.05) hatch rates

than eggs of the FOR strain deposited on the same

days, and eggs laid by SUS females on Day 5 of the

oviposition cycle, which had a significantly higher

(P < 0.05) hatch rate than eggs deposited by OPR

females on the same day. Egg hatchability in all four

strains remained >50% during the first 8 days of the

oviposition, by which time >93% of all the eggs had

been laid (Fig. 1). Generally, eggs laid subsequent to

the eighth day of oviposition showed a consistent,

of Rhipicephalus (Boophilus) microplus females that were either

hroid (PYR), or formamidine (FOR) acaricides

r the indicated strain

PYR FOR

a (25) 24.4 � 1.0 b (25) 22.4 � 1.2 a (25)

a (25) 23.2 � 0.9 b (25) 21.1 � 1.0 a (25)

a (25) 22.8 � 0.8 b (25) 21.0 � 0.7 a (25)

a (23) 22.3 � 0.8 b (25) 20.5 � 0.7 a (25)

a (23) 22.1 � 0.8 b (25) 20.4 � 0.8 a (25)

a (21) 21.9 � 0.8 b (24) 20.3 � 0.8 a (25)

a (17) 21.8 � 1.0 b (24) 20.4 � 0.6 a (25)

a (11) 21.9 � 1.0 b (22) 20.7 � 0.8 a (24)

a (9) 22.5 � 1.5 b (16) 20.7 � 0.6 a (18)

a (5) 22.2 � 1.3 b (10) 20.6 � 0.5 a (16)

a (3) 24.7 � 1.2 b (3) 21.1 � 1.1 a (10)

26.0 b (1) 20.8 � 1.3 a (5)

– 21.0 a (1)

– 23.0 a (1)

– 23.0 a (1)

a (12) 23.0 � 1.3 b (12) 21.2 � 0.9 a (15)

followed by a different letter are significantly different (P < 0.05),

OVA). Differences among means determined by Tukey’s method.

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220 217

Table 4

Mean daily percentage hatch (�S.D.) of eggs derived from four strains of Rhipicephalus (Boophilus) microplus females that were either

acaricide-susceptible (SUS) or resistant to organophosphate (OPR), pyrethroid (PYR), or formamidine (FOR) acaricides

Day eggs were laid Mean percentage egg hatch � S.D. for the indicated strain

SUS OPR PYR FOR

1 85.6 � 12.3 a (25) 80.6 � 19.8 ab (25) 86.8 � 15.4 a (25) 69.6 � 26.5 b (25)

2 91.7 � 7.4 a (24) 80.7 � 26.2 ab (25) 91.6 � 6.6 a (25) 78.8 � 19.0 b (25)

3 89.1 � 9.0 ab (24) 77.9 � 23.9 b (25) 89.3 � 8.3 a (25) 83.7 � 14.9 ab (25)

4 89.2 � 10.1 ab (24) 78.1 � 27.2 b (25) 88.1 � 8.7 ab (25) 91.4 � 8.9 a (25)

5 86.1 � 9.8 a (24) 66.9 � 32.4 b (24) 83.0 � 9.2 a (25) 86.7 � 11.0 a (25)

6 73.8 � 19.1 ab (24) 57.4 � 35.7 b (24) 72.1 � 21.3 ab (25) 81.9 � 13.7 a (25)

7 60.8 � 29.6 a (24) 55.6 � 36.7 a (20) 66.6 � 21.4 a (25) 76.7 � 21.1 a (25)

8 58.6 � 30.1 a (24) 51.0 � 39.1 a (14) 51.4 � 29.0 a (25) 55.3 � 32.5 a (25)

9 41.2 � 36.5 a (22) 47.4 � 38.9 a (11) 30.3 � 29.5 a (22) 47.4 � 39.5 a (22)

10 36.6 � 35.7 a (22) 39.9 � 41.6 a (7) 20.2 � 24.4 a (13) 47.6 � 36.7 a (18)

11 39.6 � 35.9 a (15) 28.6 � 34.0 a (6) 6.2 � 10.7 a (6) 40.5 � 37.9 a (12)

12 22.9 � 25.8 a (13) 33.4 � 47.2 a (2) 1.2 � 2.5 a (4) 18.6 � 28.3 a (9)

13 24.0 � 24.7 a (9) 0.0 a (1) 0.0 a (2) 14.3 � 37.8 a (7)

14 16.8 � 21.9 a (7) – – 20.0 � 28.2 a (2)

15 9.7 � 14.3 a (5) – – 23.5 � 23.5 a (2)

16 0.0 a (3) – – 0.0 a (1)

17 0.0 a (3) – – –

18 0.0 a (1) – – –

Overall 78.1 � 19.9 a (25) 73.7 � 21.8 a (25) 82.2 � 7.7 a (25) 80.3 � 13.8 a (25)

Numbers in parenthesis indicate n value. Means within the same row followed by a different letter are significantly different (P < 0.05), tested by

general linear model (GLM), one-way analysis of variance (ANOVA). Differences among means determined by Tukey’s method.

and often precipitous, daily decline in their hatch-

ability level.

4. Discussion

Based on the resistance scale adopted by Beugnet

and Chardonnet (1995), which reported that an RR

value of �5 was indicative of a resistant population,

all of the resistant strains evaluated in the study (OPR,

PYR, and FOR) were clearly classified as being

resistant to their corresponding acaricide (OP, P, and F,

respectively). Other studies using the same OPR and

PYR strains evaluated in this study reported higher RR

values to coumaphos (Li et al., 2005b) and permethrin

(Miller et al., 1999), respectively, but these differences

in RR values could have been the result of being

compared to a different susceptible strain than the one

used in this study. Although there has been no

previously published information on the level of

resistance of the FOR strain used in the study, analysis

of another amitraz-resistant strain reported RR values

that ranged from 13 to153 (Li et al., 2004).

All of the reproductive factors associated with the

SUS strain (preoviposition and oviposition period,

dynamics of egg incubation and egg hatch, and

numbers of eggs laid by females) were remarkably

similar to the reproductive parameters that have been

previously reported for acaricide-susceptible R. (B.)

microplus in widely divergent parts of the world, such

as Australia, Cuba, India, and the USA (Hitchcock,

1955; Cerny and de la Cruz, 1971; Bennett, 1974a;

Davey et al., 1980a; Sinha et al., 1982). These

similarities in reproductive factors associated with the

SUS strain in comparison to other populations

throughout the world suggested that the laboratory

colonization process of the SUS strain over numerous

generations had produced no obvious changes in the

reported reproductive processes of the species. This

was important because the reproductive factors of all

of the resistant strains used in this study were

compared to those of the SUS strain. Since the

reproductive parameters of the resistant strains were,

in most cases, essentially the same as the SUS strain, it

was surmised, by extension, that the laboratory

colonization process also had little or no effect on

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220218

the reproductive biology of the resistant strains. Thus,

the assumption was that any obvious differences in

reproductive biology between the SUS strain and any

of the resistant strains were not likely to be artifacts

caused by the laboratory colonization process.

It should be noted that even though engorgement

weight was not evaluated as a part of this study,

analysis conducted prior to the initiation of the study

indicated that the engorgement weight of SUS ticks

was significantly greater than any of the resistant

strains. This finding would suggest that even if all

other parameters were equal the reproductive potential

of the SUS strain would be higher than the resistant

strains because of the positive correlation between

engorgement weight and the number of eggs laid by

females. However, within the context of the objective

of the current study, results demonstrated few

differences in the reproductive dynamics between

the acaricide-susceptible strain and strains that were

resistant to either P or F acaricides. The mean

differences in preoviposition period of the FOR strain

and egg incubation period of the PYR strain in

comparison to the SUS strain, although significant,

were only 0.4 and 1.8 days apart, respectively, with a

sensitivity level of �1.0 day (eggs checked only once

each day), suggesting little biological relevance. Thus,

it would be difficult to conclude that these differences

were evidence of a reduction in fitness associated with

the resistance to P or F acaricides. Similarly, while the

PYR strain had a significantly shorter oviposition

period than the SUS strain, it would be difficult to

conclude that the shorter oviposition period exhibited

by the PYR strain put it at a selective disadvantage to

the SUS strain, since eggs deposited after the eighth

day of oviposition represented only <7% of the total

eggs laid, and the hatchability of those eggs was

dramatically lower. Consequently, the most important

conclusion that could be drawn from the results of the

study was that any reduction in fitness created by the

acquisition of resistance to either P or F acaricides

could not be attributed to any of the reproductive

parameters evaluated in the study.

The OPR strain produced more differences in

reproductive factors compared to the SUS strain than

either of the other resistant strains, although, as was

the case for the other resistant strains, some of the

differences were of minimal biological significance.

The shorter egg incubation and oviposition periods

observed in the OPR strain could hardly be considered

to be indicative of a reduction in fitness associated

with the acquisition of resistance, for the reasons that

were stated previously. The single parameter that

strongly indicated that the OPR strain was at a

selective reproductive disadvantage compared to the

SUS strain was reflected by the 30% reduction in the

mean number of eggs laid by each female. This

reduction in egg production by OPR females clearly

indicated that the reproductive potential of OPR ticks

was significantly lower than that of SUS females. This

was precisely why the use of only resistant females

within the same weight range as the SUS strain

females was so critical, because if females of distinctly

different engorgement weights had been used this

reduction may well have gone undetected. In addition

to the 30% reduction in egg numbers, the discrepancy

in reproductive potential between SUS and OPR

females was further magnified by two other factors.

First, as was stated previously, the fact that analysis

conducted prior to the initiation of the study indicated

that the engorgement weight of SUS ticks was greater

than OPR ticks means that females of the SUS strain

would likely have laid more eggs than the OPR

females even if all other factors were equal, simply

because female weight is related to egg production.

Second, even though there was no statistical difference

in the percentage egg hatch between the two strains

(SUS and OPR), the slightly lower hatch rate of the

OPR strain created an even greater discrepancy in the

number of larvae produced by the OPR strain. Thus,

the reduction in reproductive potential of the OPR

strain in comparison to the SUS strain would be at

least 34.1%, not even accounting for the difference in

egg numbers associated with a lighter engorgement

weight. Thus, the number of viable larvae that were

available to re-infest subsequent cattle would have

been dramatically lower than the number of SUS

larvae available. The results of this study were in

contrast to another study conducted on OP-resistant

ticks, which reported that the acquisition of OP

resistance did not change the reproductive potential of

the resistant ticks (Bennett, 1974b).

Under naturally occurring conditions, fitness of the

ticks in a population would be related to the

conditions to which the ticks were subjected. In the

presence of acaricide pressure susceptible ticks would

have a low degree of fitness because they would be

R.B. Davey et al. / Veterinary Parasitology 139 (2006) 211–220 219

virtually eliminated, whereas resistant individuals

would show a high degree of fitness because they

could survive the acaricide applications. Conversely,

in the absence of acaricidal pressure susceptible ticks

would likely have a high degree of fitness in relation to

resistant individuals. Thus, based on the conditions of

the current study, in a real world situation it is

probable that, in the absence of OP pressure, OP

resistance in a population would be virtually

eliminated through time because the immigration

of susceptible individuals, which have a greater

reproductive capacity, would result in a continual

reduction in the frequency of OP-resistant individuals

in the population. Thus, at some point, the application

of an OP acaricide would likely decimate the

population, at least temporarily, because of the lack

of sufficient OP-resistant individuals necessary to

maintain a stable population. However, it is highly

probable that the continued application of OP

acaricides against the strain would lead to a rapid

resurgence of OP resistance because frequently genes

that confer resistance become fixed in the population

and are rapidly expressed under repeated selection

pressure. Further studies have been planned to test

whether the above-described scenario would actually

result in elimination of an OP-resistant population of

ticks under natural conditions, in the absence of

acaricidal pressure. Additional studies are also

planned to evaluate whether there are genetic

components associated with the fitness of resistant

ticks. These future studies could be critical to the

development of management strategies that could be

used to mitigate acaricide-resistant tick populations.

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