First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics

We document the presence of a Rhipicephalus microplus tick population resistant to acaricides (organophosphates (OP), synthetic pyrethroids (SP), amitraz) and macrocyclic lactones (ML) (ivermectin). Engorged females of R. microplus were collected from a cattle farm in Veracruz, Mexico, to evaluate acaricide and ivermectin resistance. The modified larval packet test (LPT) was used to detect OP (chlorpiriphos and diazinon) and SP (flumethrin, deltamethrin and cypermethrin) resistance and the larval immersion test (LIT) to detect resistance to amitraz and ivermectin. Both, LPT and LIT were performed twice at different times with different collected samples. Mortality data with ivermectin were subjected to probit analysis to obtain lethal concentrations and resistance ratios (RR) using an ivermectin-susceptible strain (Deutch) as a reference. The R. microplus population showed resistance to all acaricides tested, with different mortalities at the discriminate dose: chlorpiriphos (1%), diazinon (24.2%), flumethrin (92.8%), deltamethrin (94.2%), cypermethrin (98.0%) and amitraz (1.5%). The studied tick population also showed resistance to ivermectin with a resistance ratio at 99% of 9.58 and 6.52 in the first and second evaluation, respectively. We report for the first time a R. microplus population in Mexico with different levels of resistance to OP, SP, amidines (Am) and ivermectin. The uncontrolled use of these products in the study area may promote the complete failure of tick control within a short period of time.     

Effect of acaricides on the activity of a Boophilus microplus glutathione S-transferase

In the present study, we report the effect of several acaricides on the enzyme activity of a Boophilus microplus recombinant glutathione S-transferase (rGST). GST was expressed in Escherichia coli and was purified with glutathione (GSH) affinity column chromatography. The kinetic constants were determined by reacting GST with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione. We report the effect of several acaricides on the enzyme activity of rGST. Some acaricides (ethion, amitraz, chlorpyrifos, DDT, cypermethrin, diazinon, ivermectin, deltamethrin and flumethrin) inhibited rGST. Contrarily, coumaphos had an activating effect. Although the accurate mechanisms of the B. microplus resistance to acaricides remain elusive, this work helps in understanding how acaricides can interact with GST.     

Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus collected from six agro-climatic regions of India

A cross sectional study was conducted to assess the prevalence of synthetic pyrethroids (SP) resistance in Rhipicephalus (Boophilus) microplus in India. Twenty-seven areas located in six agro-climatic regions were selected for the collection of engorged ticks using two stage stratified sampling procedure. Adult immersion test (AIT) and larval packet test (LPT) were optimized using laboratory reared susceptible line of R.(B.) microplus (IVRI-I) for determination of 95% lethal concentration (LC(95)) of deltamethrin (29.6 ppm in AIT and 35.5 ppm in LPT) and cypermethrin (349.1 ppm in AIT and 350.7 ppm in LPT). The AIT with a discriminating dose (2 × LC(95)) was used to detect deltamethrin and cypermethrin resistance in the field isolates of R.(B.) microplus. On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The overall prevalence of SP-resistant R.(B.) microplus among the sampled farms was 66.6% (18/27). Out of these 18 areas, resistance to deltamethrin at level I was detected in 08 areas (resistance factor=2.0-4.9), at level II in 09 areas (RF=5.2-11.8), at level III in 01 area (RF=34.9) and at level IV in 01 area (RF=95.7). The resistance to cypermethrin was detected in 16 areas and level of resistance was detected at level I in 10 areas (RF=2.06-4.64) and at level II in 06 areas (RF=5.13-9.88). The middle-gangetic and trans-gangetic plains revealed higher density of resistant ticks where intensive cross bred cattle population are reared and the SP compounds are commonly used. The data generated on acaricide resistant status in ticks will help in formulating tick control strategy for the country.     

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

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

The prevalence of pyrethroid resistance phenotype and genotype in Rhipicephalus (Boophilus) microplus in Yucatan, Mexico

A field survey was conducted to evaluate susceptibility of Rhipicephalus (Boophilus) microplus to cypermethrin on 49 farms in three areas of Yucatan, Mexico. The modified larval packet test was used to evaluate larval mortality at different cypermethrin concentrations. Dose-mortality regressions, lethal concentrations (LC(50)-LC(99)), confidence intervals and slope were estimated by probit analysis. Phenotype was defined as susceptible, tolerant or resistant when the resistance factor (RF) derived from both LC(50) and LC(99) determinations were <3, 3-5 or >5, respectively. An allele specific PCR (AS-PCR) was used to determine the frequency of a sodium channel mutation (F1550I, Phe→Ile) associated with pyrethroid resistance. Overall, 26.5%, 40.8% and 32.6% of tick populations were susceptible, tolerant and resistant to cypermethrin, respectively. A substantial inter-population variation in the level of cypermethrin response was evident (resistance factors ranged from 0.3 to 2599 and from 0.7 to >5000 when were indicated by the LC(50) and LC(99), respectively). The F1550I mutation (R allele) in R. microplus was present in all studied areas. The increasing presence of the R allele correlated well with increased levels of response indicated by both the LC(50) (r(2)=0.659, p=0.001) and LC(99) (r(2)=0.688, p=0.001) to cypermethrin. These results indicated that the F1550I mutation is a major common mechanism responsible for pyrethroid resistance in field populations of R. microplus ticks in the Mexican tropics. Both bioassay and AS-PCR showed that the prevalence of cypermethrin-resistant/tolerant R. microplus is high in Yucatan, Mexico and the relationship between the RF and the frequency of the R allele supports the role of F1550I as one of the most important mechanisms conferring pyrethroid resistance in these R. microplus populations.     

Possible risk factors on Queensland dairy farms for acaricide resistance in cattle tick (Boophilus microplus)

A case control study was carried out within a cross-sectional survey designed to investigate the management by Queensland dairy farmers of the cattle tick Boophilus microplus. Although 199 farmers were surveyed, data on acaricide resistance were only obtained from 66 farms. Multiple models were used to predict the probability of acaricide resistance associated with 30 putative risk factors. The region of the state in which the farm was located and the frequency of acaricide application were consistently associated with acaricide resistance. The risk of resistance to all synthetic pyrethroids (Parkhurst strain) was highest in Central Queensland and increased when more than five applications of acaricide were made in the previous year, when spray races were used and when buffalo fly treatments with a synthetic pyrethroid were applied frequently. The probability of resistance to amitraz (Ulam strain) was highest in Central Queensland, increased when more than five applications of acaricide were made in the previous year, and decreased on farms when a hand-spray apparatus was used to apply acaricides to cattle. The probability of resistance to flumethrin (Lamington strain) was highest in the Wide Bay-Burnett region.     

Survey of pyrethroid and organophosphate resistance in Brazilian field populations of Rhipicephalus (Boophilus) microplus: Detection of C190A mutation in domain II of the para-type sodium channel gene

The cattle tick Rhipicephalus (Boophilus) microplus causes expressive damage to livestock in Brazil and other countries. Its control is becoming more difficult due to the development of resistance in populations. Early detection of resistance can help in developing effective control strategies. This study evaluated the susceptibility of R. microplus to cypermethrin and chlorpyriphos and was the first attempt to identify the mechanism of resistance (target site insensitivity) in cattle tick populations from Minas Gerais state (Southeastern Brazil). Engorged female ticks were collected from 10 ranches within the state of Minas Gerais, and susceptibility was evaluated with the larval packet test (LPT) using technical grade cypermethrin and chlorpyriphos. It was possible to analyze LPT results of seven populations. Target site insensitivity was investigated in all 10 isolates by using molecular approaches for detection of the T2134A substitution within the domain III S6 segment and the C190A in the domain II S4-5 linker from the para-type sodium channel gene. LPT showed that all seven populations were resistant to cypermethrin with resistance ratio (RR) ranging from 16.0 to 25.0 and 85.7% were resistant to chlorpyriphos (RR=2.2-15.6). Although the T2134A mutation was not detected, the C190A mutation was highly prevalent, being present in 82-100% of the alleles sampled in field populations. A significant correlation was found between the LC50 values for cypermethrin and the frequency of the C190A mutation suggesting that it might be responsible for the phenotypic resistance detected.     

Compatibility of the fungus Metarhizium anisopliae and deltamethrin to control a resistant strain of Boophilus microplus tick

The tick Boophilus microplus causes economic impact to cattle producers and has a great capacity to develop resistance to chemical acaricides. It is very important the development of new techniques to complement the control of this parasitosis. Biological control is a promising option to maintain acceptable levels of tick populations in cattle. Therefore, the present study evaluated the association of deltamethrin and the entomopathogenic fungus Metarhizium anisopliae against B. microplus larvae resistant to pyrethroid. The synthetic pyrethroid, deltamethrin, was used at concentrations of 0.39, 0.78, 1.56, 3.12 and 6.12 ppm, M. anisopliae was used at concentrations of 10(5), 10(6), 10(7) and 10(8) conidia ml(-1). Their associations were also evaluated in in vitro tests. The assays showed that the tick strain is resistant to deltamethrin, but high mortality rates were observed when deltamethrin was associated with the entomopathogen. The larvae's mortality rates ranged from 7 to 36.5% for the different concentrations of deltamethrin, however, for the different concentrations of M. anisopliae, the mortality rates ranged from 10 to 96.9%. Mortality rates were proportional to the concentrations used for both deltamethrin and the fungus. Predominantly, the association of pyrethroid and fungus resulted in higher larvae mortalility rates than those obtained with the respective non-associated concentrations. Thus, the present study indicates that this association can be used as a tool for integrated control of the tick B. microplus.     

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

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

A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC(50)) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.     

Spatial distribution of acaricide profiles (Boophilus microplus strains susceptible or resistant to acaricides) in southeastern Mexico

The ability of Boophilus microplus strains to be susceptible (-) or resistant (+) to amidines (Am), synthetic pyrethroids (SP), and/or organo-phosphates (OP) (or acaricide profiles) was investigated in 217 southeastern Mexican cattle ranches (located in the states of Yucatán, Quintana Roo, and Tabasco). Three questions were asked: (1) whether acaricide profiles varied at random and, if not, which one(s) explained more (or less) cases than expected, (2) whether the spatial distribution of acaricide profiles was randomly or non-randomly distributed, and (3) whether acaricide profiles were associated with farm-related covariates (frequency of annual treatments, herd size, and farm size). Three acaricide profiles explained 73.6% of the data, representing at least twice as many cases as expected (P<0.001): (1) Am-SP-, (2) Am+SP+, and (3) (among ranches that dispensed acaricides > or = 6 times/year) Am-OP+SP+. Because ticks collected in Yucatán ranches tended to be susceptible to Am, those of Quintana Roo ranches displayed, predominantly, resistance to OP/SP, and Tabasco ticks tended to be resistant to Am (all with P < or = 0.05), acaricide profiles appeared to be non-randomly disseminated over space. Across states, two farm-related covariates were associated with resistance (P < or = 0.02): (1) high annual frequency of acaricide treatments, and (2) large farm size. Findings supported the hypothesis that spatial acaricide profiles followed neither random nor homogeneous data distributions, being partially explained by agent- and/or farm-specific factors. Some profiles could not be explained by these factors. Further spatially explicit studies (addressing host-related factors) are recommended.     

Acaricide resistance profiles of single- and multi-host ticks from communal and commercial farming areas in the Eastern Cape and North-West Provinces of South Africa

A field study (February 2000 to August 2001) was conducted on communal and commercial farms in the Eastern Cape and North-West Provinces of South Africa to detect the levels of tick resistance to commonly used acaricides. The larvae obtained from engorged females of the one-host tick Boophilus decoloratus, the two-host tick Rhipicepalus evertsi evertsi and the three-host ticks Amblyomma hebraeum and Rhipicephalus appendiculatus were tested against various concentrations of amitraz, chlorfenvinphos and cypermethrin using the Shaw Larval Immersion Test method. Ticks from the communal farms showed higher levels of resistance to cypermethrin and some resistance to chlorfenvinphos whilst no resistance was detected against amitraz. However, ticks from commercial farms were equally resistant to amitraz, chlorfenvinphos and cypermethrin. The B. decoloratus populations tested were considerably more resistant to all the acaricides tested than the R. evertsi evertsi, A. hebraeum and R. appendiculatus populations. This supports the hypothesis that single-host ticks develop resistance faster than multi-host ticks. This trend was recorded on most of the farms where single- and multi-host ticks co-existed. It was concluded that the use of acaricides at high frequencies and high concentrations was one of the main causes of tick resistance in the study areas. Possible factors which caused the resistance problems are discussed and acaricide management strategies recommended.     

The resistance spectrum shown by a fenvalerate-resistant strain of blue tick (Boophilus decoloratus) to a range of ixodicides

A strain of Boophilus decoloratus, resistant to fenvalerate, was subjected to larval immersion, adult immersion and stall tests using the following classes of ixodicides: organochlorines, organophosphates, a diamidide and pyrethroids. A susceptible reference strain of B. decoloratus was used for comparative purposes. The results indicated a high level of resistance to DDT and camphechlor, slight tolerance to dioxathion, chlorfenvinphos and pirimiphos ethyl, full susceptibility to bromophos ethyl and amitraz, but marked resistance to cyhalothrin, cypemethrin, deltamethrin and flumethrin. This marked resistance in the strain therefore appears to be widespread within the pyrethroid group of chemicals and may have developed as a result of organochlorine cross-resistance.     

Comparison of 3 tests to detect acaricide resistance in Boophilus decoloratus on dairy farms in the Eastern Cape Province,South Africa

The susceptibility of the larval offspring of engorged female Boophilus decoloratus, and of the engorged females, collected from cattle on the dairy farms Brycedale, Sunny Grove and Welgevind in the Eastern Cape Province, South Africa, was tested against the acaricides amitraz, chlorfenvinphos and cypermethrin. Resistance was determined by means of the Shaw Larval Immersion Test (SLIT) for larvae and the Reproductive Estimate Test (RET) and Egg Laying Test (ELT) for adults. At Brycedale the tests all indicated resistance to chlorfenvinphos, and RET and ELT indicated resistance to amitraz and emerging resistance to cypermethrin. At Sunny Grove, B. decoloratus was resistant to cypermethrin using SLIT and exhibited emerging resistance to chlorfenvinphos with SLIT and to cypermethrin with both RET and ELT At Welgevind, resistance was recorded against chlorfenvinphos (SLIT) and against cypermethrin (ELT), and emerging resistance against permethrin (RET). The results obtained with RET and ELT were generally comparable, but often differed from those obtained with SLIT. Resistance could be detected within 7 days with ELT compared to 42 days with RET and 60 days with SLIT.     

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

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

THE POTENTIAL OF SOME SYNTHETIC PYRETHROIDS FOR CONTROL OF THE CATTLE TICK (BOOPHILUS MICROPLUS)

As part of a continuing programme to aid in the development of new acaricides, the potentials of three synthetic pyrethroids, permethrin, cypermethrin and decamethrin, were assessed against a range of resistant strains of the cattle tick (Boophilus microplus). The compounds were shown to be acaricidal, with decamethrin the most effective, giving efficient control of susceptible and organo-phosphorus resistant strains of the cattle tick at concentrations as low as one fifth of those previously used for any commercial acaricide. However higher concentrations were required for satisfactory control of the DDT-resistant strain. Permethrin-selection of a field strain containing a low percentage of DDT-resistant ticks led to rapid development of resistance to a level equivalent to that of the DDT-resistant strain. Further selection increased resistance to permethrin to a higher level than that shown in the DDT-resistant strain. This increased resistance to permethrin appears to be restricted mainly to this compound and is not associated with a marked increase in resistance to cypermethrin, decamethrin or DDT. The potential of organo-phosphorus compounds to potentiate the pyrethroids when the two classes of compounds are used as mixtures against the pyrethroid-resistant and susceptible strains was confirmed.     

Prevalence and potential risk factors for amitraz resistance in Boophilus microplus ticks in cattle farms in the State of Yucatan, Mexico

Southern cattle tick, Boophilus microplus, control in the Mexican tropics is based on the application by spray, dip or pour-on of acaricides. In Mexico the first report of ticks resistant to amitraz was reported in 2002. Amitraz resistance affecting the cattle industry of Yucatan State, Mexico, has not been investigated. A cross-sectional study was carried out in order to investigate the possible association between management risk factors and the prevalence of B. microplus ticks resistant to amitraz in Yucatan, Mexico. Ninety-eight cattle farms were randomly selected and a sample of 20–30 engorged B. microplus females was collected from at least 10 cattle from each farm. Collected ticks were taken to the laboratory where the larval progeny were obtained. The modified larval immersion test was used to detect ticks resistant to amitraz. A discriminating dose of 0.0002% amitraz was used. A questionnaire was applied on each collaborating farm. For the statistical analysis a 2 × 2 contingency table was used to identify potential risk factors. The prevalence of farms with B. microplus resistant to amitraz was 19.40%. However, even though tick resistance to amitraz is a present problem there was no evidence that it was associated with the risk factors analyzed in this survey.     

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

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

A SURVEY FOR RESISTANCE IN CATTLE TICKS TO ACARICIDES

SUMMARY: A survey was made from late 1976 to late 1977 to determine the extent of resistance to acaricides in the cattle tick Boophilus microplus in Queensland. Questionnaires and requests for samples of ticks were forwarded to more than 900 randomly selected stock owners in the tick infested area which had been divided into 4 regions, Far North, Coastal North, Coastal Central and South East. The response measured by the number of tick samples tested was 43%. The prevalence of resistance to organophosphorus (OP) compounds was highest in South East where 96% of the farms had OP-resistant ticks and 95% had the Biarra strain and lowest in Far North where 12% of the farms had OP-resistant ticks and 10% had the Biarra strain which was the predominant one in all regions. The highest percentages of the Ridgelands strain (35%) and the Tully strain (30%) occurred in Coastal North. The South East had the highest percentage of Mt Alford (30%).
Although chlorinated hydrocarbons were banned for use in control of ticks in 1962, 49% of the farms in South East had some ticks resistant to dieldrin but in Far North it was only 2%. DDT-resistant ticks, which because of cross resistance to synthetic pyrethroids will affect the future use of this group, were present on 8% of the farms in both Coastal North and Coastal Central and 3% in South East and Far North.
No resistance to the amidines, chlordimeform, chloromethiuron or amitraz was found.